Oral Presentations - Arteriosclerosis, Thrombosis, and Vascular ...

3rd Annual Conference on Arteriosclerosis, Thrombosis and Vascular Biology
Arterioscler Thromb Vasc Biol. 2002;22:878
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C-Reactive Protein Is an Independent Predictor of Risk for the
Development of Diabetes Mellitus in the West of Scotland Coronary
Prevention Study
Dilys J Freeman, John Norrie, Muriel J Caslake, Allan Gaw, Ian Ford, Gordon D Lowe, Denis
S O’Reilly, Chris J Packard, Naveed Sattar. University of Glasgow, Glasgow, UK
Accumulating evidence implicates inflammation as a potential pathway in the pathogenesis of
type II diabetes. The objective of the present study was to assess the ability of C-reactive
protein (CRP) to predict the development of diabetes mellitus in the West of Scotland Coronary
Prevention Study (WOSCOPS). Baseline plasma samples for CRP measurement were available
for 5245 men of which 127 were classified, based on the American Diabetes Association (ADA)
definition of diabetes, as having a transition from normal glucose control to overt diabetes
mellitus during the study. Baseline CRP was an important predictor of the development of
diabetes mellitus in univariate analysis (hazard ratio (HR) for an increase of one standard
deviation, 1.55, 95% confidence intervals (CI) 1.32, 1.82, P0.0001). In multivariate analysis,
CRP remained as a predictor of diabetes development (HR 1.30, 95% CI 1.07, 1.58, P0.008)
independent of other predictors including baseline body mass index, fasting triglyceride and
glucose concentrations. When examined by quintiles, the highest CRP quintile was associated
with a greater than three-fold risk of developing diabetes (HR 3.10, 95% CI 1.34, 7.18) in a
multivariate analysis. We show for the first time that CRP predicts the development of type II
diabetes in middle-aged men independent of established risk factors. As CRP is very stable in
serum and the most commonly used acute phase protein in clinical practice, our observations
have clinical potential in helping to better predict individuals destined to develop type II
diabetes. They also support a role for inflammation in the pathogenesis of type II diabetes.
2
Severe Hyperlipidemia in Leptin Receptor Deficient Mice on a Background
of LDL-R or ApoE Deficiency
Alyssa H Hasty, Hitoshi Shimano, Macrae F Linton, Sergio Fazio. Vanderbilt University,
Nashville, TN; Tsukuba University, Tsukuba, Japan
Obesity, insulin resistance, and diabetes, together with hypercholesterolemia are characteristic
features of the metabolic syndrome. Leptin deficient (ob/ob) and leptin receptor deficient
(db/db) mice have long been used as models of obesity and diabetes; however, models of the
metabolic syndrome have not yet been fully developed. We have previously shown an
unexpectedly severe hyperlipidemia in ob/ob mice on a background of low density lipoprotein
receptor (LDLR) deficiency (-/-). Doubly mutant mice exhibited striking elevations in both total
cholesterol and triglyceride levels (1715 87 and 1016 172 mg/dl, respectively), at ages
3–4 months, resulting in extensive atherosclerotic lesions throughout the aorta by 6 months.
In an attempt to create a model of dyslipidemia in obese/diabetic mice that are leptin resistant
(a characteristic of human obesity) rather than leptin deficient, we have crossed db/db mice
with both LDLR-/- mice and apoE-/- mice. In the current study, we show that db/db mice on
an LDLR-/- background have a similar phenotype to the ob/ob;LDLR-/- mice with total
cholesterol levels of 753 49 mg/dl and triglyceride levels of 587 75 mg/dl at 8 weeks of
age. Interestingly, db/db mice on an apoE-/- background had cholesterol levels similar to those
on an LDLR-/- background; however, triglyceride levels were significantly lower (169 mg/dl).
The triglycerides in both groups were carried on VLDL particles; however cholesterol was only
on VLDL/IDL in the db/db;apoE-/- mice and was primarily on LDL in the db/db;LDLR-/- mice,
as demonstrated by FPLC analysis. Similarly, agarose gel electrophoresis of serum from these
mice show that both groups of mice contained pre- lipoprotein particles, but only the
db/db;LDLR-/- mice had particles. In conclusion, the over-production of triglyceride-rich
lipoprotein particles due to the obesity and diabetes related to leptin receptor deficiency,
together with impaired lipoprotein catabolism due to LDLR or apoE deficiency, results in an
extreme accumulation of atherogenic lipoproteins in a model that models human metabolic
syndrome.
Increased Whole Body Insulin-Stimulated Glucose Uptake, but Hepatic
Insulin Resistance in CD36-Deficient Mice
Jeltje R Goudriaan, Maria Febbraio, Bas Teusink, Johannes A Romijn, Louis M Havekes,
Peter J Voshol. TNO Prevention and Health, Leiden, Netherlands; Division
Hematology/Oncology, Cornell University, New York, NY; Dept. Endocrinology and Diabetes,
Leiden University Medical Centre, Leiden, Netherlands
Objectives: CD36 or fatty acid translocase (FAT) is involved in high affinity fatty acid (FA)
uptake, mainly in tissues involved in FA turnover like muscle (skeletal and heart), adipose tissue
and intestine. Furthermore, CD36 was shown to be linked to the insulin resistance syndrome
in the spontaneous hypertensive rat (SHR) which has shown to have no CD36 expression.
Methods: We measured insulin sensitivity by hyperinsulinemic, euglycemic clamp in CD36deficient
mice. Findings: Whole body insulin-mediated glucose uptake was significant higher,
108 16 vs. 81 16 mol/min.kg, in CD36-deficient vs. control mice, respectively (n5 per
group, p0.03). Interestingly, the liver of CD36-knockout mice was insulin resistant as
determined by an inability to reduce in endogenous glucose production 0% compared to 40%
in control mice. Furthermore, liver triglyceride (TG) content was significantly higher in
CD36-deficient vs. control mice, 20.4 0.2 vs. 14.2 0.3 g/mg protein, respectively, (n6
per group, p0.002). Conclusion: Decreased peripheral FA uptake in CD36 deficient mice leads
to increased whole body glucose utilization. Interestingly, increased FA flux to the liver in these
mice leads to increased liver TG content and liver-specific Downloaded insulin resistance. from
Oral Presentations
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Oral Presentations a-1
Role of SLP-76 in the Platelet Procoagulant Response to Collagen
Lorie Leo, Jorge Di Paola, Barbi A Judd, Gary A Koretzky, Steven R Lentz. University of
Iowa, Iowa City, IA; University of Pennsylvania, Philadelphia, PA
The adapter protein SLP-76 is a critical downstream mediator of signal transduction via the
platelet collagen receptor GPVI and its co-receptor FcR. Platelets from SLP-76-deficient mice
fail to undergo aggregation or granule release in response to collagen or the GPVI agonist
convulxin. We tested the hypothesis that SLP-76 is required for collagen-induced expression of
procoagulant activity in murine platelets. Washed platelets from SLP-76 null (SLP76-/-) or
heterozygous (SLP76/-) mice were activated with convulxin (144 ng/ml), thrombin (0.5 U/ml),
or ionomycin (3 uM), and surface expression of P-selectin (a marker of granule release) and
annexin V binding (a marker of procoagulant phospholipid) were determined by flow cytometry.
Aggregation was measured by optical aggregometry. As expected, convulxin induced platelet
aggregation and increased surface expression of P-selectin in SLP76/- platelets, but not in
SLP76-/- platelets (p0.01). In contrast, convulxin failed to stimulate annexin V binding to
either SLP76/- or SLP76-/- platelets. Thrombin and ionomycin stimulated surface expression
of P-selectin and annexin V binding in both SLP76/- and SLP76-/- platelets (p0.05 vs.
unstimulated platelets). Platelet procoagulant activity was measured in a prothrombinase
assay. Washed platelets were activated for 10 minutes with type I fibrillar collagen (40 ug/ml),
convulxin, or thrombin, followed by the addition of factor Va (6 nM), factor Xa (3 nM), and
prothrombin (4 uM), and the rate of thrombin generation was measured using Chromozym TH.
Collagen produced a 2-fold increase in procoagulant activity in both SLP76/- and SLP76-/platelets
(p0.001 vs. unstimulated platelets), but convulxin failed to stimulate detectable
procoagulant activity in either SLP76/- or SLP76-/- platelets. Thrombin produced a moderate
(1.2-fold) increase in procoagulant activity in both SLP76/- and SLP76-/- platelets (p0.05
vs. unstimulated platelets). These findings demonstrate that SLP-76 is not required for
collagen-induced procoagulant activity in murine platelets. Our results indicate that receptors
other than GPVI are involved in the platelet procoagulant response to collagen.
Developmental Expression of Functional Zebrafish Cyclooxygenases
Tilo Grosser, Shamila Yussuf, Ellina Cheskis, Barbara Pini, Michael A Pack, Garret A
Fitzgerald. Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia,
PA
The zebrafish is an informative vertebrate model for genetic studies, such as large-scale
mutagenesis and phenotypic screening. As study of the cardiovascular biology of cyclooxygenases
(COXs) has been limited by the critical role of COX-2 in murine reproduction and renal
organogenesis, we hypothesized that zebrafish might afford a model system in which COX
function and genetics may be investigated. The zebrafish (z) COX-1 and -2 orthologs were
cloned and showed 67% homology with the human isozymes. Prostaglandin E 2 was identified
as the predominant zCOX product by mass spectrometry and was inhibited in vivo by COX
inhibitors. The COX-2 selective compound, NS-398, was a more effective inhibitor of expressed
zCOX-2 than of zCOX-1. Zebrafish thrombocytes, like human platelets, appear to express only
COX-1. Aggregation and hemostasis were inhibited by indomethacin, a nonselective NSAID, but
not by NS-398. In contrast to extensive vascular expression of zCOX-1, vascular expression of
zCOX-2 was restricted to the carotid, pharyngeal and intestinal vasculature. Both genes were
expressed maternally in oocytes. Injection of zCOX-1 morpholino antisense oligos (100 fmoles)
into one cell stage embryos resulted in early developmental delay and growth arrest of 66
13 % embryos in shield stage (n 5; total of 231 embryos), while missense and anti-zCOX-2
morpholinos did not perturb development by morphological criteria. This effect was dose
dependent (5 to 500 fmoles) and could be rescued by simultaneous injection of zCOX-1 capped
RNA (100 pg). In summary, the vascular expression of both zCOX isozymes, the apparent
recapitulation of their human pharmacology, and the potential for gene inactivation all suggest
that zebrafish may be useful for study of this complex system.
Nitration of the Tyrosine-Based Inhibitory Motif of PECAM-1 Promotes
Binding of the Protein Tyrosine Phosphatase, SHP-2
Debra K Newman, Sara L Hoffman, Srigiridhar Kotamraju, Balaraman Kalyanaraman, Peter J
Newman. Blood Research Institute, Milwaukee, WI; Medical College of Wisconsin,
Milwaukee, WI
Peroxynitrite (ONOO-) is generated at sites of inflammation by cells that simultaneously produce
high levels of superoxide anion and nitric oxide. ONOO- reacts with tyrosine residues in proteins
to produce 3-nitrotyrosine (3-NT), which is detectable immunologically in certain diseased
tissues. 3-NT residues are poor substrates for phosphorylation by protein tyrosine kinases
(PTK), suggesting that signal transduction pathways that depend upon reversible tyrosine
phosphorylation for signal propagation might be impaired by tyrosine nitration. Platelet
Endothelial Cell Adhesion Molecule (PECAM)-1 is an inhibitory receptor with a cytoplasmic
tyrosine-based inhibitory motif (TIM). The PECAM-1 TIM, when phosphorylated at Y663 and Y686, binds to and activates the Src homology (SH) 2 domain-containing protein tyrosine phosphatase,
SHP-2. Our objective was to assess the impact of tyrosine nitration on phosphorylation
of the PECAM-1 TIM and its subsequent ability to bind SHP-2. Exposure of PECAM-1 to ONOOin
vitro resulted in its tyrosine nitration. In vitro kinase assays, using synthetic peptides
spanning PECAM-1 TIM tyrosines at position 663 or 686, confirmed previous observations that
1) both Y663 and Y686 are substrates for Src family PTK-mediated phosphorylation, and 2) Y686 is a betterby substrate guest than on April is Y663. 4, The2013 presence of 3-NT at either Y663 or Y686 blocked their
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a-2 Arterioscler Thromb Vasc Biol. Vol 22, No 5
tyrosine phosphorylation, demonstrating that nitration of PECAM-1 TIM tyrosines inhibits their
phosphorylation. In peptide-precipitation analyses, we found that both a GST fusion protein
containing the SH2 domains of SHP-2 (GST-SH2-SH2) and full-length SHP-2 bound to the
peptide containing 3-NT at Y 663, whereas neither GST-SH2-SH2 nor SHP-2 bound to the peptide
containing 3-NT at Y 686. These results are the first to demonstrate that 3-NT residues can, in
the appropriate amino acid context, serve as a docking site to recruit SH2 domain-containing
proteins. We propose that tyrosine nitration may provide an additional mechanism for
modulating the formation and function of phosphotyrosine residues in PTK-dependent signal
transduction pathways.
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Myocardin Is Expressed in Adult Aorta and Activates a Smooth Muscle Cell
Differentiation Program
Joseph M Miano, Chad M Kitchen, Jiyuan Chen, Da-Zhi Wang, Eric N Olson. University of
Rochester Medical Center, Rochester, NY; University of Texas Southwestern Medical Center,
Dallas, TX
Serum response factor (SRF) is a critical regulator of smooth muscle cell (SMC)-restricted gene
expression via its interactions with CArG elements. Several SMC-restricted genes are
attenuated or extinguished upon cell culture. However, levels of SRF protein do not decrease
from intact aorta to cultured SMC suggesting additional factors that may be down-regulated in
vitro are required for directing the genetic program of SMC differentiation. We have cloned
myocardin, a novel SRF co-activator, from rat aortic tissue. Levels of myocardin are abundantly
expressed in rat aortic media along with key SMC-restricted genes (e.g., SMMHC and
SM-Calponin). In cultured rat aortic SMC (RASMC), myocardin mRNA is virtually extinguished
as is the expression of SMMHC and SM-Calponin. To test the hypothesis that myocardin directs
a SMC differentiation program, we performed co-transfection experiments with CMVmyocardin
and various SMC promoters (SMMHC, SM-calponin, SM alpha actin, and SM22)
linked to a luciferase reporter gene. In general, myocardin activates SMC promoters greatest
in cells that exhibit little or no expression of the corresponding endogenous SMC-restricted
gene. For example, the SM22 promoter is activated several hundred-fold in Cos-7 cells (SM22
negative), but only a few fold in several SMC lines that express high levels of endogenous
SM22. Notably, however, while myocardin activates the SM-calponin promoter/intron 10-fold
in PAC1 SMC and RASMC, and does so in a CArG box-dependent manner, the same promoter
construct is minimally activated by myocardin in Cos-7 cells, which do not express
SM-calponin. Overall, SM22 and SM alpha actin, which have similarly arranged CArG boxes in
their 5’ promoter regions, are most responsive to activation by myocardin. Importantly, the
endogenous SM22 gene is turned on in 10T1/2 cells stably transfected with myocardin. These
results highlight promoter- and cell context-dependent activity of myocardin and suggest that
this SRF co-activator may be an important component of a molecular switch for the SMC
differentiation program.
Identification of a Novel Family of Secreted Proteins Highly Expressed in
Human Vascular Endothelium
Ruey-Bing Yang, Chi Kin Domingos Ng, James E Tomlinson, Laszlo G Kumuves, James N
Topper. COR Therapeutics, Inc., South San Francisco, CA
Vascular endothelial cells (EC) play a key role in a variety of physiologic and pathophysiologic
processes, such as angiogenesis, inflammation, cancer metastasis and the development of
vascular diseases. As a strategy to identify all genes expressed in human EC, large-scale EST
sequencing and expression profiling approaches were performed in human vascular EC
cultured under various stimuli (flow, cytokine, angiogenic, etc). One full-length cDNA identified
by these approaches encoded a potential secreted protein harboring a signal peptide at the
N-terminus followed by ten EGF-like domains and one CUB domain at the C-terminus (termed,
SCUBE1 Signal peptide-CUB-EGF-like domain containing protein 1). Northern and microarray
analyses demonstrate that SCUBE1 is expressed in several highly vascularized tissues such as
liver, kidney, lung, spleen and brain. The endothelial-selective pattern of expression for SCUBE1
was further confirmed by in situ hybridization on these tissues. We characterized the SCUBE1
gene product by using a transient expression system in human kidney embryonic 293 cells.
Overproduction in these cells resulted in expression of SCUBE1 protein in the conditioned
medium. Flow cytometry and immunofluorescence analyses showed that this protein could
bind to and be displayed on the cell surface. Analyses of several deletion mutants of the
SCUBE1 protein revealed that the spacer region between EGF-like and CUB domains is critical
for its secretion and cell-surface association. Furthermore, expression of SCUBE1 is depressed
in EC after IL-1 and TNF- treatment, suggesting a possible role of SCUBE1 in the
inflammatory response. A second gene encoding a homologue (designated as SCUBE2) was
also identified and appears to be expressed in an EC-specific fashion. When overexpressed,
SCUBE1 and SCUBE2 can manifest homo- and heterotypic interactions. These results indicate
that SCUBE1 and SCUBE2 define an emerging secreted protein family that is highly expressed
in human vascular endothelium.
8
component. NPAS2 is thought to be a paralogue of CLOCK, both can heterodimerize with
BMAL1 activating per and cry expression through consensus E box elements. We sought to
elucidate the mechanism by which CLOCK/NPAS2:BMAL1 heterodimers activate transcription
in the vascular clock. Co-immunoprecipitation and GST pull-down experiments demonstrate
that CLOCK, BMAL1 and NPAS2 can complex with the histone acetyltransferases p300, CBP
and PCAF. In vivo immunocytostaining reveals that p300, PCAF, CLOCK, BMAL1 and NPAS2
exhibit nuclear and perinuclear localization in hVSMC and COS-7 cells. In addition, nuclear
co-localization of CLOCK or NPAS2 with p300 was detected by dual immunoflouresence overlay
analysis. Furthermore, transactivation assays in NIH3T3 cells reveal that CLOCK:BMAL1 and
NPAS2:BMAL1 dependent E box activation is markedly enhanced in the presence of CBP and
PCAF. Importantly, histone acetylation regulatory proteins such as E1A, TWIST and the novel
histone masking complex, INHAT, inhibited basal E box mediated transcriptional activation by
CLOCK/NPAS2:BMAL1. These findings suggest that E box activation by the bHLH-PAS proteins,
CLOCK/NPAS2:BMAL1 is a HAT dependant mechanism. Blood pressure and fibrinolytic activity
exhibit a marked circadian variability, this coincides with a temporal variability in the incidence
of acute vascular events, such as myocardial infarction, sudden cardiac death and stroke. Thus
understanding how the vascular clock is regulated will be potentially important in both vascular
physiology and in clinical vascular events.
The ATP Cassette Transporter ABCA1 Regulates Monocyte Spreading,
Extravasation and Raft Formation
Gerd Schmitz, Wolfgang Drobnik, Gerhard Liebisch, Alexandra Pfeiffer, Alfred Boettcher,
Hanna Borsukova. Institute of Clinical Chemistry and Laboratory Medicine, University of
Regensburg, Regensburg, Germany
Recently, our laboratory identified ABCA1 as a central regulator of macrophage function.
Defects in ABCA1 are responsible for genetic HDL-deficiency syndromes, that are characterized
by impaired apo AI and HDL mediated cholesterol and phospholipid efflux as well as aberrant
macrophage differentiation and extravasation. In this study we have shown that cholesterol
efflux induced by apo AI or HDL3 inhibits filipodia formation in human monocytes by a process
that involves down regulation of CDC42 and other filopodia associated genes. Cholesterol
loading with E-LDL had the opposite effect and ABCA1 deficient cells were characterized by an
abnormal cytoskeleton, decreased CDC42 expression and impaired cell spreading. Goldlabelled
HDL preferentially clustered on membrane protrusions and outside of coated pits in
monocytes. In order to further investigate the relation between apo AI induced lipid efflux and
filipodia formation, we analyzed the influence of apo AI on specific lipid microdomains, called
Lubrol rafts, which may function as building units of different kinds of membrane protrusions.
In monocytes either Triton X-100 or Lubrol WX detergent resistant membranes (DRM) were
associated with the following findings: (1) Apo AI mediated cholesterol and phospholipid efflux
was exclusively derived from membrane domains that fulfil the criteria of the recently described
“Lubrol-rafts”, as they were soluble in Triton X-100 but not in Lubrol WX. (2) ABCA1 and CDC42
were present in Lubrol- but not in Triton-rafts. (3) HDL3 induced an additional cholesterol efflux
that was derived from Triton X-100 DRM, and the size of these rafts in vital monocytes was
reduced by HDL3 as assessed by epifluorescence using the raft specific stain PE-Cy5. In
contrast, cholesterol loading resulted in the formation of large confluent rafts. In summary,
these data show that different kinds of lipid rafts are affected by apo AI and HDL3 induced lipid
efflux, which likely account for the influence of these anti-atherogenic molecules on
differentiation, migration and extravasation of monocytic cells.
Diet-Induced Hyperlipidemia Causes Pulmonary Death and Accelerated
Atherosclerosis in ApoE-Null Mice Deficient in 3 Integrin
Laura Zemany, Kara Standley, Sherry Weng, Trey Coleman, Steven L Teitelbaum, Clay F
Semenkovich. Washington University School of Medicine, St. Louis, MO
Dissecting the Vascular Clock: CLOCK/NPAS2:BMAL1 Transcriptional
Activation is Regulated by the Histone Acetyltransferases p300/CBP and
9
The short term use of intravenous inhibitors of platelet integrins is useful in acute coronary
syndromes. However, chronic treatment with oral integrin inhibitors appears to promote
mortality, suggesting that inhibition of some non-platelet integrins may be detrimental. To
address the role of the 3 integrin in diet-induced vascular disease, we crossed apoE-null mice
with 3 integrin-null mice, a model for the platelet disorder Glanzmann thrombasthenia.
Ninety-six% of the 3/apoE-/- mice (110/115) survived to be weaned to a chow diet, while
only sixty-two% of the 3-/-apoE-/- mice (32/52) survived the high fat feeding associated with
suckling (P0.0001 vs 3/apoE-/-). Western diet feeding had no genotype-specific effects
on serum lipids (mean cholesterol 1096 in 3-/-apoE-/- vs 1203 in 3/apoE-/-,
P0.184). However, after 6 weeks of Western diet feeding, only 11 of 29 3-/-apoE-/- mice
were alive compared to 47 of 49 3/apoE-/- mice (P0.0001). Autopsies in 8
3-/-apoE-/- mice identified non-infectious pneumonia as the cause of death. Chow feeding
PCAF
for 6 weeks did not cause mortality. Western diet feeding for 6 weeks to 3-/- and 3/
littermates that were not apoE-null did not cause mortality. For mice in the apoE-null
Anne M Curtis, Sang-Beom Seo, Debabrata Chakravarti, Garret A Fitzgerald, Peter
background surviving Western diet feeding, atherosclerosis was 2.6 fold greater at the aortic
McNamara. Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, arch, 3.3 fold greater at the thoracic aorta, and 6.1 fold greater at the abdominal aorta in
PA
3-/-apoE-/- (n11) as compared to 3/apoE-/-(n19) littermates (all P0.0001).
These results show that accelerated diet-induced atherosclerosis can occur in mice with a
We have recently characterized a circadian clock in blood vessels. This oscillator, in addition well-defined platelet defect. They also suggest that the lack of 3 integrin may provoke
to possessing all of the components necessary to function as an autonomous peripheral lipid-induced inflammation in both the lung and the vessel wall, raising the possibility that 3
oscillator, utilizes the bHLH-PAS transcription factor Downloaded NPAS2 (MOP4) from
as ahttp://atvb.ahajournals.org/ novel core clock integrin has by beneficial guest on effects April in specific 4, 2013 tissues.
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Effects of PPAR Agonists in Apolipoprotein E-Deficient Mice Treated with
Angiotensin II
Doris M Tham, Baby Martin-Mcnulty, Yi-Xin Wang, Ronald Vergona, Mark E Sullivan, John C
Rutledge. UC Davis, Davis, CA; Berlex Biosciences, Richmond, CA
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear transcription
factors that represent a potential biological link between insulin resistance and atherogenesis.
Fibrates and thiazolidinediones, drugs commonly used to treat individuals with diabetes, are
ligands for the receptors, PPAR- and PPAR-, respectively. To examine the effects of PPAR-
or PPAR- activation on atherogenesis, we administered a diet containing either fenofibrate
(400 mg/kg/day) or rosiglitazone (20 mg/kg/day) for 30 days to male apolipoprotein E-deficient
(apo E-KO) mice that were infused with angiotensin II (Ang II, 1.44 mg/kg/day) to accelerate
atherosclerosis and induce aneurysm formation. Both PPAR-agonist treatments up-regulated
PPAR- and PPAR- mRNA expression and down-regulated monocyte chemotactic protein-1
(MCP-1), macrophage-colony stimulating factor (M-CSF), endothelial-selectin (E-selectin),
intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1)
mRNA expression in the arch, thoracic, supra-renal and infra-renal aorta. Both fenofibrate and
rosiglitazone significantly increased serum total cholesterol (TC), low density lipoproteins (LDL),
and triglycerides (TG) (p0.01). Accompanied by the changes in gene expression and
lipoprotein levels, there was also an increase in aneurysm size and atheroma formation with
fenofibrate treatment and a slight decrease with rosiglitazone treatment. These results suggest
that fenofibrate and rosiglitazone potentially have direct beneficial vascular effects as indicated
by anti-inflammatory and anti-atherogenic changes in gene regulation. However, the metabolic
effects that increased lipoproteins are associated with increased atherogenesis in the
fenofibrate-treated group. Further studies are needed to determine the balance between the
pro- and anti-atherogenic properties of PPAR activation.
Bone Marrow-Derived Endothelial Progenitors Cells Contribute to the
Recanalization and Resolution of an Existing Thrombus
Oren M Tepper, Toshinori Murayama, Marcy Silver, Marianne Kearney, Allison Hanley,
Jeffrey M Isner, Takayuki Asahara, Christoph Kalka. New York University School of
Medicine, New York, NY; St. Elizabeth’s Medical Center, Boston, MA
Objective: With increasing evidence that bone-marrow derived endothelial progenitor cells
(EPCs) contribute to neovascularization, we investigated whether EPCs play a role in thrombus
recanalization and resolution. Methods: Bone marrow (BM) cells from transgenic mice
constitutively expressing -gal under the regulation of an endothelial promoter (Tie-2) were
transplanted to irradiated athymic rats (n14). At 4 weeks post-BM transplantation, venous
thrombosis was induced by placing a vascular clamp on the inferior vena cava (IVC).
Thrombosis was easily reproduced in this model, and the clamp was removed 48 hours after
placement to restore blood flow. Localization of EPCs was identified by x-gal staining of the
tie-2/lacZ fusion transcript and counter-immunostaining with the endothelial marker CD31. To
determine the potential of EPC transplantation to enhance thrombus resolution, an additional
group of athymic rats (n12) underwent identical thrombus induction followed by injection of
either human-derived EPCs or media on days 2 and 4 post surgery. Results: BMT animals
revealed a large number of vascular channels within the thrombus, identified by positive
staining for endothelial marker CD31, which progressed over time. BM-derived EPCs were
found as early as 7 days and were capable of differentiating into mature endothelial cells lining
the vascular channels through the thrombus. BM-derived EPCs appeared in autolytic slits and
clefts in the fibrinous areas of the thrombi and also in the adventitia suggesting the migration
of EPCs from the vasa vasorum. At later time points, spindle-shaped, x-gal positive cells that
possessed morphological and immunohistological properties of mature endothelial cells lined
the channels. The treatment groups revealed that EPC therapy restores normal blood flow to
a greater extent than controls (91.2% at day 7 vs 76.4%) Conclusion: These results show that
BM-derived EPCs contribute to neovascularization in an experimental model of venous
thrombosis. In addition, systemically delivered EPCs incorporate into the vascular channels of
a thrombus and promote resolution.
14
The Inflammatory Cytokine Response of Mice Carrying the Factor V Leiden
Mutation is Regulated by Increased Production of Activated Protein C
Bryce A Kerlin, Brian Cooley, Joann Johnson, Sharon Fehrmann, Mark Zogg, Hartmut
Weiler. Medical College of Wisconsin, Milwaukee, WI; The Blood Center, Milwaukee, WI;
Blood Research Institute of The Blood Center, Milwaukee, WI
Objective: We previously described a transgenic mouse carrying a mutated thrombomodulin
(TM) gene that results in decreased TM-dependent activation of Protein C (PC). These mice are
also more susceptible to endotoxin challenge. We hypothesized that mice carrying the Factor
V Leiden (FVL) mutation would generate supra-normal levels of activated PC (APC) resulting in
down-regulation of the inflammatory response to endotoxin challenge. Methods: For APC and
Thrombin-Antithrombin complex (TAT) measurements a mixture of human thrombin (50mUnits)
and human PC (20g) was injected into the femoral vein and plasma was obtained after ten
minutes. APC and TAT levels were determined using an enzyme capture assay with a human
APC specific mAb and the Enzygnost TAT micro assay, respectively. For cytokine analysis,
plasma was collected two hours after intra-peritoneal endotoxin (5g/gm, E. coli O55:B5;
Sigma) injection. Cytokine concentrations were determined using Quantikine M mouse
immunoassays. Results: APC and TAT levels were significantly higher in mice carrying the
homozygous FVL mutation as compared to wild type (15.22.2 v. 7.92.2ng/mL, p 0.008
and 18.210.7 v. 1.71.4g/L, p 0.03 respectively). Production of the inflammatory
cytokine IL-6 was diminished in FVL mice (42.79.1 v. 59.94.3 ng/mL, p 0.01) following
endotoxin challenge. Production of IL-1 and TNF (67.619.1 v. 82.016.4 pg/mL and
26.37.4 v. 28.04.4 ng/mL respectively) wereDownloaded not significantly from
changed by the mutation.
13
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Conclusions: The mouse FVL mutation results in augmented production of APC due to
unregulated thrombin generation (demonstrated by increased levels of circulating TAT). This
endogenous augmentation of APC production appears to regulate the inflammatory response by
reducing levels of IL-6. Although not statistically significant at two hours, IL-1 production is
also slightly decreased and may be of significance at later time points. The TNF response
does not appear to be altered by this mutation. Further studies of these mice may demonstrate
other physiologic advantages conferred by FVL in an endotoxin model of systemic sepsis.
Gene Expression Profiles during Human Atherogenesis
Birgit Faber, Donald Dunbar, Darcey Black, Chris Evelo, Mat Daemen, Kitty Cleutjens.
CARIM, Maastricht, Netherlands; NV Organon, Newhouse, UK
Data on dynamic gene expression during progression of human atherosclerosis is limited. In the
present study we performed large scale expression profiling on early, advanced but stable and
ruptured atherosclerotic plaques. mRNA isolated from a pool of early stage plaques (n3) and
ruptured plaques (n3) was compared to the mRNA of stable plaques (n3) using a
micro-array cDNA chip that contained sequences coding for 8,000 genes (Human Unigene 1,
Incyte genomics Inc.). Using a threshold of 1.8-fold change in expression, we found 105 genes
upregulated and 88 genes downregulated during the early phase of atherogenesis as compared
to stable plaques. In addition, 65 genes were downregulated after plaque rupture, whereas
expression of 79 genes was enhanced after rupture of a plaque. Expression and functional
clustering resulted in the identification of several groups. Integrins and adhesion molecules
were upregulated in stable plaques. E-selectin, bone sialoprotein and osteopontin were
upregulated (3.1, 2.7, 12.7x) in stable plaques as compared to early lesions. Integrin -8,
activated leukocyte cell adhesion molecule and osteopontin showed an increase of expression
(1.9, 2.4, 3.0x) in stable plaques as compared to ruptured plaques. Phospholipase A2 group IIA
was a prominent member of the genes involved in lipid metabolism. This gene was 13 fold
downregulated during plaque progression and, as expected, upregulated (9x) after plaque
rupture. Egr1 was 2.4 fold upregulated during plaque progression and expression was further
enhanced (2.7x) after plaque rupture. Furthermore, we found over 50 EST fragments that were
differentially expressed during atherogenesis. In this study we identified a number of
functionally related groups of genes which are differentially expressed during human
atherogenesis. This set of genes may provide a better insight to the processes involved in
plaque progression and plaque rupture.
The Absence of TNF Reduces Neointimal Progression in Genetically
Modified Mice
Lena Brånén, Paul Dimayuga, Jan Nilsson, Stefan Jovinge. Department of Medicine, Malmö,
Sweden; Division of Cardiology, Los Angeles
TNF is a potent proinflammatory cytokine earlier shown to increase the extent of restenosis
in wildtype mice. This study was designed to evaluate TNF’s role in a restenotic model with
a background prone to develop atherosclerosis. Bilateral cuff-injury of the common carotid
arteries was performed on genetically modified mice. The study comprised 4 groups (n3) of
genetically modified female mice, 25 weeks of age. As a model of proatherosclerotic
environment apoE -/- mice were compared to tnf -/- apoE -/- mice. As a control and model of
nonatherosclerotic environment tnf -/- was compared to wildtype in the same injury model. The
arteries were collected three weeks after intervention, fixed in Histochoice TM and embedded in
O.C.T TM compound prior to cryosectioning. Sections were stained with haematoxylin and eosin
and external elastic lamina, internal elastic lamina, lumen, media and intima were measured
and evaluated by computer. Neointimal thickening (tnf -/- apoE -/- vs apoE -/- ,p0.026) and
medial thickening (tnf -/- apoE -/- vs apoE -/- ,p0.0087) were significantly less in the tnf -/apoE
-/- than in the apoE -/- whereas the intima/media ratio was not significantly changed
(tnf -/- apoE -/- vs apoE -/- , p0.093). In the nonatherosclerotic environment neointima was
significantly inhibited (tnf -/- vs wildtype, p0.0022) while no difference was seen in media
thickness and thus I/M ratio was significantly changed. The increased neointimal thickening
seen in apoE -/- compared to wildtype is counteracted by depletion of TNF. Thus tnf -/- apoE -/mice
has significantly less neointimal thickening than apoE -/- . Changes noted in the media
mimicked the changes in the intima when the different genetical backgrounds were compared.
Neotintimal thickening is inhibited when TNF is depleted in atherosclerotic as well as
non-atherosclerotic mice while medial thickening only inhibited when TNF is knocked out in
an atherosclerotic background. Thus TNF may play a central role in the development of
restenosis which is further accentuated in an environment prone to develop atherosclerosis.
The latter shown by the fact that not only neointima but also media was thickened.
Dual Potential of VEGF-D In Vivo
Tatiana V Byzova. Cleveland Clinic Foundation, Cleveland, OH
Oral Presentations a-3
One of the most recently characterized VEGFs, VEGF-D binds to VEGFR-2 and VEGFR-3 on
endothelial cells. Given the central role of VEGFR-2 in angiogenesis and VEGFR-3 in
lymphangiogenesis, it would be expected that human VEGF-D might induce both angiogenesis
and lymphangiogenesis in vivo. Our in vitro studies demonstrated that the fully processed form
of VEGF-D, VEGF-D homology domain (VEGF-D-HD) is an effective stimulator of the proliferation
and migration of blood vessel EC and that the migratory response of EC that express both
VEGFR-2 and VEGFR-3 in vitro is almost completely blocked by anti-VEGFR-2 antibodies
demonstrating a key role for VEGFR-2 in this process. The capacity of VEGF-D-HD to induce
angiogenesis and/or lymphangiogenesis was analyzed in two distinct in vivo models. We
characterized a new in vivo model for assessing experimental angiogenesis, the rat cremaster
muscle, that permits live video microscopy and quantitation of functional blood vessels. In this
model, a proangiogenic effect of Ad-VEGF-D-HD was evident as early as five days
post-injection. by guest Immunohistochemical on April 4, 2013 analysis of the cremaster muscle demonstrated that
15
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a-4 Arterioscler Thromb Vasc Biol. Vol 22, No 5
neovascularization induced by Ad-VEGF-D-HD, as well as by Ad-VEGF-A165 (an adenovirus
encoding the 165 isoform of VEGF-A), was comprised primarily of laminin and VEGFR-2-positive
vessels containing red blood cells, thus indicating a predominantly angiogenic response. In a
skin model, Ad-VEGF-D-HD induced both angiogenesis and lymphangiogenesis as indicated by
staining with laminin, VEGFR-2 and VEGFR-3, whereas Ad-VEGF-A165 stimulated the selective
growth of blood vessels. These data suggest that the biological effects of VEGF-D are
tissue-specific and dependent on the abundance of blood vessels and lymphatics expressing
the receptors for VEGF-D in a given tissue. The capacity of Ad-VEGF-D-HD to induce endothelial
cell proliferation, angiogenesis and lymphangiogenesis demonstrates that its potential utility for
treatment of coronary artery disease, cerebral ischemia, peripheral vascular disease, restenosis
and tissue edema should be tested in preclinical models.
18
Nitric Oxide Synthase Mediates the Vascular Protective Effect of Estrogen
on Medial Area in the Mouse Carotid Artery Injury Model
Henny Schulten, Richard Karas, Mark Aronovitz, Gary Pare, Ebo De Muinck, Michael
Mendelsohn. Cardiovascular Research Institute Maastricht, Maastricht, Netherlands;
Molecular Cardiology Research Institute, Boston, MA
We have previously shown that estrogen (E 2) inhibits the response to vascular injury in the
mouse carotid artery injury model. To determine whether the protective effects of E 2 are due
to increased nitric oxide production, we examined the effect of L-NAME, a non-selective nitric
oxide synthase inhibitor, on estrogen-mediated protection against vascular injury. Methods:
Forty-one ovariectomized adult female mice were randomized to receive vehicle alone, E 2,
L-NAME, or L-NAMEE 2. The mice were subjected to unilateral carotid artery endothelial
denudation injury. Two weeks after injury the response to vascular injury was assessed by
computerized morphometric determination of carotid artery medial area (MA). Results:
Compared to uninjured vessels (MA13.1.4 mm 2 x10 -3 ), injury induced an increase in MA in
the vehicle-treated mice (MA18.4.6 mm 2 x10 -3 ;p0.05), and this was attenuated in the
E 2-treated mice (MA15.81.1 mm 2 x10 -3 ;p0.05 vs vehicle-treated). In contrast, however,
E 2 no longer reduced injury-induced increases in MA in the L-NAME-treated mice
(MA20.01.5 mm 2 x10 -3 ;pN.S. vs L-NAME alone; p0.05 vs vehicle treated). L-NAME
alone had no significant effect on medial area following injury (MA20.81.6 mm 2 x10 -3 ;
pN.S. vs vehicle-treated). Conclusions: Inhibition of nitric oxide synthase activity abolishes
the protective effects of E 2 on medial area in the mouse carotid artery injury model. These
findings represent the first demonstration that nitric oxide mediates the vascular protective
effects of E 2 in vivo.
19
Low-Dose Aspirin Reduces Atherogenesis and Increases Plaque Stability in
LDL-Receptor Deficient Mice
Tillmann Cyrus, Siyuan Song, Lei Zhao, Colin D Funk, Lin Y Tung, Domenico Pratico.
University of Pennsylvania, Departments of Medicine and Pharmacology, Philadelphia, PA;
University of Pennsylvania, Dept. of Pharmacology, Philadelphia, PA
Pharmacological inhibition of platelet activation by aspirin is a mainstay in the prevention of
acute complications of atherosclerotic cardiovascular disease. Aspirin preferentially inhibits
platelet cyclooxygenase activity resulting in decreased production of thromboxane A2, a potent
platelet activator and vasoconstrictor, whose endogenous biosynthesis is increased in
atherosclerosis. Low-dose aspirin suppresses thromboxane A2 but does not significantly affect
prostacyclin, an anti-platelet agent and vasodilator. We wished to determine the effect of
low-dose aspirin on atherogenesis and plaque composition in Low-Density Lipoprotein
receptor-deficient mice fed a high fat diet. Compared to controls, aspirin completely inhibited
thromboxane A2 formation, but only partially reduced prostacyclin biosynthesis, and did not
affect lipid levels. This was associated with a marked decrease in circulating levels of
monocyte chemoattractant protein-1 and in vascular nuclear factor B activity. Aspirin also
significantly reduced the extent of atherosclerosis by 64 %. Lesions of aspirin-treated animals
showed 57% reduction (p0.05) in the amount of macrophage cells, 77% increase in smooth
muscle cells content (p0.05), and 23% increase in collagen (p0.05). Our results
demonstrate that in murine atherosclerosis low-dose aspirin reduces progression of atherosclerosis,
and increase stability of the atherosclerotic plaque by suppressing thromboxane A2
generation and vascular inflammation. These findings suggest that low-dose aspirin might be
rationally evaluated in the progression and evolution of human atherosclerosis.
20
Lipoprotein (a) Enhances Advanced Atherosclerosis in WHHL Transgenic
Rabbits Expressing Human Apolipoprotein (a)- A Novel Function of Lp(a) in
Vascular Calcification
Jianglin Fan, Hiroyuki Unoki, Huijun Sun, Xiaofei Wang, Tomonaga Ichikawa, Masashi
Shiomi, Santica Marcovina, Teruo Watanabe. University of Tsukuba, Tsukuba, Japan;
Institute of Experimental Animals, Kobe University School of Medicine, Kobe, Japan;
Northwest Lipid Research Laboratory, University of Washington, Seattle, WA; Saga Medical
School, Saga, Japan
High lipoprotein(a) [Lp(a)] levels form a major risk factor for the development of atherosclerosis.
The risk of elevated Lp(a) concentrations is increased significantly in patients who also have
high levels of LDL cholesterol. To test the hypothesis that increased plasma levels of Lp(a) may
enhance the development of atherosclerosis in the setting of hypercholesterolemia, we
generated Watanabe heritable hyperlipidemic (WHHL) transgenic (Trg) rabbits expressing
human apolipoprotein(a) [apo(a)] and compared the atherosclerotic lesions with those of
non-Trg WHHL rabbits. Trg WHHL rabbits had as high as 15 mg/dL of Lp(a) in plasma.
Compared to non-Trg WHHL rabbits, in which the lesions were dominated by an early-stage
lesion, i.e., fatty streak, Trg WHHL rabbits had increased Downloaded formation of from
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lesions, i.e., atheroma and fibroatheroma. Immunohistochemical study revealed that the apo(a)
deposition was often present in the lesions and colocalized with apoB. In particular, the
advanced atherosclerotic lesions in Trg WHHL rabbits were associated frequently with
calcification, which was barely evident in non-Trg WHHL rabbits. To investigate the molecular
mechanism of Lp(a)-induced vascular calcification, we examined the effect of human Lp(a) on
cultured rabbit aortic smooth muscle cells and found that Lp(a) at concentrations of 30 mg/dL
significantly increased mRNA expression of calcium-binding protein, matrix Gla protein, and
osteoblast-specific transcription factor Osf2, which was accompanied by enhanced alkaline
phosphatase activity and calcium accumulation in the culture. Our study showed for the first
time that Lp(a) accelerates advanced atherosclerotic lesion formation and may play an
important role in vascular calcification.
21
Molecular Mechanisms of Type III Hyperlipoproteinemia: The Contribution
of the Carboxyterminal Domain of ApoE2
Kyriakos E Kypreos, Ko W Van Dijk, Louis M Havekes, Vassilis I Zannis. Boston University
School of Medicine, Boston, MA; Leiden University Medical Center, Leiden, Netherlands;
TNO-PG Prevention and Health, Gaubius Laboratory, Leiden, Netherlands
Apolipoprotein E2 (apoE2) is associated with type III hyperlipoproteinemia and premature
atherosclerosis in humans. We used adenovirus-mediated gene transfer to probe the functions
of apoE in cholesterol clearance, in vivo. Infection of apoE-deficient (E -/- ) mice with 2x10 9 pfu
of adenovirus expressing the truncated form apoE2–202, normalized the cholesterol levels of
E -/- mice, without induction of hypertriglyceridemia, whereas the same dose of full-length
apoE2 increased the cholesterol levels of E -/- mice and induced hypertriglyceridemia. The
secretion of apoE2 and apoE2–202 proteins and the hepatic mRNA levels were similar.
Full-length apoE2 also induced combined hyperlipidemia in C57BL6 mice, which was
eliminated by co-infection with 2x10 9 pfu of each the apoE2 and the apoE2–202 adenovirus.
Infection of E -/- x LDL-r -/- double deficient mice with 2x10 9 pfu of apoE4–202 expressing
adenovirus did not affect the plasma cholesterol and triglyceride levels, whereas apoE4 induced
hypertriglyceridemia, and at the higher dose, it increased 3-fold the plasma cholesterol levels.
The findings suggest that a) the dislipidemia observed in E2/2 patients is not the result of
Arg1583Cys substitution, but is rather mediated by the carboxyterminal 203–299 segment of
apoE2 b) there is no significant contribution of apoE-recognizing receptors other than the LDL
receptor in the clearance of apoE-containing lipoprotein remnants. c) The truncated apoE forms
have a dominant effect in the clearance of the lipoprotein remnants, and may have therapeutic
applications in the correction of remnant removal disorders in humans.
TGF: A Key Regulator of Atherosclerotic Plaque Stability
Esther Lutgens, Philip Gotwals, Victor Koteliansky, Mat Daemen. University of Maastricht,
Maastricht, Netherlands; Biogen Inc, Cambridge, MA
We recently reported that CD40L inhibition induces a stable plaque phenotype. To unravel the
mechanism of this process, we performed cDNA expression array analysis on aortic arches of
anti-CD40L antibody and ctrl treated ApoE-/- mice (29 wks old). One of the targets was TGF,
which showed a 2.3-fold increase in mRNA expression and in immunoreactivity after CD40L
inhibition. To validate the importance of this target, recombinant soluble TGFRII (TGFRII:Fc)
which inhibits TGF signalling, was injected in ApoE-/- mice for 12 wks (50g, twice a week
intraperitoneally), in an early treatment (age 5–17, n7 TGFRII, n7 ctrl) and a delayed
treatment (age 17–29, n7 TGFRII:Fc, n7 ctrl) setting. Cholesterol, triglyceride, HDL and
LDL levels did not change. In the early treatment group, treatment resulted in a prominent
increase in inflammatory markers such as CD3, CD45 (initial lesions: 5.20.9% vs 2.10.6%,
p0.05; advanced lesions 3.30.9% vs 1.70.6%; p0.05), CD40 and CD40L. Most
profound effects were found in the delayed treatment group. Plaque area decreased 37.5%
after TGFRII:Fc treatment (p0.05). Although plaques were smaller, they exhibited an
unstable plaque phenotype. Lipid cores were 64.6% larger (54.73.8% TGFRII:Fc vs
33.22.6% ctrl, p0.05), and m content (60.92.4% TGFRII:Fc vs 43.72.7% ctrl,
p0.05) was significantly increased after TGFRII:Fc treatment. Treatment increased CD3,
CD45 (initial: 8.01.4% vs 2.01.0%, p0.05; advanced 5.70.5% vs 2.10.3%), CD40,
and CD40L positive cell content in initial and advanced lesions. The amount of fibrosis
decreased 49.6% (11.91.5% TGFRII:Fc vs 24.02.6%, p0.05), whereas SMC content did
not change. Advanced lesions of the delayed treatment group showed signs of bleeding.
Erythrocytes were observed in 20%, fibrin in 8%, iron deposition in 29% and disruption of the
endothelium in 22% of advanced lesions after TGFRII:Fc treatment vs 13%, 0%, 19% and 0%
in ctrl. These results reveal a pivotal role for TGF in the maintenance of plaque stability.
23
Oxidation of the Zinc-Thiolate Cluster of Endothelial Nitric Oxide Synthase
Triggers the Enzyme Uncoupling in Diabetes In Vivo
Ming-Hui Zou, Shao-Mei Shi, Richard A Cohen. Whitaker Cardiovascular Institute, Boston
University School of Medicine, Boston, MA
In a number of vascular disease states endothelial nitric oxide synthase (eNOS) is uncoupled
leading to generation of superoxide anions, but the mechanism of this change in function is
unknown. We present here the first evidence that peroxynitrite releases zinc from the
zinc-thiolate cluster of eNOS and forms disulfide bonds between the monomers. As a result,
disruption of SDS-resistant eNOS dimers occurs under reducing conditions. Zinc release from
purified recombinant eNOS was caused by concentrations of peroxynitrite 10 –100 fold lower
than those that oxidized tetrahydrobiopterin (BH4), an essential cofactor of eNOS. Further, zinc
depleted eNOS had lowered affinities for BH4 and its substrate, L-arginine, resulting in a loss
of the NO synthetic activity, increased NADPH oxidase activity, and generation of oxidants. A
zinc chelator mimicked these effects of peroxynitrite. Uncoupling of eNOS also occurred in
endothelial by cells guest exposed on toApril peroxynitrite, 4, 2013 or in those grown in elevated glucose, accounting for
22

the increased release of superoxide from the cells. The changes caused by elevated glucose
were associated with loss of zinc content in eNOS and were prevented by a NOS inhibitor or
superoxide dismutase, indicating that they were caused by peroxynitrite. Furthermore, eNOS
purified from diabetic low density lipoprotein (LDL) receptor deficient mice, which showed an
increased aortic lesions, had decreased NO synthetic activity, lowered zinc content, and
decreased enzyme-active eNOS dimers resulting in an increased thiol oxidation and increased
generation of oxidants. These observations indicate that in diabetes eNOS is oxidized by
peroxynitrite resulting in an enzymatic uncoupling and generation of oxidants contributing
further to the oxidant stresses in these cells. The principal mechanism appears to be oxidation
of the zinc-thiolate complex that maintains the enzyme as active dimers rather than via
oxidation of BH 4. Oxidation of zinc-thiolate cluster of eNOS, therefore, provides a novel
mechanism for modulation of the enzyme function in vivo.
Selectin-Like Bond Kinetics Promote Rapid Platelet Adhesion: The
GPIb-vWF Bond and the Impact of Type 2B Mutations
Thomas G Diacovo, Gaurav Girdhar, Avril Lawshe, David W Schmidtke, Ian J Laurenzi, Scott
L Diamond, Teresa A Doggett. Division of Newborn Medicine, Washington University, St.
Louis, MO; Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia,
PA
Rapid bond formation and dissociation are characteristic of the selectin family of adhesion
receptors that promote the capture and translocation of leukocytes on vascular endothelium.
In contrast selectins, the platelet glycoprotein receptor Ib alpha (GP Ib) mediates plateletvessel
wall interactions by binding to the A1 domain of surface-immobilized von Willebrand
factor (vWF-A1). This receptor-ligand pair, however, has been reported to have slow intrinsic
bond kinetics. We now present evidence that the GPIb-vWF-A1 bond has properties previously
identified with selectins: flow dependent adhesion and rapid and force-dependent bond
kinetics. Characterization of GPIb interactions with plasma vWF or recombinant wt A1 protein
in vitro demonstrates that a critical level of hydrodynamic flow is required to initiate adhesion
between this receptor-ligand pair. The dissociation rate constants, koff, for the GPIb vWF-A1
bond, as determined by analysis of pause times, were found to vary exponentially (4.2 0.8
s-1 to 6.9 0.3 s-1) as a function of the force applied to the bond (from 36 to 217pN). The
physiologic significance of bond mechanics in platelet adhesion was exemplified by type 2B von
Willebrand disease (vWD), a bleeding disorder resulting from the spontaneous binding of
plasma vWF to circulating platelets. Kinetic analysis of mutations associated with this disorder
revealed a loss of the shear threshold phenomenon, a 6-fold reduction in the intrinsic koff,
and a 2-fold increase in the reactive compliance of the bond suggesting a greater propensity
to breakage in response to hydrodynamic force. Thus, the increased bond lifetime observed for
type 2B vWF and the concomitant loss of the hydrodynamic threshold requirement for adhesion
demonstrate the deleterious consequences that can result from an alteration in the
biomechanical regulation of a receptor-ligand pair. Importantly, our results indicate that
evaluation of receptor-ligand interactions under physiological relevant conditions is paramount
to understanding how biomechanical properties of bonds ultimately control the process of cell
adhesion.
Increased Atherosclerosis in Apo E Deficient Mice Lacking
Macrophage-Derived ACAT1
Yan Ru Su, Dwayne Dove, Amy Major, Alyssa Hasty, Robert Falese, Macrae Linton, Sergio
Fazio. Vanderbilt University Medical Center, Nashville, TN
Acyl-coenzyme A: cholesterol acytransferase (ACAT) is an enzyme responsible for esterification
of free cholesterol to its intracellular storage form, cholesterol ester, and therefore plays an
important role in cellular cholesterol homeostasis. Two isoforms of ACAT have been cloned in
mammals: ACAT1 is preferentially expressed in macrophages, adrenal gland and kidney while
ACAT2 is mainly expressed in liver and intestine. ACAT1 catalyzes the esterification of free
cholesterol in macrophage leading to intracellular cholesterol ester accumulation, foam cell
formation therefore play a crucial role in early development of atherosclerotic lesions. Many
studies have shown that nonselective inhibition of ACAT reduces atherosclerosis. However,
previous study in our laboratory have shown that complete inhibition of ACAT1 leading to an
increase in atherosclerosis in LDL receptor deficient mice, raising the concern that the
complete absence of ACAT1 in macrophage can promote atherosclerotic lesion formation. The
objective of our current study was to determine the effect of absence of macrophage-derived
ACAT1 on atherosclerosis in apo E deficient mice using bone marrow transplantation. Our
results show that the lack of ACAT1 in apo E deficient macrophages also promotes
atherosclerotic lesion formation (89606/-46799 m 2 in ACAT1 and apoE double knockout
mice, n9; compared to 50749/-28846 m 2 in apoE deficient mice n8, p0.05).
Surprisingly, studies in peritoneal macrophages isolated from ACAT1 deficient mice showed a
30–40% reduction (p0.001) in cholesterol efflux compared to control macrophages. In
conclusion, deficiency of macrophage ACAT1 appears to have deleterious consequences to
vascular health in apoE-null animal model, suggesting that caution should be exerted in the
developing of ACAT inhibitors aimed at treating human atherosclerosis.
24
25
26
that hepatic cholesterol synthesis and hepatobiliary transport are not affected in mice lacking
ABCA1, findings incompatible with current concepts of RCT. ABCA1 is highly expressed in
peripheral tissues, including macrophages, but also in liver and intestine: its function in these
organs is unclear. Expression of the Abca1 gene is controlled by the liver X-receptor (LXR). We
have evaluated the physiological effects of LXR activation on hepatic cholesterol metabolism,
with special reference to hepatobiliary transport. For this purpose, Abca1 -/- and wildtype mice
were treated with the LXR agonist T0901317 (10 mg/kg, 5 d). Plasma cholesterol levels
increased in Abca1 -/- mice (1.11 vs. 0.50 mM) and in control mice (1.64 vs. 1.12 mM) upon
treatment. In Abca1 -/- mice, the increase was exclusively in VLDL-sized fractions, whereas in
wildtype mice VLDL and HDL cholesterol increased. Hepatic triglycerides were massively
elevated in both strains after treatment, presumably related to induction of lipogenic genes. LXR
activation strongly affected bile composition. Bile flow was identical in both strains, but bile salt
excretion was diminished in treated wildtype (-22%) and in Abca1 -/- (-46%) mice. Likewise,
biliary phospholipid output decreased upon treatment in both strains, in spite of unaffected
expression of Abcb4, encoding the hepatic phospholipid translocase. In contrast, cholesterol
output was significantly induced by treatment, leading to increases in cholesterol:phospholipid
ratios from 0.13 to 0.46 and from 0.12 to 0.42 in wildtype and Abca1 -/- mice, respectively.
Expression of Abcg5 and Abcg8, recently implicated in cholesterol transport, was induced in
livers of treated mice. In conclusion, chronic activation of LXR in mice leads to enhanced
hepatobiliary cholesterol removal independent from (ABCA1-mediated) elevation of HDL and the
presence of ABCA1 in the liver
27
Naturally Occurring Mutations of the Tangier Disease-Associated Protein,
ABCA1, Prevent the Movement of Cholesterol to the Exofacial Side of the
Plasma Membrane
Ashley M Vaughan, John Oram. University of Washington, Seattle, WA
The ATP-binding cassette protein, ABCA1, is involved in the apolipoprotein AI-mediated efflux
of excess cholesterol from cells and is mutated in patients with Tangier disease. The lack of
a functional ABCA1 protein in Tangier patients causes the absence of high density lipoprotein,
and subsequently, an increased risk for cardiovascular disease. We have recently shown that
over-expression of ABCA1 in cells leads to an increase in cholesterol oxidase-sensitive
cholesterol on the exofacial side of the plasma membrane. To examine the effect of naturally
occurring ABCA1 point mutations on protein function, we used site-directed mutagenesis to
construct ABCA1 mutants which we then expressed in cells. Four mutants were constructed,
two in the large amino-terminal extracellular loop of the protein, and one from each of the two
ATP-binding domains. All four mutants were expressed at high levels in our cell system. In
every case, expression of the mutant protein, when compared to wild-type ABCA1, lead to a
significant decrease in both apolipoprotein AI binding and apolipoprotein AI-mediated
cholesterol efflux from cells. Expression of all four mutants also failed to cause an increase in
cholesterol oxidase-sensitive cholesterol on the exofacial side of the plasma membrane. It is
tempting to hypothesize that the movement of cholesterol to the exofacial side of the plasma
membrane in ABCA1 expressing cells is essential for the subsequent apolipoprotein AI binding
and apolipoprotein AI-mediated cholesterol efflux.
Thrombin Enhances Inflammation during Cardiac I/R Injury
Oral Presentations a-5
Rafal Pawlinski, Craig Hampton, Jeanette Ennis, Patricia Andrade-Gordon, Edward Verrier,
Timothy Pohlman, Nigel Mackman. The Scripps Research Institute, La Jolla, CA; University
of Washington, Seattle, WA; R.W. Johnson Pharmaceutical Research, Spring House, PA
Ischemia-reperfusion injury has many features in common with inflammatory responses and
leads to endothelial dysfunction, microvascular collapse and impairment of blood flow,
myocardial infarction and cell death. We and others have shown that the coagulation protease
cascade contributes to injury during cardiac I/R injury. We have employed both rabbit and
mouse models of cardiac I/R injury. Inhibition of either tissue factor (TF) or thrombin reduced
infarct size whereas defibrinogenerating rabbits did not reduce infarct size. We measured the
expression of various inflammatory mediators in the rabbit and mouse models. I/R injury
induced the expression of transcription factors, such as Egr-1, adhesion molecules, such as
ICAM-1, and chemokines, such as IL-8, MCP-1 and MIP-2. We also examined chemokine
mRNA expression in mice subjected to different times of ischemia (5–30 min) or a fixed time
of ischemia (30 min) with different times of reperfusion (30 min, 1, 2, 3 and 4 hours) to
determine the kinetics of induction. Importantly, inhibition of thrombin in I/R-injured rabbits
reduced MCP-1 expression and PMN infiltration. Finally, we used PAR-1 knockout mice to test
the hypothesis that thrombin enhancement of inflammation was mediated by PAR-1. We
observed a 43% decrease (p0.01) in infarct size in PAR-1-/- mice (mean infarct /- SE, 20
/- 4, n6) compared with PAR-1/ mice (mean infarct /- SE, 35 /- 3, n6). Taken
together, these results indicate that the TF-thrombin-PAR-1 signaling pathway contributes to
inflammation in cardiac I/R injury.
Prevention of Diabetes-Induced Microangiopathy by Human Tissue
Kallikrein Gene Transfer
Increased Hepatobiliary Cholesterol Transport by Activation of the Liver
Costanza Emanueli, Bonaria Salis, Paolo Madeddu. National Laboratory INBB, Osilo, Italy
X-Receptor LXR Is Independent of ABCA1 in Mice
Microvascular remodelling and insufficiency contributes to end-organ damage in diabetes. The
Torsten Plösch, Tineke Kok, Vincent W Bloks, Rick Havinga, Martin J Smit, Giovanna
lack of effective therapy for the treatment of microangiopathy urges to seek new strategies of
Chimini, Albert K Groen, Folkert Kuipers. Department of Pediatrics, University Hospital
vascular protection and repair. We evaluated if human kallikrein (HK) gene therapy can prevent
Groningen, Groningen, Netherlands; Centre d’Immunologie, INSERM-CNRS, Marseille,
or rescue microangiopathy in limb skeletal muscle in streptozotocin (STZ)-induced diabetic
France; Department of Gastroenterology, Academic Medical Center, Amsterdam, Netherlands mice. We found that, after an initial increase in vascularity, diabetic mice develop progressive
capillary and arteriole loss and rare faction, due to apoptotic cell death. This was associated
The ATP binding cassette transporter ABCA1 is essential for HDL formation and therefore with remodelling of arterioles of various luminal diameter, impaired response to acetylcholine
considered rate-controlling for reverse cholesterol Downloaded transport (RCT). Recently, from
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we demonstrated or reperfusion, by guest shortage on ofApril muscular 4, cGMP 2013content
and ultimately muscular damage. Chronic
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a-6 Arterioscler Thromb Vasc Biol. Vol 22, No 5
insulin supplementation ensured adequate control of hyperglycemia and preserved in part
endothelium-dependent vasodilation, yet did not prevent vascular pathology. In contrast,
application of gene therapy with HK at early phases of diabetes (14 days) halted the progression
of microangiopathy, as a result of neovascularization and inhibition of apoptosis. The beneficial
action occurred no matter mice had been treated or not with insulin, which indicates that HK
is effective independently from imposed metabolic control of the disease. Vascular effects of
HK were limited to the site of injection, thus discounting the risk of inappropriate vascular
growth in distant organs. The improved limb microcirculation allowed for an accelerated
hemodynamic recovery during post-ischemic reperfusion. Finally, we documented that
application of HK gene therapy at a later stage (60 days) with the attempt to rescue established
microangiopathy results in a marked increase in muscular capillarity, which implies vascular
regeneration. Our discoveries indicate that HK may be a useful tool for the treatment of
microvascular disease in diabetics.
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Megakaryocyte Endocytosis and Intracellular Trafficking of Plasma-Derived
Factor V Results in a Physically Unique Platelet-Derived Cofactor
Weston R Gould, Paula B Tracy. University of Vermont College of Medicine, Burlington, VT
Platelet-derived factor Va (FVa) originates from plasma via megakaryocyte endocytosis of
circulating factor V (FV). Despite the identical site of synthesis, platelet-derived FVa is a unique
substrate for several enzymes. For example, in contrast to the plasma-derived cofactor,
platelet-derived FVa is resistant to phosphorylation at Ser692 catalyzed by casein kinase II
(CKII). These data suggest that, subsequent to its endocytosis by megakaryocytes, specific
intracellular trafficking events lead to modifications that yield a cofactor resistant to
phosphorylation at this site. Consequently, purified plasma and platelet-derived factor Va heavy
chains (HC) were isolated by SDS-PAGE and subjected to digestion with trypsin such that the
resulting fragments could be identified by MALDI-TOF mass spectroscopy. Such analyses
allowed the identification of a fragment with m/z of 2087.8 corresponding to residues 685–701
(LEPEDEESDADYDYQNR) in the plasma-derived FVa HC, which, subsequent to phosphorylation
by CKII, lead to a 80 Da shift of this fragment directly demonstrating the incorporation of
phosphate into Ser692. Despite its resistance to phosphorylation at this site, MALDI-TOF
analysis of the platelet-derived cofactor demonstrated that Ser692 was not modified
suggesting that, post-endocytosis, any alteration to the protein that inhibits phosphorylation,
must occur at an alternative location. Additional analyses of the heavy chain of platelet-derived
FVa identified a fragment at m/z of 679.5 unique to the platelet-derived molecule that
corresponds to the addition of an N-acetylglucosamine (GlcNac) at Thr402 (DTLK GlcNac)
indicating for the first time, that the platelet-derived cofactor is physically distinct from the
plasma-derived molecule although the functional consequence of this alteration has yet to be
defined. However, the current study offers strong support of the concept that subsequent to
endocytosis by megakaryocytes, plasma-derived FV undergoes retrograde transport to the
trans-Golgi network and is retailored post-translationally to yield a unique platelet-derived
cofactor.
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HoxB5 Transcriptionally Regulates the Vascular Endothelial Growth Factor
Receptor KDR/flk-1 In Vitro and In Vivo
Yaxu Wu, Cam Patterson. Carolina Cardiovascular Biology Center, University of North
Carolina at Chapel Hill, Chapel Hill, NC
KDR/flk-1 is a receptor tyrosine kinase that participates in angiogenesis by serving as a
receptor for vascular endothelial growth factor. It is also the earliest known marker for
hemangioblasts and is required for endothelial and hematopoietic differentiation during
development. We have studied the regulation of KDR/flk-1 as a model for understanding
transcriptional events that control angiogenesis and endothelial cell differentiation. Using
DNase I footprinting and gel shift analyses, we identified a novel AT-rich 20-bp sequence in the
first intron of the mouse KDR/flk-1 gene that was bound by nuclear proteins in an endothelial
cell-specific fashion. Using transgenic mice, we demonstrated that a 4-bp mutation in this
element abolished endothelial-specific expression of a reporter gene under control of the
KDR/flk-1 promoter/enhancer. We used a yeast one-hybrid approach to clone the nuclear
protein(s) responsible for binding to this element. 5 positive clones were identified in a screen
of a mouse embryo library with this element; 2 of these clones encoded the homeodomain
protein HoxB5. HoxB5 bound to the wild-type KDR/flk-1 element, but not to a mutated
sequence, demonstrating the specificity of this interaction. In transient transfection assays,
HoxB5 was able to transactivate the KDR/flk-1 promoter 8–10 fold. Until now, the function of
HoxB5 has been largely obscure. Our present results provide strong evidence that HoxB5 is
required for KDR/flk-1 expression in vivo and in vitro, via a novel cis-acting element. This
unexpected finding suggests that this homeodomain protein is a likely candidate transcription
factor that regulates hemangioblast differentiation and angiogenesis.
Bmx Mediates TNF-Induced Inflammatory Angiogenesis through
Association with TNFR2
Ping An, Wang Min. University of Rochester, Rochester, NY
As a proangiogenic factor, TNF promotes inflammatory angiogenesis in progression of
rheumatoid arthritis through up-regulation of a large number of angiogenic factors such as
VEGF, bFGF and MCP-1. However, the signaling of TNF-induced angiogenesis is not fully
understood. Bmx/Etk, a member of Btk/Tec non-receptor tyrosine kinase family is highly
expressed in endothelial cells and exerts a variety of bioactivities related with proliferation and
migration through interaction with various signal complexes. Recently we showed that activity
of Bmx, JNK kinases and JNK-dependent gene expression were increased dramatically in
arthritic joints of hTNF transgenic mice. These data prompted us to examine the role of Bmx
in inflammatory angiogenesis. Here, we showed Downloaded that Bmx constitutively from
interacts with and is
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activated by TNFR2 but not TNFR1. In contrast, the dominant negative form of Bmx (Bmx-DN)
abolishes activation of JNK induced by TNF, but not by overexpression of TRAF2, indicating that
Bmx and TRAF2 activate JNK through different pathways. Overexpression of both the wild type
(WT) and the constitutively active (SK) Bmx activate JNK, while the dominant-negative form
(DN) and the kinase domain deletion mutant (DK) of Bmx inhibits TNF-induced JNK activation.
Mapping studies show that SH3 domain in Bmx and the 16 amino acids within the C-terminus
of TNFR2 (non-TRAF2-binding domain) are required for the interaction of the two proteins. We
further investigated the biological activities of Bmx with two in vitro angiogenic models: a
three-dimensional culture in collagen gel and a migration assay of EC. Results show that
Bmx-SK facilitated, while Bmx-DK blocked, TNF-induced tube formation and “wound healing”
of EC. Taken together, our data suggest Bmx is a critical mediator involved in TNF-induced
angiogenesis and may provide a novel approach for treatment of angiogenesis dependentdiseases
such as atherosclerosis, cancer and rheumatoid arthritis.
Decreased Mural Cell Proliferation Is Associated with Impaired Vascular
Development in the NG2 Knockout Mouse
Ugur Ozerdem, William B Stallcup. The Burnham Institute, La Jolla, CA
Confocal imaging with endothelial (CD31, flk-1, CD105), pericyte (alpha-SMA, PDGF betareceptor),
cardiomyocyte (MLC2a, MLC2v), and nuclear markers was utilized to compare
wild-type (wt) and NG2-knockout (ko) mice to reveal a functional role for the NG2 proteoglycan
in cardiovascular development and hyperoxia-induced retinal angiogenesis. BrdU labeling was
used to quantify proliferation of vascular cell types. We find that retinal angiogenesis is reduced
in the ko mouse. At P17 the mean number of vascular nuclei in ischemic angiogenic retinal
tufts is 54.9 per histological section in the ko retina versus 119.8 per section in the wt retina
(p0.0019 for 10 wt eyes (50 sections) and 10 ko eyes (50 sections)). Decreased vascular cell
proliferation was noted: 38.3% of endothelial cells incorporate BrdU in wt mice versus 22.8%
in ko mice (p0.0147); 45.2% of wt pericytes label with BrdU versus 18.7% in ko mice
(p0.0068). The endothelial cell/pericyte ratio is 1.161 in wt eyes versus 4.065 in ko eyes
(p0.0011 for 2 wt eyes (10 sections) and 2 ko eyes (10 sections)). At E10 the NG2-null heart
is distinguished from wt heart by a 46.4% ventricular enlargement (p0.0068), a 114.6%
increase in the ratio of inflow tract (IFT)/outflow tract (OFT) diameter (p0.0001), and a 64%
increase in the ratio of IFT/OFT wall thickness (p0.0039). Trabeculation is reduced in the ko
ventricle. In wt ventricle, 67.6% of cardiomyocytes label with BrdU versus 26.9% in ko
ventricle. In wt OFT, 53.5% of cardiomyocytes label with BrdU versus 27.6% in ko OFT. In wt
IFT, 27.5% of cardiomyocytes are BrdU-positive versus 33.6% in ko IFT. This pattern is
significant, since ventricle and OFT normally exhibit high levels of NG2 expression, while IFT
is low in NG2. Conclusions: NG2-null mice exhibit diminished angiogenesis, decreased BrdU
uptake by vascular cells (especially pericytes), and reduced pericyte investment of endothelial
tubes. A larger but less trabeculated ventricle and a decrease in OFT size in the ko embryo may
be due to reduced cardiomyocyte proliferation in these areas.
Massive Xanthomatosis and Widespread Tissue Accumulation of
Macrophages in Hyperlipidemic Mice Lacking ABCA1
Robert J Aiello, Omar L Francone, Dominique J Brees, Patricia-Ann Bourassa, Lori J Royer,
Saralyn Lindsey, Timothy M Coskran. Pfizer Inc, Groton, CT
The discovery of ATP-Binding Cassette Transporter A1 (ABCA1) and its role on High density
lipoproteins (HDL) biogenesis has brought considerable attention to the potential for enhancing
its activity as a therapeutic means to affect atherosclerosis. Despite the complete absence of
HDL and tissue accumulation of macrophages in homozygous patients with Tangier Disease
and mice lacking Abca1, the contribution to Abca1 to the development of atherosclerosis
remains unclear. In this study we have examined whether the absence of Abca1 is
accompanied by an increased susceptibility to atherosclerosis. In the setting of severe
hypercholesterolemia caused by a deficiency of apolipoprotein E (apoE-/-) mice, the additional
absence of Abca1 led to a marked alteration on the distribution of tissue macrophages with
prominent accumulation in skin, resulting in severe xanthomatosis. Complete necropsy
demonstrated age-related infiltration of macrophages in the uterus, stomach, lymph nodes,
kidneys and lungs. The absence of ABCA1 did not affect the development and progression of
atherosclerotic lesions in mice fed a chow or high fat diet despite the near complete absence
of HDL and apolipoprotein AI (apoAI).
Oxidized LDL and 12/15-Lipoxygenase Stimulate Actin Polymerization in
Macrophages and Affect Phagocytosis of Apoptotic Cells
Yury I Miller, Joseph L Witztum. University of California, San Diego, La Jolla, CA
Atherosclerotic tissue is characterized by accumulation of oxidized LDL (OxLDL) and by many
apoptotic cells despite the presence of macrophages, which normally efficiently engulf these
moieties via scavenger receptors. Actin polymerization (formation of F-actin) is necessary for
phagocytosis. Macrophages in murine atherosclerotic lesions express high levels of 12/15lipoxygenase
(LO). Thus, we examined if LO and OxLDL could affect macrophage actin
polymerization and phagocytosis. Digital deconvolution microscopy was used to image LO and
F-actin in macrophages, and FACS was used to quantify F-actin levels. When macrophages
were exposed to apoptotic cells, LO translocated from the cytosol to the cell surface and
colocalized with F-actin of emerging filopodia. Inhibition of the LO activity by PD 146176 or
disruption of the 12/15-LO gene down regulated F-actin formation in response to apoptotic
cells, as shown by FACS analysis. Addition of LO products, such as 13(S)-HODE provoked
F-actin formation in vitro and in cell lysates. When macrophages were incubated with OxLDL
or minimally modified LDL (mmLDL), instead of apoptotic cells, enhanced actin polymerization
was also observed. The levels of F-actin were increased by 20% in 10 minutes and 100% in
8 hours. by Morphologically, guest on April the macrophages 4, 2013 appeared extensively spread. In contrast to
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macrophages phagocytosing apoptotic cells, LO activity seemed not to be obligatory for the
response to OxLDL (or mmLDL). Most importantly, macrophages pre-incubated with mmLDL
exhibited a decreased ability to phagocytose apoptotic thymocytes. We speculate that OxLDL
and mmLDL carry oxidized lipids similar to the products of 12/15-LO catalysis. In turn, these
oxidized lipids promote exhausting actin polymerization in macrophages, interfering with
subsequent phagocytic activity. These findings could provide part of the explanation as to why
apoptotic cells are not efficiently cleared from atherosclerotic tissue.
Induction of Monocyte Chemotactic Activity by Leptin
Farhad Parhami, Jason Hwang, Susan Hama, Mohamad Navab, Yin Tintut, Linda L Demer.
UCLA Division of Cardiology, Los Angeles, CA
Leptin, the product of the ob gene, regulates food intake, energy expenditure, and other
physiological functions of the peripheral tissues. We have previously demonstrated leptin
receptor in mouse artery wall, leptin protein in atherosclerotic lesions of ApoE null and LDL
receptor null mice, and osteoinductive effects of leptin on calcifying vascular cells. Since
human monocytes express leptin receptor(s), and leptin causes the activation and proliferation
of these cells, we investigated other effects of leptin on human peripheral blood monocytes that
may impact atherogenesis. Treatment of these cells with 1 g/ml human recombinant leptin
resulted in formation of structures resembling lamellipodia of migratory cells. Furthermore,
leptin (0.5–4 g/ml) caused a dose-dependent increase in monocyte chemotactic activity in a
Boyden chamber chemotaxis assay (control2.50.5; leptin: 0.5 g/ml5.21; 1 g/
ml7.50.5; 2 g/ml11.52; 4 g/ml19.03 monocytes/field SD; p0.05 for
control vs. all leptin concentrations). Western blot anaylsis of STAT3 activation in monocytes
showed no induction of STAT3 phosphorylation by 1–4 g/ml leptin after 5–30 minutes of
treatment, whereas the positive control IL-6 (50 ng/ml) induced STAT3 activation in those cells.
In addition, cultured human peripheral blood monocytes expressed leptin mRNA and protein as
assessed by Northern blot analysis and ELISA, respectively. These results show that leptin may
be a chemotactic factor for monocytes, and its presence in the atherosclerotic lesion may
further promote monocyte transmigration into the artery wall. We hypothesize that this
observation may also at least in part explain the previously reported resistance of leptin and
leptin receptor deficient mice to atherosclerotic lesion formation.
Evidence for Involvement of the Plasminogen Activator System in the
Aetiology of Plaque Destabilization and Rupture
Kevin G Carson, Aernout Luttun, Helen Williams, Peter Carmeliet, Christopher L Jackson.
Bristol Heart Institute, University of Bristol, Bristol, UK; Center for Transgene Technology and
Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium
The brachiocephalic artery of the apolipoprotein E knockout mouse (apoE-/-) has been
established as a site of predilection for atherosclerotic plaque rupture. The aim of this study
was to test the hypothesis that the plasminogen activator system may be involved in the
process of plaque destabilization and rupture. Double knockout mice, deficient in the genes for
apoE and Plasminogen Activator Inhibitor-1 (apoE-/-:PAI-1-/-) on a mixed C57Bl6/129SvJ strain
background were created by intercrossing PAI-1-/- and apoE-/- mice. Eleven of these
apoE-/-:PAI-1-/- mice and twelve apoE-/- controls were fed a high fat diet, and after 25 weeks
they were terminated and perfused. Serial sections were taken throughout the length of the
brachiocephalic artery. These were examined for the presence of plaque ruptures, and
additionally, the thickness of the fibrous cap, the proportion of the plaque occupied by the lipid
core, and the number of fibrous layers within the plaque (previous healed ruptures) were also
determined. There were significantly more plaque ruptures in the apoE-/-:PAI-1-/- mice than
in the apoE-/- controls (6 out of 11 compared to 1 out of 12, p 0.027, Fisher’s exact test).
Furthermore, in those that had plaque ruptures, the lipid core occupied a significantly greater
proportion of the plaque than in those without (44 /- 3 % compared to 33 /-3%,p 0.01,
unpaired t-test with Welch correction), and there were significantly more fibrous layers within
the plaque (4.9 /- 0.3 compared to 3.4 /- 0.5, p 0.02, unpaired t-test with Welch
correction). These results suggest that the plasminogen activator system plays an important
role in the process of plaque destabilization and rupture. They also demonstrate that this model
has important correlates with human lesions, in that the proportion of the plaque occupied by
lipid and the presence of previous healed ruptures both act as markers of instability.
Plasmin-Induced Gene Expression: Possible Mechanisms and Potential
Implications
Usha R Pendurthi, Samir Mandal, Elizabeth Crump, Mylinh Ngyuen. UT Health Center at
Tyler, Tyler, TX
Proteases involved in coagulation and fibrinolysis play an important role in pathophysiology,
such as wound healing, tissue remodeling and tumor metastasis. Our recent studies show that
a number of proteases involved in clotting and fibrinolysis, such as VIIa, thrombin and plasmin,
induced the expression of Cyr61, a gene that encodes extracellular matrix signaling protein that
was shown to regulate cell migration and proliferation, in fibroblasts. In the present study, we
have investigated possible mechanisms by which plasmin upregulates Cyr61 gene expression
and the ability of plasmin to induce cell proliferation. To evaluate a posssible involvement of
specific protease activated receptors (PARs) in plasmin-mediated gene induction, we investigated
the ability of plasmin to induce Cyr61 expression in mouse fibroblasts that lack (or
express) specific PARs. Plasmin effectively induced Cyr61 gene expression in both wild-type
and PAR-2 deficient cells but not in PAR-1 deficient cells. Transfection of PAR-1 deficient cells
with PAR-1 plasmid conferred plasmin responsiveness to these cells. However, in addition to
PAR-1, an additional cell associated factor may be necessary for plasmin-induced gene
expression since COS-7 cells, which has functional PAR-1 and PAR-2, failed to respond to
plasmin. PD 98059, a specific inhibitor of p44/42 Downloaded MAPK, inhibited plasmin-induced from
Cyr61 gene
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Oral Presentations a-7
expression. Consistent with this, plasmin is shown to induce p44/42 MAPK phosphorylation in
fibroblasts. Plasmin (up to 100 nM) failed to induce cytosolic calcium mobilization in WI-38
cells. In additional studies, we investigated the effect of plasmin on fibroblast proliferation using
3H-thymidine incorporation assays. The data show that plasmin (50 nM) increased 3Hthymidine
incorporation by 3-fold. In conclusion, our data suggest that plasmin-induced gene
expression involves PAR-1 and is mediated through p44/42 MAPK pathway, independent of
Ca2-signaling. The ability of plasmin to upregulate the expression of Cyr61, and probably
other growth factor-like molecules, which promote cell proliferation and migration, adds a new
dimension to how plasmin plays a role in tissue remodeling
39
VEGF and bFGF Alter the Urokinase Receptor (uPAR) Associated Proteolytic
Activity on the Surface of Human Umbilical Vein Endothelial Cells
Gerald W Prager, Judit Mihaly, Florian Gruber, Bernd Binder. University of Vienna, Austria,
Vienna, Austria
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) play an
important role in the angiogenic process. Urokinase type plasminogen activator receptor (uPAR)
is tightly regulated by these cytokines in ECs and itself plays an important role for the
angiogenic process that is dependent on local proteolytic activity. Using flow cytometry we
could confirm previous data that VEGF or bFGF upregulate expression of uPAR in a time and
dose dependent manner via the MEK/ERK pathway, starting 8 hrs. after addition of growth
factors. We could inhibit this upregulation by using PD98059, a specific inhibitor of MEK-1. For
the first time we can show here an additional immediate short term effect of these growth
factors (GF) on the distribution of uPAR within the cell. When VEGF (50ng/ml) or bFGF (10ng/ml)
was added to the cells, a redistribution of uPAR was seen. The GFs induce internalization of
surface uPAR after 30 min with a maximum after 4 hrs. To proof a possible LRP dependent
internalization process, we used the Receptor Associated Protein (RAP), a specific inhibitor of
LDL-receptor family mediated internalization and found that the redistribution was RAP
dependent. To further delineate the initial events by GFs, we analyzed cell surface bound
urokinase by fluorometric cell assay and ELISA, using antibodies recognizing the inactive
pro-uPA as well as the active form uPA, respectively. We found a loss of pro-uPA on the cell
surface upon VEGF stimulation, while total uPA antigen followed the changes in uPAR.
Internalization of uPAR and activation of uPA in response of VEGF was absent in the presence
of PI3-kinase inhibitors, but could be restored adding exogenous active uPA to the PI3-kinase
inhibited cells. Because a specific inhibitor of gelatinases (MMP-2, MMP-9) could inhibit the
redistribution of uPAR and the loss of pro-uPA induced by growth factors in ECs, this indicates
that the MMP-protease dependent pro-uPA activation by VEGF is the part being PI3-kinase
dependent. Such mechanism might play a role in redistribution of uPAR to the leading edge and
fine tuning of surface bound proteolytic activity.
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Identification of Cell Adhesion Sites in Angiopoietin-1 and Angiopoietin-2
Timothy R Carlson, Kwesi O Mercurius, Yuezhong Feng, Milan Mrksich, Godfrey S Getz, Alex
O Morla. University of Chicago, Chicago, IL
Angiopoietin-1 (Ang-1) knockout animals are embryonic lethal and display profound defects in
angiogenesis. Angiopoietin-2 (Ang-2), an Ang-1 homologue, is upregulated in the vasculature
of many human tumors and has been shown to potentiate angiogenesis induced by VEGF and
bFGF in various angiogenesis models. However, the precise mechanism of angiopoietin action
remains unknown. Previously, we showed that cells adhere to both Ang-1 and Ang-2 and that
this adhesion is mediated primarily by 1 and v5 integrins. Herein, we report the
identification of sequences of Ang-1 and Ang-2 that support cell adhesion. The amino acid
sequences of Ang-1 and Ang-2 are about 60% identical and both contain an N-terminal
coiled-coil domain and a C-terminal fibrinogen-like domain. Cells plated onto surfaces coated
with recombinant fibrinogen-like domain do not adhere, even though this domain binds Tie2,
an endothelial-specific receptor tyrosine kinase. On the other hand, recombinant coiled-coil
domains of Ang-1 and Ang-2 produced in E. Coli support adhesion of fibroblasts and endothelial
cells. To further localize the cell binding sites, 100 amino acid polypeptides were produced
and tested for adhesivity. We find that Ang-1 (103–202) supports strong cell adhesion and
spreading. Ang-1 (21–120) and Ang-2 (21–121) demonstrate weak adhesive activity, while
Ang-1 (182–284) and Ang-2 (103–202 & 183–282) appear non-adhesive. To further localize
the cell binding sites, Ang-1 (103–202) was spanned with eleven 15mer peptides, and each
peptide was immobilized to an inert surface and tested in adhesion experiments. Two of these
peptides support cell adhesion and spreading comparable to that observed on surfaces
presenting a control Arg-Gly-Asp sequence. Thus, we have identified cell adhesion sites within
Ang-1 and Ang-2. Interestingly, the sites are physically separable from the sequences required
to bind Tie2.
Involvement of RhoA/Rho Kinase-Signaling in VEGF-Induced Endothelial
Cell Migration and Angiogenesis in Vitro
Geerten P Van Nieuw Amerongen, Amanda Versteilen, Erna A Peters, Rick Van Leeuwen,
Pieter Koolwijk, Victor W Van Hinsbergh. VU University Medical Center, Amsterdam,
Netherlands; TNO-Prevention& Health, Leiden, Netherlands
Vascular Endothelial Growth Factor (VEGF) potently stimulates endothelial cell (EC) migration
and angiogenesis. Reorganization of the F-actin cytoskeleton and cell-matrix adhesion is
essential in these processes. In the present study we investigated whether RhoA and its
downstream target Rho kinase are involved in the VEGF-induced EC changes. VEGF induced a
rapid and prolonged reorganization of the F-actin cytoskeleton in ECs, manifested by the
formation of stress fibers and focal adhesions. This was accompanied by a targeting of RhoA
to the cell membrane. 5 g/mL C3 transferase and 10 mol/L Y-27632, specific inhibitors of
RhoA andby Rho guest kinaseon respectively, April 4, 2013 completely abolished the VEGF-induced cytoskeletal
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a-8 Arterioscler Thromb Vasc Biol. Vol 22, No 5
changes. Inhibition of Rho kinase had no effect on basal EC migration in response to
mechanical wounding, but prevented the VEGF-enhanced EC migration. In an threedimensional
in vitro model for angiogenesis treatment with C3 transferase reduced the mean
tube length of the capillary-like tubular structures formed in response to VEGF/TNF- by
40.2 13.6 % (mean SD, n8, p0.01). A similar, dose-dependent reduction in mean
tube length was observed in Y-27632-treated ECs. However, at a maximally inhibiting
concentration of 10 mol/L Y-27632, the number of the onsets of tube-like structures that
started to form in response to VEGF/TNF- increased. These data indicate a dual role of Rho
kinase in the formation of tubular structures. Rho kinase activity is necessary for the proper
ingrowth of ECs in the three-dimensional matrix, and restricts the number of ECs that start to
develop tubular structures . Thus, the VEGF-induced cytoskeletal changes in ECs are mediated
by RhoA and Rho kinase and activation of RhoA and Rho kinase is involved in the VEGF-induced
in vitro EC migration and angiogenesis.
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Cadherin Expression Regulates Vascular Smooth Muscle Cell Proliferation
Sarah J George, Christopher L Jackson, Raymond C Bush, Gianni D Angelini, Andrew C
Newby. University of Bristol, Bristol, UK
To date little is known of the role of cadherin expression in vascular smooth muscle cells
(VSMCs) in vascular disease. In this study the role of cadherins in VSMC proliferation, which is
an important component of atherosclerosis, restenosis after angioplasty and vein graft
neointimal thickening, was examined. The expression of cadherins in balloon injured rat
carotids (n3 per timepoint) and the effect of perturbing cadherin function on VSMC
proliferation in aortic rings (n4 per condition) in vitro were determined. Balloon injury
significantly reduced the number of VSMCs positive for R-cadherin by 32%, 52% and 23% at
0.25, 24 and 48 hours after injury, respectively, and for T-cadherin by 65% and 35% at 0.25
and 24 hours after injury, respectively. In contrast, E-cadherin expression was not present prior
to injury but was significantly increased at 24 and 48 hours after injury. These changes in
cadherin expression coincided with the initiation of medial VSMC proliferation and detection of
nuclear -catenin. In addition, the association of cadherin expression and proliferation was
demonstrated by the observation that all R-cadherin negative and E-cadherin positive VSMCs
were positive for proliferation. Inhibition of classic cadherins with a HAV peptide and a
R-cadherin neutralizing antibody significantly increased proliferation whilst an E-cadherin
neutralizing antibody significantly decreased proliferation. These results suggest that regulation
of cadherin expression and -catenin signaling may be essential to permit medial VSMC
proliferation. Alterations in cell-to-cell adhesion through cadherins may therefore play an
important role in neointimal thickening and restenosis.
Proteomics Inventory of Atherosclerosis-Specific Endothelial Cell
Membrane and Caveolar Proteins Suited for Vascular Targeting
Richard R Sprenger, Dave Speijer, Hans Pannekoek, Anton J Horrevoets. Academic Medical
Center, Amsterdam, NH, Netherlands
Atherosclerosis is initiated by lipid accumulation in the intima of large vessels at sites of
disturbed blood flow. Endothelial cell caveolae are involved in transcytosis and signal
transduction, and their protein composition varies as a result of (patho-) physiological
conditions, including changes in blood flow and inflammation. A direct proteomics approach is
used to identify atheroma-specific caveolar proteins of endothelial cells (EC) and their possible
causative role in lesion formation. The caveolar and total cell protein composition of EC exposed
to atherogenic stimuli, including TNF, VEGF, IL-1 and laminar shear stress, were explored
using 2D-gel electrophoresis. When studying total cell lysates, their complexity was reduced by
using differential solubilization methods. Caveolar proteins were isolated from resting and
stimulated EC using both buoyant density and cationic silica methods. Comparison of a large
series of 2D-maps resulted in the isolation of 50 proteins that are differentially expressed
under specific conditions. Selected proteins were subjected to mass spectrometry (MALDI-TOF
and Q-TOF Tandem-MS), leading to the identification of novel resident caveolar proteins, in
addition to the expected known (trans-) membrane, signal transduction and GPI-linked proteins.
In addition to this approach, luminal plasma membrane and caveolar protein 2D maps were
also generated under various conditions after surface biotinylation and subsequent fractionation.
This lead to the isolation of several surface exposed proteins, which are suitable for
targeting, and their identity is currently being established. These results show that EC protein
expression and in particular caveolar protein composition is substantially changed as a result
43
of activation of endothelial cells by pro-atherogenic stimuli. Identification of surface exposed
proteins will be essential for vascular targeting to sites of atherosclerotic lesion formation.
Translation State Array Analysis Reveals Control of Gene Expression at
Translational Checkpoints in Stimulated Endothelial Cells
Douglas I Schmid, Andrew S Weyrich, Guy A Zimmerman, Larry W Kraiss. University of
Utah, Salt Lake City, UT
Background: Gene expression is controlled at multiple checkpoints. While transcriptional
mechanisms are well documented in stimulated human endothelial cells, relatively little is
known about post-transcriptional regulation. We approached this issue using TSAA, which
couples polysome fractionation with cDNA array analysis to estimate the efficiency of
translation for individual mRNA transcripts (Zong et al, PNAS, 1999). Methods: Ribosomal
preparations from quiescent (CON) or serum-stimulated (6 hours, SER) human umbilical vein
endothelial cells were separated by gradient centrifugation into monosome-associated,
under-translated (UT) and polysome-associated, actively translated (AT) fractions. Labeled
mRNA from these fractions was then hybridized to microarrays containing 4,000 cDNA
sequences and analyzed by phosphorimaging. Change in translation state (TS) was calculated
as TS [SER(AT/UT)] / [CON(AT/UT)]. Significant redistribution of an mRNA transcript
between UT and AT fractions (0.4 TS 2.5) implies distinct translational regulation. TS values
1 suggest translation for that transcript is not separately regulated. Results: Hybridization
signals for 946 of 4,000 genes were above background. TSAA revealed that TS values for
these 946 genes were normally distributed around a value of 1. TS values for 68 (7%)
genes suggested independent translational regulation. Many of these genes regulate cell
growth or are components of energy/biosynthetic pathways integral to cell division. Six hours
after serum stimulation, 37 genes showed translational activation (TS 2.5, including CDC34,
UMP synthase and citrate synthase) and 31 were translationally repressed (0.4 TS, including
protein phosphatase 2A and CDK6). Conclusions: Activated endothelial cells regulate expression
of a significant number of genes (7%) at translational checkpoints, consistent with
studies in 3T3 cells and yeast. Many of these genes are likely to be critical in endothelial repair
and response to injury, implying important roles for translational regulation in determining
vascular phenotype.
Changes in Atherosclerotic Plaque Protein Expression Profiles: A
Proteomics Analysis
Marjo M Donners, Sylvia Heeneman, Luc Van Den Akker, Monique J Verluyten, Mat J
Daemen. Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht,
Maastricht, Netherlands; Maasland Hospital, Sittard, Netherlands
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Rupture of atherosclerotic plaques is the predominent underlying process in the pathogenesis
of acute coronary syndromes and peripheral vascular disease. Understanding the complex
changes taking place in plaque progression will eventually lead to a better understanding of the
underlying mechanism. Proteomic technology is a powerful tool to describe changes on the
level of protein expression and modification patterns. In this study, we used a proteomic-based
approach to characterize changes in protein expression in different types of human
atherosclerotic plaques. Human carotid plaque specimens were obtained from patients
undergoing endarterectomy. Tissues were immediately divided on ice so that side-to-side
tissue was either used for plaque classification or snap-frozen in liquid nitrogen (used for
protein analysis). Protein separation was achieved using 2-dimensional electrophoresis (2-DE).
The first dimension was performed using IPG-strips (24 cm, pH 4–7) followed by large SDS gel
electrophoresis (12%) and silver-staining. 2DE protein maps were made of pooled tris-extracts
of 3 advanced stable plaques and pooled tris-extracts of 3 ruptured plaques. The stable and
ruptured plaque extracts were prepared, processed and stained in five replicate gels.
Quantification of matching efficiencies revealed that around 80% of all spots (1200 of 1500
per gel) were matched on the five replicate gels (same sample, gels produced, run and stained
in parallel). An area containing 380 spots was selected for further analysis. Eighty-nine proteins
were detected only in the advanced stable lesions, while 67 were found exclusively in ruptured
plaques. Other spots showed differences in intensity, and need further quantification. The
proteins that were differentially expressed in either stable or ruptured lesions are being
identified using MALDI-MS technology. In conclusion, proteomics is a powerful tool to assess
reproducible differences in protein expression associated with atherosclerosis, which will
provide important insights in the mechanisms of plaque progression and rupture.
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P47
Effect of Adenosine A 1 Receptor Agonist R-Phenylisopropyladenosine on
Left Ventricular Function and Heart Rate in Rats
Guo Jin-Ai, Dai Cheng-Xian, Jing Chen. No. 466 Hospital of People’s Liberation Army,
Beijing, China
Objective To determine the regulative effects of adenosine A 1 receptor agonist
R-phenylisopropyladenosine(R-PIA) on the heart rate(HR) and left ventricular function in rats.
Methods Forty male Wistar rats were randomly divided into five groups (n8 ): Group normal
saline; Group R-PIA 0.03mg/kg; Group R-PIA 0.04mg/kg; Group R-PIA 0.05mg/kg; Group R-PIA
0.03mg/kg plus DPCPX (an adenosine A 1 receptor antagonist) 0.2mg/kg. After being anesthetized,
rats were inserted plastic cannula into left ventricular (LV) cavity through carotid artery
,and the hemodynamics indexes of LV were continuously monitored and processed by Cardio2
pressure signal processing software. At different point before and after subcutaneous
administration of the drugs , the parameters of HR , LV-PSP, LV-DP, dp/dt max and RPP were
recorded . The data were processed by SPSS 8.0 statistics analysis software . Results 1) A
dose-dependent negative chronotropic effect on the heart was produced by R-PIA in
anaesthetized rats; The decrease of HR was started from 5 min after R-PIA subcutaneous
injection and got the lowest value during 15–30 min after administration of R-PIA . The effect
of R-PIA on HR gradually disappeared during 90–120 min . 2) The temporary inhibition of LV
function induced by R-PIA went to maximum during 15–30 min and tended to weaken at 60
min after administration of the drug . 3) The RPP, an index reflecting myocardial oxygen
requirement, was significantly reduced by R-PIA. Conclusions 1) This study validated again
that adenosine A 1 receptor agonist R-PIA could bring on a dose-dependent negative
chronotropic effect and negative inotropic effect on the heart of rat. 2) R-PIA’s effect of
reducing myocardial oxygen requirement could last for a longer time than its negative inotropic
effect, and those could be of much benefit to myocardial protection induced by R-PIA.
Increased Levels of LDL Oxidation in Patients with Familial
Hypercholesterolemia and in End-Stage Renal Disease Patients on
Hemodialysis
Lambertus J Van Tits, Jacqueline De Graaf, Sebastian J Bredie, Pierre N Demacker, Paul
Holvoet, Anton F Stalenhoef. University Medical Center Nijmegen, Nijmegen, Netherlands;
Catholic University of Leuven, Leuven, Belgium
Patients with familial hypercholesterolemia (FH) and patients with end-stage renal disease
(ESRD) undergoing dialysis suffer from accelerated atherosclerosis. Oxidation of LDL is crucial
in atherogenesis. Objectives: In the present study, we determined LDL oxidation level of
isolated LDL of 11 male patients with FH, 15 male ESRD patients on hemodialysis, and 15
age-matched male normolipidemic healthy controls. FH patients were without lipid-lowering
medication for at least 4 weeks and were reassessed after two-year cholesterol-lowering
therapy with statins. Methods: LDL oxidation level was measured by ELISA using monoclonal
antibody 4E6 to oxidized LDL (oxLDL) as the capture antibody, and anti-human apoB antibody
for detection, and expressed as percentage oxLDL. Findings: In FH patients and in ESRD
patients on hemodialysis, plasma LDL oxidation levels were significantly elevated compared to
controls (4.9 1.3; 3.7 2.0; 1.7 0.6%, respectively). Within each group of subjects, LDL
oxidation level was not associated with history of CVD nor with smoking. Furthermore, in
neither group a significant correlation was found between plasma concentration of LDL
cholesterol and LDL oxidation level. After cholesterol-lowering therapy, LDL oxidation level in
FH patients had not changed significantly and remained elevated compared to controls, despite
a reduction of LDL cholesterol by 55% on the average. Also absolute plasma oxLDL
concentrations, obtained by multiplying LDL oxidation level with plasma LDL cholesterol
concentration, were significantly higher in FH patients before and after cholesterol-lowering
therapy and in ESRD patients on hemodialysis than in controls (489 145; 189 122; 100
65; 59 27 moles/L, respectively). Conclusion: In conclusion, we show that both in FH
patients and in ESRD patients on hemodialysis, oxidation level of circulating LDL is increased,
and that cholesterol-lowering therapy with statins does not normalize elevated LDL oxidation
levels in FH patients. Increased LDL oxidation levels might be involved in accelerated
atherosclerosis in FH and in ESRD.
Interaction of Aggregated LDL with Cultured Human Coronary Artery
Endothelial Cells
Bin Zhao, Wei Huang, Wei-Yang Zhang, Howard S Kruth. Section of Experimental
Atherosclerosis, NHLBI, NIH, Bethesda, MD
OBJECTIVE: It is known that macrophages take up aggregated LDL (AgLDL), a form of LDL
found in atherosclerotic lesions. The purpose of the present investigation was to determine
whether cultured human coronary artery endothelial cells (HCAEC) also interact with AgLDL.
RESULTS: Both human monocyte-macrophages and HCAEC showed cell-association of LDL
aggregated by treatment with sphingomyelinase. After incubation with 50 ug/ml 125I-AgLDL for
1 day, cell-associated 125I-AgLDL was 394 and 455 ug/mg cell protein for macrophages
and HCAEC, respectively. When incubated with 50 ug/ml 125I-LDL, macrophages and HCAEC
showed very low levels of cell-associated 125I-LDL, 0.40 and 2.10.3 ug/mg, respectively.
Interestingly, the microtubule inhibitor, nocodazole, decreased HCAEC cell-associated 125I- AgLDL by 52%, but did not decrease macrophage cell-associated 125I-AgLDL. Degradation of
125 125 125 I-AgLDL (i.e., medium TCA-soluble I) compared with I-LDL was increased 37-fold (from
20 to745 ug/mg) in macrophages, but wasDownloaded not increased forfrom HCAEC (131 and 111
Poster Presentations
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P49
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Poster Presentations a-9
ug/mg). Thus, macrophages and HCAEC degraded 65% and 22% of the total accumulated
125 I-AgLDL (cell-associated degraded), respectively. In contrast to cell-associated 125 I-
AgLDL, nocodazole decreased 125 I-AgLDL degradation in macrophages by 38% (from 745 to
464 ug/mg), but did not affect 125 I-AgLDL degradation in HCAEC. This means that although
both macrophages and HCAEC can take up and degrade AgLDL, microtubules functioned
differently in this process for each cell type. Examination of HCAEC cultures by phase
microscopy showed that AgLDL was bound to the surface of some but not all cells. Some
HCAEC strains showed low levels of cell-associated 125 I-AgLDL and these same cell strains
showed very few endothelial cells that bound AgLDL. CONCLUSIONS: HCAEC process AgLDL
differently than macrophages by showing relatively high levels of 125 I-AgLDL cell-association
that was nocodazole-sensitive, but low levels of 125 I-AgLDL degradation, which was
nocodazole-resistant. In addition, HCAEC strains show cell to cell and strain to strain
heterogeneity in their interaction with AgLDL.
Lipoprotein Lipid Loading and Cytokine Response in Macrophages
Marie Lindholm, Jenny Persson, Jan Nilsson. Lund University, Malmö, Sweden
It is well established that macrophage uptake of modified LDL results in foam cell formation,
cytokine signaling and most probably propagation of atherosclerotic plaques. However, massive
lipid droplet formation and a foam cell like appearance may also be caused by incubation of
macrophages with other lipoproteins. The main objective of the current study has been to study
how such lipid loading affect macrophage inflammatory response. Human monocyte-derived
macrophages have been pre-incubated with fresh LDL, vortex-aggregated LDL (AgLDL) or
VLDL, which increase intracellular cholesterol esters or triglycerides, respectively. Cells were
differentiated for five days before commencing the experiment, then pre-treated with
respective lipoprotein for 16 h. Cells were incubated for 4 h with fresh media with or without
TNF- .Cytokine concentrations have been measured in conditioned media and lipid content in
the cells (n10). It appears as if the lipoproteins have differential effects on both basal and
stimulated cytokine secretion. Pre-treatment with VLDL increase basal secretion of IL-1 with
230% compared to control, and decrease secretion of TNF- by 57% and IL-8 by 70%. AgLDL
have a less pronounced but similar pattern of effects on cytokine secretion (160, 51 and 50%,
respectively), as does native LDL (70, 44 and 50%, respectively). The cells were also stimulated
with TNF- after pre-incubations with lipoproteins. VLDL-treatment decreased stimulated IL-8
secretion by 32% compared to control, whereas LDL and AgLDL had mild increasing effects.
In contrast, AgLDL had a strong enhancing effect on stimulated IL-1 secretion (80% more
than control) compared to LDL and VLDL (8 and 40%). In conclusion, it appears as if LDL,
AgLDL and in particular VLDL have specific effects on basal and cytokine-stimulated
macrophage inflammatory signals in vitro. This is of interest as VLDL clearance is slow in
diabetic patients and it has long been recognized that this may be causative to the increased
risk for atherosclerosis in these individuals.
P51
Overexpression of Antioxidant Enzymes Reduces OxLDL-Induced Monocyte
Adhesion to Mouse Endothelial Cells
Zhongmao Guo, Hong Yang, Felicia Rymundo, Arlan Richardson. Meharry Medical College,
Nashville, TN; University of TX Health Science Center at San Antonio, San Antonio, TX
Adhesion of monocytes to endothelium plays a critical role in atherogenesis. In the present
study, we determined the effect of overexpressing antioxidant enzymes on the adhesion of
monocytes to endothelial cells (EC) and the expression of EC adhesion molecules. ECs were
obtained from the aorta of wild-type mice and transgenic mice overexpressing Cu/Znsuperoxide
dismutase (SOD), catalase or both Cu/Zn-SOD and catalase. The ECs were
incubated with native LDL, CuSO4-oxidized low-density lipoprotein (oxLDL) or culture medium
alone (control) for 2 hours and then incubated with mouse monocytes for 1 hour. Adhesion of
monocytes to ECs was determined by measuring the myeloperoxidase activity or the number
of monocytes firmly attached to ECs. The expression of EC adhesion molecules was determined
by measuring the protein level of vascular cell adhesion molecule 1 (VCAM-1) and monocyte
chemotactic protein 1 (MCP-1) after the ECs were incubated with native LDL, oxLDL or culture
medium alone. Results from this study showed that oxLDL significantly increased monocyte
adhesion to ECs and significantly increased the protein level of VCAM-1 and MCP-1 in ECs,
overexpression of antioxidant enzymes significantly reduced oxLDL-induced monocyte adhesion
to ECs and significantly reduced the level of EC adhesion molecules. For example, after
ECs were treated with oxLDL, the number of monocytes attached to the ECs obtained from
transgenic mice overexpressing Cu/Zn-SOD, catalase or both Cu/Zn-SOD and catalase was
50%, 30% and 70% less, respectively as compared to that attached to ECs obtained from
wild-type mice. The protein level of VCAM-1 and MCP-1 in ECs obtained from mice
overexpressing Cu/Zn-SOD and/or catalase was significantly lower than that in ECs obtained
from wild-type mice. These results suggest that reactive oxygen species, such as superoxide
and hydrogen peroxide, play a role in oxLDL-induced monocyte adhesion to ECs. This work is
supportedby in part guest by AHA on grant April 0030239N 4, 2013and
NIH grant 1P30AG13319.
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a-10 Arterioscler Thromb Vasc Biol. Vol 22, No 5
P52
Conformational Activation and Receptor Co-Association of GpIIb/IIIa on
Platelets
Stefan Barlage, Alexandra Pfeiffer, Antonia Wimmer, Gregor Rothe, Gerd Schmitz. Institute
for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg,
Germany
Analysis of the fluorescence resonance energy transfer between pairs of fluorochrome labeled
antibodies enables the analysis of receptor conformation or receptor clustering. The alphaIIb/
beta3 integrin (GpIIb/IIIa) is expressed on platelets, where it acts upon conformational activation
as a fibrinogen receptor, playing a critical role in platelet aggregation. GpIIb/IIIa antagonists
have been accused to cause occasional thrombocytopenia, probably via drug induced platelet
activation or immunogenic neoepitopes. Binding of the GpIIb/IIIa receptor antagonist MK-383
induced a GpIIb/IIIa conformation which differed from both the resting and the ADP-activated
receptor but there was no effect on P-selectin expression, indicating that MK-383 binding did
not induce general platelet activation via activated GpIIb/IIIa receptors. The interaction of
GpIIb/IIIa with other surface receptors may further modulate the signaling associated with
GpIIb/IIIa activation. Moreover, hypercholesterolemia or hypertriglyceridemia are characterized
by an increased in vivo activation of platelets and an altered membrane fluidity as determined
by pyrenedecanoic acid monomer/excimer distribution. It is tempting to speculate that
functional receptor co-operation, probably within cholesterol/sphingolipid rich membrane
domains is a major regulatory principle of platelet activation. We could demonstrate activation
dependent changes in the composition of a GPIIb/IIIa associated receptor cluster. GPIIb/IIIa is
associated with CD36 ex vivo. Upon activation, however, CD36 is separated from the complex
and the Fcgamma-receptor II (CD32) is included into the cluster. Concomitantly, upon
activation, CD32 associates to an integrin associated protein (CD9). In summary, the interaction
of GpIIb/IIIa with other receptor molecules or integrin associated molecules may represent an
important regulatory mechanism of platelet activation.
P53
p38 MAP Kinase Regulates Interleukin-1 Synthesis by Dissociating mRNA
from TIA-R in Activated Platelets
Stephan Lindemann, Neal D Tolley, Thomas M McIntyre, Guy A Zimmerman, Andrew S
Weyrich. Human Molecular Biology and Genetics, Salt Lake City, UT
We have previously demonstrated that platelets synthesize IL-1 (Interleukin-1) in an
activation-dependent manner. Here we delineate the specific signaling pathway that regulates
IL-1 synthesis by platelets. Activated platelets synthesize the majority of pro-IL-1 by 8 hours
and subsequently process this precursor into its mature form (between 200-1000 pg/ml) in a
concentration and agonist-dependent manner. When platelets are pretreated with the p38
MAP-kinase inhibitor SB203580, but not an inactive scrambled derivative, IL-1 synthesis is
completely abrogated (below 10 pg/ml). p38-dependent IL-1 synthesis is consistent with a
rapid increase in p38 MAP kinase phosphorylation that is sustained over an 8 hour time period.
Neutralization of p38 MAP kinase activity also blocks eIF4E (eukaryotic Initiation Factor-4E)
phosphorylation. However, it does not prevent eIF4E from binding IL-1 mRNA in activated
platelets. In a search for other RNA binding proteins that may regulate the translation of IL-1
mRNA in a p38-dependent fashion, we isolated proteins bound to poly-adenylated mRNAs that
were extracted with oligo-dT beads. In these studies, we found that platelet activation
increased the number of proteins bound to mRNAs compared to resting cells. In addition,
separation of the proteins by 2-dimensional SDS-PAGE demonstrated that pretreatment of the
activated platelet suspensions with the p38 MAP kinase inhibitor captured 2 additional
RNA-binding proteins. Predictions based on the molecular weight and PI of these proteins
indicated that they were from the TIA family of translational repressing RNA-binding proteins.
Immunocytochemistry and western analysis confirmed the presence of TIA-R in platelets and
subsequent studies demonstrated that TIA-R binding to IL-1 mRNA is markedly increased in
activated platelets pretreated with the p38 MAP kinase inhibitor. These studies suggest that the
p38 MAP kinase pathway modulates RNA binding to translational repressing TIA proteins. They
also suggest that p38 MAP kinase inhibitors may be useful clinical tools for inhibiting cytokine
synthesis by aggregated platelets.
P54
Are Routine Investigations for Hypercoagulable Disorders Justified in Acute
Ischemic Stroke?
Majaz Moonis, Timea Kovats. UMass Medical School, Worcester, MA
Introduction. Indications for hypercoagulable work-up in acute ischemic stroke are unclear.
Under current recommendations hypercoagulable work-up is not recommended if other risk
factors present might explain the basis of the stroke. However a hypercoagulable workup is
expensive. Under these circumstances, is it useful to screen patients with acute ischemic
stroke for hypercoagulable states? Methods. We performed a retrospective cohort study of all
consecutive patients admitted with acute ischemic stroke between January, 1999-March,
2001.The objective was to determine the prevalence of hypercoagulable risk factors and their
concomitant association with other vascular risk factors. Work-up in all cases included Lupus
Anticoagulant, Anticardiolipin antibodies, Antithrombin 111, protein S and C deficiency, Factor
V Leiden, Prothrombin 20210A gene mutation and Fibrinogen. Results.Of the 283 patients ,
hypercoagulable work-up had been done in 53(18.7%)and a positive result was seen in
23(43.4%). Antithrombin 111 ,Protein S and Protein C deficiency were seen in 52%, 17.3% and
21.7% respectively. Lupus Anticoagulant and Anticardiolipin antibodies were detected in
30.4%. Elevated fibrinogen levels were evident in 13%. In patients with abnormal results stroke
types were as follows: 52.3%had embolic, 17.3% thrombotic,13.2% lacunar and 17.3%
indeterminate. In 32(60.2%) other concomitant risk factors were identified. Diagnosis of
hypercoagulable states led to a significant increase in the percentage treated with anticoagulants:
heparin (9%) and warfarin (22%). Death in 5 patients was associated with 2
Antithrombin 111 levels. Conclusions. Hypercoagulable Downloaded states appear from
to be more common than
http://atvb.ahajournals.org/
currently believed and co-exist with other stroke risk factors including underlying cardiac
disease.In patients with large vessel non-cardioembolic strokes where anticoagulation is
usually not anticipated, evaluation for a hypercoagulable state may effect therapeutic decisions.
This may reduce stroke recurrence and other co-morbid conditions such as deep vein
thrombosis and pulmonary embolism. Comparative results with studies will be discussed.
P55
Functional Properties of the Endothelial and Media Layers of the Human
Tissue-Engineered Blood Vessel
Murielle Remy-Zolghadri, Guillaume Grenier, Karina Laflamme, Lucie Germain, Francois A
Auger. Laval University, Quebec, QC, Canada
We have reported the production of a human TEBV by the self-assembly approach as the first
step in the development of a small diameter vascular prosthesis. We present herein the
characterization of physiological functions of the endothelial and media layers. The role of
endothelial cells (EC) in the hemostatic balance and inflammatory process was assessed by
quantification of vWF, thrombomodulin (TM) and proadhesive molecules. The function of the
reconstructed media (RM) was analyzed by the detection of contractile proteins in SMC, the
accumulation of extracellular matrix and their organization in the media. The vasocontractile
response of the RM to endothelin was also evaluated. Results show that the endothelium (50
000 5400 EC/cm2) presents a basal secretion of procoagulant vWF that increases twice after
stimulation. Eighty percent of EC express the anticoagulant TM at any time of the maturation
process. Twenty four hours after seeding, 1.26 3.3 and 2.3 2.2 % of cells express
E-selectin and ICAM-I, respectively. As expected, cells up-regulate the expression of these
molecules when stimulated. Concerning the RM, results show that the tension is a key factor
for SMC survival and maturation since cells in a loose environment dedifferentiate. In contrast,
when a tension is applied, SMC become aligned in the axis of the tension and a compaction
of the ECM occurs. From a cellular point of view, SMC up-regulate their secretion of collagen
IV whereas collagen I is down-regulated. Differentiation markers increase as a function of
culture time and ECM remodeling. RM respond to endothelin (EC50 of 5nM) and SMC express
Et-A receptors since contraction was diminished by BQ-123 antagonist. In summary, the
endothelium displays anticoagulant properties associated with no proinflammatory characteristics.
The vascular media contains differentiated SMC aligned in an organized ECM. Moreover,
SMC are able to react to endothelin by expressing Et-A receptors. Taken together, these results
show that cells in the TEBV possess physiological functions normally present in blood vessels,
indicating the potential of these living tissue constructs as vascular prosthesis.
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P57
Cyclooxygenase-2 Is Induced in Monocytes by PPAR and Oxidized Alkyl
Phospholipids from Oxidized LDL
Thomas M McIntyre, Aaron V Pontsler, Andy St Hilaire, Gopal K Marathe, Guy A
Zimmerman. University of Utah, Salt Lake City, UT
LDL oxidation and monocyte infiltration of the vessel wall underlie atherogenesis. These cells
express cyclooxygenase-2, but the mechanism by which oxidized LDL stimulates
cyclooxygenase-2 transcription is unknown. Circulating human monocytes do not contain
PPAR, but we find that engagement of their surface ICAM-3 induces PPAR message and
protein accumulation within a few hours. Oxidatively-fragmented phospholipids isolated from
oxidized LDL, a synthetic oxidized alkyl phospholipid (azPC) that is a potent PPAR agonist, or
rosiglitazone induced cyclooxygenase-2 expression and enhanced PGE 2 secretion. Phospholipase
A 1 and A 2 digestion demonstrates that oxidized alkyl phospholipids, and not oxidized fatty
acids, were the relevant agonists. Cyclooxygenase-2 induction only occurred in monocytes
containing PPAR. The cyclooxygenase-2 PPRE was required for induction by oxidized
phospholipids, and these lipids, azPC, and rosiglitazone stimulated full length and (Cox-2
PPRE) 3-luciferase reporters. The cyclooxygenase-2 PPRE responded strongly to PPAR
agonists, less well to WY14643 at concentrations selective for PPAR, while higher
concentrations of 15-deoxyPGJ 2 and ciglitazone were inhibitory. 15-deoxyPGJ 2 inhibited
PMA-induced cyclooxygenase-2 expression by PPRE-dependent and -independent mechanisms.
Thus PPAR is rapidly induced in primary monocytes by appropriate outside-in
signaling. This allows them to respond to previously undetectable agonists in oxidized LDL.
Vascular cells such as endothelial cells and smooth muscle cells constitutively express
PPARgamma and are not subject to this form of regulation. Cyclooxygenase-2 and PGE 2
secretion are induced - not inhibited - by selective PPAR agonists that includes oxidativelyfragmented
phospholipids in oxidized LDL.
Ceramide Upregulates Nitric Oxide Synthase Expression in Human
Endothelial Cells
Huige Li, Peter Junk, Christian Burkhardt, Thomas Wallerath, Ulrich Förstermann, Andrea
Huwiler, Josef Pfeilschifter. Johannes Gutenberg University Mainz, Mainz, Germany;
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am
Main, Germany
Ceramide is a sphingolipid second messenger that can be formed by hydrolysis of membrane
sphingomyelin by sphingomyelinase. Recent studies have indicated that ceramide can induce
vasodilation and can activate endothelial NO synthase (eNOS) through a Ca2-independent
mechanism. The aim of this study was to examine whether ceramide modifies the expression
of eNOS gene. Methods: eNOS mRNA and protein expression in human umbilical vein
endothelial cells (HUVEC) and in HUVEC-derived EA.hy 926 endothelial ce lls were analyzed by
RNase protection assay and Western blot, respectiviely. A 1.6-kb human eNOS promoter
fragment by cloned guest before on the April luciferase 4, 2013 gene was transfected transiently into EA.hy 926
P58

endothelial cells to determine the effect of ceramide on eNOS promoter activity. Results:
N-hexanoyl-D-erythro-sphingosine (C6-ceramide) increased the expression of eNOS mRNA and
protein in a concentration- and time-dependent manner. A significant increase of the eNOS
mRNA was already observed after a 3 h incubat ion with 10microM C6-ceramide; the
stimulation was maximal at 9 h. A9hincubation of the cells with 1 and 10microM of
C6-ceramide upregulated the eNOS mRNA level to 150% and 231% of control, respectively.
C6-ceramide enhanced the eNOS promoter activity and did not affect the stability of the eNOS
mRNA. Incubation of the cells with C8-ceramides and sphingomyelinase increased the eNOS
mRNA expression as well. The known downstream signaling pathways of ceramide include
activation of protein kinase C (PKC), mitogen-activated kinase (MAPK) and/or the protein
phosphatase 2A (PP2A). The eNOS induction by ceramide was neither affected by PD 98059
(10microM), an inhibitor of the MAPK pathway, nor by the PKC inhibitors Go6983 (1microM) and
GF 109203X (1micr oM). It was, however, reduced by the PP2A inhibitors okadaic acid (10nM)
and calyculin A (10nM). Conclusion: These results suggest that ceramide increases eNOS
transcription through activation of PP2A. This finding m ay be helpful for understanding the role
of ceramide in atherogenesis.
Vascular Smooth Muscle Cell Proliferation Requires Dismantling of
N-Cadherin Cell-Cell Contacts
Elizabeth B Uglow, Gianni D Angelini, Andrew C Newby, Sarah J George. University of
Bristol, Bristol, UK
Vascular smooth muscle cell (VSMC) proliferation is an important component of cardiovascular
disease. Cadherin-mediated cell-to-cell contacts inhibit proliferation in cancerous cells.
Cadherins are a family of transmembrane glycoproteins that bind to the actin cytoskeleton
through interacting with -catenin. Disruption of this linkage leads to release of -catenin into
the cytoplasm where it is either degraded or translocated to the nucleus where it up-regulates
genes involved in proliferation. In this study the involvement of the N-cadherin-catenin complex
in VSMC proliferation was determined. Human saphenous vein VSMCs were quiesced for 72
hours in serum-free media and then proliferation was stimulated by incubation in 10% foetal
calf serum (FCS) or 5ng/ml platelet-derived growth factor (PDGF) for 24 hours. N-cadherin and
-catenin expression was determined by methods including semi-quantitative and quantitative
RT-PCR, Western blotting and immunocytochemistry. Stimulation of proliferation by FCS (n4)
and PDGF (n3) significantly reduced the amount of N-cadherin protein detected compared to
quiescent VSMCs by 57% and 87%, respectively. This was not due to reduced mRNA synthesis
or increased endocytosis. However, stimulation of proliferation caused release of N-cadherin
fragments into the culture media, suggesting the involvement of proteolytic shedding. Indeed,
incubation with a matrix-degrading metalloproteinase inhibitor significantly reduced shedding.
Total levels of -catenin and phosphorylation status were not affected by stimulation of
proliferation but its localization was (n3). The detection of peri-nuclear -catenin was
increased in VSMCs treated with FCS compared to quiescent VSMCs. We hypothesize that
proteolytic shedding of N-cadherin permits nuclear translocation of -catenin, which in turn
induces the expression of cell cycle genes and proliferation. We propose that inhibition of
N-cadherin shedding and -catenin signaling may be clinically useful for reduction of VSMC
proliferation.
Relative Importance of TNF- Receptor I vs. Receptor II in Leukocyte
Adhesion to TNF--Treated Mouse Endothelial Cells In Vivo
Unni M Chandrasekharan, Murat Unsal, Paul E Dicorleto, Maria Siemionow. Cleveland Clinic
Foundation, Cleveland, OH
Tumor necrosis factor- (TNF-) binds to two cell surface receptors, namely TNF- receptor-I
(TNFR-I) and TNF- receptor-II (TNFR-II). TNFR-I is thought to be functionally dominant, and
few discreet physiological functions for TNFR-II have been identified. Activation of endothelial
cells (EC) by TNF- induces the expression of various cell adhesion molecules, and regulated
expression of these adhesion molecules mediates the rolling, firm adhesion and transmigration
of leukocytes during an inflammatory reaction. We have approached the question of specific
TNFR-II activity in vivo using a cremaster muscle-flap model and intra-vital microscopy with
wild-type mice (WT) and mice that are null for either one or both of the TNFR genes. We have
discovered that leukocyte adhesion to TNF--stimulated EC was solely mediated through
TNFR-II. Local application of TNF- (2 ng/ml) to the cremaster muscle-flap of WT mice induced
two temporal peaks (one at 30–60 min and one at 90–135 min) for leukocyte rolling and a
single peak (at 90–135 min) for leukocyte adhesion. TNF- induced a 6.40.9 fold increase
in leukocyte adhesion in WT mice. In TNFR-II null mice this activity was completely abolished.
In contrast, leukocyte adhesion induced by TNF- in TNFR-I null mice was not reduced.
Leukocyte rolling was partially compromised in both the TNFR null mice. Double null mice did
not respond to TNF-. Histology of cremaster tissues from WT mice after 3hofTNF-
treatment showed abundant infiltrated leukocytes in the interstitial space. This infiltrate was not
present in either TNFR-II null or TNFR-I null mice. We conclude that TNFR-II activation alone is
responsible for TNF--induced leukocyte adhesion to mouse EC, and both TNFR-I and TNFR-II
are involved in TNF--induced leukocyte rolling. Further, both the TNF- receptors are critically
involved in leukocyte transmigration from blood vessels to the extravascular space.
Molecular Basis for Prostaglandin E2-Regulated Endothelial Cell Motile
Response to Wounding
Huimiao Jiang, Aaron Ponstler, Kelley Murphy, Stephen Prescott, Guy Zimmerman, Thomas
McIntyre. Human Molecular Biology Genetics, Salt Lake City, UT; Huntsman Molecular
Biology Genetics, Salt Lake City, UT; Huntsman Cancer Institute, Salt Lake City, UT
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Poster Presentations a-11
PGE2 receptors on endothelial cells and their effect on endothelial cell migration are unknown.
RT-PCR estimation of message suggested that human umbilical vein endothelial cells (HUVECs)
express three of the four PGE2 receptor subtypes, EP2, EP3 and EP4. Real-time PCR showed
EP2 and EP4 messages were quantitatively induced in wounded monolayers as HUVECs began
to migrate into the denuded area. Flow cytometry showed increased expression of EP4 protein
on the surface of endothelial cells recovered from wounded monolayers. Immunohistochemistry
revealed that cells at the wound edge were especially bright when stained with fluorescent
anti-EP4 antibody. Exogenous PGE2 facilitated HUVECs migration in an in vitro endothelial
wound repair assay. This effect was mediated through EP2 and/or EP4 receptors, because EP2
and EP4 agonists facilitated HUVEC migration as well as PGE2. Enzyme-linked immunosorbant
assay showed that endogenous PGE2 secretion was increased with an increase in the level of
monolayer wounding, and that this increased secretion of PGE2 occurred in a cyclooxygenase-2
dependent way. These data document autocrine and paracrine effects of PGE2 on HUVECs
migration and regulation of key receptor systems by wounding. These results, in turn, provide
a mechanism for eicosanoid-mediated angiogenic responses in vivo.
The Roles of Fibrinogen and Apolipoprotein(A) in Diabetic Coronary
Atherosclerosis
Christoph Saely, Stefan Aczel, Peter Langer, Zeynep Vetter, Heinz Drexel. VIVIT-Institute,
LKH Feldkirch, Feldkirch, Austria
Background: Atherothrombotic disease involves atherogenic and thrombogenic episodes.
Because diabetic atherosclerosis typically exhibits thrombotic lesions interest has focused on
prothrombotic factors like fibrinogen and apolipoprotein(a) (apo(a)). Although cohort studies
have shown diabetes to be a prothrombotic state, little data exist for the diabetic patient with
established coronary atherosclerosis. Materials and Methods: We therefore performed a large
angiographic study involving 406 consecutive patients undergoing coronary angiography, of
whom 148 were hyperglycemic: 56 had established diabetes mellitus, 35 fasting plasma
glucose 125 mg/dl, 57 HbA1c 6.1%; and 258 patients fulfilled none of the three criteria
(normoglycemic). First, the three hyperglycemic subgroups were compared to the normoglycemic
patients by a Mann-Whitney U-test. Then we investigated by logistic regression analysis
the association of thrombogenic factors with the presence of coronary atherosclerosis. Results:
Serum levels of apo(a) did not differ significantly between any of the three hyperglycemic
subgroups and the normoglycemic patients. Also, if the three sugbroups were pooled together,
there was no significant difference between hyperglycemic and normogylcemic patients in
apo(a). In contrast, all three subgroups of hyperglycemic patients exhibited increased fibrinogen
levels (mean 373, 364, and 358 mg/dl vs. 329 mg/dl in normoglycemic patients, p values 0.06,
0.012, and 0.005). In the logistic regression model, fibrinogen - but not apo (a) - proved
predictive of the presence of coronary atherosclerosis. The predictive value of fibrinogen
remained significant upon inclusion of classical risk factors (age, gender, alcohol consumption,
smoking, hypertension, triglycerides, HDL and LDL cholesterol). Conclusions: We conclude that
fibrinogen is increased in diabetic patients with coronary atherosclerosis, and that fibrinogen
levels are strongly and independently predictive of the presence of coronary atherosclerosis. In
contrast, apo(a) levels are neither increased in diabetic patients nor predictive of the presence
of coronary atherosclerosis.
P63
Inhibition of Human Pulmonary Vascular Smooth Muscle Cell Proliferation
and Migration by BIIB722, a Novel Na/H Exchange Inhibitor
Dongmei Wu, Jean Marie Stassen, Henri Doods. Mount Sinai Medical Center, Miami Beach,
FL; Boehringer Ingelheim Pharma KG, Biberach, Germany
Abnormal growth of vascular smooth muscle cells (VSMCs) is seen in various pathologic
conditions such as hypertension, atherosclerosis and restenosis. Na /H exchange (NHE) has
been proposed to be involved in the regulation of the cell growth. The present study
investigated the effect of a novel NHE1 selective inhibitor, BIIB722 ((N-(Aminoiminomethyl)-4-
[4-(1H-pyrrol-2-ylcarbonyl)-1-piperazinyl]-3-(trifluormethyl)-benzamid) on human pulmonary
vascular smooth muscle cell proliferation and migration. BIIB722 produced concentrationdependent
inhibition of human pulmonary VSMC proliferation stimulated by growth factors, as
monitored by measuring cell count. Fifty percent inhibition (IC50) was achieved at 30.69
0.97 M. BIIB722 also produced concentration-dependent inhibition of DNA synthesis, as
examined by measuring BrdU incorporation into DNA, IC50 value was 42.56 1.20 M. Cell
growth inhibition was not due to cell death, as demonstrated by the measurement of
intracellular lactate dehydrogenase release, Caspase 3 activity, and by the reversibility of
inhibition upon washing. Flow Cytometry analysis revealed that BIIB722 arrests the proliferation
of human pulmonary SMCs at the G0/G1 phase of the cell cycle. At 60 M, BIIB722 blocked
the G1/S transition completely. In a cell migration model, BIIB722 concentration-dependently
inhibited human pulmonary VSMCs migration (0 to 60 M). The results support the hypothesis
the NHE1 plays an important role in the regulation of human pulmonary VSMC growth. BIIB722
inhibits VSMC proliferation by blocking G1/S transition of the cell cycle. Accordingly, BIIB722
may have therapeutic potential in the clinical treatment of diseases related to the abnormal
growth of vascular smooth muscle cells such as (pulmonary) hypertension, atherosclerosis and
restenosis.
P64
Vascular Thoracic-Outlet-Syndrome with Occlusion of Left Subclavian and
Left Cubitan Artery Because of Cervical Ribs
Jörg Nossen, Thomas Vierzigmann, Erich Lang. Waldkrankenhaus St. Marien, Erlangen,
Germany
Background: Thoraco-cervical neurovascular compression syndromes can be classified into
Endothelial cells migration is crucial in wound healing and angiogenesis. Prostaglandin E2 neurological (80–90%) and vascular (10–20%) thoracic-outlet-syndrome (TOS). Case report:
(PGE2), a product of cyclooxygenase, induces angiogenesis Downloaded in vivo. from
The expression http://atvb.ahajournals.org/
pattern of We reportby about guest a 27-year on April old white 4, 2013 woman with coldness and painful necrosis of left
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a-12 Arterioscler Thromb Vasc Biol. Vol 22, No 5
finger-tips. Angiography revealed occlusion of left subclavian as well as left cubitan artery.
Initially we tried embolectomy by fogarty catheter. Afterwards we treated with GP IIb/IIIa
receptor antagonists and weight-adjusted low molecular heparin. Further evaluation by plain
x-ray revealed left and right cervical ribs. Magnetic resonance tomography visualized
topographic relation between left cervical rib and occluded left subclavian artery. Occlusion of
left cubitan artery was considered as embolism. Now angiography evaluated unsuspected right
subclavian vein and artery and left subclavian vein in neutral position but completely
compression of right subclavian vein and artery and left subclavian vein by elevation and
abduction. Further therapy consisted of transaxillary resection of left cervical and first rib.
Occlusion of left subclavian artery was bypassed. Resection of right cervical and first rib is
recommended. Conclusions: Vascular thoraco-cervical compression syndromes because of
congenital anomalies are rare. Sometimes first symptomes may be seen after childhood. In
case of occlusion or embolism of veins and arteries of upper extremities this diagnosis should
be taken into consideration, too.
Pulsatile Flow-Induced Angiogenesis: Role of Gi3 Subunits
John P Cullen, Shariq Sayeed, Nicholas G Theodorakis, James V Sitzmann, Eileen M
Redmond. University of Rochester Medical Center, Rochester, NY
Shear stress is the primary driving force for control of blood vessel architecture and a
correlation has been reported between blood flow and angiogenesis in a variety of animal
models. Both pertussis toxin (PTX) sensitive (Gi 1,2,3) and insensitive (Gq) proteins have
been implicated in the transduction pathways necessary for shear stress-stimulated signaling
in endothelial cells (EC). We investigated the role of G proteins in pulsatile flow-induced
angiogenesis using a perfused transcapillary system incorporating bovine aortic EC (BAEC)
transiently transfected with or without mutant constructs of the G protein signaling pathway.
BAEC were exposed to static (0 ml/min) or flow conditions (1.2 ml/min - 67.0 ml/min,
corresponding to a shear stress of 1.4 dyn/cm 2 - 19.2 dyn/cm 2 ) for various periods of time (2
- 24 hours). Following exposure, angiogenesis was measured as tubule formation on Matrigel.
Pulsatile flow increased angiogenesis, in a temporal- and force-dependent manner, by approx.
4-fold (16 hrs, 13.2 dyn/cm 2 ) when compared to BAEC cultured under static conditions. These
effects of pulsatile flow were totally inhibited in the presence of PTX (100 ng/ml). Transfection
with the inhibitory mutant of the subunit of Gi3 (i3-G202T) resulted in a decrease in the
flow-induced angiogenic response (517.7 67.0% v 349.7 116.0%, n5) while
transfection with a constitutively activated mutant of the subunit of Gi3 (i3-Q204L) led to
an increase in the response (270.8 13.1% v 508.6 124.9%, n3) when compared to
BAEC transfected with empty vector. The pulsatile flow-induced angiogenic response was
significantly increased by 118% (p0.05) in BAEC transfected with a 194 amino-acid peptide
responsible for binding the subunit (ARKct) (270.8 13.1% v 590.7 97.5%, n3).
These results suggest that pulsatile flow-induced angiogenesis is mediated via PTX sensitive
G proteins. The partial inhibition and augmentation of flow-induced angiogenesis by transfection
with i3-G202T and i3-Q204L, respectively, provides strong evidence for the involvement
of Gi3 subunits in this response.
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Analysis of Inflammatory Gene Induction by Oxidized Phospholipids In Vivo
by Quantitative Real Time RT-PCR in Comparison with Effects of LPS
Alexandra Kadl, Joakim Huber, Florian Gruber, Valery N Bochkov, Bernd R Binder, Norbert
Leitinger. University of Vienna, Vienna, Austria
Oxidized phospholipids have been shown to play a major role in the development of
atherosclerosis, when they accumulate in the vessel wall. Purpose of this study was to
investigate the biological activity of oxidized phospholipids in vitro and in vivo, to demonstrate
their contribution in chronic inflammation. We analyzed expression of inflammatory genes that
had been shown relevant for atherosclerosis in cultured HUVEC and U937 cells three hours after
stimulation with oxPAPC or LPS as well as in various organs of female C57/Bl6-mice that
underwent intravenous injection, using real time RT-PCR. In accordance with previous studies
we found that in HUVEC OxPAPC induced EGR-1, MCP-1 and in liver JE and HO-1. In addition
we found elevated expression of JE in heart, of HO-1 in HUVEC, U937, aorta, lung and blood,
of EGR-1 in HUVEC, liver and heart, of TF in U937, lung and blood. Pointing to an important
difference in cell activation by LPS and OxPAPC, we demonstrate that LPS induces all tested
genes but HO-1, whereas HO-1-expression was strongly induced by oxPAPC. In contrast,
E-Selectin was induced in HUVEC and in all organs by LPS, but not by oxPAPC. We show that
the oxPAPC-induced HO-1-expression in blood can be blocked by pre-injection of a
PAF-receptor-antagonist, indicating a possible receptor-mediated effect. We conclude that
oxidized phospholipids are biologically active in vivo and exert a specific response inducing a
pattern of genes that is different from that induced by LPS. In addition, we demonstrate that
light cycler technology is a proper tool to investigate inflammatory gene induction in vivo.
HELP LDL-Apheresis and Its Effect on Lipoprotein-Associated
Phospholipase A 2
Patrick M Moriarty, Cheryl A Gibson, Nam Kim, Robert L Wolfert. University of Kansas
School of Medicine, Kansas City, KS; diaDexus, Santa Clara, CA
Background. Elevated levels of lipoprotein-associated phospholipase A2 (Lp-PLA2) appear to be
a strong and independent risk factor for coronary heart disease and may prove to have a direct
role in atherogenesis. We undertook this study to evaluate Lp-PLA2 changes associated with the
Heparin-induced Extracorporeal Low-density Lipoprotein Precipitation (HELP, B. Braun) system,
a LDL-apheresis procedure. Method. We conducted a prospective trial of 6 patients. All patients
were treated with LDL apheresis on an alternate week basis. We evaluated Lp-PLA2 levels and
lipid profiles immediately before and after lipid apheresis. Results. All 6 non-obese patients
(2M; 4 F; mean age 58 years), had cardiovascular Downloaded disease and severe from
hypercholesterolemia
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(LDL 200 mg/dl) resistant to diet and pharmacotherapy. Table 1 displays the results for the
pre/post comparison of Lp-PLA 2 and lipid values. Conclusion. As shown, a single LDL apheresis
treatment resulted in a 19.5% decrease in Lp-PLA 2 levels. Additionally, preliminary results
indicated that LDL apheresis might result in a significant long-term decrease of Lp-PLA 2 levels
despite lipid values rebounding to pre-treatment levels. Additional studies of the impact of LDL
apheresis mediated changes on Lp-PLA 2 and its implications for atherogenesis and risk
assessment are needed.
P68
Discordance of Endothelial Nitric Oxide Synthase in the Arterial Wall and
Its Circulating Products: Interactions with Redox Metabolism
Jian Wang, Diane Warzecha, Jane Vandeberg, Xing Li Wang. Southwest Foundation for
Biomedical Research, San Antonio, TX
Although peripheral arteries and circulating factors have been frequently used to assess
systemic or central arterial, such as coronary artery, endothelial nitric oxide synthase (eNOS)
functions, no direct evidence to support that they are related. We explored relationships in
eNOS levels among coronary, aortic, carotid and brachial arteries, and relationships with redox
metabolisms. In 40 baboons (Papio hamadryas), we found no correlations in eNOS or NOx levels
among all four arteries (R: 0.02–0.19, P0.05). While eNOS were high in both coronary
(133.7810.9 pg/mg protein) and aortic arteries (168.5316.32 pg/mg protein), they were
low in brachial (82.885.5 pg/mg protein) and carotid (89.837.15 pg/mg protein) arteries.
Arterial eNOS were not correlated with plasma NOx either. However, coronary and aortic eNOS
were positively correlated with corresponding arterial total antioxidant status (TAS) (R0.638,
P0.001, and R0.615, P0.0001). Coronary eNOS was also negatively associated with
coronary advanced glycation end products (AGE) (R-0.454, P0.003). Furthermore, there
were significant associations in TAS levels between plasma and arterial wall extracts
(R:0.344–0.369, P0.05) and among arteries of different anatomic sites (R:0.637–0.877,
P0.001). While arterial TAS were negatively correlated with corresponding arterial AGE, TAS
in arterial wall and plasma were positively associated with plasma AGE. Our study shows that
eNOS is differentially expressed in different anatomic sites and is not associated with plasma
NOx, suggesting that brachial eNOS may not be used to assess coronary eNOS. The positive
or negative associations between eNOS and TAS or AGE, especially in coronary artery, indicate
an important role of eNOS in arterial wall redox balance.
Magnetic Resonance Angiography of Mouse Aorta at 12 Tesla
Stuart M Grieve, Robin P Choudhury, Juergen E Schneider, Paul J Cassidy, Stefan
Neubauer, Kieran Clarke. Oxford Cardiac Research Group Magnetic Resonance Unit,
Department of Biochemistry, University of Oxford, Oxford, UK; John Radcliffe Hospital,
University of Oxford, Oxford, UK
OBJECTIVE: Recent advances in techniques of genetic manipulation mean the phenotypical
characterisation of transgenic mice has become increasingly important. This study investigates
the potential of high-field MRI as a tool to measure aortic dimensions and flow in mice. A
spin-echo MRA sequence (SE-MRA) has been developed and shows promise as a method for
quantifying cross-sectional areas and flow rates in the aortic arch and descending aorta.
METHODS: Mice were imaged at 12 T under isoflurane anaesthesia. SE-MRA was used to
obtain images of approximately 100 m resolution at several positions along the aorta. Flow
profiles were imaged using a presaturation method. RESULTS: Figs A and B show SE-MRA
images of mouse vasculature in vivo. Fig. A shows the aortic arch, Fig. B is a section through
the aortic root. Fig C shows a section of the descending aorta acquired 15 ms after a
presaturation slab was applied. The velocity flow profile across the vessel can be clearly
observed with a maximum velocity of approximately 3.5 cm/s. CONCLUSION: SE-MRA shows
promise as a tool for the investigation of vasculature in mice.
Simvastatin Selectively Inhibits the Expression of Tissue Factor and
Monocyte Chemotactic Protein-1 in the Aorta of Older Apo E-/- Mice
without Lowering Plasma Lipids
Florian Bea, Erwin Blessing, Monica I Shelley, Michael E Rosenfeld. University of
Washington, Seattle, WA
Recent studies suggest that some of the beneficial effects of HMG-CoA reductase inhibitors
(statins) in reducing cardiovascular disease endpoints may be independent of their capacity to
lower plasma lipids. To further test this hypothesis and to determine whether statin treatment
alters geneby expression guest on within April established 4, 2013 atherosclerotic lesions, simvastatin (50 mg/kg/d) was
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added to normal chow and fed to 30 week old, male apo E -/- mice for 12, 18 and 24 weeks.
Chow-fed apo E -/- mice develop advanced atherosclerotic lesions in the aorta and the
innominate and carotid arteries by 30 weeks of age. Furthermore, chronic simvastatin
treatment does not lower plasma cholesterol levels in these older mice. Quantitative real-time
PCR was used to measure expression of the mRNA for tissue factor (TF) and monocyte
chemotactic protein-1 (MCP-1) in the aorta of each mouse. Expression of TF was reduced 3 fold
by 12 weeks (n10), 4 fold after 18 (n10) weeks and 7 fold after 24 weeks (n7) as
compared to non-treated control mice (n 4–7). The content of activated TF was also reduced
in advanced lesions in the innominate arteries as evaluated histologically following binding of
labeled factor VIIa. There were no significant differences in the aortic expression of MCP-1 after
12 and 18 weeks. However, 24 weeks after treatment MCP-1 mRNA levels were reduced 3 fold
in comparison to control. In conclusion, these data suggest that chronic administration of
simvastatin to older apo E-/- mice inhibits the expression of pro-thrombotic/pro-inflammatory
genes within established atherosclerotic lesions by a mechanism that is independent of
lowering plasma lipids.
Induction of Glutathione Synthesis in Macrophages by Oxidized
Low-Density Lipoproteins Involves Transactivation of Consensus
Antioxidant Response Elements
Florian Bea, Francesca N Hudson, Terrance J Kavanagh, Michael E Rosenfeld. University of
Washington, Seattle, WA
The accumulation of oxidized low-density lipoproteins (oxLDL) by macrophages leading to
conversion to foam cells is a seminal event in the development of atherosclerosis. However,
excessive accumulation of oxidized LDL induces oxidative stress in the cells and causes a
compensatory increase in the synthesis of the endogenous antioxidant glutathione (GSH).
Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in GSH synthesis and is comprised
of a catalytic subunit (GCLC) and a modifier subunit (GCLM), products of separate genes.
Treatment of RAW cells with oxidized LDL (30 g/ml) induces increased expression of both
GCLC (8X) and GCLM (16X). The increase in mRNA occurs via increased transcription as
demonstrated with luciferase promoter reporter constructs for both subunits. The promoters for
both GCLC and GCLM contain consensus antioxidant response elements (AREs). Electrophoretic
mobility shift assays revealed induction of nuclear factor binding to these AREs following
treatment of the RAW cells with oxLDL. Nuclear factor binding to the AREs is diminished by a
single-base pair substitution in the core sequence, and additional site-directed mutagenesis of
the GCLM ARE results in a further decrease of luciferase activity following treatment with
oxLDL. Supershift analyses revealed that ox-LDL induces binding of the transcription factors
Nrf1 and Nrf2 to the GCLM ARE. These data suggest that AREs play a direct role in mediating
the induction of GSH synthesis by oxLDL and in protecting macrophages against oxidized lipid
induced oxidative stress.
Elevated Levels of C-Reactive Protein are Associated with Arterial
Endothelial Activation and Development of Transplant Coronary Artery
Disease
Carlos A Labarrere, Joshua B Lee, David R Nelson, Mohammed H Al-Hassani, Steven J
Miller, Douglas E Pitts. Methodist Research Institute at Clarian Health, Indianapolis, IN;
Cleveland Clinic Foundation, Cleveland, OH; Department of Transplantation, Clarian Health,
Indianapolis, IN
Objectives: Intercellular adhesion molecule-1 (ICAM-1) expression on arterial endothelium and
elevated levels of serum soluble ICAM-1 are implicated in the development of transplant
coronary artery disease (CAD). We investigated whether C-reactive protein, known to stimulate
ICAM-1, was associated with increased ICAM-1 levels and subsequent development of CAD.
Methods: Using a sandwich ELISA, we measured C-reactive protein and ICAM-1 levels in serial
serum samples (4.3 0.1/patient) obtained during the first 3 months post-transplantation from
109 heart transplant patients. Matching endomyocardial biopsies were studied immunohistochemically
for arterial endothelial ICAM-1. Serial coronary angiograms (3.2 0.2/patient) were
assessed for CAD. We analyzed data using Cox and logistic regression. Findings: We found a
significant correlation (p 0.002) between elevated C-reactive protein levels and arterial
endothelial ICAM-1 expression within endomyocardial biopsies. We also found a significant
relationship between C-reactive protein and soluble ICAM-1 concentrations soon after
transplantation (p 0.001). Early elevated C-reactive protein levels were associated with
development (p 0.002), severity (p 0.002), and progression (p 0.001) of CAD, as well
as with graft failure (p 0.02). Conclusion: Elevated C-reactive protein concentrations during
the first 3 months after transplantation are associated with arterial endothelial ICAM-1
expression and elevated serum soluble ICAM-1 levels. Elevated C-reactive protein concentrations
are also associated with subsequent development, severity, and progression of CAD and
with graft failure. C-reactive protein levels can be used to identify heart transplant patients at
risk of developing CAD and graft failure.
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Physical Fitness Does Not Protect from the Prothrombotic Effects of Acute
Dynamic Exercise in Middle-Aged Subjects
Eileen E Mackie, Mandy Dawson, Allison McKenzie, David Hughes, Stewart W Hillis.
University of Glasgow, Glasgow, UK; Western Infirmary, Glasgow, UK
Acute dynamic exercise stimulates coagulation, fibrinolysis and thrombocytosis.However the
benefits of physical fitness on the balance of haemostasis is controversial.This study aimed to
assess the haemostatic changes with exercise in a group of trained subjects in the age group
at risk of acute coronary events. Methods: 21 soccer referees peformed a ramp protocol
exercise test. Blood sampling was performed pre, Downloaded immediately post and from
30 mins post exercise.
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Poster Presentations a-13
Samples were analysed for platelet count, Prothrombin time(PT), Activated Partial Thromboplastin
Time(APTT), Fibrinogen, Tissue Plasminogen Activator(tPA). Flow cytometry using
CD62(p-selectin) and antifibrinogen antibodies was used to assess platelet activation at rest
and in response to ADP, collagen and epinephrine. Results: Expressed as mean(SEM).Statistical
analysis was performed using paired t-tests. The mean age of participants was 39.2(5.1)years.
Total platelet count increased immediately post exercise (228.2(40.5),278.6 (48.9), p0.001)
remaining elevated at 30 minutes. APTT was reduced immediately post exercise
(32.15(3.1),29.7(3.94)p0.001) with further reduction seen at 30 minutes
(32.15(3.1),28.4(3.31),p0.001). %CD62 expression increased post exercise
(0.688(0.52),1.42(1.3)p0.008) with no significant difference seen at 30 minutes.%Antifibrinogen
expression increased post exercise (5.19(4.31), 13.01(14.24)p0.017) with a
further increase seen at 30 minutes (5.19 ( 4.31), 20.47(26.8) p0.02). Significant increases
were seen post exercise in response to epinephrine. tPA increased immediately post exercise
(0.47(0.85),6.28(4.45)p0.001) returning to baseline by 30 minutes. Conclusion: Despite the
protective effects of fibrinolysis, the changes in coagulation and platelet activation persist at 30
minutes when tPA has returned to baseline levels. In contrast to previous studies in young
athletes, this suggests that in an older population, physical fitness does not protect against the
prothrombotic effects of acute dynamic exercise and that the potential risks persist well into
the recovery period.
P74 WITHDRAWN
The Effect of Heparin and Insulin on Cell Damage and bFGF Levels in
Glucose-Treated Cultured Endothelial Cells
Linda M Hiebert, Anil K Mandal. University of Saskatchewan, Saskatoon, Canada; University
of Florida, Jacksonville, FL
Clinical observations suggest that heparan sulfate depletion may contribute to endothelial cell
(EC)injury associated with diabetes. Basic fibroblast growth factor (bFGF) activity, required for
tissue repair, is dependent on heparan sulfate. Our objectives were to determine if heparin
and/or insulin would reduce EC injury due to high glucose (HG) and to examine the role played
by bFGF. Porcine aortic ECs were treated with 30 mM glucose or mannitol for 14 days. In a
separate study, heparin (5 g/ml) and/or insulin (2 U/ml) were added to medium of HG-treated
ECs for 10 to 13 days. To assess injury, percent (%) viable cells was determined by trypan blue
exclusion and lactate dehydrogenase (LDH) release to medium was determined by spectrophotometry.
bFGF was determined by ELISA. Data was analysed using a one-way ANOVA. %
viable cells decreased and LDH (expressed as % of control on Day 2) increased in HG treated
vs. control ECs with differences increasing over time (control 85%, 300; mannitol 82%, 333;
HG 73%, 505; HG vs. control p0.02, 0.03 for % viable cells and LDH respectively on Day 14).
On days 10 and 13, heparin and/or insulin significantly prevented the decrease in % viable cells
(control 95%, HG 90%, HG heparin 93%, HG insulin 93%, HG heparin insulin 96%;
p 0.002, 005, 0.01, 0.0002 for HG vs. control, HG heparin, HG insulin, HG heparin
insulin respectively) and LDH release except for insulin (HG 173, HG heparin 106, HG
insulin 138, HG heparin insulin 110% of control; p 0.006, 0.002, 0.08, 0.005 for HG
vs. control, HG heparin, HG insulin, HG heparin insulin respectively). In cell
suspensions, HG decreased bFGF while insulin and insulin plus heparin increased bFGF levels
in HG treated ECs (control 2.6, HG 1.6, HG heparin 1.9, HG insulin 4.2, HG heparin
insulin 6.5 pg/mg protein; p 0.03, 0.04, 0.0002, 0.002 for HG vs. control, HG heparin,
HG insulin, HG heparin insulin respectively). bFGF in media was one-hundredth the
concentration in cell suspensions and did not differ between groups. Results suggest that
exogenous heparin and insulin preserve cellular bFGF providing protection against glucose
induced EC damage.
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P77
Angiotensin AT1 Receptor Signaling Modulates Reparative Angiogenesis
Induced by Limb Ischaemia
Costanza Emanueli, Paolo Madeddu. Cardiovascular Medicine and Gene Therapy Section
National Laboratory INBB, Osilo, Italy
The concept that angiotensin II (Ang II) exerts pro-angiogenic activity is not universally
accepted. We evaluated whether inhibition of the renin-angiotensin system (RAS) would
influence reparative angiogenesis in a model of limb ischaemia. Perfusion recovery following
surgical removal of the left femoral artery was analysed by laser Doppler flowmetry in mice
given the ACE inhibitor ramipril (1 mg/kg per day), the AT1 antagonist losartan (15 mg/kg per
day) or candesartan (10 mg/kg per day), or vehicle. Muscular capillarity was examined at
necroscopy. Ramipril-induced effects were also studied under combined blockade of kinin B1
and B2 receptors. Furthermore, the effects of ischaemia on AT1 gene expression and ACE
activity were determined. In untreated mice, muscular AT1a gene expression was transiently
decreased early after induction of limb ischaemia, whereas AT1b mRNA was upregulated. ACE
activity was reduced in ischaemic muscles at 1 and 3 days. Gene expression of AT1 isoforms
as well as ACE activity r eturned to baseline by day 14. Spontaneous neovascularization allowed
for complete perfusion recovery of the ischaemic limb. Reparative angiogenesis was negatively
influenced by ramipril (P0.02), losartan (P0.01), or candesertan (P0.001), leading to
delayed and impaired post-ischaemic recovery (50 –70% less compared with controls).
Ramipril-induced effects remained unaltered under kinin receptor blockade. The present study
indicates that (a) Expression of Ang II receptors and ACE activity are modulated by ischaemia,
(b) ACE-inhibition or AT1 antagonism impairs reparative angiogenesis, and (c) Intact AT1
receptor signalling is essential for post-ischaemic recovery. These results provide new insights
into the role of the RAS in vascular biology and suggest cautionary use of ACE inhibitors and
AT1 antagonists by guest in peripheral on April ischaemia. 4, 2013

a-14 Arterioscler Thromb Vasc Biol. Vol 22, No 5
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Upregulation of Endothelial PAI-1 and MMPs during Ischemia-Reperfusion
Injury: Role of TLR-4
Suzanne M Nicholl, S S Okada. University of Rochester Medical Center, Rochester, NY
Ischemia-reperfusion (I/R) injury induces vascular endothelial injury and compromises the
homeostasis of fibrinolytic/proteolytic functions, which contributes to ischemic heart disease.
Albeit necessary the consequences of flow restoration, which is at least in part a misguided
inflammatory response, lead to a series of cellular events, such as protease production, at the
vascular level, resulting in clinical complications. The Toll-like receptors, such as TLR-4, play
an important role in the response to inflammatory stimuli and may stimulate plasminogen
activators and MMPs via NF-kB activation. We hypothesized that I/R injury predisposes blood
vessels to thrombosis via up-regulation of EC PAI-1 and MMPs respectively, and that this
upregulation is in turn mediated by enhanced EC TLR-4 expression. Human umbilical vein
endothelial cells (HUVECs) were exposed to control conditions (static or low flow (0.9
dynes.cm-2)) for 24 hours or control conditions followed by 4 hours of high flow (25
dynes.cm-2) in a perfused transcapillary culture system. Levels of PAI-1 were increased by
40% (119874 of 85828) and 723% (102153 of 12407), using casein zymography and Western
analysis, respectively. Levels of pro-MMP-9 (92 KDa), active MMP-9 (84 KDa), pro-MMP-2 (72
KDa) and active MMP-2 (62 KDa) were then assessed by gelatin zymography. Levels of
pro-MMP-9, pro-MMP-2 and active MMP-2 expression were increased by 1060% (10860 of
936), 209% (80142 of 25896) and 1481% (3337 of 211), respectively, during I/R injury. Basal
levels of TLR-4 were increased by I/R injury, as assessed by Western analysis, in contrast to
basal levels of Ik-Ba expression, which were decreased. This suggests that nuclear
translocation of NF-kB has occurred. Blockade of TLR-4 protein decreased the levels of
pro-MMP-9, active MMP-9, pro-MMP-2, and active MMP-2 by 20% (4226 of 5266), 26% (9194
of 12498), 30% (32665 of 46988) and 73% (1417 of 5312) respectively. In conclusion, I/R
injury causes an increase in expression levels of PAI-1 and MMP-2 and MMP-9, the increase
in expression of MMPs is partially enhanced by TLR-4.
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A Novel Truncated Estrogen Receptor Alpha Isoform in Human Endothelial
Cells: Role in Acute versus Transcriptional Responses
Gemma A Figtree, Hugh Watkins, Keith M Channon. Molecular Cardiology, Wellcome Trust
Centre for Human Genetics, Oxford, UK
The identity of the receptor involved in acute eNOS activation by estrogen and its relationship
to the classical receptor alpha (ER-66) remain unclear. RT-PCR and sequencing was used to
identify an alternatively spliced estrogen receptor alpha (ER) transcript in human endothelial
cells (HUVEC, hMEC, and EA.926) that encodes a truncated 46 kDa ER (ER-46) originally
observed by the Gannon laboratory in breast cancer cell lines. Western blotting demonstrated
a 46 kDa ER in EA.926 cells that was identical in size to the recombinant protein coded for by
ER-46 cDNA in transfected Cos cells. Confocal microscopy revealed that a proportion of both
ER-66 and ER-46 were localised outside the nucleus, and mediated specific cell-surface
binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding and competition
studies. Both ER-alpha isoforms colocalized with eNOS and mediated acute eNOS activation as
detected by the NO-sensitive DAF fluorescence. Prolonged estrogen exposure resulted in a loss
of eNOS association, and complete nuclear localization. Transfection experiments using
ERE-luciferase constructs demonstrated negative regulation by ER-46 on ER-66 mediated
transcriptional activity in endothelial cells. This is the first demonstration of exon 1 splicing of
ER-alpha in human cardiovascular tissue. ER-46 is similar to ER-66 in its subcellular
localization and ability to mediate estrogen binding at the cell surface. Its participation in
estrogen-induced eNOS activation, but inability to mediate transcriptional responses in
endothelial cells suggests a unique role of this exon 1 truncated ER-alpha in this cell type.
Ruptured Plaques from Apolipoprotein E Knockout Mice Exhibit
Characteristics Associated with Ruptured Human Plaques
Helen Williams, Kevin G Carson, Jason L Johnson, Christopher L Jackson. University of
Bristol, Bristol, UK
The apolipoprotein E knockout (apoE KO) mouse fed a high-fat diet is a well-established model
of atherosclerotic plaque progression, and has been previously reported to suffer atherosclerotic
plaque rupture in the brachiocephalic artery (Johnson and Jackson 2001, Atherosclerosis,
154:399–406). ApoE KO mice were fed high fat diet containing 21% lard and 0.15%
cholesterol. Mice were either sacrificed and perfuse-fixed at time-points of 6, 9 and 12 months,
or died suddenly during the study. Sections were cut along the length of the vessel and stained
with Haematoxylin and Eosin, or Miller’s Elastin stain. Plaques were examined for presence of
rupture, before comparison of ruptured and stable plaques using image analysis. Data are
presented as meanstandard error. Of the 36 mice studied, 17 had a ruptured atherosclerotic
plaque in the brachiocephalic artery. In plaques exhibiting a current plaque rupture there was
a significant increase in the number of buried caps within the lesion, a decrease in fibrous cap
thickness, and an increase in lipid core size, compared to plaques showing no rupture (see
table). These data confirm the presence of ruptured plaques in the brachiocephalic artery and
show that the plaques share characteristics with those found in human coronary arteries.
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P81
CD40L Deficiency Results in Diminished CD4 CD25 Lymphocytes: Possible
Relation with Stability of Atherosclerosis?
M L Smook, P Heeringa, E Lutgens, M J Van de Gaar, J G Damoiseaux, M J Daemen, J W
Cohen Tervaert. Clinical and Experimental Immunology, CARIM, University Maastricht,
Maastricht, Netherlands
Atherosclerotic lesions contain activated T cells, mainly of the Th1 subset, suggesting that T
cells contribute to the pathogenesis of atherosclerosis (AS). Prevention of CD40/CD40L
interaction results in tendency towards T H2 responses and reduced AS and plaque stability in
AS prone mouse strains. We hypothesize that the plaque stability is due to differences in the
T cell repertoire since CD40/CD40L interactions are essential in T cell priming, activation and
differentiation. To test this hypothesis, we analyzed the T cell repertoire in lymphoid organs and
peripheral blood of AS prone ApoE-/- mice that lack expression of CD40L (ApoE-/-CD40L-/-) by
flowcytometry. The CD4, CD45RB, CD25 markers were used in conjunction with the population
markers CD3 and CD8. The results of the ApoE-/-CD40L-/- mice were compared with ApoE-/and
CD40L-/- mice. Compared to ApoE-/- mice, ApoE-/-CD40L-/- and CD40L-/- mice showed
a marked absolute and relative decrease in the CD4 CD25 population, that was accompanied
with an increase in the CD45RB high :CD45RB low ratio. However, the CD40L deficiency did not
result in altered CD3 T cell counts or a change in T cell CD4:CD8 ratio. These preliminary
results show that reduction of AS in ApoE-/-CD40L-/- mice is associated with a decrease in the
CD4 CD45RB low CD25 T cell population, i.e. regulatory T cells. We postulate that the decrease
in regulatory T cells upon inhibition of CD40/CD40L interaction results in a shift in Th1/Th2
balance towards Th2 eventually resulting in a more stable plaque phenotype. To further
establish the role of T cells in AS plaque stability, CD40L-/- T cells will be transferred into AS
prone mice.
Expression of the Macrophage Marker CD68 in Mouse Aortic Smooth
Muscle Cells Is Regulated by Cholesterol Loading
James X Rong, Robin P Choudhury, Eugene Trogan, Edward A Fisher. Mount Sinai School
of Medicine, New York, NY
Background. Lipid laden, SMC-derived foam cells have been demonstrated in atherosclerotic
plaques, however, little is known about the phenotypic changes at the molecular level as the
cholesteryl ester content of SMC increases. Furthermore, it is not known whether any such
changes would be reversible if there were lipid removal, as may occur with aggressive statin
treatment or elevation of HDL. Methods and Results. Mouse aortic SMCs were loaded with
cholesterol using cholesterol:methyl--cyclodextrin complex (10 ug/ml). Cellular cholesterol
content increased 2-fold to 46336 ug/mg cellular protein, with most in the cholesteryl ester
fraction. Foam cell formation was demonstrated by accumulation of intracellular, oil-red-O
stained lipid droplets. By immunohistochemistry, smooth muscle -actin staining decreased,
whereas staining of CD68, a characteristic macrophage marker, visibly increased after
cholesterol loading. Cholesterol efflux was then induced by methyl--cyclodextrin, and the
number of lipid droplets was reduced. CD68 staining was decreased and -actin staining was
unchanged after cholesterol removal. To examine these changes at the molecular level, total
RNA was isolated before (baseline), after cholesterol loading, and after cholesterol removal.
HMGCoA Reductase mRNA level decreased after cholesterol loading and was partially restored
to baseline after removal. Real-time quantitative PCR revealed that -actin mRNA was 5% of
baseline after cholesterol loading, but CD68 mRNA increased 7-fold after cholesterol loading,
and declined to 4-fold over baseline after removal, respectively, consistent with the findings by
immunohistochemistry, thereby demonstrating that the changes at the protein level were
regulated by mRNA levels. Conclusions. Cholesterol loading of SMCs results in phenotypic
changes that appear to be macrophage-like. These changes appear to be regulated at the RNA
level and are partly reversible by increasing cholesterol efflux. The results imply that a larger
fraction of foam cells observed in vivo may be of SMC origin and their phenotypic changes
could contribute to plaque instability.
Apolipoprotein J / Clusterin Deficiency Exacerbates Diet-Induced
Hypercholesterolemia and Alters Apolipoprotein E Distribution among
Lipoproteins in Mice
Norman A Granholm, Scott E Street, Eddy Konaniah, Kari Theurer, David Y Hui. Univ
Cincinnati, Cincinnati, OH
The role of apolipoprotein (apo) J / clusterin in plasma cholesterol homeostasis remains an
enigma. This study compared plasma lipid levels in apoJ(/) FVB/N wild type mice and
apoJ(-/-) mice on FVB/N background after feeding either a chow or atherogenic (ath) diet for
13 wk. We found no significant difference in plasma cholesterol levels between apoJ(/) and
apoJ(-/-) mice in plasma cholesterol levels when fed the chow diet. After 3 wk of ath diet, the
apoJ(-/-) mice have a significantly higher plasma cholesterol level compared to the apoJ(/)
group (44747 vs 333129 mg/dL, n6, P0.0153). This significant difference persisted for
the duration of the study. Analysis of the lipoprotein profile by FPLC demonstrated that the
increase in plasma cholesterol in apoJ(-/-) mice occurred principally in the VLDL fraction, with
a moderate increase in IDL-LDL. ApoJ was found to be associated with both the IDL-LDL and
HDL lipoproteins in apoJ(/) mice but absent in the apoJ(-/-) mice under both dietary
conditions. ApoE was found primarily in the VLDL fractions of both apoJ(/) and apoJ(-/-)
mice when fed the chow diet. However, when fed the ath diet, the apoE profile was dramatically
different between the apoJ(/) and apoJ(-/-) mice. In the apoJ(/) group, apoE appeared
with the VLDL and diminished gradually through the IDL-LDL fractions. In apoJ(-/-) mice, apoE
was trimodally distributed: there are distinct and similar peaks in the VLDL, IDL-LDL, and HDL
fractions. These results indicate that the lack of apoJ alters apoE distribution, possibly by
increasing apoE association with higher density lipoproteins and preventing its redistribution to
VLDL. The alteration of apoE distribution in apoJ(-/-) mice may account for the increased diet
induced hypercholesterolemia observed in these animals. Thus, apoJ plays an indirect role in
lipoproteinby metabolism guest on by April modulating 4, 2013 apoE distribution among lipoproteins.
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A Role for C/EBP in Hepatic Transcription of the Gene Encoding TAFI
Michael B Boffa, Jeffrey Hamill, Rebecca Dillon, Michael E Nesheim, Marlys L Koschinsky.
Queen’s University, Kingston, ON, Canada
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Thrombin activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that
appears to play a role in regulating the balance between the activities of the coagulation and
fibrinolytic cascades. Plasma concentrations of TAFI are largely genetically determined and vary
in the population by almost an order of magnitude. Emerging data suggest that TAFI
concentrations constitute a risk factor for thrombotic disorders and that TAFI is a positive acute
phase reactant. Currently, no information exists concerning the mechanisms underlying control
of TAFI gene expression. Using a computer algorithm to locate consensus transcription factor
binding sites, we identified overlapping potential binding sites for the liver-enriched transcription
factors C/EBP and hepatic leukemia factor (HLF) in the TAFI promoter. Based on the known
sequence requirements for binding of the respective transcription factors, single nucleotide
mutations were introduced into the TAFI promoter that would be expected to abolish either
C/EBP or HLF binding or both. The mutant promoter sequences were used to construct
luciferase reporter plasmids that were transiently transfected into HepG2 cells. Mutations that
would be expected to abolish C/EBP binding alone or both C/EBP and HLF binding drastically
reduced TAFI promoter activity (to 10 –20% of wild-type) while the mutation that would be
expected to abolish HLF binding alone had no effect. Gel mobility shift assays and supershift
assays revealed that this site was able to bind C/EBP present in nuclear extracts prepared from
HepG2 cells and adult rat liver; C/EBP was the predominant binding isoform in the HepG2
nuclear extracts (consistent with the fetal-like phenotype of this cell line) while both C/EBP
and C/EBP bound in the adult rat liver nuclear extracts. Importantly, binding site mutations
that decreased TAFI promoter activity also abolished C/EBP binding. These results were
corroborated using nuclear extracts from a non-hepatic cell line (Baby Hamster Kidney (BHK))
overexpressing C/EBP isoforms; these experiments also showed that C/EBP, which is induced
in liver during the acute phase, also is capable of binding to the TAFI promoter C/EBP site.
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Fabry Disease in Mice Is Associated with Age-Dependent Susceptibility to
Vascular Thrombosis
Peter F Bodary, Yuechen Shen, Susan R Wild, Akira Abe, James A Shayman, Daniel T
Eitzman. University of Michigan, Ann Arbor, MI
Fabry disease is an x-linked lysosomal storage disorder due to deficiency of -galactosidase
A activity that results in widespread accumulation of neutral glycosphingolipids. Renal failure,
neuropathy, premature myocardial infarction and stroke occur in patients with this condition
primarily due to progressive deposition of glycosphingolipids in vascular endothelial cells. The
clinical consequences of Fabry disease suggest that vascular thrombosis may play a prominent
role in the pathogenesis of this disease, however the vasculopathy associated with Fabry
disease has not been extensively studied. To determine if Fabry mice are susceptible to
vascular thrombosis, mice deficient in -galactosidase A were studied in a photochemical
thrombosis model. In this model of carotid artery thrombosis, Fabry mice displayed a
progressive age dependent shortening of the time to occlusive thrombosis following vascular
injury that correlated with progressive accumulation of the glycosphingolipid, Gb3, in the
arterial wall (Figure). No age-dependent changes in thrombosis were observed in wild-type
mice. Therapies designed to reduce the accumulation of Gb3 were also tested in this
thrombosis model. Both substrate deprivation therapy and enzyme replacement therapy were
ineffective in attenuating the prothrombotic response despite a reduction in vascular Gb3.
These studies reveal a potent vascular prothrombotic phenotype in Fabry mice that is resistant
to therapies designed to reduce Gb3 accumulation.
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Antibody Inhibition of V and 3 Integrins but Not Inherited
3-Integrin-Deficiency Protects Mice from Developing Intimal Hyperplasia
after Vascular Injury
Susan S Smyth, Hsiming Yu, Barry S Coller, Ernane Reis. University of North Carolina,
Chapel Hill, NC; The Rockefeller University, NY, NY; Mount Sinai School of Medicine, NY, NY
Percutaneous coronary interventions are increasingly used to treat coronary artery disease, but
restenosis still limits their utility. The 3 integrins (IIb3 and V3) have been implicated in
events that may contribute to the development of intimal hyperplasia and restenosis after
vascular injury. In a mouse model of endoluminal femoral artery injury in which endothelial
denudation is followed by sequential platelet deposition and leukocyte accumulation, 3integrin-deficient
mice are not protected from the development of intimal hyperplasia. Since
inhibitors of V3(IIb3) reduce intimal hyperplasia after vascular injury in other animal
models, we performed bilateral arterial injury in wild-type Downloaded mice; in wild-type from
mice treated with
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a control hamster F(ab)’ 2, an anti-mouse V F(ab)’ 2, or an anti-mouse 3 F(ab)’ 2; and in 3-/mice.
Mice received antibody (2 mg/kg, ip) 1 h prior to surgery and then daily for 9 days.
Intimal, medial and luminal areas were measured four weeks after injury. The mean
intima/media ratio in the mice treated with anti-V or anti-3 F(ab)’ 2 were reduced by 60%
compared to the control antibody-treated wild-type mice (see Table). The mean luminal area
in the mice treated with antibody to V or3 was 1.5 times greater than the luminal area
in untreated and control antibody-treated wild-type mice. The difference in response to arterial
injury in mice lacking 3 integrins on an inherited basis and wild-type mice treated with
inhibitors of 3 integrins may be due to upregulation of compensatory molecules in 3-/- mice
or an effect of the anti-3 antibody on distinct integrins.
Inhibition of Tumor Growth Using an Antisense Morpholino Oligomer to
Integrin v Subunit
Michelle A De Sousa, James D Bird, Carla A London, Patrick L Iversen, Gayathri R Devi,
David H Farrell. Oregon Health & Science University, Portland, OR; AVI BioPharma, Corvallis,
OR
The microvascular endothelial cell integrin and fibrinogen receptor, v 3, is highly up-regulated
during angiogenesis, which occurs during such processes as wound healing and neovascularization
of tumors. Our laboratory is investigating the role of v 3 and its interaction with fibrin
during angiogenesis, and studying possible anti-angiogenic therapies directed against v 3.
Direct binding inhibitors of v 3 such as monoclonal antibodies and synthetic peptides have
been used by other investigators to inhibit v 3 and prevent angiogenesis. Our approach
involved the inhibition of angiogenesis by down-regulation of expression of the v 3 protein
itself. This was achieved in a murine xenograft model by using a specific 20-mer
phosphorodiamidate morpholino oligomer (PMO) directed against the mouse v mRNA. The
PMOs represent a novel antisense structural type, wherein the deoxyribose moiety of DNA is
replaced with a six-membered morpholine ring and the charged phosphodiester internucleoside
linkage is replaced with a phosphorodiamidate linkage. The PMOs are neutral and avoid
a variety of potentially significant limitations observed with the earlier generation of ionic
phosphorothioate structural type oligomers. Nude mice were injected subcutaneously with
human glioblastoma cell line U87MG. Established tumors were treated intratumorally with
saline or 300 g antisense oligomer, or a scrambled version of the oligomer. Tumors of mice
injected with the v antisense oligomer showed decreased average volume and mass
compared to scrambled oligomer- and saline-injected controls. 50% of the test animals
showed actual tumor regression. Those mice that showed tumor regression also had the lowest
ratios of v to total protein, indicating that inhibition of v expression correlated with inhibition
of tumor growth. These results suggest that the PMO antisense inhibition of v 3 gene
expression is sequence-specific and causes inhibition of tumor formation in vivo.
Regulation of PAI-1 Promoter Activity by SREBP
Poster Presentations a-15
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John A Schoenhard, Layton H Smith, Douglas E Vaughan. Vanderbilt University Medical
Center, Nashville, TN
Although HMG-CoA reductase inhibitors consistently improve endothelial-dependent vasodilation,
their effects on other aspects of endothelial function are less well defined. Reduction of
serum cholesterol levels and inhibition of intracellular isoprenylation are proposed mechanisms
by which statins may limit plasminogen activator inhibitor-1 (PAI-1) expression and thus
improve vascular fibrinolytic balance. In this study, we tested the alternative hypothesis that
activated sterol response element binding protein (SREBP) may regulate PAI-1 expression. This
was accomplished using PAI-1 promoter / luciferase reporter constructs in transfected
endothelial cells. Overexpression of constitutively active SREBP1a induced -3.4 and -0.8 kb
PAI-1 promoters by 11.5- and 9.5-fold, respectively (P 0.001). While further truncation of the
PAI-1 promoter to -0.4 kb eliminated SREBP1a responsiveness, fusion of the distal PAI-1
promoter (-0.8 to -0.4 kb) to a minimal PAI-1 promoter (-52 to 76 bp) conferred novel
SREBP1a inducibility to this construct, demonstrating that sequence elements located between
-0.8 and -0.4 kb are both required and sufficient for SREBP1a mediated activation. Indeed, this
region contains three potential SREBP binding sites, namely a consensus sterol response
element (-766 to -757) and two E-boxes (-680 to -675, -569 to -564), the latter of which we
have defined as responsive to other bHLH transcription factors including BMAL1, BMAL2, and
TFE3. Mutation of these sites, however, failed to obviate SREBP1a induction of the PAI-1
promoter, indicating that activated SREBP may induce PAI-1 expression by an indirect or novel
mechanism. Balanced against previous studies that have focused on mechanisms by which
statins may limit PAI-1 expression independent of SREBP activation, our results suggest that
SREBP activation may enhance PAI-1 expression, thus providing an explanation for inconsistent
results obtained so far from clinical studies concerning the effects of statins on plasma PAI-1.
Furthermore, our results suggest that lipid lowering therapies more narrowly targeted to SREBP
activation by mayguest not capture on April potential 4, fibrinolytic 2013 benefits of statin therapy.
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a-16 Arterioscler Thromb Vasc Biol. Vol 22, No 5
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Anti-Atherogenic Effect of Schistosoma Mansoni Infection in Apolipoprotein
E Knockout Mice
Christopher L Jackson, Ronald G Stanley, Michael J Doenhoff. Bristol Heart Institute, Bristol,
UK; University of Wales Bangor, Bangor, UK
OBJECTIVES Over 200 million people in the tropics are infected with schistosomes. Infections
are heaviest during the first two decades of life and then generally decrease, but many people
retain a residual worm burden into old age. The stable relationship that is eventually
established between host and parasite in older people could be an adaptation by which the host
derives some benefit from the parasite. In order to test this hypothesis, we have used an
experimental animal model to test whether schistosomiasis has an effect on atherosclerosis.
METHODS Male and female apolipoprotein E knockout mice were fed a diet containing 21%
lard and 0.15% cholesterol from 8 weeks. Two weeks after initiation of high-fat feeding half
of the animals were infected with 25 Schistosoma mansoni cercariae; the remainder of the
animals were uninfected controls. Eighteen weeks after infection mice were terminated with
perfusion exsanguination and fixation. RESULTS Control animals had typical lesions in the
brachiocephalic arteries: large eccentric plaques containing large lipid cores with extensive cell
death and destruction, covered by a fibrous cap. Atherosclerotic plaques in the brachiocephalic
arteries of mice with 16 week patent schistosome infections were reduced in size by
approximately 50% compared with uninfected controls (values given as mean/-SEM; control,
n15, 165180/-25620 um2; infected, n14, 68080/-20820 um2: p0.05). The lesions
consisted of a small lipid-rich region covered by a fibrous cap, with a necrotic core. Infected
animals also had lower total plasma cholesterol concentrations (control, n15, 100/-5
mmol/L; infected, n14, 60/-4 mmol/L: p0.001). CONCLUSIONS Infection of apolipoprotein
E knockout mice with Schistosoma mansoni decreases both plasma cholesterol
concentration and atherosclerotic lesion size.
Electrical Stimulation of Blood Vessels to Increase Tissue Plasminogen
Activator Production: A Novel In Vivo Approach to Prevent and Treat
Thrombosis
Salah N Almohammed, Amer M Amer, Adam B Wienfeld, Saleh M Shenaq. Baylor College of
Medicine, Houston, TX
Objectives: Overcoming arterial and venous thrombosis presents a great challenge for
physicians of all disciplines. Currently available therapeutic and preventive modalities range
from aspirin to recombinant thrombolytic agents. Although these medications reduce the
adverse impact of thromosis, untoward side effects remain a great concern. Tissue
plasminogen activator (t-PA) is an effective anti- thrombotic peptide produced by the vascular
endothelial cells. The aim of this study is to evaluate the effect of direct electrical stimulation
(DES) on increasing t-PA level and activity for thrombus formation prevention and lysis. Material
and Methods: Human endothelial cell culture has shown increased t-PA production after
alternating current (AC) electrical stimulation. For the in-vivo studies, 31 NZW male rabbits
were used to evaluate t-PA level after DES. The left femoral artery of each rabbit in the
stimulation groups was cleaned, isolated and stimulated with 15 volts for 5 or 45 minutes using
specific electrode design. Phosphated buffered saline aliquots were then ( 5 min., 24 hrs and
48 hrs after DES ) incubated into the lumen of the femoral artery and harvested after 30
minutes. t-PA level, activity and t-PA mRNA were then tested for using ELIZA, activity assay and
rt-PCR, respectively. Arterial sections were also taken to test for tissue damage using light and
electron microscopy. Results and Conclusion: In all DES samples, t-PA level, activity and mRNA
were increased. This increase was detected immediately after stimulation and as late as 48 hrs
after stimulation. Increasing the duration of DES from 5 min. to 45 min resulted in 2 fold
increase in t-PA level and activity. No significant arterial wall damage was noticed using light
and electron microscopy. Direct electrical stimulation has shown to increase t-PA level and
activity in cell culture and in-vivo. Such encouraging results have led us to start the second
phase of applying this novel local approach for the prevention and treatment of thrombosis.
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N-3 Fatty Acid-Stimulated Degradation of Apoprotein B100 in Hepatic Cells
Involves Lipid Peroxidation
Meihui Pan, Arthur I Cederbaum, Christopher Cardozo, Kevin J Williams, Edward A Fisher.
Mount Sinai School of Medicine, New York, NY; Thomas Jefferson University, Philadelphia,
PA
We have shown that polyunsaturated n-3 fatty acids (DHA or EPA) stimulate pre-secretory
degradation of apoB100 in a post-ER compartment of hepatic cells, through a process involving
PI3 kinase, but not proteasomes, lysosomes, or cell-surface re-uptake (Fisher EA et al. J Biol
Chem 276:27855, 2001). To further explore the underlying mechanisms, we incubated rat
hepatoma cells or primary hepatocytes with the n-3 fatty acid DHA complexed to BSA, while
blocking candidate cellular mediators. Inhibitors of the major classes of intracellular proteases
had no significant effects on DHA-stimulated apoB100 degradation. N-3 fatty acids are
precursors to eicosanoids, but aspirin (which blocks their formation) had no effect. Interestingly,
treatment of cells with the Ca chelator EGTA or BAPTA blocked DHA-stimulated
apoB100 degradation. There was, however, strong evidence against a role for calcium.
Chelation of intracellular Ca with BAPTA-AM, pharmacologic blockage of Ca channels,
depletion of the ER-calcium pool, and increasing Ca influx with an ionophore each had no
effect. Because EGTA also chelates Fe, and DHA is subject to Fe-dependent
peroxidation, we examined the role of this lipid modification. 1) Desferroxamine (DFX), a
chelator of Fe, the antioxidant vitamin E (VE), and the synthetic antioxidant BHT, each
inhibited DHA-stimulated apoB100 degradation by 60%. 2) DHA incubation, compared to
BSA, resulted in elevated cellular levels of TBARS (a standard index of lipid peroxidation), which
were reduced by co-treatment with EGTA, DFX, VE, or BHT. 3) DHA treatment resulted in the
formation of high molecular weight apoB complexes, Downloaded consistent withfrom the known ability of lipid
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peroxides to promote protein aggregation. Summary: DHA-stimulated degradation of apoB100
is a post-ER process involving PI3 kinase and also lipid peroxidation, leading to modifications
of apoB100 and its destruction by a novel proteolytic mechanism.
Decreased Modified Lipoprotein Uptake in Macrophages Lacking Both
Scavenger Receptor A I/II and CD36
Vidya V Kunjathoor, Maria Febbraio, Lorna Andersson, Roy Silverstein, Mason W Freeman.
Massachusetts General Hospital, Boston, MA; Weill Medical College of Cornell University,
New York, NY
The scavenger receptor family of multi-ligand binding proteins comprises 6 subgroups, termed
SR-A through SR-F. The SR-A type I and type II receptors and CD36 (a member of the SR-B
family) bind modified forms of low density lipoprotein (LDL) leading to cholesterol ester
accumulation and macrophage foam cell formation. Mice lacking either SR-A or CD36 have
been generated and both groups show diminished modified lipoprotein uptake. CD36 null mice
have markedly diminished but not absent atherosclerotic lesion formation, whereas the effect
on atherosclerosis of the loss of SR-A has varied with the strain of mice used in the analysis.
To further characterize the importance of these receptors in lipid uptake and atherosclerosis
and to assess compensatory mechanisms responding to their loss, we have crossed
SR-A(I/II)null and CD36 null mice. The resulting double null mice appeared healthy and were
derived in the expected Mendelian ratios. Serum cholesterol levels were not significantly
different in wild ty pe (825 mg/dl) and double null mice (10325 mg/dl). Macrophages
derived from the double knockouts, when stimulated with lipopolysaccharide produced
equivalent amounts of pro-inflammatory cytokines as compared to wild type animals (TNF,
wild type 5.5 0.36 ng/mg protein; double null 8.10.4 ng/mg protein and IL-6, wild
type 42.80.55 ng/mg protein; double null 503.2 ng/mg protein, n3 mice per group).
In contrast, a 96% dec rease in the degradation of acetylated LDL was measured in peritoneal
macrophages taken from the SR-A/CD36 deficient animals as compared to wild type mice (wild
type 27,2926,920 ng/mg protein, CD36-/- 6,817655* ng/mg protein, MSR-/-
1,7253 08* ng/mg protein, double null 786201* ng/mg; * indicates p0.05 versus wild
type). This uptake was substantially less than that measured in either SR-A-/- or CD36-/- mice.
Reduction in oxLDL degradation and foam cell formation were also measured. These data
suggest that alternative scavenger receptors do not compensate for the loss of SR-A and CD36.
The double null mice have been bred into the apoE null strain of C57BL/6 mice and studies on
the effect of atherosclerosis are now in progress.
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Apolipoprotein L-1 (apoL-1) Levels and the Frequency of an apoL-1 Allele
are Increased in Patients with CAD and Hyperglycemia
Timothy S Albert, Philippe N Duchateau, Samir S Deeb, Clive R Pullinger, Min H Cho, Mary
J Malloy, John P Kane, B Greg Brown. University of Washington School of Medicine,
Seattle, WA; Cardiovascular Research Institute, University of California, San Francisco, CA
Objectives: The function of the recently described apoL family of apolipoproteins is still unclear.
ApoL-1, a plasma protein member of this family, is found on VLDL and HDL particles and is
elevated in high triglyceride (TG) states. We measured apoL-1 levels, and genotyped for the
Lys166Glu apoL-1 polymorphism, in subjects enrolled in the HDL Atherosclerosis Treatment
Study (HATS) in order to characterize this new apolipoprotein in a population with low
HDL-cholesterol (HDL-C35 mg/dL) and CAD. Methods: ApoL-1 levels on- (TapoL-1) and off-
(BapoL-1) therapy were quantified by ELISA in 137 of 160 pts enrolled in HATS, a 3-year
angiographic trial of lipid lowering therapy and antioxidant vitamins. Regressions of BapoL-1
with baseline variables (age, fasting glucose (FG), hyperglycemia (HG diabetes or impaired
FG (FG110–125 mg/dL)), body mass index, smoking or hypertension history, insulin, and
lipid/apolipoprotein levels) and TapoL-1 with change in coronary stenosis (%S) were
completed. The Lys166Glu variant of the apoL-1 gene was analyzed for by restriction
fragmentation (n141) after our initial findings in order to assess for associations of the Glu
allele with apoL-1 level and HG. Findings: ApoL-1 was positively and independently related to
HG (p0.001) and VLDL-TG (p0.028) by multivariate analysis (multiple r0.37, p0.0001).
ApoL-1 (meanSD) corrected for VLDL-TG was 50% higher in HG (n35) than normal
glycemic (NG, n101) pts (15.113.2 vs. 9.98.1 g/mL, p0.008). No association
between TapoL-1 and %S was seen (r0.05, p0.52). The Glu allele (frequency0.204)
was not independently associated with apoL-1 level (p0.18), however, its frequency was
2-fold higher in HG (n33) than NG (n108) pts (0.318 vs. 0.167, 2 7.20, p0.007).
Conclusions: ApoL-1 does not appear to be pro-atherogenic for the population as a whole. HG
pts with low HDL-C and CAD, however, have independent elevations of apoL-1 and an
increased frequency of a less common apoL-1 allele. These findings lead us to speculate that
apoL-1 may play a unique role in diabetes and/or diabetic CAD, possibly contributing to the
dyslipidemia of diabetes.
P95
The LXR Ligand T0901317 Induces Severe Hypertriglyceridemia and Very
Low HDL in the DB/DB Diabetic Mouse
Jeffrey W Chisholm, Scott A Mills, Prabha N Ibrahim, Richard M Lawn. CV Therapeutics,
Palo Alto, CA
Liver-X-Receptor (LXR) ligands are currently under investigation as potential therapeutic agents
for the treatment of low HDL common in both non-diabetic and diabetic humans. T0901317
(Tularik), a selective LXR ligand, has been shown to raise ABC1 and HDL levels in non-diabetic
animal models while causing a moderate hypertriglyceridemia through increased SREBP1c
expression. However, the effect of LXR ligands in a diabetic background have not been
investigated. T0901317 was administered i.p. (50 mg/kg/d) for 12 days to diabetic DB/DB mice
(7 wk old). Analysis of plasma lipids revealed that T0901317 treatment caused massive
increases by in plasma guest total on April cholesterol 4, 2013 (3.4X) and triacylglycerols (17X) with an unexpected
P93

severe reduction in HDL (90%). Hepatomegaly (liver/body ratio 3.6X) and lipid accumulation
were evident. Lipoprotein analysis by FPLC confirmed that T0901317 treatment nearly
eliminated HDL and caused a severe VLDL accumulation. Gene expression analysis of the LXR
target genes SREBP1c and ABC1 showed that levels were unchanged in the liver and
significantly induced in both the intestine and adipose tissue. Hepatic apo CIII, lecithin:
cholesterol acyltransferase (LCAT) and hepatic lipase expression were reduced 69, 88 and 87%
respectively, while both hepatic and intestinal apo A-I and apo CII expression were unchanged.
Hepatic lipoprotein lipase (LPL) was increased (3.8X) while there were no changes in the
expression of either LPL or hormone sensitive lipase in adipocytes. This data suggests that HDL
production may be impaired due to decreased LCAT synthesis and that the hypertriglyceridemia
may be due to either increased VLDL triacylglycerol production or decreased VLDL remnant
uptake. Together, these results strongly suggest that a diabetic background may exacerbate
the lipogenic effects of the LXR ligand T0901317 leading to a severe hypertriglyceridemia,
hepatotoxicity and a resulting loss of plasma HDL.
Expression of Scavenger Receptor BI in Macrophages Stimulates
Esterification of Plasma Membrane Cholesterol
Zhi H Huang, Theodore Mazzone. Rush Presbyterian and St Luke’s Medical Center, Chicago,
IL
SR-BI expression has been shown to facilitate the bi-directional movement of sterols between
cells and extracellular acceptors; perhaps related to a reorganization of plasma membrane lipid
domains. The aim of this study was to investigate whether SR-BI expression influenced the
subcellular disposition of endogenous membrane cholesterol in macrophages. We constitutively
expressed SR-BI in J774 macrophages or CHO cells at 4 and 7 fold higher levels compared to
control cells. The rate of new cholesterol synthesis was higher in both macrophages and CHO
cells (3.9, and 2.1 fold increase respectively, both p0.05), as a result of increased SR-BI
expression; likely due to increased sterol efflux mediated by SR-BI. However, increased SR-BI
expression also enhanced incorporation of 14 [C]oleate into cholesterol ester in J774 macrophages
(2.6 fold increase, p0.05) but not in CHO cells. Experiments utilizing selective labeling
of plasma membrane sterol with 3 [H]cholesterol at 15 o C indicated that the source of cholesterol
for increased cholesterol ester synthesis in SRBI-expressing macrophages was derived from
plasma membrane. Macrophages with increased SR-BI expression showed a 4.6 fold increase
(p0.01) in 3 [H]cholesterol ester formation. There was no increase of plasma membrane
cholesterol esterification in CHO cells with increased SR-BI expression. Addition of 25hydroxycholesterol
to macrophages eliminated the difference in oleate incorporation into
cholesterol ester between control and SRBI-expressing cells, but the increase of plasma
membrane cholesterol esterification persisted. When acetate was used to monitor cholesterol
ester synthesis, SR-BI expression increased synthesis (3.3 fold higher, p0.05) in macrophages
and this increase was maintained after stimulation of sterol efflux with -CD (3.1 fold
higher, p0.05). These results suggest that expression of SR-BI in macrophages facilitates the
movement of cholesterol between plasma membrane and endoplasmic reticulum compartments.
This enhanced transport could be related to the previously noted re-ordering of
cholesterol within subdomains of cell plasma membrane.
P97
Evidence for Matrix Metalloproteinases and Silent Plaque Rupture in the
Development of Unstable Atherosclerotic Plaques in Male Apolipoprotein E
Deficient Mice
Jason L Johnson, Sarah J George, Andrew C Newby, Christopher L Jackson. University of
Bristol, Bristol, UK
The rupture of an atherosclerotic plaque with associated thrombosis is the main underlying
cause of myocardial infarction and stroke. We aimed to determine the involvement of matrix
metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of MMPs (TIMPs)
during early plaque development and progression in our apolipoprotein E knockout mouse
(ApoE -/- ) model of plaque rupture. Six-week-old male and female animals (C57/Bl6;Sv129)
were fed a high-fat diet for 8 weeks and culled. Blood was taken for cholesterol analysis.
Animals were perfusion fixed and the brachiocephalic artery, aortic arch, aortic root and heart
were removed. Serial sections were examined by H&E, EVG, sirius red and immunocytochemistry
for MMPs and TIMPs, smooth muscle cells (SMC), macrophages, MCP-1, T-cells and
cleaved-PARP for apoptosis. In situ zymography was performed to detect MMP activity. Male
mice had statistically greater total cholesterol levels when compared to females (451v202
mmol/l; p0.02). The brachiocephalic artery and aortic sinus of female mice (n6) contained
small fatty streaks consisting of lipid-laden SMC-derived foam cells. Macrophage-rich
atherosclerotic plaques were identified in the brachiocephalic artery, aortic sinus, aortic arch
and coronary arteries of male mice (n12). 66.6% (8 out of 12) of brachiocephalic lesions
exhibited signs of silent plaque rupture at shoulder regions, characterised by breaks in the
internal elastic lamina and associated thrombus incorporation within the plaque. Increased
MMP activity and MMP-3, -7, -9, -13 & -14, TIMP-2 & -3 expression was detected,
predominantly in the macrophage rich shoulder regions. Foam cell emigration with associated
thrombus, apoptotic cells and MCP-1 expression was also observed. Increased MMP activity,
foam cell emigration, and apoptosis occur in silent rupture of early male ApoE -/- brachiocephalic
atherosclerotic plaques. Healed and subsequent silent plaque ruptures may result in
increased plaque burden and therefore, increase the propensity for occlusive plaque rupture as
observed in this model.
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P96
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Poster Presentations a-17
P98
Oxidized Low Density Lipoprotein Upregulates CXCR4 Expression in Human
CD4 T Cells
Ki Hoon Han, Jong Min Song, Cheol Whan Lee, Jae Joong Kim, Seong Wook Park,
Seung-Jung Park. Asan Medical Center, Seoul, South Korea
Oxidation of low density lipoprotein and the infiltration of circulating CD4 T cells into the
vascular wall are detected from the early stage of atherogenesis. The chemokine receptor
CXCR4 is expressed in CD4 T cells within the peripheral blood and atheroma. Smooth muscle
cells, endothelial cells and macrophages in human atherosclerotic plaques strongly express
stromal derived factor (SDF-1) and the SDF-1 - triggered activation of CXCR4 induces the
chemotaxis of T cells and possibly contributes to the T cell - mediated immunomodulation in
the plaque. This study results demonstrated that the oxidized low density lipoprotein (OxLDL)
upregulated CXCR4 expression in human CD4 T cells. The treatment of CD4 T cells isolated
from the peripheral blood with OxLDL enhanced the amounts of both CXCR4 transcripts and
proteins up to 4 fold in a dose (-10ug/ml) and time (-72hours) dependent manner. Among
the components of OxLDL, lysophosphatidylcholine (lysoPC) showed the identical properties in
the regulation of CXCR4 expression, whereas POVPC or KLH-conjugated phosphorylcholine did
not affect the expression level of CXCR4. The positive regulatory effect of OxLDL and lysoPC
on CXCR4 was nearly completely abolished by the incubation with CAPE (10 ug/ml), a NFkB
inhibitor. The inhibition of tyrosine kinase and protein kinase C did not attenuate the effect of
OxLDL on CXCR4 expression. Pretreatment of human CD4 T cells with lysoPC (10
ug/ml/24hrs) promoted the SDF-1 - induced migration 3 fold. The production of proinflammatory
cytokines i.e. IL-2 and TNF-alpha from anti-CD3-immobilized CD4 T cells after SDF-1
costimulation was enhanced up to 4 fold by the pretreatment of the cells with lysoPC.
Costimulatory effect of SDF-1 to increase surface expression of CD25 and CD154 (CD40 ligand)
in anti-CD3-immobilized CD4 T cells was not obvious in this study. However, the activation
of CXCR4 by SDF-1 significantly prevented the OxLDL - induced downregulation of CD28. These
study results suggest that OxLDL in atherosclerotic lesion may directly and indirectly regulate
the functional activity of CD4 T cells, which may play an important role in the progression
of atherosclerosis.
P99
Absence of CCR2 Receptors in Bone Marrow-Derived Cells Decreases
Angiotensin II Induced Atherosclerosis and Abdominal Aortic Aneurysms in
ApoE Deficient Mice
Punnaivanam Ravisankar, Lisa A Cassis, Stephen Szilvassy, Alan Daugherty. University of
Kentucky, Lexington, KY
Angiotensin II (AngII) infusion promotes atherosclerosis and abdominal aortic aneurysm (AAA)
formation in hyperlipidemic mice. AngII-induced vascular disease is characterized by increased
arterial infiltration of monocytes and lymphocytes. A potential mediator of this infiltration is
monocyte chemoattractant protein (MCP-1), a CC group chemokine that exerts its effect
predominantly via CCR2 receptors. To determine the role of CCR2 in AngII-induced vascular
pathology, we created chimeric apoE-/- x CCR2/ and apoE-/- x CCR2-/- mice by bone
marrow transplantation. Eight week old male C57BL/6 ApoE-/- recipient mice were lethallyirradiated
(900 rads) and transplanted with bone marrow from either CCR2/ or CCR2-/-
C57BL/6 ApoE-/- donor mice (CCR2/ recipients n9; CCR2-/- recipients n12). Mice
were permitted 6 weeks for bone marrow repopulation, then fed a high-fat diet and infused
subcutaneously with AngII (1000 ng/kg/min) using Alzet osmotic pumps for 28 days. AngII
infusion increased systolic blood pressure equally in CCR2/ and CCR2-/- groups. Serum
concentration of total cholesterol was decreased by 21% (P0.009) in the CCR2-/- group, but
there was no difference in triglycerides. Cholesterol was decreased in VLDL and LDL fractions
in the CCR2-/- group. Serum MCP-1 concentrations were not different between the groups.
CCR2 deficiency decreased AngII-induced atherosclerosis in both the aortic root (64%, P
0.002) and thoracic aorta (33%, P0.006). AAA formation was decreased in the CCR2-/- group
(78% in CCR2/; 8% in CCR2-/-, P0.002). In conclusion, these data demonstrate that
absence of CCR2 receptors in bone marrow derived cells reduces AngII-induced atherosclerosis
and AAA in ApoE -/- mice.
P100
Human (CETP X ApoB-100) Double Transgenic Mouse: Model of Profound
Hyperinsulinemia, Obesity, Mild Hyperlipidemia and Limited Atherosclerosis
Kathryn K McMahon. Texas Tech University Heatlh Sciences Center, Lubbock, TX
Human hyperinsulinemic pre-diabetes are prone to atherosclerosis. Current hypotheses
suggest this is due to increased vascular inflammatory responses to elevated blood lipid
oxidation. Indeed, humans develop either or both type 2 diabetes and atherosclerosis with
chronic relatively mild excesses of cholesterol and triglycerides. A mouse model, the human
CETP X human ApoB-100 double transgenic mouse (dTrG), has been shown to develop type 2
diabetes, hyperinsulinemia, hyperlipidemias and hypertension. We hypothesized that this model
would also develop atherosclerosis. To test this hypothesis, we fed male control (C57Bl/6) or
dTrG mice a either a chow (4% fat/no cholesterol) or 34% fat/0.1% cholesterol (high fat) diet
for 16 weeks (at least 3 mice/group). Blood samples were taken monthly to assay plasma
insulin, glucose, total cholesterol and triglyceride levels. Body weights were determined
weekly. At the end of the feeding regime, aortas and hearts were evaluated for atherosclerotic
plaque formation. At the initiation of the feeding regimes, the dTrG mice had mildly elevated
(i.e. less than 2-fold) insulin, glucose, and cholesterol levels and triglyceride levels were
elevated by 3-fold. At the end of the feeding regime, dTrG fed chow had insulin levels over
6-fold higher than control mice fed chow and glucose, cholesterol and triglyceride levels were
less than 2-fold higher than controls. Control mice and dTrG mice fed the high fat diet had
insulin levels over 33- and 46-fold higher than the control/chow mice, respectively. The high
fat-fed mice (control and dTrG) had glucose levels less than 2-fold above the control/chow
mice, cholesterol by guest levelson of 3- April and 4-fold 4, 2013 above control/chow mice and triglyceride levels 2- and

a-18 Arterioscler Thromb Vasc Biol. Vol 22, No 5
4-fold above control chow mice. Body weights increased in both control and dTrG mice fed the
high fat diet by 41% and 59% respectively. Atherosclerosis plaque formation was not different
in the control or dTrG mice fed the chow or high fat diets. In conclusion, this dTrG mouse model
is hyperinsulinemia, obese and mildly hypercholesterolemia but does not have elevated
atherosclerotic plaque formation.
P101
Transforming Growth Factor- and Fibronectin Expressions after Balloon
Catheter Injury in Diabetic Rats and the Effects of Losartan
Zheng L Li, Fu X Hua, Li C Guang, Wang Jun, Yi Lijing Q Xia. Second Affiliated Hospital of
Hebei Medical University, Shijiazhuang, China; Institute of Pharmacology, Henan Medical
University, Zhengzhou, China
Aim: To study the mechanisms of the high restenosis rate of the patients with diabetes mellitus
after percutaneous transluminal coronary angioplasty (PTCA) and the influences of losartan on
excellular matrix constituents(EMC)formation and deposition. Materials and methods: The
thoracic aortae of 16 diabetic rats induced by streptozotocin and 8 normal rats were injured by
balloon. All rats were divided randomly into three groups:control ,diabetes mellitus(DM), and
losartan. Losartan group were treated with losartan 20mg.kg -1 .d -1 . 2 weeks after the balloon
injury, all thoracic aortae were excised. Transforming growth factor receptors (T R)1 ,2 and
fibronectin(FN)were determined by RT-PCR ,Northern blotting and immunohistochemical
analysis. HPLC-RIA was used to measure angiotensin(Ang)2 level in artery. Results: We found
that expressions of T R2,FN mRNA and protein in DM group is increased by
20%,21%,32%,45% respectively than control group.The level of Ang 2 in DM group was
enhanced by 22% than control group. There was no difference in the expression of T R1mRNA
and protein between control and DM groups. Administration of losartn could reduce expressions
of T R2, FN mRNA and protein by 60%,53%,47%,65% respectively than DM group.It also
could reduce the level of Ang 2 by 36% than DM group. Conclusions: It is suggested that there
is an interaction between T R2and the pathogenesis of diabetic restenosis after PTCA.
Losartan can suppress the expression of T R2 in artery.
P102
Evaluation of Carotid Calcification as a Screening Marker for Coronary
Artery Stenoses
Jörg Nossen, Thomas Vierzigmann, Erich Lang. Waldkrankenhaus St. Marien, Erlangen,
Germany
Background: Ultrasound is an established and widespread method for examination of carotid
arteries concerning calcified plaques and stenoses. Questionable is if hard plaques in the
cerebral arteries are a surrogate screening marker for stenoses of coronary arteries. Methods:
139 patients underwent cardiac catheterization with selective coronary angiography (technique
of Judkins) and ultrasound examination (B-mode) of carotid arteries. In case of calcified
plaques number and distribution among the extracranial cerebral arteries were measured.
Coronary angiograms were analyzed for disease severity and extent (number of main vessels
with 50% stenosis). Conclusions: Ultrasound of carotid arteries is a simple screening marker
to improve predictive value of risk factor-based multivariate models.
Circulation Is Established in a Step-Wise Pattern in the Mammalian
Embryo
Kathleen E McGrath, Anne Koniski, James Palis. University of Rochester, Rochester, NY
P103
The common site and time of origin of the first embryonic endothelial and blood cells within
the yolk sac suggests that hematopoietic and vascular fates are closely intertwined.
Furthermore, the forming vascular system is the site of blood synthesis in the early embryo. To
better understand the relationship between the hematopoietic and vascular systems, we
investigated the establishment of the circulation in mouse embryos by examining various
vascular beds for the presence of yolk sac-derived primitive erythroblasts and definitive
hematopoietic progenitors. Our studies reveal that small numbers of erythroblasts first enter the
embryo proper at 6–8 somite pairs (sp) (embryonic day 8.25 (E8.25)) which correlates with the
proposed onset of cardiac function. Hours later (E8.5), increasing numbers of red cells have
entered the embryo proper, however, there is still a predominance (10-fold greater number) of
erythroblasts in the yolk sac. The number of red cells continues to expand in the embryo proper
reaching a steady state of approximately 40% of total cells between 26–30 sp (E9.75). During
this early stage of circulation (between 6 and 25 sp), erythroblasts are not distributed
throughout the embryo’s vasculature, rather they gradually progress though specific vascular
beds. The establishment of a robust circulation at E9.75 correlates with vascular remodeling
suggesting that vessel arborization and/or smooth muscle recruitment are required. We also
examined the distribution of committed hematopoietic progenitors within the developing mouse
conceptus. Prior to E8.25, all progenitors were found in the yolk sac. When normalized to
circulating erythroblasts, there was still a significant enrichment (20 to 5-fold) of progenitors
in the yolk sac compared to the embryo proper from E9.5 to E10.5. These results suggest that
the yolk vascular network remains a site of progenitor Downloaded synthesis and/or from
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even as the fetal liver becomes a hematopoietic organ. We conclude that a functional vascular
system develops gradually and that specialized vascular-hematopoietic environments exist
after circulation becomes fully established.
Circulating Activated Platelets Promote Leukocyte Recruitment to
Atherosclerotic Lesions of Atherogenic Mice
Yuqing Huo, Klaus Ley. University of Virginia, Charlottesville, VA
P104
Occurrence of activated circulating platelets and platelet-leukocyte aggregates in the individuals
with atherosclerosis has been found for several decades. However, it is unknown whether
they are just makers or active players. Here, we investigated the roles of activated platelets in
the interactions of leukocytes with atherosclerotic lesions of apoE-/- mice in a novel in vivo
model. ApoE-/- mice fed with western diet for 8 weeks were able to develop atherosclerotic
lesions on the external branch of carotid arteries. Using epifluoresence intravital microscopy,
it was rare to observe leukocytes labeled with rhodomine 6G interacting with atherosclerotic
lesions, consistent with chronic inflammation-the nature of atherosclerosis. Interactions of
leukocytes with atherosclerotic lesions were dramatically promoted following perfusion of
activated mouse or human platelets but not the resting ones. These interactions occur mainly
on early atherosclerotic lesions, shoulders, but not on the centrals of established lesions.
Blockade of endothelial P-selectin inhibits leukocyte adhesion on atherosclerotic lesion by 55%
while blocking platelets P-selectin almost completely prohibited these interactions mediated by
activated platelets. Activated platelets also were not able to cause leukocytes deficient in
P-selectin ligand, PSGL-1 to interact with atherosclerotic lesions. Examination using SEM
showed that the number of leukocytes on atherosclerotic lesion after activated platelet
perfusion was 30–50 times higher than that after perfusion of activated platelets without
P-selectin. Platelets were found to bind on most of the leukocyte adhering on atherosclerotic
lesions. These results suggest that activated platelets can serve as active players in
atherosclerosis. Molecules including platelet P-selectin and leukocyte PSGL-1 are crucial for
the involvement of activated platelets in atherosclerosis. The findings clarify the molecular
mechanism involved in contribution of platelets to atherosclerotic lesions and suggest potential
new approaches to curbing the development of atherosclerosis lesions.
P105
Endotoxin Activates Pro-Inflammatory Cytokine and Superoxide Production
in Human Blood Vessels: Relevance to Atherosclerosis
James B Rice III, Lynn Stoll, Wei G Li, Neal L Weintraub. University of Iowa Hospitals and
Clinics, Iowa City, IA
Atherosclerosis, the leading cause of death worldwide, is a chronic inflammatory disorder. The
sources of vascular inflammation in atherosclerosis, however, remain to be elucidated. One
potentially important source of vascular inflammation is endotoxin, a cell wall component of
Gram-negative bacteria. Plasma endotoxin level of 50pg/ml has recently been identified
as a powerful, independent risk factor for the development of atherosclerosis. Potential
mechanisms whereby endotoxin could contribute to atherosclerosis include increased production
of superoxide and pro-inflammatory cytokines. Accordingly, human saphenous vein
explants were treated with low levels of endotoxin (10 ng/ml). Release of the cytokines
interleukin-8 (IL-8) and monocyte chemoattractant peptide-1 (MCP-1), key in recruitment of
inflammatory cells to atherosclerotic lesions, was measured by ELISA. To detect superoxide in
situ, tissues were stained with hydroethidine (HE) and examined by laser confocal microscopy.
Measurable increases in IL-8 and MCP-1 release were observed at endotoxin concentrations
as low as 30 pg/ml, well within the range of levels that have been associated with an increased
risk for atherosclerosis. Treatment with 100 pg/ml endotoxin resulted in approximately 7-fold
increase in IL-8 release over basal levels; similar results were obtained in cultured human
coronary artery smooth muscle cells. HE fluorescence was dose-dependently increased
throughout the vessel wall by a 4-h incubation with 1–10 ng/ml endotoxin, indicating increased
superoxide levels. We conclude that clinically-relevant concentrations of endotoxin activate
inflammatory cytokine and superoxide production in human blood vessels, suggesting a
putative mechanistic link between endotoxin, vascular inflammation, and atherosclerosis.
P106
In Baboon Smooth Muscle Cells, Interleukin-1 Inhibits PDGF-BB Induced
Migration through a Cox2 Dependent Pathway
Michael J Englesbe, Jessie Deou, Alexander W Clowes, Guenter Daum. University of
Washington, Seattle, WA
Objective: Interleukin-1 (IL-1) is a proinflammatory cytokine that is present in the injured
and atherosclerotic vessel wall. Since PDGF plays an important role in the pathogenesis of
vascular injury, we studied the effects of IL-1 on PDGF-BB induced baboon smooth muscle
cell (SMC) migration. Methods: SMC migration was assayed with a modified Boyden
microchemotaxis chamber, as previously described. Data is expressed as the fold increase in
migration relative to controlSEM. Findings: IL-1 (0.1ng/ml) inhibits PDGF-BB (10ng/ml)
induced migration (IL-1 PDGF BB: 2.230.10 vs. PDGF BB control: 3.800.12). This
IL-1 induced inhibition is markedly attenuated by treatment with a Cox2 specific inhibitor,
NS398 (10M) (PDGF BB NS398: 3.260.27 vs. PDGF BB IL-1 NS 398: 3.040.10).
The IL-1 effect is also relieved by the p38-MAP kinase inhibitor, SB20358 (3M) (PDGF BB
SB20358: 3.190.23 vs. PDGF BB IL-1 SB20358: 3.550.40), indicating that Cox2
induction may be mediated by p38-MAP kinase. By Western blot analysis we demonstrate that
IL-1 mediated Cox2 induction is inhibited by SB20358. Conclusion: IL-1 inhibits PDGF-BB
induced SMC migration. This inhibition occurs through IL-1 induced Cox2 expression via a
p38-MAP by kinase guest dependant on April signaling 4, 2013 pathway.

P107
Cardiac Ankyrin Repeat Protein Expression in Human and Murine
Atherosclerotic Lesions: Activin A Induces CARP in Smooth Muscle Cells
Vivian De Waard, Tanja A Van Achterberg, Nicholas J Beauchamp, Hans Pannekoek, Carlie
J De Vries. Academic Medical Center, Amsterdam, Netherlands
Cardiac ankyrin repeat protein (CARP) is a transcription factor related protein. By using
extensive serial analysis of gene expression (SAGE) we previously identified CARP as one of the
genes induced under atherosclerotic conditions both in cultured endothelial cells and vascular
smooth muscle cells (SMCs). In the present study, we investigate the expression of CARP in
human and murine atherosclerotic lesions by in situ hybridization. CARP expression is observed
in endothelial cells lining human atherosclerotic plaques, whereas lesion macrophages are
devoid of CARP. Furthermore, we establish that CARP mRNA and SM -actin antigen
co-localize in a subset of neointimal SMCs, while no CARP mRNA is encountered in medial
SMCs. Since the CARP mRNA-expressing neointimal subset of SMCs is distinct from neointimal
SMCs that synthesize the activation marker osteopontin, it is deduced that CARP is specifically
expressed in (re)differentiating intimal SMCs which become quiescent. Finally, we show that
activin A, a member of the TGF superfamily that enhances (re)differentiation of SMCs, induces
CARP expression in SMCs. It is argued that our data support the view that CARP is involved in
(re)differentiation of SMCs during atherogenesis.
P108
Induction of SM22 Alpha Promoter Activity by Vasopressin and
Suppression by Platelet-Derived Growth Factor Involve Distinct Regulatory
Elements in Vascular Smooth Muscle Cells
Nihal Kaplan-Albuquerque, Chrystelle Garat, Vicki Van Putten, Raphael A Nemenoff.
University of Colorado Health Sciences Center, Denver, CO
Development of VSMC involves the coordinated induction of a number of smooth muscle
specific markers. SM22alpha has been used as a marker to study the differentiated state of
VSMC. Under certain pathophysiological states, VSMC decrease expression of contractile
proteins, and convert to a more proliferative phenotype. Relatively little is known about the
pathways controlling regulation of SM22 expression by circulating factors. In this study, control
of SM22 expression in adult VSMC was examined. High basal SM22 mRNA levels and promoter
activity in cultures of rat VSMC were markedly inhibited by PDGF. Stimulation with AVP
increased SM22 mRNA levels and caused a 2–4-fold increase over basal promoter activity,
which was blocked by PDGF. SM22 promoter has three CArG elements, one E-box and two GC
boxes. Expression of a fragment containing only one CArG box was sufficient to drive AVP
stimulated promoter activity. Mutations in near CArG and GC boxes reduced the basal and AVP
stimulated promoter activity but had no effect on suppression by PDGF. Similarly, overexpression
of SRF enhanced the basal and AVP stimulated activity of fragments with CArG
boxes but did not affect PDGF suppression. In contrast, over-expression of Sp1 inhibited the
promoter activity. By mutational analysis and EMSAs, the GC box did not appear to be the site
for Sp1 binding. However, Sp1 bound to a GC-rich region 3’ to the GC box. Expression of gain
of function Ras and Rac1, but not RhoA and Cdc42, potently suppressed SM22 promoter
activity. Inhibition of p38 MAP kinase and PI-3 kinase inhibited AVP stimulated promoter
activity, whereas, inhibition of ERKs had no effect. None of these agents affected PDGF-induced
suppression. These data indicate that in the regulation of SM22 expression, Vasoconstrictors
appear to act through a pathway that involves p38 MAP kinase and PI-3 kinase, whereas PDGF
suppression may be mediated through Ras and Rac1. These signaling pathways are likely to
act through distinct transcription factors, which either lead to induction of promoter activity
(SRF) or suppression (Sp1).
Shear Stress Induction of p21 waf1/cip1 Rescues Endothelial Cells from
Apoptosis During Cobalt Chloride-Simulated Hypoxia
Carlo Gaetano, Stefania Mattiussi, Fabio Martelli, Laura M Barlucchi, Annalisa Antonini,
Roberta Palumbo, Barbara Illi, Corrado Cirielli, Chiara Nicolò, Roberto Testi, Lucia Testolin,
Francesco Osculati, Giulio Pompilio, Maurizio C Capogrossi. Istituto Dermopatico
dell’Immacolata, Roma, Italy; Istituto Cardiologico “Fondazione Monzino”, Milano, Italy;
Istituto Cardiologico “Fondazione Monzino”, Roma, Italy; Università degli Studi “Tor
Vergata”, Roma, Italy; Università degli Studi di Verona, Verona, Italy
P109
Hypoxia was simulated by addition of CoCl2 to human umbilical vein endothelial cells (HUVEC)
cultured in absence of growth factors. After 12 hours of this treatment (T0), flow cytometry,
ELISA and pulse field analyses revealed the initial onset of an apoptotic process. At T0, HUVEC
were exposed to laminar shear stress (SS) (15 dyne/cm2 /sec-1 ) for additional 2 to 12 hours or
kept in static condition (ST). SS induced cell cycle arrest in G0/G1 preventing accumulation of
cells with sub-2N DNA content, chromatin fragmentation and poly(ADP-ribose)polymerase
(PARP) cleavage while cells in ST underwent significant DNA degradation. In this experimental
setting, targeting p53 tumor suppressor by papilloma E6 protein expression significantly
reduced the number of apoptotic cells detected in hypoxic ST culture at any time point. Notably,
in wild-type as well as p53 deficient HUVEC, SS progressively increased p21Waf1/Cip1 protein
levels reaching a peak between 4 to 6 hours. In order to investigate its functional role, a sense
(Sp21) and antisense p21Waf1/Cip1 (Asp21) were expressed in cells kept in ST or in presence of
SS by recombinant adenoviruses (AdCMV.Sp21 and AdCMV.ASp21) infection. Remarkably,
AdCMV.Sp21 infected HUVEC, cultured in ST, were protected from death to a similar extent as
mock infected cells exposed to laminar SS. Conversely, the expression of ASp21 significantly
reduced SS protection of HUVEC from apoptosis. These results show that p21Waf1/Cip1 induction,
occurring in presence of SS, is required for preventing human endothelial cells to undergo
apoptosis during hypoxia .
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P110
Analysis of the Tissue Factor Pathway Inhibtor Gene and Antigen Levels in
Relation to Venous Thrombosis
Ali A Nekoo. University of Leeds, Leeds, UK
Poster Presentations a-19
TFPI inhibits tissue-factor induced coagulation. The major part of TFPI is releasable by heparin.
We recently found eight patients with thrombosis and low levels of heparin-releasable TFPI. The
aims were to investigate the TFPI gene for mutations. A transition of G to A coding for
Valine264Methionine in the heparin-binding domain was found. The Val264Met polymorphism
had an allele frequency of 3% in 96 healthy individuals. A silent polymorphism was identified
in TFPI exon IV (T®C), which does not alter Tyrosine 56. Apart from Val264Met which was
detected in one out of the eight patients, no other mutations in the TFPI gene were found in
patients with low heparin-releasable TFPI. Analysis of Val264Met in 317 patients with DVT and
292 controls showed no association between Val264Met and DVT. However a study of total
TFPI antigen levels in 122 DVT patients and 126 controls demonstrated an association between
TFPI levels and venous thrombosis (p0.0001). These results provide an evidence for a relation
between venous thrombosis and Total TFPI level as a possible risk factor, whereas they do not
support a link between DVT and mutations in the nine exons of the TFPI gene.
P111
Human Adipose Stromal Cells Express the Angiogenic Factor VEGF and Its
Receptor VEGFR-2
Jalees Rehman, Jingling Li, Catharine A Williams, Sebastian Bekkers, Robert V Considine,
Keith L March. Indiana University and Indiana Center for Vascular Biology, Indianapolis, IN
Background: The delivery of stem and progenitor cells to augment angiogenesis is emerging
as a novel therapy. One obstacle is the limited availability of such cells for autologous delivery.
The recent discovery that the adipose stromal fraction contains pluripotent stem cells suggests
that it may be a source of cells for therapeutic angiogenesis. We examined the ability of
cultured adipose stromal cells to secrete the angiogenic factor VEGF (Vascular endothelial
growth factor) and express the stem cell marker AC133 as well as VEGF receptor VEGFR-2
(KDR). Methods: Subcutaneous adipose tissue biopsies were obtained from obese patients. The
adipose stromal fraction was separated from the adipocyte fraction by centrifugation. Cells
were cultured in either DMEM (Dulbecco’s Modified Eagle Media) or EGM2-MV (Endothelial
Growth Media-2 MV, Clonetics). To determine VEGF production, cell cultures were switched to
media without supplemental growth factors for 48 hrs. Supernatants were subsequently
assayed for VEGF by ELISA. The cell surface expression of VEGFR-2 and the stem cell marker
AC133 were determined by flow cytometric analysis. Results: The VEGF concentration in the
supernatants of adipose stromal cells cultured in DMEM was 526 pg/ml as compared to 279
pg/ml when cultured in endothelial media. The percentage of cells expressing the VEGF
receptor KDR was 2.4% in DMEM and 1.45 % in endothelial media. The endothelial media
favored the expression of the stem cell marker AC133 with 1.5% positive cells when compared
to only 0.6% in the DMEM. Conclusions: These experiments demonstrate for the first time that
stromal cells obtained from the easily accessible subcutaneous adipose tissue are able to
secrete VEGF and also express the VEGF receptor VEGFR-2. Furthermore, the stem cell marker
AC133 can be found in the adipose stromal fraction, thus supporting previous reports that the
adipose stromal fraction contains pluripotent stem cells. These findings suggest that
subcutaneous adipose stromal cells may be an attractive candidate for autologous cell delivery
in patients with athersclerosis to augment angiogenesis.
P112
Transcriptional Profile of Human Endothelial Cells Exposed to Fluid Shear
Stress
Scott M Wasserman, Fuad Mehraban, Laszlo Komuves, James E Tomlinson, Ying Zhang,
Frank Spriggs, James N Topper. Stanford University, Stanford, CA; CuraGen Corporation,
New Haven, CT; COR Therapeutics, South San Francisco, CA
Hemodynamic forces generated by blood flow play an integral role in vascular homeostasis. In
vitro biomechanical forces such as fluid shear stresses, modulate endothelial phenotype, in
part, through changes in gene expression. We employed the high throughput expression
profiling technique GeneCalling® to evaluate the mRNA transcript profile of human umbilical
vein endothelial cells exposed to a steady laminar shear stress at 10 dynes/cm2 (an arterial
magnitude of shear stress). A total of 35,985 bands, corresponding to cDNA fragments resulting
from digests with 93 pairs of restriction enzymes, were detected. Four hundred eighty four
bands demonstrated a greater than two fold change in expression (up/down) at 24 hours of
laminar shear stress compared with the no flow control in triplicate experiments. Differentially
expressed bands were queried against sequence databases, resulting in the identification of
108 distinct genes. Approximately 15% of these represent species whose response to shear
stress has been previously demonstrated in endothelial cells. The remaining genes constitute
known and uncharacterized species, many of which have not been described in vascular
endothelium. The characterized genes fall into a limited number of functional categories that
include stress response modifiers, genes involved in cellular metabolism, transcription factors
and signaling molecules, regulators of the cell cycle, and growth factors. Immunohistochemistry
and in situ hybridization on tissue microarrays were utilized to evaluate the in vivo
expression of a number of these genes in the vascular endothelium. Preliminary analyses
demonstrate that indeed these genes are expressed in the endothelium in vivo. Quantitative
PCR analyses performed on a subset of genes has confirmed their shear stress regulation. The
in vitro regulation of these genes by prolonged, steady laminar shear stress and their in vivo
endothelial expression suggest that these transcripts represent a set of the genes responsible
for the establishment and maintenance of endothelial phenotype in vivo. Current efforts are
directed at by understanding guest on the April role(s) 4, of 2013 these genes in the vascular wall in vivo.

a-20 Arterioscler Thromb Vasc Biol. Vol 22, No 5
Lipid-Coated SPIO: Introducing a Novel Tracer for MR Imaging of
Macrophages Infiltration in Vulnerable Atherosclerotic Plaque
Maziar Azadpour, Chinnaswamy Jagannath, Daniel Chan, Ward Casscells III, James T
Willerson, Morteza Naghavi. Center for Vulnerable Plaque Research at the University of
Texas-Houston and the Texas Heart Institute, Houston, TX; Department of Molecular
Pathology, University of Texas-Houston Health Science Center, Houston, TX; Division of
Oncology, University of Colorado Health Science Center, Denver, CO
P113
SPIO (super paramagnetic iron oxide) is a MRI contrast media in the form of nano-particles
consisting a central core of iron oxide coated by colloidal polysaccharide mainly dextran. We
have previously shown that dextran coated SPIO including the commercially available SPIO
(Feridex) are avidly taken up by macrophages, however, their uptake was followed by a
significant rise in intracellular oxidative enzyme activity mainly due to respiratory burst
associated with digestion of dextran in macrophages. Here we report a novel liposomal SPIO.
We hypothesize that lipid-coated SPIO particles enter into macrophages through a different
pathway than dextran-coated SPIO allowing more uptakes with less oxidative stress. Method:
Mouse peritoneal macrophages were isolated in the culture medium and incubated with
different SPIOs for 4 hours and then washed. After 24 hours production of nitric oxide (NO) was
measured in supernatant by Greiss reagent. In a second series of experiments fluorescentlabeled
SPIO (FL-SPIO) was added to macrophages in the presence of two inhibitors of manose
receptor, dextran and mannan. Intracellular retention of FL-SPIO was measured after 2 hours
in four different groups, macrophages with SPIO, SPIO and dextran, SPIO and mannan and no
SPIO as control. Results: See Figure 1 (p0.05). Also FL-SPIO studies showed that mannan a
known inhibitor of mannose receptor significantly inhibited macrophage uptake of dextran
coated SPIO but not lipid coated SPIO. Conclusion: Liposomal SPIO are promising candidate for
MR contrast enhanced imaging of macrophage infiltration in vulnerable plaque.
P114
Evidence of Macropinocytosis in Atherosclerotic Lesions of White Carneau
Pigeons
Nancy L Jones, Marie A Plyler, Dawn C Schwenke, Mark C Willingham. Wake Forest
University School of Medicine, Winston-Salem, NC
Macropinocytosis, occurs constitutively, but is up-regulated in macrophages (M) by growth
factors and phorbol esters. Macropinosomes (MP), are classified as phase-bright, fluid-filled
organelles forming primarily at the base of membrane ruffles (diameters 0.2 -5.0 m).
Recently, AcLDL and OxLDL were shown to stimulate macropinocytosis and be internalized in
part via MP by pigeon monocyte-derived M (Jones and Willingham, Anatomical Record,
255:57–68). In this study, we considered whether MP formed within M of atherosclerotic
lesions. Lesions from the thoracic aorta immediately anterior to the celiac bifurcation of
hypercholesterolemic random bred White Carneau pigeons were incubated in vitro with the fluid
phase indicator Dextran-biotin. After a pre-incubation with buffer, aortic segments were
incubated with Dextran-biotin (10,000 or 500,000 MW, 1 mg/ml) with either buffer, AcLDL (20
g protein/ml) or M-CSF (150 ng/ml) for 1 hr 37°C. Using peroxidase-streptavidin to detect the
presence of biotin-dextran, we found that many M foam cells in the lesions had dextran-filled
organelles consistent with MP 0.2 - 5.0 m. M foam cells within lesions incubated without
AcLDL or M-CSF showed some MP, indicating a basal level of MP in lesion. Equivalent aortic
segments incubated with either AcLDL or M-CSF had more Dextran-filled MP. This study
demonstrates that macropinocytosis occurs in M within atherosclerotic lesions in vitro and
suggests that macropinocytosis may occur in M within atherosclerotic lesions in vivo. Figure:
In Vitro Macropinosome Formation in Lesions. Aorta was pre-incubated for 30 min, and
subsequently, was incubated with dextran-biotin (500,000 MW) with AcLDL for 1 hr 37°C.
Nnucleus, arrowMP. Bar10m
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P115
Versican Content Is Dynamically Regulated during Expansion and
Regression of Flow-Dependent Neointimal Thickening in Baboon Vascular
Grafts
Jens W Fischer, Richard D Kenagy, John Sandy, Stephanie Lara, Alexander W Clowes,
Thomas N Wight. Institute of Pharmacology, University Kiel, Kiel, Germany; Division of
Vascular Surgery, University of Washington, Seattle, WA; Shriners Hospital for
Children-Research, Tampa, FL; Department of Pathology, University of Washington, Seattle,
WA; Hope Heart Institute, Seattle, WA
An experimental model of graft healing was used to analyze changes in the composition of
extracellular matrix during expansion and regression of the neointima. In baboon aortoiliac
grafts, the extent of neointimal thickening is dependent on the blood flow, which can be
modulated experimentally by the construction of arteriovenous fistulas. In this model, normal
flow induces neointimal thickening whereas high flow induces regression of this thickening.
During neointimal expansion, the subendothelial part of the neointima is enriched in
proteoglycans and stains intensely for versican. This versican-rich region also shows the
highest proliferative activity of smooth muscle cells (SMC). In contrast, the deeper part of the
neointima contains other components of the ECM as well such as type 1 collagen, biglycan and
fibronectin and exhibited considerably less proliferative activity of SMCs. Neointimal regression
in response to high flow was associated with a dramatic loss of proteoglycan as detected by
histochemical staining. Furthermore, immunostaining with an antibody that recognizes a
cleavage product of the core protein of versican shows intense staining of this region and little
staining of the expanded neointima under normal flow. The present data support the hypothesis
that accumulation or removal of proteoglycans, in particular versican, mediate adaptation of
neointimal area and lumen size in response to changes in shear stress.
Gene Knockouts for Dopamine Beta-Hydroxylase (DBH), 1B- and
1D-Adrenoceptors (AR) Suggest 1-AR-Mediated Trophic Action in
Carotid Injury and Pulmonary Hypertension (PH)
James E Faber, Caroline L Szymeczek-Seay, Susanna Cotecchia, Gozoh Tsujimoto, Hua
Zhang. University of North Carolina, Chapel Hill, NC; University of Lausanne, Lausanne,
Switzerland; Natl Children’s Med Res Ctr, Tokyo, Japan
P116
In previous cell/organ culture & in vivo studies we have shown: norepinephrine (NE) has direct
hypertrophic effects on smooth muscle cells & adventitial fibroblasts of rat aorta & carotid; this
effect is strongly augmented after balloon injury to contribute significantly to wall growth,
lumen loss, & inward remodeling; antagonist studies suggest mediation by 1A- & 1B- but
not 1D-, 2D/A or -ARs that are also expressed in both media & adventitia (Zhang & Faber,
Circ Res 89(8),2001; Erami et al, FASEB J 15 (5):A947,2001). Herein, a model of outward
remodeling was used in knockout (KO) and wildtype mice. Carotid was over-distended by
pressure, followed by air drying of endothelium. This causes increased (inc) lumen area, medial
& adventitial area & thickness, and external elastic lamina circumference. DBH-KO prevented
these inc, except for adventitial inc [50% less inc (p.05)]. 1B-KO prevented all inc. In
contrast, 1D-KO only attenuated inc medial area & thickness (by 65%, p.05), possibly due
to small decrease (dec) arterial pressure in 1D-KO only. To test for a NE trophic contribution
to PH, mice were given 21days of 10% FIO 2. DBH-KO & 1B-KO did not significantly dec rt.
ventricular systolic pressure (RVP) or hypertrophy (RVH), but hematocrit (HCT) inc WT in both
KOs (p.05), suggesting less pulmonary vascular resistance (morphometry in progress). 1B
involvement is consistent with induction of its promotor by hypoxia (Eckhart, Faber et al, PNAS
94:9487, 1997). 1D-KO dec RVP by 40% (p.05) & RVH by 50% (but p.05), while HCT inc
similarly; morphometry should indicate if this reflects the small dec in arterial pressure in
1D-KO. These data suggest NE and 1-ARs may have a direct trophic role in injury-induced
vascular remodeling.
P117
A Role for Glutathione Reductase in OxLDL-Induced Macrophage Oncosis
Reto Asmis, Jim G Begley. University of Kentucky, Lexington, KY
Electron microscopy studies suggest that in human atherosclerotic lesions oncosis not
apoptosis is the predominant mode of macrophage death. While macrophage apoptosis has
been studied extensively, the mechanism of macrophage oncosis is poorly understood. We
showed previously that oxidized LDL (OxLDL) induces oncosis in macrophages and
macrophage-derived foam cells. Here we show that in human macrophages OxLDL promotes
intracellular oxidative stress and caspase-3 activation within 4hofstimulation. Both oxidative
stress and activation of caspase-3 continued to increase over 24 h. After 24 h, macrophage
lysis was detected and continued to increase thereafter. Both OxLDL-induced oxidative stress
and macrophage lysis, but not caspase-3 activation were inhibited by the water-soluble
antioxidant Trolox. Conversely, neither the caspase-3 inhibitor z-DEVD-fmk or the general
caspase inhibitor z-VAD-fmk blocked OxLDL-induced macrophage lysis, demonstrating that
caspase activation was not required for OxLDL-induced macrophage oncosis. OxLDL also
promoted the accumulation of oxidized glutathione and increased protein S-glutathiolation.
Pretreatment of macrophages with low doses of the glutathione reductase inhibitor 1,3-bis[2-
Chloroethyl]-1-nitrosourea (BCNU) in the absence of OxLDL did not induce cell lysis. However,
BCNU pretreatment potentiated OxLDL-induced cell lysis and protein S-glutathiolation.
Pretreatment of macrophages with high doses of BCNU increased levels of oxidized glutathione,
promoted protein S-glutathiolation and induced cell lysis in the absence of OxLDL. Our results
suggest that the oxidative inactivation of glutathione reductase may play an important role in
OxLDL induced-macrophage by guest on April oncosis. 4, 2013

P118
Antioxidant Vitamin Intake, Smoking, and Statin Use Predict Paraoxonase
Activity
Gail P Jarvik, Clement E Furlong, Thomas S Hatsukami. University of Washington Medical
Center, Seattle, WA
Paraoxonase (PON1), an esterase physically associated with high-density lipoprotein (HDL), has
been shown to inhibit low-density lipoprotein (LDL) and HDL oxidation. The oxidized products
of LDL have been implicated in vascular disease. PON1 activity appears to be primarily under
genetic control with some environmental modification and is a predictor of vascular disease.
Dietary antioxidants vitamin C and E scavenge free-oxygen radical products that may depress
PON1 activity. The vitamin C and E intake of male Caucasian subjects (N184) was estimated
using a standardized food survey using linear regression. With covariates, vitamin C or E
intakes were significant positive predictors of PON1 activity for the hydrolysis of paraoxon
(POase activity) and diazoxon (DZOase activity). Smoking status and statin use were
independent predictors of activity. Vitamin C or E intake, smoking status and stain use
accounted for 10% of the variance in PON1 activity that was not attributable toe PON1192
and PON1–108 polymorphisms. PON1 activity, which is primarily genotype-dependent, varies
with antioxidant vitamins, cigarette smoking, and statin drug use. Because PON1 activity is a
better predictor of vascular disease than currently described genetic variation in PON1, further
studies of environmental influences on PON1 activity and additional PON1 genetic variants is
warranted. Paraoxonase activity may be Paraoxonase activity may be a modifiable vascular
disease risk factor.
P119
Coronary Endothelial Apoptosis and Inflammatory Response Induced by
Ischemia-Reperfusion is Blocked in Mice Lacking Gene Encoding Guanylyl
Cyclase A
Takehiko Izumi, Yoshihiko Saito, Ichiro Kishimoto, Kazuwa Nakao. Kyoto University Graduate
School of Medicine, Kyoto, Japan
Coronary endothelial apoptosis is involved in myocardial tissue injury during ischemiareperfusion
(I/R). We have previously shown that the secretion of natriuretic peptides (NPs) from
the heart is markedly augmented during I/R. However, it is not known whether NPs contribute
to development of inflammatory process and endothelial cell apoptosis during I/R. First we
showed the activation of endogenous guanylyl cyclase (GC)-A, a biologically active receptor for
ANP and BNP, leads to acceleration of inflammation and endothelial apoptosis in vivo.
Myocardial infarct size after I/R was smaller in hearts from GC-A -/- mice after ligation of left
anterior descending artery for 30 minutes followed by reperfusion for 2 days. The decrease was
associated with the decrease in infiltrated PMNs, in the expression of endothelial P-selectin and
in the activity of NF-B. Second, TUNEL positive staining was detected in vascular endothelial
cells in vivo. The number of TUNEL positive cells was significantly decreased in GC-A -/- mice
to approximately 31% of that in wild-type mice. Western blot analysis showed that both active
caspase 8 and 9 levels were apparently decreased in GC-A -/-, suggesting that Fas signaling
pathway is involved. Third, to determine whether ANP directly induces endothelial cell
P-selectin expression and apoptosis during I/R, we examined the effect of ANP in combination
with hypoxia-reoxygenation (H/R) on P-selectin and apoptosis of endothelial cells in vitro.
Cultured human umbilical vein endothelial cells (HUVECs) were exposed to 0.5% O 2 hypoxia for
18 hrs in the absence or the presence of 10 -6 M ANP followed by 3 hrs of reoxygenation. In the
absence of ANP, H/R increased the percentage of TUNEL positive cells and P-selectin
expression. Treatment with ANP in combination with H/R further increased in both apoptosis
and P-selectin expression. These results suggest that the activation of ANP/BNP-GC-A signaling
pathway induces NF-B activation and leads to inflammatory change and apoptosis in coronary
endothelial cells during myocardial ischemia-reperfusion.
P120
PDGFR- Interferes with PDGFR- Function Inhibiting PI3K Activation in
Bovine Aortic Smooth Muscle Cells
Roberta Palumbo, Carlo Gaetano, Maria Cristina Fidoli, Giorgio Scita, Pierpaolo Di Fiore,
Carl-Hendrix Heldin, Maurizio C Capogrossi. Istituto Cardiologico Fondazione “Monzino”,
Milano, Italy; Istituto Dermopatico dell’Immacolata, Roma, Italy; Istituto Europeo di
Oncologia, Milano, Italy; Ludwig Cancer Center, Uppsala, Sweden
Migration of vascular smooth muscle cells (SMC) is a key event in the formation of neointima
and the Platelet Derived Growth Factor (PDGF) is known to play a key role in this process. In
this study, we investigated in vitro the role of PDGF receptors and isoforms and the
regulation of intracellular signaling mediators activated in response to PDGFAA and PDGFBB
stimulation of SMC. We found, in a series of Boyden chamber assay, that SMC migration was
activated in response to PDGFBB but not PDGFAA (301.7 and 60.9 cells/field respectively,
p0.05). However, both PDGF receptor (PDGFR-) and PDGF receptor (PDGFR-) were
found phosphorylated. In order to investigate the functional role of these molecules transient
transfections were performed in SMC with dominant negative (dn) PDGFR- or dnPDGFR-
expression vectors that, respectively, increased or inhibited SMC migration in response to
recombinant PDGF-BB. In addition, SMC migration was significantly reduced by overexpression
of a wild type PDGFR-. Notably, a similar effect was obtained co-stimulating SMC with both
PDGF-AA and -BB recombinant molecules (PDGF-AA 71, PDGF-BB 405, PDGF-AABB
153 cells/field, P0.001). Consistently, PDGF-AA and -BB co-stimulation inhibited by 40 to
60% lamellipodia formation compared to PDGF-BB alone. These data indicate that the two
PDGF ligands, through their receptors, play different and apparently opposing roles regulating
SMC chemotaxis. To explore further those molecules involved in these processes a series of
functional assays were performed in SMC to evaluate PI3K and ERK1 and 2 MAP kinase (MAPK)
activity in presence of PDGF-AA and -BB ligands (10 ng/ml). We found that, while PDGF-AA and
-BB alone equally stimulated phosphatidylinositol -4,5- biphosphate converting capacity of PI3K
and MAPK phosphorylation, these effects were completely Downloaded abolishedfrom in SMC co-stimulated by
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Poster Presentations a-21
both ligands. In conclusion, PDGF-AA and PDGFR- function may interfere with the PDGF-BB
dependent SMC chemotaxis by inhibiting PDGFR- signaling at the level of PI3K and MAPK.
Therefore PDGFR- activation may be important preventing SMC accumulation during
neointima formation.
P121
PAI-1 Antigen and Activity Modifications after Vitamin E Supplementation
in Type 2 Diabetic Subjects Are Dependent on PAI-1 Genotype
Daniela Mari, Raffaella Coppola, Roberto Testa, Anna Rita Bonfigli, Cristina Sirolla, Ivano
Testa, Silvana Manfrini, Elisabetta Sacchi, Claudio Franceschi. IRCCS Maggiore Hospital and
Department of Internal Medicine, University of Milano, Milano, Italy; INRCA, Department of
Gerontology Research, Ancona, Italy; Institute of Internal Medicine, Ancona, Italy;
Immunology and Transfusion Department L. Sacco Hospital, Milano, Italy; INRCA,
Department of Gerontology Research, Ancona, Italy, Ancona, Italy
Background. PAI-1 4G/5G polymorphism is a predisposing factor to arterial thrombosis.
Epidemiological studies have shown that environmental and genetic factors act in a synergistic
way to determine PAI-1 plasma levels. In particular, the 4G polymorphism of PAI-1 gene
promoter seems to enhance the expression of PAI-1 causing a condition of pathological
fibrinolysis. Objectives. As type 2 diabetes mellitus is a known cause of increase in PAI-1
plasma levels and vitamin E supplementation is able to lower these levels, we wanted to verify
whether the 4G/5G gene polymorphism may be important in these changes. Twenty-eight type
2 diabetic patients were enrolled (19 males and 9 females, mean age 61.35.8 years. The
guanine insertion/deletion polymorphism 4G/5G in the promoter of the PAI-1 gene was
evaluated. These patients were treated with vitamin E (500 IU/die) for 10 weeks. PAI-1 antigen,
PAI-1 activity, and the main fibrinolytic parameters were evaluated at baseline and after 5, 10
and 30 weeks. Results. As expected decreases were detected for PAI-1 antigen levels and
PAI-1 activity between baseline and the 10th week (p 0.01) followed by an increase at the
30th week. Patients with 4G/4G genotype showed the same profiles as the patients with 4G/5G
genotype while those with 5G/5G genotype had significant differences with respect to 4G/4G
and 4G/5G genotype (p 0.01). These data evidenced that type 2 diabetic patients with at least
one 4G allele showed different decrease in PAI-1 plasma levels compared to patients who were
homozygous for the 5G allele. In conclusion,decreased PAI-1 plasma levels occur during
vitamin E supplementation and this effect is modulated by a common insertion/deletion
polymorphism in the PAI-1 promoter.
P122
Effects on Atherosclerosis by Omapatrilat and Candesartan in ApoE-/- Mice
Grace Noh, Tarek Ackad, Willa A Hsueh, Ronald E Law, Alan R Collins. UCLA, Los Angeles,
CA; Astra Zeneca, San Diego, CA
We investigated the role of Omapatrilat (OMA, an ACE/NEP inhibitor) and Canesartan (CND) on
the formation and progression of atherosclerosis in male apoE-/- mice in the presence or
absence of angiotensin II (AngII) infusion. To examine the role of OMA and CND on the
progression of early xanthoma formation, 3-month-old mice were placed on 1)chow, 2)chow
with OMA or 3)chow with CND for a period of 12 weeks. Both OMA and CND significantly
decreased systolic blood pressure as measured by tail cuff (101 mmHg control vs 72 mmHg
OMA and 74mmHg CND; p0.01). Both drugs also inhibited the formation of fatty streaks
determined by the en face method by 70% (3.2% control vs 1.1% OMA and 1.2% CND;
p0.01). To investigate the role of OMA and CND on atherosclerosis during AngII-induced
hypertension, 3-month-old mice were placed on the same dietary groups as above and infused
with 2.5g/kg/min AngII for 4 weeks. Infusion of AngII increased systolic blood pressure in both
chow (164mmHg;p0.05) and OMA (149 mmHg;p0.05) groups while CND decreased the
blood pressure(89 mmHg;p0.05) compared to non-infused controls (104 mmHg). The
infusion of AngII dramatically increased both the rate and progression of atherosclerosis in the
chow (30%;p0.01) and OMA (37%;p0.01) groups, which was completely ameliorated by
CND (0.5%: control 0.6%). The lesions showed a greater complexity similar to human lesions
having necrotic lipid cores and fibrous caps with a proteoglycan rich matrix. This indicates that
NEP has no effect on the development of atherosclerosis in the presence of AngII. To determine
whether CND could cause regression of pre-existing lesions, 6-month-old apoE mice were
placed into one of three groups: 1)baseline controls, 2)3 months continued chow or 3)3 months
on chow/CND. The apoE mice had 4.1% atherosclerotic lesions at 6 months of age. The
inclusion of CND in their water inhibited the further development in the percentage of the aortic
surface covered by lesions (4.3%) compared to the 9-month-old chow fed group
(11.8%;p0.05). Both of these drugs exhibit important atherosclerotic protective effects.
P123
Arterial Permeability is Increased in Mice with Hyperhomocysteinemia
Evoked by Methionine Supplementation
Ussama B Zaid, Adam E Mullick, John C Rutledge, J D Symons. University of California
Davis, Davis, CA; University of Utah, Salt Lake City, UT
Increasing evidence indicates that elevated plasma homocysteine concentrations initiate and/or
contribute to cardiovascular disease. The mechanisms responsible for the atherogenic effects
of hyperhomocysteinemia, however, are unclear. Previously we showed in rats that hyperhomocysteinemia
evoked by folate-depletion increased carotid arterial permeability compared to
animals that consumed standard chow. The possibility exists, however, that the methods used
to elevate homocysteine (i.e., folate-depletion) contributed to these deleterious permeability
changes. In the present study, we hypothesized that hyperhomocysteinemia evoked by
methionine-supplementation increases carotid arterial permeability. After weaning, C57Bl/6J
mice consumed standard rodent chow and regular water (CON) or water supplemented with
0.5% L-methionine (HHC). At 163 weeks of age blood samples were obtained via cardiac
puncture, and permeability to a reference macromolecule (dextran; 4,400 MW) was assessed
in carotidby arteries guest using on quantitative April 4, 2013 fluorescence microscopy. While plasma homocysteine

a-22 Arterioscler Thromb Vasc Biol. Vol 22, No 5
(M) was higher (p0.05) in HHC (397) vs CON (175) mice, liver folate concentrations (g
folate/g liver) were similar between groups (111 and 121, respectively). Further, the rate
of macromolecule accumulation (ng/cm 2 /min) was greater (p0.05) in arteries from HHC
(3.950.22, n8 vessels) vs CON mice (2.870.17, n13 vessels). These data support our
hypothesis and indicate that hyperhomocysteinemia, in the presence of normal folate,
increases arterial permeability. Since increased vascular permeability could facilitate lipoprotein
accumulation in the arterial wall, our findings provide evidence for the atherogenic
potential of hyperhomocysteinemia. Supported in part by AHA Scientist Development Grant
0130099N (JDS)
P124
Systemic Smooth Muscle Impairment in Patients with Erectile Dysfunction
and no Other Clinical Cardiovascular Disease
Daniel R Kaiser, Kevin Billups, Carol Mason, Rebecca Wetterling, Alan Bank. St. Paul Heart
Clinic, St. Paul, MN; The Epicenter for Sexual Health and Medicine, St. Paul, MN
Erectile dysfunction (ED) is now recognized as being vascular in origin in the majority of
patients. ED may be an early marker of atherosclerosis in patients without clinical cardiovascular
disease. We assessed systemic vascular structure and function in patients without clinical
cardiovascular disease and with ED of vascular origin as documented by penile Doppler
ultrasound. Thirty patients with ED (age 47 yrs) and 25 age-matched normal (NL) control
subjects (46 yrs) underwent tests including fasting lipid profile (cholesterol, LDL, HDL, TG,
Lp(a), homocysteine) and measurements of vascular parameters including: 1) carotid and
brachial artery diameter (Cd and Bd), intima-media thickness (Cimt and Bimt), compliance (Cc
and Bc) , and distensibility (Cdist and Bdist), 2) aortic pulse wave velocity (PWV), 3) coronary
calcification, and 4) brachial artery endothelium dependent and independent vasodilation.
Results: There were no significant differences in baseline demographics (height, weight, BP) or
lipid values between the 2 groups. Heart rate was significantly increased in the ED group (64
vs. 60 bpm, p 0.05). Structural vascular parameters including coronary calcium score 5
(27%–8 of 30 vs. 20%–4 of 21), diameter (Cd, 7.14 vs. 7.30 and Bd, 4.59 vs. 4.48, mm), and
IMT (Cimt, 0.65 vs. 0.62 and Bimt, 0.41 vs. 0.40, mm) were similar in ED and NL, respectively.
Functional vascular parameters of compliance (Cc , 100.58 vs. 111.26 and Bc, 9.20 vs. 9.37,
x10–3 mm2/mmHg), distensibility (Cdist, 2.47 vs. 2.67 and Bdist, 0.54 vs. 0.59, x 10 –3
mmHg-1), and PWV (7.80 vs. 7.88, m/s) were similar between ED and NL. There was a trend
for decreased brachial artery flow mediated vasodilatation (FMV) (2.78 vs. 1.80, %, p0.11)
in ED. The response to nitroglycerin (NTG) (13.03% vs. 17.83%, p 0.05) was significantly
impaired in ED vs. NL, respectively. In addition, there was a significant correlation between FMV
and NTG in ED (r 0.60, p0.005) but not in NL (r 0.03, pNS) Conclusions: These data
show that ED may be an early marker for the development of systemic cardiovascular disease
and may manifest as an abnormality of the NO/cGMP pathway.
P125
Atorvastatin Inhibits Hypercholesterolemia-Induced Cellular Proliferation
and Bone Matrix Production in the Rabbit Aortic Valve
Nalini M Rajamannan, Malayannan Subramaniam, Margarett Springett, Thomas Sebo, Marek
Niekrasz, Joseph McConnell, Ravinder Singh, Neil Stone, Robert O Bonow, Thomas C
Spelsberg. Northwestern University, Chicago, IL; Mayo Clinic, Rochester, MN; Northwestern
University, Chicago, MN; Mayo Clinic, Rochester; Northwestern University, Chicago
Intro: Recently, aortic valve disease has been associated with elevated cholesterol levels. We
tested a model of experimental hypercholesterolemia for effects on atherosclerosis and bone
matrix expression in the aortic valve(AV). Methods: To test this hypothesis we examined the
effects of cholesterol (chol)in the rabbit AV with and without atorv. Rabbits (n48) were
studied: Group I (n16) normal diet, Group II (n16) 1% chol diet, and Group III (n16) 1%
chol diet atorv. The valves were examined with hematoxylin and eosin and masson trichrome
stains. Chol and C-reactive protein (hsCRP) levels were obtained. Immunohistochem stain and
digital image analysis (DIA) were performed and evaluation for protein expression and
quantification of proliferating nuclear cell antigen (PCNA) (%area) macrophage cell (RAM11) and
osteopontin (OPN). Semi-quant RT-PCR was performed for the osteoblast (OB) bone markers
OPN, and OB transcription factor (Cbfa1). Results: Immunostain for macrophage and PCNA
revealed an increase in the chol group compared to the atorv group. Immunostain and
immunogold labeling for OPN demonstrated an increase in the chol treated rabbits and a
decrease in OPN protein expression in the atorv group. Similarly, the osteoblast specific marker
genes OPN and Cbfa1 were increased with chol feeding and were reduced by atorv. Concl:
These findings of increased macrophage infiltration, PCNA levels, and bone matrix markers in
the aortic valve during experimental hyperchol support the concept that degenerative aortic
valve disease is a proliferative, atherosclerotic process that is associated with the expression
of an osteoblast-like phenotype in the AV. The inhibition of this process by atorv suggests this
may be modulated by pharamacologic intervention.
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P126
Influence of Mild Hypothermia on Tissue Plasminogen Activator-Induced
Clot Lyses in Carotid Thrombosis
Kamel Chair, Dong-Wei Gao, Michael W Dae. University of California, San Francisco, San
Francisco, CA
Background: Mild hypothermia reduces infarct size in experimental models, inhibits platelet
aggregation in vitro, and may be a useful adjunct to reperfusion therapy in patients with acute
ischemic syndromes. Little is known, however, about the effects of mild hypothermia on the
action of thrombolytic agents in vivo. We studied the effects of mild hypothermia on tissue
plasminogen activator (TPA)-induced thrombolysis in the rabbit carotid thrombosis model.
Methods: Thrombus formation was achieved by electrical injury to the carotid artery. 60
minutes after thrombotic occlusion, confirmed by Transonic flow probe, TPA (9mg/kg) was
administered intravenously to normothermic (38°C, n7) and hypothermic (32°C, n7)
animals using a front-loaded protocol over 90 minutes. Hemodynamics and regional
temperature (close to the carotid artery) were monitored continuously. Hypothermia was
induced by cold saline perfusion through a closed loop balloon thermo-exchange catheter
(Radiant Medical Inc.), via the esophagus. Time to reperfusion, incidence of reocclusion, and
incisional blood loss were measured. Results: Regional temperature in the hypothermic animals
was 30.4Â3.5°C vs. 38Â0.8°C in the normothermic animals (p.01). There was no
significant difference in median time to reperfusion in hypothermic versus normothermic
animals (107 min vs. 106 min, pns). The incidence of reocclusion tended to be higher in the
normothermic group than the hypothermic group (5/7 vs. 2/7), while incisional blood loss was
greater in the normothermic animals (41Â27 g vs. 14Â10 g, p.05) in a limited time of
observation. Conclusion: Mild hypothermia did not significantly affect the TPA-induced clot lytic
process, but showed fewer propensities for bleeding and reocclusion as compared to
normothermia. The combination of TPA-induced thrombolysis and mild hypothermia may be
applied clinically as a therapeutic strategy in the setting of acute ischemic syndromes.
P127
Carotid Artery Stiffness in an Elderly Multi-Ethnic Population: The Northern
Manhattan Stroke Study
Tanja Rundek, Mitchell S Elkind, John Pittman, Bernadette Boden-Albala, Ralph L Sacco.
Columbia University, Neurological Institute, and New York Presbyterian Hospital, New York,
NY
Objective: To assess common carotid artery stiffness (CAS) in an elderly, multi-ethnic
population. Background: Carotid artery stiffness recently has been introduced as a potential
marker of atherosclerosis and a risk factor for stroke. Information regarding CAS for the elderly
from different race-ethnic backgrounds is limited. Methods: CAS was assessed as part of the
Northern Manhattan Cohort, a population-based study of stroke-free individuals. The systolic
(SD) and diastolic diameters (DD) for the right CCA from 5 B/M-mode registrations were
measured (mm) and averaged. Strain (% change in diameter), stress-strain elastic modulus
(E [systolic blood pressure (BP) - diastolic BP] / strain), and stiffness ( ln [systolic BP /
diastolic BP] / strain) were calculated. Results: CAS was analyzed in 210 subjects (mean age
68 years; 61% women; 54% Hispanic, 22% Black, 19% White). The mean systolic CCA
diameter was 6.93 1.31, and diastolic CCA diameter was 6.46 1.27. Systolic and diastolic
diameters were significantly smaller in women then in men (SD women 6.70 1.22 vs. men 7.31
1.36, p0.002; DD women 6.26 1.17 vs. men 6.81 1.35, p0.004), especially for white
women (SD 6.57 0.99, DD 6.09 1.10 vs. non-White women: SD 6.86 1.22, DD 6.42 1.19), and
Hispanic men (SD 7.18 1.34, DD 6.73 1.32 vs. non-Hispanic men: SD 7.65 1.92, DD 6.94 1.13).
Increased stiffness and decreased strain was observed in those older than 75 years and
Caucasian women and Hispanic men. The mean values of parameter E were not associated
with age, gender or ethnic background. In a multivariate linear regression model, female
gender (p0.009) and systolic blood pressure (p0.0001) were the most significant
independent predictors of carotid artery stiffness. Conclusion: Increased systolic blood
pressure and gender (women) are independent predictors of greater stiffness of the carotid
artery wall. Non-invasive high-resolution B-mode ultrasound assessment of both functional and
structural properties of the carotid artery may be a useful method to assess subclinical
atherosclerosis and vascular risk among elderly subjects.
Role of PDGF-B in Lymphocyte Activation and its Implications in
Atherogenesis
Jingjing Tang, Koichi Kozaki, Paul Martin, Per Lindahl, Andrew Farr, Christer Betsholtz,
Elaine W Raines. University of Washington, Seattle, WA; Fred Hutchinson Cancer Research
Center, Seattle, WA; University of Goteborg, Goteborg, Sweden
P128
To test the role of PDGF in vivo and in atherogenesis, we have generated chimeric ApoE null
mice lacking PDGF-B in their circulating cells lethal irradiation of ApoE-/- recipients and
transplantation with fetal liver cells from ApoE-/- /PDGF-B / or -/- embryos. Analysis of
early fibrous cap lesion formation (35 weeks) suggested an enhanced inflammatory response
and a decrease in smooth muscle cell accumulation in PDGF-B null chimeras. This was
associated with a 20–30% decrease in total B cell numbers (B220 cells) in their spleens, a
20–40% increase in the number of T cells (CD4 and CD8 cells) and a more activated
CD4 T cell population (more CD25, less CD62L). The changes in splenic B and T cells
appear constant from 19 to 45 weeks of age, and the decrease in total B cell numbers does
not appear to be due to a block in B cell development from proB (B220/CD43), preB
(B220/BP-1) to IgM bearing B cells. Despite decreases in B220 cell numbers, ApoE-/-/PDGF-
B-/- chimeras demonstrate an increase in antibody levels to oxidized low-density lipoprotein
(LDL) that is associated with an increase in smooth muscle fibrous cap formation at 45 weeks.
ApoE-/-/PDGF-B/-/- mice also show a preferential increase in total IgG2a levels in serum, which
is characteristic of a Th1 helper T-cell response. An elevated Th1 response in the
ApoE-/-/PDGF-B-/- by guest chimeras on April is further 4, 2013 supported by an increase in CCR5 mRNA levels assayed

y RNase protection analysis and an increase in IFN-gamma secreting CD4 T cell population
in splenocytes upon ex vivo challenge with MDA-LDL. This modulation of the immune response
in the ApoE-/-/PDGF-B-/- chimeras may contribute to their altered lesion formation. Supported
by NIH HL18645 and NIH 5T32HL07828.
P129
Angiographic Characteristics of Coronary Atherosclerosis in Patients with
Diabetes Mellitus
Stefan Aczel, Christoph Saely, Werner Benzer, Hannes Holzmueller, Wolfgang Fuchs, Peter
Langer, Heinz Drexel. VIVIT-Institute, LKH Feldkirch, Austria
Background:It is well established that coronary artery disease confers higher mortality in
diabetic than in non-diabetic subjects. The mechanisms causing this worse prognosis are
unclear. The notion that diabetic patients develop more diffuse and more peripheral
atherosclerosis could explain the poor outcome, but this pattern has been described rather in
type 1 than in type 2 diabetes. Methods: We performed a large-scale angiographic study
involving 757 consecutive patients referred to coronary. Extent of coronary atherosclerosis CA
was defined as the number of significant lesions (50 %), severity of CA as the mean
percentage of individual stenosis, and diffuse sclerosis as nonfocal, nonsignificant irregularity
of the coronary artery wall. According to the glycemic status patients were divided into 4
groups: established type 2 diabetes (n 128), fasting plasma glucose (FPG)125 mg/dl
(n60), HbA1c6.1 % and FPG126mg/dl (n99), and patients with none of the 3 criteria
(normoglycemic, n464). Results: Extent of CA was higher in the hyperglycemic than in the
normoglycemic patients (1.56 vs. 1.50 vs. 1.63 vs. 1.30). The 3 hyperglycemic groups did not
differ significantly by extent. Therefore they were pooled together and opposed to the
normoglycemic subjects. Extent of CA proved significantly increased in hyperglycemic subjects
(1.57 vs. 1.30, p 0.02). The severity of CA was comparable in all the groups (79.77 vs. 79.89
vs. 76.46 vs. 78.52% mean stenosis). The prevalence of diffuse coronary sclerosis also did not
differ significantly between the groups (70% vs. 75 % vs. 65 % vs. 66%). Conclusions: Thus
we can conclude that diabetic patients express a pattern of multifocal significant atherosclerotic
lesions whereas the severity of CA is not affected by the glycemic status. Moreover,
contrary to the current view, patients with type 2 diabetes do not have more diffuse coronary
disease than non-diabetic patients with CA. In summary, coronary atherosclerosis in diabetic
patients is specifically characterized by a significantly increased number of distinct focal
lesions. This pattern of CA is a ready explanation for increased mortality in diabetic patients.
P130
Hepatic Secretion but not the Synthesis of apo B-100 is Inhibited in apo
B-100-Only Mice Heterozygous for an apo B-38.9-Specifying Mutation
Zhouji Chen, Robin L Fitzgerald, Gustav Schonfeld. Washington University School of
Medicine, St Louis, MO
Apolipoprotein (apo) B-truncation-specifying mutations of the apo B gene (Apob) cause familial
hypobetalipoproteinemia (FHBL) in humans and Apob-modified mice. Kinetic studies on
heterozygous FHBL humans consistently show that production rates of apo B-100, the product
of the unaffected Apob allele, are reduced by 70%, instead of the expected 50%. To generate
a suitable mouse model to study the underlying mechanism, we crossbred our recently
reported apo B-38.9 mice with apo B-100-only (Apob 100/100 ) mice to produce mice doubly
heterozygous for apo B-100- and apo B-38.9-specifying alleles (Apob 100/38.9 ). The Apob 100/38.9
mice displayed typical FHBL phenotypes including a 75% reduction in plasma apo B-100 levels.
Two and 4 h after the simultaneous intravenous injection of 35 S-methionine/cysteine and Triton
WR-1339, the amounts of 35 S-labeled apo B-100 accumulated in plasma of Apob 100/38.9 mice
were only 25% and 10% of those of Apob 100/100 mice, respectively. Likewise, on continuous
metabolic labeling of cultured primary hepatocytes in the absence and presence of oleic acid
(OA) (0.5 mM), secretion rates of 35 S-labeled apo B-100 from the Apob 100/38.9 cells were 55%
and 70% lower than those of the Apob 100/100 cells, respectively. Apo B-100 synthetic rates in
the Apob 100/38.9 hepatocytes were reduced by 55% regardless of OA supplementation.
Pulse-chase studies showed that a larger proportion of newly synthesized apo B-100 was
subjected to intracellular degradation in Apob 100/38.9 than in Apob 100/100 hepatocytes. Furthermore,
while OA significantly inhibited intracellular apo B-100 degradation in Apob 100/100
hepatocytes, it was ineffective in Apob 100/38.9 hepatocytes. Together, these results provide
evidence for an inhibitory role for the apo B-truncation mutation in the secretion of apo B-100,
the product of the unaffected Apob allele, in an FHBL mouse model.
P131
An Ovary-Intact Mouse Model that Mimics Peri-Menopause in Women
Loretta P Mayer, Patrick J Devine, Patricia J Christian, Samuel L Marion, Gina M Buss,
Carole L Banka, Cheryl A Dyer, Patricia B Hoyer. University of Arizona, Tucson, AZ; Northern
Arizona University, Flagstaff, AZ; La Jolla Institute for Molecular Medicine, La Jolla, CA
Current menopause animal models rely on ovariectomized animals. However, this model does
not accurately represent the physiology of an ovary-intact menopausal woman. The menopausal
ovary is follicle-deplete and primarily composed of interstitial cells. Previous studies
have determined that repeated dosing of mice with the occupational chemical,
4-vinylcyclohexene diepoxide (VCD), selectively destroys primordial and primary ovarian
follicles by acceleration of the natural process of atresia resulting in pre-mature ovarian failure.
This study was designed to characterize an ovary-intact mouse with premature ovarian failure.
Female B6C3F1 mice (d28, n8/group) were dosed with VCD (160mg/kg, i.p.) for 15d (d43).
Ovaries were collected (d64) and prepared for histological evaluation. VCD treatment resulted
in ablation of primordial and primary follicles, and a reduction (p0.05) in secondary (6.6% of
control) and antral (37.7% of control) follicles. Compared with controls, circulating follicle
stimulating hormone (FSH), a clinical index of menopausal status, was increased (11.2ng/ml,
VCD; 1.7ng/ml, control, p0.05). VCD treatment did not alter body, adrenal, kidney or spleen
weights; however, ovarian and uterine weights were Downloaded smaller (p0.05). from
There was no evidence
Poster Presentations a-23
of liver damage as there was no elevation of plasma alanine aminotransferase (ALT) or
aspartate aminotransferase (AST), and histopathology of the liver in VCD-treated animals did
not differ from controls. Total plasma cholesterol was not altered in VCD-treated mice. Ovarian
interstitial cells express luteinizing hormone (LH) receptor as well as SR-BI, a scavenger
receptor that sequesters cholesterol for progestin and androgen production, and receptor
staining colocalized on interstitial cells, as visualized by immunoflourescence. Cultured cells
from ovaries of VCD-treated animals produced progesterone and androstenedione, which could
be stimulated by LH. These results demonstrate that the VCD-treated mouse reflects the
follicular content and hormonal status of the peri-menopausal woman. This model will likely be
useful for investigation of the development of cardiovascular disease in women as they age.
Angiotensin II Accelerated Atherosclerosis Is Attenuated in
Osteopontin-Deficient Mice
Dennis Bruemmer, Alan R Collins, Ulrich Kintscher, Grace Noh, Yuri Ozawa, Eckart Fleck,
Ronald E Law, Willa A Hsueh. UCLA, Los Angeles, CA; German Heart Institute, Berlin,
Germany
P132
Osteopontin (OPN) is a large acidic phosphoprotein containing the arginine-glycine-aspartate
(RGD) tripeptide integrin binding motif. OPN acts as an adhesion substrate and migration
stimulus by interaction with several integrins; these effects result in activation of cell signaling
pathways and regulation of gene expression. Overexpression of OPN has been identified in
atherosclerotic lesions in nondiabetic and diabetic animal models. Angiotensin II (AngII) is a
potent inducer of OPN expression in the vessel wall. We hypothesized that OPN may be a key
player in the early and late changes associated with development and progression of
AngII-accelerated atherosclerosis. To test this hypothesis we bred OPN-/- with ApoE-/- mice to
develop ApoE-/-OPN/- heterozygote mice deficient in OPN. Three month old male ApoE-/-
OPN/- heterozygote mice and ApoE-/-OPN/ littermate controls were infused with 2.5
microg/kg/min AngII for four weeks. Both groups of mice developed marked hypertension
(systolic 160 mmHG vs. 102 mmHG in control animals). En face analysis of the thoracic aorta
revealed a significant decrease in atherosclerotic lesions in OPN/- heterozygotes compared
to OPN/ animals (5.5 0.8 n7 vs. 15.3 1.3 n4, p0.01). Real time PCR analysis
confirmed that OPN mRNA expression in aortae of OPN/- mice was decreased by
approximately 50% compared to littermate controls. Lipid profiles were similar between
OPN/ and OPN/- mice. Peritoneal macrophages from OPN/- mice exhibited an
impaired chemotactic response towards MCP-1 in transwell migration assays which could
contribute to the diminished atherosclerosis observed in these animals after AngII-infusion.
These data suggest that OPN is an important proatherogenic factor in the vessel wall.
Approaches designed to inhibit OPN expression and/or adhesion in the vasculature may provide
a novel therapeutic strategy against atherosclerosis.
P133 WITHDRAWN
P134
Progress with the Calibration of a 3F Near Infrared Spectroscopy Fiber
Optic Catheter for Monitoring the pH of Atherosclerotic Plaque: Introducing
a Novel Approach for Detection of Vulnerable Plaque
Tania Khan, Babs Soller, Mohammad Madjid, James T Willerson, Samuel W Casscells III,
Morteza Naghavi. University of Massachusetts, Worcester, MA; Division of Cardiology,
University of Texas-Houston Health Science Center, and Texas Heart Institute, Houston, TX
Near-infrared (NIR) spectroscopy has been proposed to characterize structural properties of
vulnerable plaques (VP). We hypothesized that localization of VP can be enhanced by
physiological factors such as low pH, high temperature (T), NO, hypoxia, and oxyradicals, which
shift NIR spectra. Previously, we have shown that inflamed regions of plaque have lower pH.
Therefore, we chose pH to calibrate our spectroscopy catheter. Methods: Different probe sizes
were used. Eventually, using a unique miniaturized fiber optic side-viewing catheter, we
demonstrated the feasibility of performing the correlation. 5 human carotid endarterectomized
plaques were collected and placed immediately in a humidified, 37 C controlled T glove-box
type incubator. Optical reflectance spectra (ORS)(400 –1100 nm) were taken with the prototype
catheter connected to a spectrometer. 17 tissue pH readings were done (using microelectrodes)
and correlated with matching ORS. Partial Least Squares multivariate calibration
techniques were used. Results: Probe miniaturization localized the optical spectra and allowed
previously undetected quantitative differences to be detected in the heterogeneous plaque. The
range of the electrode pH was 6.83 to 7.54. The R2 of the determination of tissue pH from the
optical NIR calibration was 0.63 and the Root Mean Squared Deviation (RMSD) was 0.14 pH
units. Conclusion: This feasibility study suggests that plaque pH can be determined with NIR
spectroscopy both ex vivo and in vivo. Further improvements in signal-to-noise ratio is required
to meet the goal of detection of VP by pH.
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a-24 Arterioscler Thromb Vasc Biol. Vol 22, No 5
Measurement of 15-Deoxy--12,14-PGJ 2 in Human Urine by
LC-Electrospray Ionization-MS
Leslie C Bell-Parikh, Helen Zhou, John A Lawson, Garret A Fitzgerald. University of
Pennsylvania, Center for Experimental Therapeutics, Philadelphia, PA
P135
The cyclopentenone prostaglandin 15-deoxy--12,14-PGJ 2 (J-M) is a dehydration product of
the cyclooxygenase (COX) metabolite PGD 2. Recent studies using micromolar quantities of J-M
suggest that it functions as an endogenous ligand for PPAR-, a protein that is abundant in
macrophages and adipocytes and is believed to modulate lipid metabolism. High levels of J-M
have been assigned additional diverse signaling functions, including a contribution to the late
resolution phase after inflammatory stimuli. To date, there is scant evidence that J-M is actually
formed in vivo. We have developed an assay utilizing liquid chromatographic-negative
ion-electrospray ionization-mass spectrometry (MS) to analyze human urine for evidence of
J-M biosynthesis. This assay is linear from 1–50 pg J-M/mL sample (r0.99) and the limit of
detection is 5 pg/mL. A urinary compound was identified that co-purifies with a 2 H 4-J-M
internal standard through silica-gel TLC and RP-HPLC. Tandem MS analysis reveals that two
major transition ions characteristic of J-M are generated at the expected relative abundances
during collision-induced fragmentation of the peak. Basal levels of J-M in urine were calculated
to be 7.02.7 pg/mg creatinine (n8). No gender difference was observed. Urinary J-M was
decreased by 80% by COX isoform nonselective NSAIDs or by selective COX-2 inhibition with
celecoxib, suggesting that COX-2 is the dominant source of J-M biosynthesis in healthy
individuals. Bacterial LPS is known to induce COX-2 induction in vitro. LPS was administered
to volunteers at a dose sufficient to stimulate expression of both COX isoforms ex vivo and to
markedly increase urinary metabolites of prostacyclin and thromboxane (J. Clin. Invest. (2000)
105:1473–1482). LPS failed to evoke a detectable increment in J-M biosynthesis either
coincident with peak formation of other prostanoids or as a delayed response 16 –24 hr later.
J-M is formed in picomolar quantities, deriving predominantly from COX-2 in humans. The
cellular source of J-M biosynthesis does not appear to be activated by LPS in humans and J-M
formation is not evident during the late resolution phase after this inflammatory stimulus.
Oxidative Damage and Inflammation are Separate Pathways for Early
Coronary Calcification
P136
David R Jacobs Jr, Myron D Gross, Russell Tracy, Cora E Lewis, Stephen Sidney, Kiang Liu,
Cay Loria, Michael W Steffes. University of Minnesota, Minneapolis, MN; University of
Vermont, Colchester, VT; University of Alabama, Birmingham, AL; Kaiser Permanente,
Division of Research, Oakland, Oakland, CA; Northwestern University Medical School,
Chicago, IL; National Heart, Lung, and Blood Institute, Bethesda, MD
Oxidative damage and inflammation may promote or accelerate the underlying pathophysiology
of atherosclerosis. In small studies, F2-isoprostanes (products of arachidonic acid peroxidation)
were found in atherosclerotic plaque and in oxidized LDL. CRP, a general marker of
inflammatory activation, has been shown to relate to risk of CHD. In year 15 of the Coronary
Artery Risk Development in Young Adults (CARDIA) study (ages 33–45), concentration of
plasma F2-isoprostanes were assayed by gas chromatography/mass spectrometry (n1357),
C-reactive protein (CRP) by immunoassay (n 2800), and coronary calcification (CaC) was
assessed by computed tomography (189 male and 81 female cases with some calcification).
F2-isoprostanes were weakly associated with CRP i n women (r0.11, p.003), but not in
men (r0.01). Per standard deviation, adjusting for race and clinical center, the odds ratio for
having some vs. no CaC was 1.35 (p0.01) in men and 1.20 (p0.01) in women for
F2-isoprostanes. The odds ratio was 1.2 5 (p0.01) in men and 0.98 (p0.9) in women for
CRP. These odds ratios persisted in models that included both F2-isoprostanes and CRP, as
well as in models that included smoking, HDL-C, LDL-C, triglycerides, body mass index (BMI),
and systolic blood press ure. Findings for CRP and CaC in women should be interpreted with
caution because they had few CaC cases. This study suggests that in these young adults
F2-isoprostanes, a direct measure of lipid oxidation, are linked to atherosclerosis at least partly
by a pathway other than inflammation or hyperlipidemia.
Additive Effects of the Mutations in the 3-Adrenergic Receptor and
Uncoupling Protein-3 Promoter Genes on Abdominal Fat and Glycemic
Control in Response to Mild Weight Loss
Yangsoo Jang, Jong Ho Lee, Oh Yoen Kim, Ha Jung Ryu, Hyun Young Park. Cardiovascular
Genome Center, Yonsei University, Seoul, Korea; Dept of Food & Nutrition, College of
Human Ecology, Yonsei University, Seoul, Korea
P137
The search for candidate genes for overweight and its related metabolic syndromes has been
actively studied. This study determined whether the Trp64Arg mutation in 3-adrenergic
receptor (3AR) gene and -55C3T mutation in the promoter region of uncoupling protein-3
(UCP3) have association with abdominal fat lossDownloaded and insulin-resistant from
glucose metabolism in
http://atvb.ahajournals.org/
response to mild weight loss. One hundred twenty five overweight (body mass index25kg/m 2 )
subjects with coronary artery disease or metabolic syndrome (abdominal obesity, dyslipidemia
or high blood pressure) completed the 12 weeks weight loss program by low calorie diet
(-300kcal/d). The subjects were divided into 4 groups according to their 3AR and UCP3
promoter genotype: no mutation (control; n42), only C3T mutation in the UCP3 promoter
(n48), only Trp64Arg in the 3AR (n17), and both mutations (n18). Despite similar 5%
loss of initial body weight in 4 groups, the control showed reductions in visceral (8%, P0.01)
and subcutaneous (7%, P0.05) fat areas at the levels of L1 and L4, and subjects with only
UCP3 variant showed a decrease in those areas (4%, P0.05) at L1 level. However, subjects
with both mutations showed 7% reduction in mid-thigh muscle without any reduction in fat
area. In response to weight loss, the fasting insulin levels decreased to the same extent across
the four groups but only control subjects showed a decrease in insulin response area during
oral glucose tolerance test (OGTT). Fasting glucose levels and glucose response area during
OGTT improved to the similar extent except subjects with mutation in both genes. The results
suggest that a combination of the Trp64Arg mutation in the 3AR and -55C3T mutation in
the UCP3 promoter may be associated with difficulties in losing fat and improving glycemic
control in response to mild weight loss.
P138 WITHDRAWN
P139 WITHDRAWN
P140
Significant Relation Between Carotid Atherosclerosis and Apolipoprotein E
Polymorphism Independent of Classical Risk Factors in a Large-Scale
Japanese General Population Evaluated by High Resolution
Ultrasonography: The Suita Study
Shunroku Baba, Tomohiro Katsuya, Toshifumi Mannami, Nozomu Inamoto, Kazuhiko
Ishikawa, Masayuki Fukuda, Yuko Koyama, Yoshihiro Kokubo, Jun Ogata. National
Cardiovascular Center, Suita, Japan; Osaka University Medical School, Suita, Japan
Apolipoprotein E (apoE) polymorphism was determined with TaqMan PCR method and carotid
artery ultrasonography was done for the 3,125 subjects randomly selected from Japanese
general urban population aged 30 to 86 years. Relations between apoE polymorphism and
ultrasonographic findings were examined. Carotid atherosclerosis was evaluated using
atherosclerotic indexes of intimal-medial thickness (IMT), plaque number (PN), and plaque
score (PS). IMT was measured in mean IMT (MIMT), and max IMT (IMTMAX). The risk factors
monitored at the same time were serum levels of total cholesterol (TCHOL), high density
lipoprotein cholesterol (HDLC), triglyceride (TG), plasma glucose (FPG), blood pressure, height,
weight and drinking and smoking habits. Blood was sampled more than 5 hours fasting.
Comparisons were done for all the subjects, by sex, and by age groups of 60 years (younger
group) and 60 years (older group). As the results, prevalences per 100 of E2/2, 2/3, 2/4,
3/3, 3/4, 4/4 were observed as 0.4, 8.5, 0.9, 72.6, 16.6, 1.0, respectively which are similar to
those in other Japanese populations. The comparison of ultrasonography findings showed
significant difference between polymorphisms in MAXIMT for all subjects and younger men
(p0.05), all men (p0.01), PN for all subjects, all men, and younger men (all p0.05), and
older women (p0.01) and PS for all subjects, all men, and older women (all p0.01), and
younger men (p0.05). The findings are from better to worse in the order of E2/4,4/4,2/3,3/
3,3/4,2/2 mostly. Analysis of covariance was done with the covariant factors of age, sex,
smoking, drinking, systolic blood pressure, body mass index, TCHOL, HDLC, TG, and FPG. The
significance was found for MIMT in all women (p0.05), for IMTMAX in all subjects (p0.01)
and all men (p0.05), for PN in all subjects and all women (both p0.001), and for PS in all
subjects (p0.01) and all women (p0.05). The present result suggests the significance of
apoE genetic polymorphism as an independent atherogenic risk factor.
Reflex Sympathetic Nerve Activation Enhances Platelet Functions and
Reduces Fibrinolytic Activity in Patients with Coronary Heart Disease
Kan-Ichi Otowa. Graduate School of Medical Science, Kanazawa University, Kanazawa,
Japan
P141
Objectives: It has been reported that sympathetic hyperactivity associates with excessive
platelet aggregation. However, the effect of reflex sympathetic excitation on platelet functions
and fibrinolytic activity in humans remain unknown. The purpose of this study was to examine
the effectby of guest reflex sympathetic on April activation 4, 2013on
muscle sympathetic nerve activity (MSNA),

hematocrit, platelet functions, coagulation and fibrinolytic activity in patients with coronary
artery disease. Methods: This study included 10 patients with coronary heart disease (mean
age 56.66.7 years). Patients with diabetes mellitus, congestive heart failure, and neurological
diseases were excluded. MSNA was measured using microneurography. Lower body negative
pressure (LBNP) was applied at -40mmHg for 30 minutes. Blood samples for measurements
of hematological parameters were obtained at baseline and the end of LBNP. Results: During
LBNP, red blood cell count, hematocrit and MSNA (from 25.63.7 to 48.23.0 bursts/min)
were significantly increased (p0.01, respectively). LBNP increased fibrinogen from
251.018.8 to 276.125.1 mg/dl (p0.01), and decreased thrombin-antithrombin complex
from 5.32.0 to 2.10.4 ng/ml (p0.063), and prothrombin fragment from 0.770.09 to
0.640.05 nmol/l (p0.066). LBNP significantly increased active plasminogen activator
inhibitor 1 (PAI-1) from 4.00.7 to 5.11.0 IU/ml (p0.05), in 6 patients. Thrombomodulin
remained unchanged during LBNP. There was significant correlation between MSNA and active
PAI-1 (r0.82, p0.01). Conclusions: These results suggest that reflex sympathetic activation
enhances platelet functions and reduces fibrinolytic activity in patients with coronary heart
disease, which may contribute to the development of thrombosis.
P142
Effects of Ovariectomy on Aggregation, ATP Secretion, and Expression of
Matrix Metalloproteinase (MMP) in Platelets of Female Pigs
Muthuvel Jayachandran, Whyte Owen, Virginia Miller. Mayo Clinic and Foundation,
Rochester, MN
Objective: Platelet activation and secretion of growth factors contribute to the vascular
responses of injury. MMP-2 stimulates platelet aggregation while MMP-9 prevents aggregation
of human platelets. Sex steroid hormones regulate matrix metalloproteinases (MMPs) in
reproductive tissues but effects on MMPs in platelets are unknown. Therefore, experiments
were designed to determine if loss of ovarian hormone affects aggregation and MMP
expression in platelets from female pigs. Methods: Blood was collected from 7-month-old,
gonadally intact (n6) and ovariectomized (for 4 weeks, n6) female pigs to determine total
blood platelet number and percent of reticulated platelets, platelet aggregation and ATP
secretion in intact platelets from platelet-rich plasma; expression of MMP-2, MMP-9, tissue
inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2 were determined in isolated platelet
lysate. Results: Numbers of circulating platelets did not change with ovariectomy but platelet
turnover (percent of reticulated platelets) increased. Platelet aggregation and ATP secretion
also increased significantly with ovariectomy. Active forms of MMP-2 and MMP-9 increased
(p0.05) significantly whereas TIMP-1 and TIMP-2 expression did not change significantly.
Conclusion: Results indicate that platelet turnover and MMP-2 and MMP-9 expression are
regulated by ovarian hormones. Increased expression of MMPs was associated with increased
platelet aggregation and ATP secretion. The MMP (MMP-2/MMP-9) systems in porcine platelets
may alter platelet-platelet and platelet-vessel wall interactions. Increased platelet aggregation
and secretion would contribute to vascular remodeling at sites of vascular injury.
Evidence for a Novel Adhesion Site on GPIIb/IIIa
P143
Ciara A Mc Manus, Steven Kerrigan, Marc Devocelle, Desmond J Fitzgerald, Dermot Cox.
Royal College of Surgeons in Ireland, Dublin, Ireland; University of California San Francisco,
San Francisco, CA
Soluble fibrinogen binding of platelets is mediated by activated GPIIb/IIIa. However, platelets
can also bind immobilised fibrinogen via an unactivated GPIIb/IIIa confirmation. A novel platelet
integrin recognition sequence - NGR was previously identified by phage display and found to
have a high affinity for the vitronectin receptor, v 3. NGR is found on both the and chains
of fibrinogen and we have previously shown that it inhibits resting platelet adhesion to
fibrinogen while vitronectin antagonists have no effect. However, NGR had no effect on either
activated platelet adhesion or on aggregation. We investigated whether resting GPIIb/IIIa could
interact with fibrinogen via a NGR dependent mechanism. 96 well plates were coated with
fibrinogen (20g/ml) or biotin-NGR (20g/ml) for two hours and blocked. Gel filtered platelets
were incubated with peptide for 30 minutes. The number of adherent platelets was determined
by acid phosphatase content. Drug binding to GPIIb/IIIa using anti GPIIIa antibodies, mAb1 and
mAb2 was quantified by flow cytometry. Platelets were incubated with biotin-NGR, chemically
crosslinked and immnuoprecipitated. GPIIb was identified in the immunoprecipitate by western
blot. Resting platelets bound immobilsed biotin-NGR which was inhibited by NGR (500M,
89%0.094) . GPIIb/IIIa antagonist, tirofiban (500nM) only partially inhibited (49%0.005,)
while inhibiting adhesion to fibrinogen (80%0.06). Tirofiban inhibits mAb2 (80%1) but not
mAb1 (15%1.8) binding to GPIIIa while NGR had no effect on either antibody (1%1.8,
3%2.8). When incubated with tirofiban and NGR mAb2 binding was 69%1.2. Biotin-NGR
showed minimal binding to COS cells. However, when transfected with GPIIb/IIIa, NGR was
shown to bind clearly to COS cells. In conclusion, platelets bind directly to NGR. This is
mediated in part by GPIIb/IIIa as platelet binding to NGR is inhibited partially by GPIIb/IIIa
antagonist and NGR crosslinks to GPIIb/IIIa. However, as NGR dos not inhibit tirofiban binding
it would suggest a non-competitive interaction. This suggests resting GPIIb/IIIa interacts with
immobilised fibrinogen via a NGR dependent binding site distinct from the small molecule
binding sites.
Poster Presentations a-25
ical relevance of these findings was supported by the facts that failing human myocardial
VLDL-R was phosphorylated to significant level compared to control tissues. Further study on
the role of phosphorylated VLDL-R in diabetes-induced cardiomyopathy in the myocardium of
streptozotocin induced diabetic rats has indicated significantly greater levels of phosphorylated
VLDL-R in diabetic rats than normal controls. In order to elucidate the role of VLDL-R
phosphorylation in diabetes induced cardiomyopathy and also to characterize the cells within
heart tissue involved in the phosphorylation process, cardiomyocytes were isolated from normal
mouse heart tissue using collagenase treatment. The cardiomyocytes phenotype was
confirmed by the presence of myocardial actin and the absence of smooth muscle cell actin
by immunocytochemical staining. The cultured cardiomyocytes were treated with either 0.15
M PMA for 2 hours or 16.5 mM glucose for 6 hours in the presence or absence of 30 nM of
PK-C ****223’3f NEEDS TO BE ADDED TO TIMES NEW ROMAN GREEK FONT****II inhibitor,
LY379196. The relative ligand binding activity of the VLDL-R in the cell extracts as assessed
by RAP-ligand blotting indicated a diminished ligand binding and this effect was blocked by
PK-C II inhibitor. Furthermore, PK-C II was translocated to membrane following PMA or
glucose treatment and this effect was blocked by PK-C inhibitor. These observations suggest
that in hyperglycemic condition cardiomyocyte VLDL-R phosphorylated through PK-C II
dependent phosphorylation pathway and leads to decreased VLDL-R function. These studies
further support our hypothesis that the PK-C II dependent phosphorylation of VLDL-R may play
a role in diabetes-induced cardiomyopathy.
P145
High Dose is More Effective than Low Dose of Atorvastatin in the Removal
from Plasma of Chylomicrons in Hypercholesterolemic Subjects: Study with
Artificial Emulsions
Raul D Santos, Andrei Sposito, Rosangela Amancio, Jose A Ramires, Raul Maranhao. Heart
Institute University of Sao Paulo Medical School, Sao Paulo, Brazil
Chylomicrons (CM) and their remnants that are removed from plasma by the LDL receptor
pathway have been implicated in atherogenesis. The objective of this study was to evaluate the
effects of low and high doses of atorvastatin (ATORVA) upon the plasma kinetics of a CM-like
emulsion in primary hypercholesterolemia. We studied 42 subjects (age 30 –76 years) with
average LDL-C of 170 mg/dl and triglycerides (TG) of 200 mg/dl. Patients were randomized to
a 6-week treatment period with placebo (n15), ATORVA 10 mg (n 17) or ATORVA 40 mg
(n 13). The CM-like emulsion labeled with 14C-cholesteryl oleate (14C-CO) and 3H-triolein
(3H-TO) was injected after a 12 hour fast and blood samples were collected during 60 minutes
in order to determine the radioactive decay curve. The kinetics of 14C-CO and 3H-TO
respectively evaluate emulsion remnant removal and peeling of TG fatty acids from the
emulsion. The radioisotopes residence times (RT) in minutes as well as the lipolysis index (LI)
in % were calculated. Results: The higher the ATORVA dose greater were the effects on plasma
lipids and on kinetic parameters (table 1). Conclusions:ATORVA increased the removal from
plasma of the CM-like emulsions in a dose dependent maner.The reduction in the LI with
ATORVA treatment suggests that CM-like emulsions were removed with little lipolysis. This may
have antiatherogenic implications.
P146
Arterial Compliance: A Diagnostic Marker for Cardiovascular Diseases?
Bonni Syeda, Michael Gottsauner-Wolf, Stefan Denk, Phillip Pichler, Dietmar Glogar.
University of Vienna, Vienna, Austria
Background: Previous studies have shown atherogenesis to be related with increased vessel
stiffness. Measures of the arterial compliance can be performed non-invasively from pressure
pulse contour analysis of arterial waveforms. This prospective study aims to analyze to what
extent vessel compliance can reflect the angiographic coronary artery status. Methods: Large
and small artery elasticity indices (LAEI and SAEI respectively, in ml/mmHgx10) were measured
in 151 patients on the radial artery with the HDI device (PulseWave, CR-2000, Eagan, USA). All
patients were classified into diffuse-CAD (coronary artery disease) (defined as stenosis
length15mm), focal-CAD (defined as stenosis length between 1 and 15mm) or no-CAD.
P144 Findings: Both LAEI and SAEI were reduced in the diabetic group (LAEI: 11.22.9 versus
The Ligand Binding Activity of the Very Low-Density Lipoprotein Receptor 13.44.5, p0.006; SAEI: 3.71.6 versus 4.72.4, p0.01). Inverse association was seen
(VLDL-R) in Isolated Cardiomyocytes is Regulated through Glucose-Induced between age and LAEI (r -0.41; p0.001) and SAEI (r -0.38; p0.001). No-CAD was found
PK-C II Dependent Phosphorylation
in 31 patients, focal-CAD in 64 patients and diffuse-CAD in 56 patients. Mean LAEI were
13.83.5, 13.74.7 and 11.33.5 in the groups no-CAD, focal-CAD and diffuse-CAD
Ramasamy Sakthivel, Timothy Kern, Nicholas Ziats, Kenneth Margulies, Keith McCrae. Case respectively (p0.004), (no-CAD versus diff use-CAD: p0.04; focal-CAD versus diffuse-CAD:
Western Reserve University, Cleveland, OH; Temple University, Philadelphia, PA
p0.009). Respective SAEI values were 5.62.5, 5.02.1 and 3.11.6 (p0.001), (no-CAD
versus diffuse-CAD: p0.001; focal-CAD versus diffuse-CAD: p0.001). Multivariate analysis
Our previous studies on the regulation of human VLDL-R demonstrated that the ligand binding r evealed SAEI (p0001), hypercholesterolemia (p0.005) and male gender (p0.001) to be
affinity of VLDL-R is regulated through PK-C dependent Downloaded phosphorylation. from
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pathophysiolog- diagnosticby markers guest ofon theApril type of4, vessel 2013 disease. Interpretation: SAEI was found to be an

a-26 Arterioscler Thromb Vasc Biol. Vol 22, No 5
independent marker of diffuse coronary disease. Thus compliance measurements may be used
for early identification of patients with diffuse atherosclerotic processes of the coronary
arteries.
Granzyme B Protein is Present in Advanced Atherosclerotic Plaques
Jonathan C Choy, Paul C McDonald, Brian W Wong, Janet E Wilson, Bruce M McManus.
McDonald Research Laboratories / The iCAPTURE Centre, St. Paul’s Hospital / Providence
Health Care - University of British Columbia, Vancouver, BC, Canada
P147
Background: Increased apoptosis of smooth muscle cells (SMC) may decrease the stability of
atherosclerotic plaques by reducing the synthesis of certain extracellular matrix components.
Studies have suggested that FasL may contribute to SMC apoptosis in atherosclerotic plaques.
However, the role of granzyme B and the related arm of the cytotoxic immune response in
modulating apoptosis and plaque rupture have not been examined. Thus, to initiate studies on
the granzyme B pathway, we used immunohistochemistry to establish the presence and locale
of granzyme B protein in atherosclerotic arteries. Methods: Formalin-fixed, paraffin-embedded
human coronary arteries from patients with mild and advanced atherosclerosis were
immunohistochemically stained for granzyme B and smooth muscle alpha-actin. Granzyme B
staining was scored semi-quantitatively on a 1–5/5 scale. Terminal deoxynucleotide
transferase dUTP nick end labeling (TUNEL) was utilized, in conjunction with morphological
analysis, to identify apoptotic cells, and was quantitated using Image Pro Plus. Statistical
evaluation included a non-parametric t-test. Results: Arteries with advanced atherosclerosis
had significantly more TUNEL-positive cells than those with mild disease (p0.02). Granzyme
B immunoreactivity was largely absent in arteries with early atherosclerosis, but was prominent
in the intima and media of arteries with advanced atherosclerosis (p0.02). In the intima,
staining of granzyme B was localized mainly to lipid-rich regions around the atheromatous core
and stained intracellularly in foam cells. Serial sections stained for smooth muscle alpha-actin
suggested that granzyme B-positive foam cells were largely of smooth muscle cell origin. Also,
granzyme B localized to smooth muscle cells in the media of vessels with advanced disease.
Staining of serial sections with granzyme B and TUNEL indicated that many granzyme
B-positive cells in the intima and media were TUNEL-positive. Conclusion: Granzyme B
localization to apoptotic cells is associated with increased severity of atherosclerosis,
suggesting that granzyme B-mediated apoptosis contributes to plaque rupture and vascular
remodeling.
P148
Small, Dense LDL (sdLDL) is a Common Characteristic for the 3 Major
Lipoprotein Phenotypes of FCHL
Amir F Ayyobi, Sandra H McGladdery, Marguerite J McNeely, Melissa A Austin, John D
Brunzell. University of Washington, Seattle, WA
Hypertriglyceridemia (HTG) may lead to the presence of sdLDL. However, treatment of HTG with
gemfibrozil in FCHL has been shown to reduce peak LDL density, with no effect on sdLDL mass.
Lipoprotein (LP) fractions in FCHL have been characterized, but not the LP cholesterol
distribution among FCHL phenotypes. Affected subjects were identified from previously
described families and were segregated based on plasma TC and TG into IIA (n14), IIB
(n19), and IV (n29). The LP cholesterol distribution was determined over 38 fractions
obtained by density gradient ultracentrifugation. As expected, FCHL patients with HTG (IIB and
IV) (A and B) had higher cholesterol levels in VLDL than IIA, while IIA showed higher cholesterol
in the big, buoyant LDL (bbLDL)(fractions 10–20) and HDL. LDL-C was higher in IIB than IV (C).
Notably, the majority of the increase in LDL-C was associated with bbLDL rather than sdLDL
(fractions 6–9)(A to C). The change in LPs between phenotypes was due to VLDL and bbLDL.
Mass of sdLDL cholesterol for each group was IIA 5319, IIB 5914, IV 4714 (mg/dl).
Although the differences in LP profile were variable due to differences in LP phenotype, sdLDL
remains one of the most prominent characteristics in common for the three different LP
phenotypes.
P149
SR-BI and CD36 Mediate the Uptake of OxLDL through Distinct Pathways
Agnès Boullier, Felicidad Almazan, Yury Miller, Simone Green, Oswald Quehenberger.
University of California, San Diego, La Jolla, CA
Receptor-mediated endocytosis of oxidized LDL is believed to be a key event in the lipid-loading
of macrophages and accumulation of foam cells in the arterial wall, the hallmark of
atherosclerotic lesions. Macrophages express several scavenger receptors that mediate the
interaction with modified forms of LDL. We have recently shown that both SR-BI and CD36 bind
OxLDL with high affinity. We now present evidence indicating that these two receptors take up
OxLDL via different mechanisms. Although, both SR-BI and CD36 ectopically expressed bound
OxLDL, only SR-BI transfected cells produced proteolytic degradation products. This came as
a surprise since CD36 is clearly involved in the uptake and degradation of OxLDL by
macrophages. Lipid degradation products of OxLDL are known to activate the nuclear hormone
receptor PPAR.Therefore, to determine whether CD36 is sufficient for internalization but
delivers OxLDL to cytoplasmic sites with restricted Downloaded access for degradation, from
we employed a
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reporter system consisting of transfected cells expressing either CD36 or SR-BI and the
PPARresponse element. Unlike the SR-BI transfected cells, the CD36 transfected cells did not
mediate internalization and degradation, evidenced by the absence of a reporter signal. To
further examine whether CD36 can internalize but not degrade OxLDL, we incubated
transfected cells with OxLDL and analyzed for cytoplasmic lipid inclusions. This treatment
resulted in intracellular lipid droplets in SR-BI but not in CD36 transfected cells. We previously
demonstrated that SR-BI does not mediate selective uptake of lipids from OxLDL. To further
rule out this possibility, we stained the transfected cells with EO6 and MB47, two antibodies
that recognize oxidized phospholipids and apoB, respectively. Incubation of SR-BI transfected
cells with OxLDL resulted in an intracellular staining that was similar for both antibodies and
indicated uptake of the whole particle. Taken together these results suggest that CD36 and
SR-BI mediate the uptake of OxLDL by different mechanisms. We propose that for function as
an internalizing receptor the formation of a complex between CD36 and other membrane
component(s) may be necessary
P150
Histaminemia, Depleted Ascorbate, and Oxidative Stress Predispose to
Acute Coronary Syndrome
Sanjeev Hasabnis, Sanda Clejan, Shankar Japa, James Talano. Tulane University Health
Sciences Center, New Orleans, LA
BACKGROUND: Mast cells are prevalent in the shoulder of unstable atheromas; cardiac mast
cells secrete proteases capable of activating matrix metalloproteinases. Histamine is essential
in the inflammatory cascade of the unstable plaque. Ascorbate depletion has been correlated
with histaminemia which impairs endothelial-dependent vasodilation. This study evaluates
whether oxidative stress as measured by isoprostanes (PGF2-) coupled with an inflammatory
state characterized by histaminemia predisposes patients to acute coronary syndrome (ACS).
METHODS: Whole blood histamine, serum ascorbate, and serum PGF2- levels were drawn on
48 patients with ACS as determined by standard diagnostic criteria, 46 patients with stable
coronary artery disease (CAD), and 50 age and sex matched normal subjects (NML). Samples
were taken at 8:00 am in the fasting state in the CAD and NML groups and within 24 hours
of presentation to the emergency room in the ACS group. RESULTS: See table 1 below.
STATISTICAL ANALYSIS: Data were analyzed by stepwise discriminant and Spearman’s Rank
correlation coefficient. A significant relationship exists between histamine and PGF2-. As
PGF2- rises above 60 pg/mL, an exponential increase in histamine occurs in both ACS and
CAD groups. A significant inverse relationship exists between ascorbate and histamine in the
ACS versus NML groups *(P 0.05) and the CAD versus NML groups #(P 0.05).
CONCLUSION: Histamine and isoprostane levels increase in CAD and ACS patients. Mast cell
activation and lipid oxidation generated during atherosclerosis manifest this inflammatory
response. Accelerated isoprostane formation and depleted ascorbate paired with histaminemia
may be active in CAD and predispose patients to acute coronary syndrome.
P151
Identification of Anti-GP IIb-IIIa Antibodies in Individuals Receiving an Oral
and/or Intravenous Platelet GP IIb-IIIa Antagonist
Lisa K Jennings, Steven M Slack, Lou Ann Keith, Mary V Jacoski, Sophie Combe. University
of Tennessee, Memphis, TN; University of Memphis, Memphis, TN; Aventis Pharmaceuticals
Inc, Bridgewater, NJ
Objectives: This objective of this study was to develop an ELISA assay to identify anti-GP IIb-IIIa
antibodies in the plasma collected from individuals participating in Phase II clinical trials of a
novel oral and intravenous (IV) platelet GP IIb-IIIa antagonist, RPR109891. Plasma from
individuals exhibiting significant reductions in platelet counts following receipt of drug were
compared with those who did not. Methods: Purified human platelet GP IIb-IIIa in the absence
or presence of RPR109891 or its active metabolite, RPR109039, was adsorbed to the wells of
microtiter plate. Plasma samples were added to buffer with or without the platelet antagonist.
After a 2 hour incubation, a secondary HRP-conjugated goat anti-human antibody was added
to each well. Color was developed and the optical densities (ODs) were measured at 405 nm.
Statistical Analysis: The normality of the experimental data was verified using the Kolmogorov-
Smirnov testing procedure. A single-factor ANOVA was used to compare results from the
various treatments. Significant differences (p 0.01) were identified among the treatments
using the Student-Neuman-Keuls multiple comparison procedure. Findings: A total of 80
plasma samples from individuals receiving the oral formulation of RP R109891 were tested. Of
these, 14 (17.6%) exhibited ODs greater than one standard deviation (SD) above the average
normal OD and 7 (8.8%) exhibited ODs greater than two SDs above the average normal OD.
In the study evaluating the IV formulation of t he antagonist, 11 patient plasma samples were
tested and none was positive for anti-GPIIb-IIIa antibodies. In the study in which individuals
received both the oral and IV drug formulations, 34 samples were tested and 6 (17.7%)
exhibited ODs greater than one SD above the average normal OD and 1 (2.9%) exhibited an OD
greater than two SDs above the average normal OD. Conclusion: The ELISA technique
developed in this study successfully identified anti-GP IIb-IIIa antibodies in plasma from
individuals who received a platelet antagonist and who exhibited severe reductions in platelet
counts. by guest on April 4, 2013

P152 WITHDRAWN
Oxidized Phospholipids Inhibit Angiogenesis In Vitro but Stimulate
Angiogenesis In Vivo: Potential Influence of Monocyte/Macrophages
Karol E Watson, Leslie A Caromile. UCLA School of Medicine, Los Angeles, CA
P153
Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) are a group of
oxidized phospholipids which have been shown to be the active compounds in minimally
oxidized LDL (MM-LDL). Emerging evidence suggests that lipoproteins may modulate
angiogenesis, we therefore tested the ability of Ox-PAPC to affect parameters of angiogenesis.
In our studies, Ox-PAPC inhibited in vitro proliferation, migration and tube formation of
endothelial cells. Adding exogenous recombinant VEGF at 100ng/ml restored EC proliferation
and migration to baseline. To confirm inhibition of angiogenesis by Ox-PAPC we performed an
in vivo angiogenesis assay. Surprisingly, Ox-PAPC stimulated angiogenesis in vivo (figure 1).
Further studies in vitro demonstrated that Ox-PAPC also stimulated VEGF production by
monocyte/macrophages. We conclude that Ox-PAPC directly inhibits angiogenesis in vitro, but
that in vivo this is overcome by the stimulation of VEGF produced by monocyte/macrophages.
These results may explain the co-localization of both atherosclerosis and angiogenesis in vivo
and form the basis for neovascularization of atherosclerotic plaques.
P154
Regulation of Class A Scavenger Receptor-Mediated Cell Adhesion by Gi/o Signaling Pathways
Steven R Post, Cecelia Gass, Stuart Rice, Dejan Nikolic, Reto Asmis. University of Kentucky,
Lexington, KY
Class A macrophage scavenger receptors (SRA) are multifunctional receptors with roles in
modified lipoprotein uptake and cell adhesion. Although not typically associated with a
signaling function, incubation of macrophages with acetylated LDL (AcLDL) was shown to
activate several intracellular signaling cascades. Activation of these signaling cascades by
AcLDL was inhibited by pertussis toxin (PTX) indicating that SRA activates a G i/o protein.
Recently, we reported that in mouse peritoneal macrophages (MPM), G i/o activation positively
regulated SRA-mediated lipoprotein uptake. The finding that SRA-mediated lipoprotein uptake
was PTX-sensitive led us to hypothesize that SRA-mediated cell adhesion might be similarly
regulated. Because macrophages express multiple classes of scavenger receptors, we
expressed SRA in HEK cells, cells that lack endogenous SRA expression. Similar to our results
using MPM, we found that SRA-mediated AcLDL uptake by transfected HEK cells was reduced
by 35% following PTX treatment. Using a standard adhesion assay in the presence of serum,
we found that cells expressing SRA displayed enhanced cell adhesion which was reduced by
42% following PTX treatment. The ability of the SRA antagonist polyinosine to reduce cell
adhesion to the level observed with HEK cells lacking SRA confirmed that the increased cell
adhesion was mediated by SRA. To rule out the potential involvement of integrins, adherant
cells were incubated in Ca 2 -free media. Under these conditions, 72% of cells that expressed
SRA remained attached, whereas only 4% of the cells that lacked SRA remained attached.
However, following PTX treatment only 36% of SRA expressing cells remained attached.
Because the actin cytoskeleton plays an active role in cell adhesion, we used phalloidin to stain
F-actin in HEK cells. We found that cells expressing SRA developed a phenotype characterized
by fine filopodial extensions; a phenotype similar to that observed in primary macrophages and
in cells transfected with activated Cdc42. Overall, our results demonstrate that the ability of
SRA to enhance cell adhesion is PTX-sensitive and correlates with changes in the actin
cytoskeleton.
P155
Pulse Pressure: Is it an Age-Independent Predictor of Mortality in Patients
with Angiographic Coronary Disease?
Keshav Chander, James S Zebrack, Tami L Blair, Robert R Pearson, Joseph B Muhlestein,
Benjamin D Horne, Jeffrey L Anderson. University of Utah, Salt Lake City, UT; LDS Hospital,
Salt Lake City, UT
Objective: Wide pulse pressure (PP) has been proposed as a stronger predictor of mortality than
systolic (SP) or diastolic blood pressure (DP). PP is known to widen with age; and its prognostic
value independent of age is uncertain. We evaluated its predictive value for mortality; alone,
and together with age and other standard riskDownloaded factors in a large, from
prospectively followed
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Poster Presentations a-27
population with documented coronary artery disease (CAD). Methods: We prospectively studied
1370 consecutive patients with angiographically defined CAD (1 stenosis 70%). Baseline
pulse pressure together with other standard risk factors was recorded. During 2.8Â1.2 years
of follow-up, 173 patients died (12.6%). Predictors of mortality were assessed using Cox
regression analyses. Findings: In univariate analysis, PP was a highly significant predictor of
mortality (relative hazard [RH]1.10, 95% CI1.03 - 1.18 / 10 mmHg,p0.005). In
multivariate analysis with SP and DP, the predictive value of PP improved (RH1.29 [1.11 -
1.50] / 10 mmHg, p0.0008). PP correlated moderately with age (Pearson†s correlation
coefficient 0.381, p.001). When forced into bivariate survival analysis, age (RH1.87 [1.56
- 2.24] / decade, p.0001), but not PP (RH0.99[0.92 - 1.07]) was significantly predictive of
mortality. Multivariate analysis (stepwise, backward conditional) including PP with the standard
risk factors of age, sex, diabetes, history of [h/o] hyperlipidemia, h/o hypertension, h/o
smoking, and family history was performed. The age(p.0001), h/o hyperlipidemia (p.0006),
and diabetes (p.002), but not PP came out as independent predictors. Conclusion: PP is a
strong univariate predictor of mortality in patients with CAD. However, it was not found to be
an independent predictor when adjusted for age in our population. Thus, the independent
predictive value of PP remains to be established for secondary risk assessment.
P156
Oxidized Phospholipids Inhibit Inflammatory Effects of Endotoxin Both In
Vitro and In Vivo
Valery N Bochkov, Alexandra Kadl, Joakim Huber, Florian Gruber, Bernd R Binder, Norbert
Leitinger. Inst. of Vascular Biology and Thrombosis Research, Univ. of Vienna, Vienna,
Austria
Minimally modified LDL and their active components - oxidized phospholipids - are known to
regulate expression of inflammatory genes in endothelial cells (EC). Oxidized 1-palmitoyl-2arachidonoyl-
sn-glycero-3-phosphorylcholine (OxPAPC) is known to up-regulate tissue factor
and interleukin-8 in human EC, but on the other hand to suppress induction of E-selectin after
stimulation of EC by LPS or TNF. In this work, we have studied mechanisms and in vivo
relevance of this inhibitory action. We have found that OxPAPC only partially inhibited elevation
of E-selectin levels in TNF or IL-1-stimulated human umbilical vein endothelial cells. In
contrast, OxPAPC completely blocked LPS-induced up-regulation of E-selectin, ICAM-1 and
VCAM-1. The inhibitory effect of OxPAPC could not be overcome by raising LPS concentration
100-fold above saturation, suggesting that the mechanism of inhibition is more complex than
competition between OxPAPC and LPS for the LPS receptor(s). The anti-endotoxin effect of
OxPAPC was not mimicked by unoxidized PAPC, lysoPC or arachidonic acid. OxPAPC inhibited
activation of the NFB pathway, namely LPS-induced phosphorylation and degradation of IB,
activation of p65/DNA binding and stimulation of 5xNFB-luciferase reporter construct. In
C57BL/6 mice injected intraperitoneally with LPS, simultaneous injection of OxPAPC inhibited
accumulation of blood-borne cells in the peritoneal cavity, expression of E-selectin mRNA in
peritoneal tissue, heart and aorta, and blocked oedema formation in peritoneal tissue. Finally,
we have found that OxPAPC significantly reduced lethality in mice injected with high doses of
LPS. We hypothesize that oxidized phospholipids accumulating at the sites of acute bacterial
inflammation due to high concentrations of reactive oxygen species generated by neutrophils,
can function as a negative feedback to down-regulate the process of acute inflammation. Thus,
oxidized phospholipids represent a novel class of compounds with anti-endotoxin activity which
potentially can be used for treatment of Gram-negative sepsis.
P157
Heterodimerization of the and Isoforms of the Human Thromboxane
Receptor
Paul Sullivan, Emer M Smyth. University of Pennsylvania, Philadelphia, PA
Two splice variants of the human thromboxane receptor (TP) have been identified. TP and
TP are 89% homologous, have similar ligand binding and signaling properties but
demonstrate divergent patterns of sequestration. Heterodimerization of highly homologous G
protein-coupled receptors has been reported, but has not been tested for TP/TP. HEK 293
cells were stably transfected with hemagglutinin (HA) tagged TP (HEK-TP), myc tagged TP
(HEK-TP) or both (HEK-TP/), and heterodimerization examined. Protein complexes were
cross-linked (2mM DSP, 20 min), cell membranes prepared and solubilized in 2% digitonin and
TP immunoprecipitated with an anti-Myc antibody. Immunoprecipitates were resolved by
SDS-PAGE and HA-TP revealed with an HRP-anti-HA antibody. HATP was present in
anti-Myc immunoprecipitates from HEK-TP/ , but not HEK-TP, demonstrating that
co-expression of TP with TP led to the formation of TP/ heterodimers. TP signaling was
examined by measurement of inositol phosphate (InsP) generation. IBOP, a TP agonist,
stimulated InsP generation in all three cell lines (Table 1). The isoprostane iPF2-III, a free
radical catalyzed by guest product on April of arachidonic 4, 2013 acid present in atherosclerotic plaque, mediates

a-28 Arterioscler Thromb Vasc Biol. Vol 22, No 5
TP-dependent cardiovascular effects in vivo but binds to the recombinant TP isoforms with
extremely low affinity. InsP generation stimulated by iPF 2-III was greatly increased in
HEK-TP/ compared to HEK-TP or HEK-TP (Table 1). These data support the formation of
TP/ heterodimers facilitating TP-mediated ligation of iPF 2-III.
P158
Multiple Inositol Polyphosphate Phosphatase Is an Important Regulator in
Vascular Smooth Muscle Cells and Suppresses Neointimal Hyperplasia
after Endothelial Denudation
Nicole R Ross, James Bruns, Michelle Zeleny, David G Kuhel, David Y Hui. University of
Cincinnati, Cincinnati, OH
Multiple inositol polyphosphate phosphatase (MIPP) is an enzyme that hydrolyzes inositol
polyphosphates such as InsP 4, InsP 5, and InsP 6. This enzyme is expressed in a multitude of
tissues including liver, kidney, and brain. In view of previous studies implicating InsP 4, InsP 5,
and InsP 6 in signaling pathways that are activated during cell migration and proliferation, this
study was undertaken to explore the potential involvement of MIPP in vascular response to
arterial injury. We compared the expression of MIPP in carotid arteries of a mouse strain
(C57BL/6) that has minimal neointimal hyperplasia after endothelial denudation with one
(C57L/J) that develops massive neointima after arterial injury. Semi-quantitative RT-PCR of RNA
isolated from the injured arteries of these animals showed a 2.5 fold higher expression of MIPP
in C57BL/6, the resistant strain, in comparison with that in the neointima-prominent C57L/J
mice. In situ RT-PCR hybridization identified medial smooth muscle cells as the cell type in the
arteries expressing MIPP. The role of MIPP in limiting smooth muscle cell response to
injury-induced migration and proliferation was confirmed by in vitro experiments demonstrating
a higher expression of MIPP in quiescent smooth muscle cells in comparison with cells
stimulated with PDGF. Taken together, these results suggest that MIPP is an important
regulator of neointimal hyperplasia after endothelial denudation by inhibiting intracellular
signaling events that lead to smooth muscle cell migration and proliferation.
P159 WITHDRAWN
P160
Altered Vascular Smooth Muscle Cell (VSMC) Sensitivity to the Proliferative
Effects of IGF-1 and Nitric Oxide (NO) in Young Spontaneously
Hypertensive Rats (SHR)
Brian P Nolan, Sadaf Waqar, Patti Senechal, Cynthia A Standley, Paul R Standley.
Midwestern University, Glendale, AZ
Vascular medial thickening, a hallmark of hypertension and restenosis, is associated with VSMC
hypertrophy and hyperplasia. Although the precise mechanisms responsible for these
associations are elusive and multi-faceted, we have shown that dynamic strain-induced
regulation of autocrine IGF-1 and NO reciprocally modulate VSMC proliferation in vitro.
Therefore, we investigated potential IGF-1 and NO abnormalities in young (10 wk) SHR and WKY
animals and their respective VSMC ex vivo. SHR had increased mean arterial pressures (1732
vs. 1283 mmHg, n24, p0.05), but similar pulse pressures (312 vs. 303 mmHg;
p0.05) vs. WKY rats. SHR also exhibited increased aortic wall thickness vs. WKY: 52316
vs. 35517m; p0.05. No significant differences were seen in plasma NO x
(WKY0.480.11 vs. SHR0.580.18 M) or plasma IGF-1 (WKY100728 vs.
SHR95326 ng/ml). Early passage aortic VSMC from SHR displayed enhanced basal
proliferation vs. WKY as shown by a decreased half-time to confluence: 392 vs. 603 hours,
p0.05. Underlying this enhanced proliferation was decreased SHR VSMC sensitivity to the
antiproliferative NO donor DETA-NO (ID 50: SHR 27020 M; WKY 15011 M;
p0.05). SHR VSMC were not sensitive to exogenous IGF-1 proliferative effects, while WKY
VSMC exhibited a dose-dependent increase in proliferation with IGF-1 (10 -10 to 10 -7 M). Ex vivo
VSMC secretions of IGF-1, NO x, and cGMP under normal culture conditions are shown in the
table. Together, these data suggest that VSMC hypertrophy / hyperplasia in early hypertension
is not reflected by imbalances in plasma IGF-1 / NO nor by altered VSMC secretion of these two
hormones. Rather, reduced SHR VSMC sensitivity to NO is responsible, in part, for the observed
hyperproliferation seen in early stages of hypertension. This reduced NO sensitivity appears to
outweigh depressed SHR sensitivity to the proliferative effects of IGF-1.
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P161
Chlamydia pneumoniae (Cpn) Induces Hypercoagulability in Patients with
Symptomatic Carotid Artery Disease
Tryfon Vainas, Harrie Kurvers, Werner Mess, Rick De Graaf, Rajaa Ezzahiri, Mat Daemen,
Cathrien Bruggeman, Peter Kitslaar. Maastricht University Hospital, Maastricht, Netherlands
Background and Purpose - Transcranial Doppler monitoring of the ipsilateral middle cerebral
artery during carotid endarterectomy offers the opportunity to study plaque instability and
hypercoagulation in-vivo, through registration of, (i) atherothrombotic emboli dislodging from an
unstable carotid plaque before removal of the plaque, (plaque-related emboli, PE), and (ii)
emboli related to thrombus-formation at the endarterectomy site after removal of the plaque
and restoration of flow (thrombosis-related emboli, TE). Our study was designed to clarify
whether infection with Cpn as shown by the presence of Cpn IgA antibodies is correlated with
plaque instability and/or hypercoagulability in patients with carotid artery disease. Methods -
Cpn IgA-antibodies were assessed in 53 patients with symptomatic carotid artery disease
undergoing carotid endarterectomy. The removed carotid plaques were studied microscopically
to assess histological signs of plaque instability, i.e., fibrous cap rupture and/or luminal
thrombosis. PE and TE were registered in 43 patients. Data were analyzed with Fisher’s exact
test. Results - Histological plaque instability (43%) correlated with the occurrence of PE (23%,
p0.003) but not TE (26%). IgA seropositivity (57%) was significantly associated with TE
(p0.014), but not with PE nor with histological plaque instability. Conclusions - Active
infection with Cpn as shown by the presence of Cpn IgA-antibodies is associated with
embolization after removal of carotid plaque and restoration of flow. Since these micro-emboli
represent platelet aggregations and are related to cerebrovascular complications, our data
suggest that Cpn infection contributes to cerebrovascular events through induction of
hypercoagulability.
P162
A Single Injection with Cytomegalovirus Antigens Is Sufficient to Increase
Atherosclerotic Lesion Size and T- Cell Influx in ApoE -/- Mice
Inge Vliegen, Frank Stassen, Gert Grauls, Cathrien Bruggeman. University Maastricht, the
Netherlands, Maastricht, Netherlands
Previous studies documented that cytomegalovirus (CMV) infection aggravates the atherosclerotic
process. Here we studied whether multiple infections further exacerbate the atherosclerotic
outcome and whether this effect is dependent on the viability of the virus. Methods: Eight
week old apoE -/- mice (8/group) were injected once with 5x10 4 pfu MCMV or UV-MCMV. In
a separate group, mice were injected once every month during 7 months. Two weeks after the
last injection mice were sacrificed. Control mice were sacrificed at similar time points. Salivary
gland, liver, lungs and aortic arch were collected and imbedded in paraffin. Atherosclerotic
lesion size and xanthomas on top of and adjacent to more mature lesions were determined on
hematoxylin-eosin stained aortic arch longitudinal sections. T-cell influx was determined in all
tissues. Differences were considered statisticaly significant if P0.05 (1-way ANOVA). Results:
A single MCMV infection resulted in a 2.3-fold increase in atherosclerotic lesion size and a
significant increase in T-cell number. Also, a significant increase in T-cell influx was observed
in internal organs. UV-MCMV injection also increased lesion size and T-cell number. In the
internal organs T-cell number was not altered. Following 7 monthly infections the number of
xanthomas was significantly enhanced only in MCMV-infected mice while lesion sizes were not
different between groups. At this time point T-cell numbers were normalised to control levels
in all tissues from both groups. Conclusions: Here we demonstrate that in addition to viable
MCMV, UV-MCMV also exacerbates the atherosclerotic process. This may result from a local
stimulation of the already ongoing inflammation in the atherosclerotic plaque since no
increased T-cell influx was observed in other internal organs in the UV-MCMV group. However,
after recurrent infections only viable MCMV affected atherosclerotic lesions by increasing the
number of xanthomas. This may be the result of a more efficient clearance of UV-MCMV by the
immune system when compared with MCMV, since viable MCMV possesses numerous immune
escape mechanisms.
Cox-2 Dependent Prostacyclin Formation is Regulated by Low-Density
Lipoprotein Cholesterol In Vitro
Layton H Smith, Olivier Boutaud, Matthew Breyer, Jason D Morrow, John A Oates, Douglas
E Vaughan. Vanderbilt University Medical Center, Nashville, TN
P163
The reduction of plasma low-density lipoprotein is associated with reduced risk of myocardial
infarction, stroke, and death. Some of this clinical benefit may be derived from an improvement
in endothelial-dependent vasodilation. In the present study, we examined the effects of LDL
reduction on cyclooxygenase activity and prostacyclin (PGI2) production. Human umbilical vein
endothelial cells (HUVECs) exposed to reduced concentrations of LDL demonstrated increased
prostacyclin production in a dose-dependent manner (0.750.2ng/ml to 2.60.2 ng/ml;
p0.0001). This alteration in PGI2 production did not result from LDL-induced changes in PGI2 synthase activity. However, selective inhibition of cyclooxygenase-2 (COX-2), but not COX-1,
blocked PGI2 production under low cholesterol conditions. Addition of exogenous cholesterol
induces dose-dependent reductions in endothelial COX-2 expression as measured by RT-PCR
and by western blotting. Pre-treatment of cells with actinomycin-D reduced COX-2 derived PGI2 production by 45.9% (0.550.09 to 0.250.08 ng/mL). Taken together, these observations
indicate that endothelial PGI2 production is regulated by cholesterol at the transcriptional level,
and that cholesterol-sensitive transcriptional pathways regulate COX-2 expression in vascular
tissues by guest on April 4, 2013

P164
Pulsatile Shear Stress Regulates Inflammatory Responses in the Arterial
Bifurcations
Tzung K Hsiai, Mohamad Navab, Srinuvasa Reddy, Linda L Demer. UCLA School of
Medicine, Los Angeles, CA
Introduction: The focal nature of the atherosclerotic lesions demonstrates the importance of
hemodynamics, specifically, shear stress, in regulating the biological activities of endothelial
cells(EC). By virtue of high spatial and temporal resolution, micro shear stress sensors offer an
entry point to investigate the mechanisms by which unidirectional pulsatile flow downregulates
the inflammatory responses, whereas oscillating flow with a zero mean shear stress
upregulates MCP-1 and, thus, monocytes binding to EC in the arterial bifurcations. Methods:
A pulsatile flow channel with parallel plate in the testing section was designed to deliver arterial
flows representing known vascular conditions in the bifurcations. Micro shear stress sensors,
comparable to an elongated EC, were fabricated by nanotechnology and microfabrication.
These sensors were embedded in the upper wall of parallel plate to measure shear stess real
time at a frequency response of 1 kHz. Bovine aortic endothelial cells (BAECs), which form a
confluent monolayer on the bottom of the parallel plate, were exposed to three flow conditions
at 60 cycles/min: 1)pulsatile flow with high shear stress temporal gradient(/t293
dyn/cm 2 sec), with mean shear stess 50 dyne/cm 2 ; 2)low/t71, with identical mean
shear stress; and 3) reversing oscillating flow at 0 /- 5 dynes/cm 2 . After 4 hours of flow
exposure,RT-PCR and quantification were performed for MCP-1 mRNA expression. Monocyte
adhesion assay was also performed. Results: Pulsatile flow at high /t downregulated
MCP-1 expression by 33/-8%, and low/t downregulated by 15/-4%, but oscillating
flow upregulated MCP-1 by 13/-5%. High/t reduced monocyte binding to BAEC by
64/-3% compared with static condition, and low/tby31/-3%, whereas oscillating flow
increased monocyte binding by 22/- 2%. We hereby provide the first evidence linking direct
shear stress measurement with inflammatory responses via MCP-1 mRNA expression and
monocyte binding to ECs.
P165
Gene Expression During Atherogenesis in ApoE-/- Mice: A Micro-Array
Based Study
Esther Lutgens, Birgit Faber, Chris Evelo, Gordon Porter, Mat Daemen, Kitty Cleutjens.
University of Maastricht, Maastricht, Netherlands; Incyte Inc, Palo Alto, CA
To identify the regulation of known and new pathways during atherosclerosis, we performed
large scale expression profiling on RNA isolated from aortic arches of ApoE-/- mice and C57Bl6
mice fed a normal chow diet or Western type diet for 3, 4.5 and 6 months. mRNA was amplified
and hybridized in duplicate to a cDNA chip reporting 9000 genes (Mouse Unigene 1, Incyte
genomics Inc.) and compared to the co-hybridized reference (ApoE-/- mice fed a normal chow
diet for 3 months). To identify genes involved in plaque regression, an additional group of
ApoE-/- mice, in which the Western type diet was replaced after 4.5 months by a normal chow
diet (for 1.5 months), was investigated. In total, 499 genes were differentially expressed
2-fold in at least one group compared to the reference. We found genes that were only
upregulated in early atherosclerosis (3 and 4.5 month normal chow/ western diet), such as fatty
acid synthetase. The majority of genes was differentially expressed during the progression of
atherosclerosis, and expresion levels increased even further when atherosclerosis progressed
(3/4.5/6 month normal chow/diet). Functional clustering revealed upregulation of many genes
involved in collagen turnover such as TIMP, cathepsin B/D/H/L/S/Z, MMP2 and 12 and
pro-collagen type I, and some cellular matrix markers such as actin, zyxin, tenascin and fibulin.
Furthermore, we observed increased expression of inflammatory genes like IFN regulatory
factor 15, gm-CSF 2 receptor, VCAM and CD44, as well as growth factors like EGF-1, TGF and
GDF 15. Also genes involved in signal transduction, such as MAPKKKK1, and genes involved
in lipid metabolism such as scavenger receptor, apo-B as well as embryonic genes such as
homeobox C6, LIM homeobox protein 1 and hairy enhancer of split 5 were upregulated.
Interestingly, genes involved in calcification such as MGP and cartilage oligomeric matrix
protein, or oxidative stress, like SOD, were only upregulated after 6 months of normal chow or
Western type diet. During plaque regression, we found upregulation of several genes including
ephrin 4 and serum amyloid 3, whereas arginine vasopressin and dihydrofolate reductase were
downregulated.
P166
Synergistic Effect of Urotensin II with Mildly Oxidized LDL on Vascular
Smooth Muscle Cell Proliferation
Takuya Watanabe, Takashi Katagiri, Rajbabu Pakala, Claude R Benedict. Showa University
School of Medicine, Tokyo, Japan; University of Texas-Houston Health Science Center,
Houston, TX
Objectives: Urotensin II (UII) found in coronary atheroma is the most potent vasoconstrictor
known to date. Mildly oxidized LDL (moxLDL) contributes to atherogenesis and plaque
formation. We assessed the effect of UII and its interaction with moxLDL, highly oxidized LDL
(oxLDL), and their oxidative components, i.e., reactive oxygen species (ROS), lysophosphatidylcholine
(LPC), or 4-hydroxy-2-nonenal (HNE) on vascular smooth muscle cell (VSMC)
proliferation. Methods: Growth-arrested VSMCs were incubated in serum-free medium with
different concentrations of LDL, moxLDL, oxLDL, hydrogen peroxide (H2O2, as a donor of ROS),
LPC, or HNE with or without UII. 3H-Thymidine incorporation into DNA was measured as an
index of VSMC proliferation. Results: UII stimulated 3H-thymidine incorporation in a dosedependent
manner with a maximal effect at a concentration of 50 nM (161%). Low
concentrations of UII potentiated the mitogenic effect of LDL (108 to 242%), oxLDL (129 to
302%), moxLDL (120 to 345%), H2O2 (177 to 226%), LPC (115 to 332%), and HNE (142 to
299%). In combination with moxLDL (100 ng/mL), nonmitogenic concentration of UII (10 nM)
induced a greater synergistic interaction (345%) compared with same dose of endothelin-1
(287%) or angiotensin II (277%). The synergistic Downloaded interaction between from
UII and moxLDL was
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Poster Presentations a-29
partially inhibited by anti-Gq/11 antibody (2.5 L/plate of 2 mL), EGF receptor tyrosine kinase
inhibitor erbstatin A (10 M), or intracellular free radical scavenger NAC (400 M) and
completely inhibited by c-Src tyrosine kinase inhibitor radicicol (10 M), PKC inhibitor
Ro31–8220 (0.1 M), or MEK inhibitor PD098059 (10 M). Conclusions: Our results suggest
that UII acts synergistically with moxLDL in inducing VSMC proliferation via the c-Src/PKC/
MAPK pathway, which may explain the relatively rapid progression of atherosclerosis in
patients with hypertension and hypercholesterolemia.
LDL Receptor Knockout Mice Have a Normal VLDL Production Rate
John S Millar, Cyrille Maugeais, Ilia V Fuki, Daniel J Rader. University of Pennsylvania,
Philadelphia, PA
P167
INTRODUCTION: In vitro studies have shown that primary hepatocytes from LDL receptor (LDLR)
knockout mice have increased production rates of triglyceride (TG) and apolipoprotein (apo) B.
These studies indicated that that the LDLR may modulate hepatic apoB production. We
addressed the question of whether the LDLR affects hepatic apoB production in LDLR -/- mice
in vivo. METHODS: We performed studies in LDLR -/- and wild type mice and in LDLR -/- and
LDLR /- mice on an apobec1 -/- background. Metabolic studies were conducted by
simultaneous injection of mice with Triton WR1339 and [35S]-methionine. TG and apoB
production rates were determined by calculating the rate of increase in plasma TG and [35S]
incorporation into apoB over time. RESULTS: Total cholesterol and TG levels were significantly
higher in the LDLR -/- group compared to wild type mice. There were no differences between
LDLR -/- (n5) and wild type (n6) mice in plasma TG production rates determined following
Triton WR1339 injection (116 66 vs. 132 39 mg/kg/hr, respectively, p.76). There were
no differences between LDLR -/- (n5) and wild type (n6) mice in the secretion of VLDL
apoB-100 (73 67 vs. 66 52 cpm/ul/hr, p.86) or apoB-48 (62 32 vs. 85 51
cpm/ul/hr, p.45). We next studied TG and apoB production rates in LDLR -/- and LDLR /mice
on an apobec1 -/- background. LDLR -/- mice had baseline total cholesterol and TG levels
that were significantly higher than those from the LDLR /- group. There were no differences
in the secretion of total TG from LDLR -/- (n9) and LDLR /- (n10) groups (96 43 vs.
83 24 mg/kg/hr, respectively, p.44). The secretion of VLDL apoB100 was not different
between the LDLR -/- (n4) and LDLR /- (n5) groups of apobec1 -/- mice (387 115 vs.
354 105 cpm/ul/hr, p.67). CONCLUSION: In this in vivo study the production rate of plasma
TG and VLDL apoB was not different between LDLR -/- and receptor competent mice. This was
found in mice that make apoB100 and B48 and those that express apoB100 only. These results
indicate that the LDLR is not involved in determining the hepatic apoB production rate in vivo.
Deficiency of Apolipoprotein CI in Macrophages Increases Cholesterol
Esterification and Reduces Cholesterol Efflux
P168
Patrick C Rensen, Dianne J Delsing, Miek C Jong, Sabine J Van Dijk, Bas Teusink, Anton J
Horrevoets, Hans M Princen, Louis M Havekes. TNO-PG, Gaubius Laboratory, Leiden,
Netherlands; AMC, University of Amsterdam, Amsterdam, Netherlands
Apolipoprotein CI (apoCI) has been shown to be an important modulator of several steps in lipid
metabolism. We have previously shown that apoCI overexpression severely reduces adipose
tissue stores in mice, and fully protects against the development of obesity in genetically obese
ob/ob mice by inhibiting the esterification and storage of free fatty acids. Since apoCI mRNA
expression has been demonstrated to be upregulated (85-fold) during monocyte to macrophage
differentation, the aim of the present study was to examine whether apoCI is also involved in
lipid homeostasis in macrophages. In situ hybridization studies showed that apoCI is expressed
at high levels within macrophages present in primary atherosclerotic lesions in the aortic valve
area of hyperlipidemic apoE3-Leiden mice, whereas low expression levels were detected in
non-atherosclerotic controls. To investigate the significance of this finding, thioglycollateelicited
peritoneal macrophages were isolated from wild-type and apoC1-/- mice. Incubation of
48 h-cultured cells with acetylated (Ac) LDL (50 g/ml; 24 h) followed by coincubation with
3 3 -/-
H-oleate (0.1 mM; 0–2 h) resulted in an increased H-oleate accumulation in apoC1
macrophages (80%; P0.05). Separation of lipids by high performance thin layer chromatography
showed that this effect was caused by an enhanced 3H-oleate esterification into
cholesteryl esters (74%; P0.005) and triglycerides (31%; P0.05). Incubation of macrophages
with 3H-cholesteryl oleate-labeled AcLDL (50 g/ml; 48 h), followed by extensive
washing and incubation in the presence of wild-type mouse serum (5% v/v; 3 h) resulted in a
40% reduced cholesterol efflux from apoC1-/- macrophages as compared to wild-type cells,
which is similar to the reducing effect of apoE-deficiency on efflux (40%) as demonstrated
using apoE /- macrophages. From these data we conclude that the expression level of apoCI in
macrophages affects fatty acid esterification and cholesterol efflux, suggesting that apoCI may
have a protective role in atherogenesis.
Deficiency of Glutathione Peroxidase-1 Sensitizes to Endothelial
Dysfunction in Hyperhomocysteinemic Mice
P169
Sanjana Dayal, Kara L Brown, Christine J Weydert, Larry W Oberley, Erland Arning, Teodoro
Bottiglieri, Frank M Faraci, Steven R Lentz. The University of Iowa, Iowa City, IA; Baylor
University Medical Center, Dallas, TX
Hyperhomocysteinemia has been proposed to impair endothelial function through oxidative
inactivation of endothelium-derived nitric oxide. To explore the role of oxidative stress in
endothelial dysfunction during hyperhomocysteinemia in vivo, we tested the hypothesis that
deficiency of cellular glutathione peroxidase (GPx-1) enhances susceptibility to endothelial
dysfunction in hyperhomocysteinemic mice. GPx-1-deficient mice were crossbred to the
C57BL6 genetic background, and then interbred to generate littermates that were wild type
(Gpx1/), heterozygous (Gpx1/-), or homozygous (Gpx1-/-) for the mutated Gpx1 allele. At
the time of by weaning, guest mice on were April placed 4, 2013 on either a control diet or a methionine-rich diet for 17

a-30 Arterioscler Thromb Vasc Biol. Vol 22, No 5
weeks. Plasma total homocysteine was elevated (18–23 umol/L) in mice fed methionine-rich
diet compared with mice fed control diet (5–6 umol/L), and was not influenced by Gpx1
genotype. Hepatic GPx activity tended to be lower in Gpx1/ mice fed methionine-rich diet
(29963 U/mg) than in Gpx1/ mice fed control diet (34747 U/mg). GPx activity in
Gpx1/- mice was similar on control diet (17742 U/mg) and methionine-rich diet (18649
U/mg). GPx activity was undetectable in Gpx1-/- mice fed either diet (p0.01 vs. Gpx1/
mice). In mice fed control diet, maximal relaxation of the aorta to the endothelium-dependent
dilator acetylcholine was similar in Gpx1/, Gpx1/-, and Gpx1-/- mice. In mice fed the
methionine-rich diet, maximal relaxation to acetylcholine was selectively impaired in Gpx1-/mice
compared with Gpx1/ mice (736% vs. 902%; p0.05). No differences in
vasorelaxation to nitroprusside or papaverine (endothelium-independent dilators) were observed
between Gpx1/ and Gpx1-/- mice fed either diet. These findings demonstrate that
deficiency of GPx-1 sensitizes to endothelial dysfunction in mice with moderate hyperhomocysteinemia,
and provide support for the concept that endothelial dysfunction in hyperhomocysteinemia
is mediated in part through oxidative mechanisms.
P170
Rosiglitazone has Beneficial Effects on Lipid Metabolism and Insulin
Resistance in obob Mice Overexpressing Human Apolipoprotein C1 (ApoC1)
Martin Muurling, Vivian E Dahlmans, Ronald P Mensink, Johannes A Romijn, Louis M
Havekes, Peter J Voshol. TNO - Prevention and Health, Leiden, Netherlands; Maastricht
University, Maastricht, Netherlands; Leiden University Medical Center, Leiden, Netherlands
Objectives: Leptin deficiency in obob mice leads to severe obesitas and insulin resistance. We
have previously shown that homozygous overexpression of human ApoC1 on an obob
background protects against obesitas and its associated metabolic aberrations. This protection
was explained by impairment of peripheral fatty acid uptake caused by ApoC1. Homozygous
ApoC1 overexpressing mice have a rather severe phenotype. We have therefore tested the
effect of the moderate (heterozygous) ApoC1 expression on an obob background, and
compared those effects with another treatment of insulin resistance i.e. Rosiglitazone (ROSI).
Since ApoC1 reduces fatty acid uptake and WAT formation, whereas ROSI does the opposite,
we tested whether ROSI is effective in obob mice overexpressing ApoC1. Furthermore we
determined the effects of ROSI treatment on peripheral glucose and FA uptake in ApoC1
overexpressing mice. Methods: We determined the effect of ROSI (100 mol per kg food)
treatment on plasma lipids, glucose and insulin levels in obob (n12) and obob/ApoC1 (n12)
mice. Furthermore, insulin sensitivity and glucose tolerance test were performed to determine
the effectiveness of ROSI on insulin sensitivity. Finally, tissue-specific glucose and FA uptake
were measured under hyperinsulinemic euglycemic clamp conditions in both genotypes.
Findings: ROSI treatment resulted in similar body weight increase in both genotypes (obob:
9.82.4 g vs. 14.73.5g(P0.05), obob/ApoC1: 9.72.8 g vs. 18.62.0g(P0.05)).
Furthermore, plasma levels of glucose (obob: 8.92.0 mM vs. 6.80.4 mM (P0.05),
obob/ApoC1: 17.45.9 mM vs. 8.41.0 mM (P0.05)) and insulin (obob: 4.72.1 ng/ml vs.
0.90.2 ng/ml (P0.05), obob/ApoC1: 5.93.0 ng/ml vs. 1.40.5 ng/ml (P0.05))
decreased due to ROSI treatment. Finally, treatment with ROSI improves insulin action and
increases insulin-stimulated glucose uptake mainly in heart muscle. Conclusion: Although
ApoC1 affects body weight and fuel metabolism, it does not affect the actions of ROSI
significantly. Therefore, ApoC1 and ROSI appear to act on different pathways.
P171
Retrospective Study on Common Ambulatory Used Drugs and Warfarin
Interactions
Yun Lu, Luanne Sojka, Tracy Veronen, Katie Won. Hennepin County Medical Center,
Minneapolis, MN; University of Minnesota, Minneapolis, MN
PURPOSE: This study aimed to determine the onset and severity of common ambulatory drugs
(amoxicillin, amiodarone, augmentin, azithromycin, ciprofloxacin and co-trimoxazole) and
warfarin interactions. METHOD: Retrospective reviewing of Hennepin County Medical Center
Anticoagulation Clinic patient charts (total of 300 per year) between December 1997 to
December 2000, all INR readings were analyzed during the defined durations. The defined
durations were from the first day to 40th week after the addition of amiodarone, or from the
first day to 28th day after the addition of the identified antibiotics. RESULTS: After the addition
of Amiodarone, significant INR elevation were observed in 60% (27/45) clinic visits during first
4 weeks. Among clinic visits from the 16th week to the 24th week, 56% (31/55) INR elevations
were reported. Average INR elevation with amiodarone addition during the defined observation
period was 30%. Within one month after the addition of amoxicillin, 7.6% (4/54) INR readings
were supratherapeutic. This was compared with 14% (5/54) INR readings elevation after the
addition of augmentin. While for azithromycin, there were 14.3% (6/42) INR readings were
supratherapeutic after the initiation of azithromycin. On the other hand, 60% supratherapeutic
INR were reported (27/45) after the addition of ciprofloxacin. After the addition of cotrimoxazole,
14% supratherapeutic INR were reported (4/28) patient visits. CONCLUSIONS:
Amiodarone and warfarin interaction peaks in the first month and again during the 16th-24th
weeks after the addtion of amiodarone. The second peak of amiodarone warfarin interaction
has not been reported in literature. It suggests more frequent INR monitorings are also
important when serum amiodarone level reaches their static status. Higher incidences of
augmentin ciprofloxacin, co-trimezole and warfarin interactions indicates that frequent INR
monitoring is necessary for the first two weeks after the addition of the antibiotics, esp. in the
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Factor VIIa Stimulates Endothelin-1 Synthesis via Protease-Activated
Receptor-2
Amarjit S Sethi, Delphine M Lees, Julie A Douthwaite, Roger Corder. William Harvey
Research Institute, London, UK
P172
Objective: Endothelial dysfunction, including a prothrombotic state, frequently precedes clinical
manifestations of coronary artery disease. Inflammation plays a pivotal role in atherogenesis
and initiates prothrombotic changes by upregulation of tissue factor (TF), which binds and
activates Factor VII (FVII) to FVIIa. As endothelin-1 (ET-1) is strongly implicated in atherogenic
processes we investigated whether FVIIa could cause the further propagation of endothelial
dysfunction by stimulating ET-1 synthesis. Methods: The effect of activated recombinant FVII
(rFVIIa) on ET-1 synthesis was assessed on bovine aortic endothelial cells under basal
conditions and after pre-treatment with tumor necrosis factor-alpha (TNF). For comparison, the
responses to FVII, Factor X (FX), activated Factor X (FXa) or a combination of rFVIIa and FX were
also tested. Results: On TNF pre-treated cells rFVIIa produced a concentration dependent
increase in ET-1 release (p0.001) with a concordant increase in ET-1 mRNA levels. Without
TNF pre-treatment, rFVIIa had no effect. Inactivation of rFVIIa with a specific active-site inhibitor
completely blocked the rFVIIa response showing that this effect was dependent on the
proteolytic activity of rFVIIa. As the protease-activated receptor-2 (PAR2) can be activated by
rFVIIa, we tested the role of PAR2 in ET-1 synthesis. SLIGKV, a PAR2 agonist, increased ET-1
synthesis in TNF pre-treated cells. More importantly, application of SLIGKV during TNF
pre-treatment led to desensitisation with no further response to PAR2 stimulation. This also
abolished the effect of rFVIIa indicating that PAR2 mediated this response. TF was stimulated
by TNF with peak mRNA levels at 1h and maximum activity at 2h. TNF followed by FVII
increased ET-1 release (p0.001) but FX had no effect. FXa increased ET-1 release but this
did not reach significance. The combination of rFVIIa 1pM and FX 1U/ml significantly increased
ET-1 release compared to TNF/rFVIIa 1pM (p0.01) or TNF alone (p0.05). Conclusion:
Activation of PAR2 by FVIIa leads to ET-1 synthesis. This has considerable relevance for
atherogenesis when inflammation and prothrombotic changes occur together.
P173
Interferon- Deficiency Enhances Angiotensin II-Induced Abdominal Aortic
Aneurysm Formation in Apolipoprotein E Deficient Mice
Victoria L King, Lisa A Cassis, Alan Daugherty. University of Kentucky, Lexington, KY
Angiotensin II (AngII) induced atherosclerosis and abdominal aortic aneurysm (AAA) formation
in hyperlipidemic mice is associated with an inflammatory response that is characterized by
recruitment of macrophages and T lymphocytes to the arterial wall. T lymphocytes recruited to
mouse atherosclerotic lesions are commonly considered to be Th1 orientated, as defined by the
predominant secretion of the cytokine interferon-gamma (IFN-). To define the effects of IFN-
on the development of AngII-induced vascular disease, we defined the effect of IFN-
deficiency in apoE-/- mice. Compound apoE -/- x IFN--/-mice were developed in a C57BL/6
background. AngII (1000 ng/kg/min) or saline was infused subcutaneously for 28 days into 8
week old male apoE-/- and apoE -/- x IFN- -/- mice. In apoE -/- x IFN- -/- mice infused
with AngII, rupture of AAA resulted in 50% (3 of 6) mortality within 10 days. All the mice that
died exhibited AAAs. There was no mortality in apoE -/- mice infused with AngII. At 28 days,
100% (3 of 3) of the remaining IFN- deficient apoE -/-mice developed AAAs compared to a
20% (1 of 5) incidence in apoE-/- mice. In addition, the AAA in IFN- exhibited pronounced
remodeling and dilation compared to wild type mice. These findings suggest that IFN-
protects against the mortality and morbidity of AngII induced AAA in apoE-/- mice. Therefore,
AngII induced recruitment of T lymphocytes plays a critical role in the development of vascular
pathology.
P174
The Macrophage Scavenger Receptor Macrosialin does not Play a Direct
Role in Oxidized LDL Metabolism
Zhenze Zhao, Maria C De Beer, Deneys R Van der Westhuyzen, Willem J De Villiers.
University of Kentucky, Lexington, KY
Murine macrosialin and its human homologue CD68 are heavily glycosylated transmembrane
proteins expressed specifically in macrophages and macrophage-related cells. Macrosialin is
predominantly a late endosomal protein but is also found on the cell surface where it is reported
to bind oxidized LDL. Here we show upregulation of macrosialin in the livers of atherosclerosissusceptible
C57BL/6 mice fed an atherogenic high fat diet. Upregulation is rapid with increased
mRNA expression evident after 3 days and a 10-fold increase achieved by a 5 week dietary
regimen. Increased mRNA production is accompanied by increased protein expression.
Macrosialin upregulation occurs specifically, but is also in part due to macrophage infiltration
into the liver, as measured by F4/80 expression. We also found that resident mouse peritoneal
macrophages incubated with oxidized LDL showed 1.5 to 2-fold upregulation of macrosialin
mRNA and protein expression. This specific upregulation of macrosialin in Kupffer cells by a
pro-inflammatory atherogenic high-fat diet could indicate a compensatory protective role in
atherogenesis. Adenoviral-mediated expression of macrosialin in cultured cells resulted in
oxidized LDL binding as illustrated by ligand blotting. We were however unable to show in
classical binding studies that macrosialin acted as a receptor for oxidized LDL despite
significant surface expression. We also found that adenoviral-mediated hepatic over expression
of macrosialin did not affect the plasma lipoprotein profiles of mice fed an atherogenic high fat
diet known to generate inflammatory lipoprotein molecules. This evidence does not support a
direct roleby for guest macrosialin on April in oxidized 4, 2013 LDL metabolism.

P175
Gene Transfer with Vascular Endothelial Growth Factor Improves Regional
Blood Flow and Regional Contractility in a Dose-Dependent Manner in a
Chronic Ischemic Pig Heart Model
Michael K Hsin, Mark H Danton, Richard Ammer, Kathryn Q Flores, Rita G Laurence, Tom
Aretz, Lawrence H Cohn, Lishan Aklog. Harvard Medical School, Boston, MA
BACKGROUND: Although gene therapy using plasmid encoding Vascular Endothelial Growth
Factor (pl-VEGF) has been shown to promote angiogenesis in ischemic myocardium in animal
studies, the optimal dose has not been established. We sought to determine whether the
functional benefit of pl-VEGF 165 gene transfer is affected by the dose administered. METHOD:
22 pigs (15–20 kg) underwent placement of ameroid constrictors to the proximal circumflex
arteries to produce chronic ischemia. 6 weeks later the animals underwent: (1) sham
thoracotomy (n7) or direct intramyocardial injection of pl-VEGF 165 to the ischemic zone
using (2) 4.5mg/pig (High Dose, HD, n7) or (3) 0.45mg/pig (Low Dose,LD, n8). 8 weeks
later, the following were measured: REGIONAL MYOCARDIAL BLOOD FLOW (RMBF) with
radioactive microspheres, and REGIONAL CONTRACTILITY assessed by measuring Fractional
Area Change (FAC) with epicardial piezoelectric crystal pairs placed in the ischemic zones. Data
was taken at rest and under rapid atrial pacing. RESULTS: In the High Dose group, with rapid
pacing, mean RMBF in the ischemic zone was 2.7 times higher compared to the sham group,
and the FAC was significantly higher than sham. There was no significant difference between
the Low Dose group and sham group at rest or paced, or in the High Dose group at rest.
CONCLUSION: Plasmid VEGF gene transfer improves regional blood flow and contractility in a
dose-dependent manner in chronically ischemic pig myocardium.
Fas (CD95) Sensitizes Macrophages to Oxidized-LDL Induced Death
Thomas Q Nhan, W C Liles, Alan Chait, Stephen M Schwartz. University of Washington,
Seattle, WA
P176
In vitro studies of macrophage death in response to oxidized LDL (oxLDL) were undertaken as
a model for the formation of the necrotic core of the atherosclerotic plaque. We showed that
thioglycollate-elicited mouse peritoneal macrophages avidly incorporate both oxLDL and
acetylated LDL (acLDL) to become foam cells. OxLDL-treated macrophages, but not acLDLtreated
macrophages, showed nearly 100% death with characteristics consistent with
apoptosis, including cell surface phosphatidylserine exposure, intracellular caspase-3 activity,
cleavage of caspase-3 substrates, and DNA fragmentation as shown by TUNEL assay.
Surprisingly, the p17 cleaved form of caspase-3, the activated molecule normally found in
dying cells, was also present in both control and acLDL-treated macrophages. However,
caspase-3 activity was only detected in oxLDL-treated macrophages. The amount of the
cleaved form of caspase-3 was reduced by in vitro blockade of FasL with antibody (MFL3) and
was absent in lpr macrophages, which lack functional Fas (CD95). Moreover, lpr macrophages
resisted oxLDL-cytotoxicity. The naturally occurring Fas-FasL induction of caspase-3 cleavage
in living macrophages may, therefore, represent an important physiologic mechanism that
sensitizes them to the cytotoxicity of oxLDL. The resulting cleavage of caspase-3 in
macrophages is necessary but not sufficient for oxLDL-induced macrophage death. These data
suggest that macrophages may exist in an undead state, that represents a functional state
dependent on caspase activation. This state appears to be necessary for death in response to
oxLDL.
P177
Intracellular Metal Chelation Inhibits TNF-Induced Expression of Adhesion
Molecules and MCP-1 in Human Aortic Endothelial Cells
Weijian Zhang, Balz Frei. Linus Pauling Institute, Oregon State University, Corvallis, OR
Endothelial activation and monocyte adhesion are initiating steps in atherogenesis thought to
be caused, in part, by oxidative stress. Because redox-active transition metal ions, such as iron
and copper, can cause generation of reactive oxygen species and initiation and propagation of
lipid peroxidation, they may also cause endothelial activation. Therefore, the objective of the
study was to investigate the effects of the iron-chelator desferrioxamine (DFO) and the
copper-chelator neocuproine (NC) on TNF-induced expression of adhesion molecules and
monocyte chemoattractant protein-1 (MCP-1) in human aortic endothelial cells (HAEC). We
found that pre-incubation of HAEC for 16 hr with NC (0.1- 0.5 mmol/L) or DFO (0.01- 0.1
mmol/L), but not iron-saturated DFO, dose-dependently inhibited TNF-induced (10 U/ml)
protein expression of E-selectin, VCAM-1 and ICAM-1. DFO at a concentration of 0.1 mmol/L
inhibited TNF-induced E-selectin, VCAM-1 and ICAM-1 expression by 742.5%, 843.3%
and 591.7%, respectively; and 0.5 mmol/L NC inhibited expression of these adhesion
molecules by 933.6%, 843.0% and 894.0%, respectively (P0.01 compared with TNF
alone, n 3). In contrast, no inhibitory effect was observed when the cells were co-incubated
with DFO plus TNF, or treated for only 1 hr with NC or with bathocuproinedisulfonic acid, a
membrane-impermeable copper-chelator, before addition of TNF. DFO and NC also inhibited
TNF-induced upregulation of mRNA levels of all three adhesion molecules, as well as MCP-1.
Addition of cycloheximide completely abolished the inhibitory effect of NC, but not DFO,
indicating that new protein synthesis was required for NC to exert its inhibitory effect.
Furthermore, pre-incubation with DFO and NC (0.5 Downloaded mmol/L) inhibitedfrom TNF-induced activation
http://atvb.ahajournals.org/
Poster Presentations a-31
of the transcription factor NFB by 34% and 45%, respectively. In conclusion, our data indicate
that intracellular transition metals play an important role in cytokine-induced endothelial
activation. Therefore, intracellular metal chelation might be a novel strategy to prevent and
treat atherosclerosis and other inflammatory conditions.
P178
Increased Consumption of Polyunsaturated Fat, Vitamin E and Carotenoids
Enhances Brachial Arterial Vasoreactiviy in Men with Coronary Artery
Disease
Oh Yoen Kim, Ji Young Kim, Jong Ho Lee, Yangsoo Jang, Seok Min Kang. College of
Human Ecology, Yonsei University, Seoul, Korea; Cardiovascular Genome Center, School of
Medicine, Yonsei University, Seoul, Korea
Background: This study aimde to determine whether the isocaloric replacement of cooked
refined rice with polyunsaturated fatty acid (PUFA)-rich muffin, composed of walnut, seaweed,
sesame oil, and wheat flour in lunch meal reduces coronary artery disease (CAD) risk factors,
such as endothelial dysfunction and lipid peroxidation in CAD patients. Methods & Results:
Sixty-one male patients with CAD were randomly assigned to either a daily PUFA-rich muffin
group or a cooked refined rice group for 8 week. When compared to 307g cooked refined rice,
90g PUFA-rich muffin was isocaloric as 450kcal, but it contained different composition of
nutrients. 307g cooked refined rice provided 95g carbohydrate, 7.8g protein, 1.5g fat, 0.31g
fiber, 0.86mg vitamin E, 9.45g folate, 1.29g saturated fatty acid (SFA), 1.72g monounsaturated
fatty acid (MUFA) and 1.84g PUFA. On the other hand, 90g PUFA-rich muffin provided 42g
carbohydrate, 6.2g protein, 28.9g fat, 2.6g fiber, 3.29mg vitamin E, 10.79g folate, 138RE
vitamin A, 563g -carotene, 8.08g SFA, 7.21g MUFA and 11.99g PUFA. The muffin group
showed the reduction in urinary 8-epiprostaglandin F 2 (PGF 2) (57091pg/mg creatinine vs
48177) without altering body weight and energy intake after 8 week. The baseline urinary
PGF 2 was inversely related to brachial arterial vasoreactivity, that is, flow mediated dilation
(FMD) (r-0.419, P0.017) and nitroglycerin mediated dilation (NMD) (r-0.553, P0.002),
respectively. In the muffin group, FMD and NMD were increased from 4.30.7% to 6.30.9
(P0.05) and from 6.00.7% to 10.60.9 (P0.001) respectively after 8 week. Conclusion-
:Increased intake of PUFA, vitamin E and carotenoids via the isocaloric replacement of cooked
refined rice with PUFA-rich muffin in a meal exhibited significant beneficial effect on brachial
arterial vasoreactivity and on lipid peroxidation in CAD patients. This effect is likely to
substantially reduce the risk factors for CAD.
P179
Identification of a Pro-Inflammatory CD36-Associated Signal Transduction
Pathway in Macrophages
Kathryn J Moore, Joseph El Khoury, Lea A Medeiros, Andrew D Luster, Mason W Freeman.
Massachusetts General Hospital, Boston, MA
The class B scavenger receptor CD36 has been implicated in a wide variety of physiological
processes, including atherosclerosis, angiogenesis, lipid metabolism, malarial pathogenesis,
tissue homeostasis and Alzheimer’s disease. However, despite its identification more than a
decade ago, the physiological events regulated by CD36 ligation remain largely unknown. We
have now identified a pro-inflammatory CD36 signal transduction pathway that is induced in
macrophages. We demonstrate that -amyloid binds to CD36 on macrophages and microglia,
inducing the activation of the Src kinase Lyn and p44/42 mitogen-activated protein kinase.
These signaling events require CD36 expression, as -amyloid does not induce p44/42
phosphorylation in macrophages derived from CD36-/- mice. Furthermore, using macrophages
derived from mice containing targeted mutations in Src kinase family members, we
demonstrate that Lyn, but not Fyn, is essential for CD36 activation of p44/42 MAPK. Interruption
of this CD36 signaling cascade in macrophages abrogates pro-inflammatory responses to
-amyloid including the production of reactive oxygen species and cytokines. CD36-/macrophages
stimulated with -amyloid produced approximately 75–80% less TNF- and
MCP-1 as compared to similarly treated wild type macrophages. Comparable decreases of
TNF- and MCP-1 were also observed through inhibition of p44/42 MAPK using 5 M
PD98059 and in Lyn-/- macrophages, suggesting that interruption at any step of this signaling
cascade abrogates these inflammatory responses. In summary, we have identified a -amyloid
induced signaling cascade involving the sequential activation of CD36-Lyn-p44/42 MAPK, and
resulting in the production of macrophage pro-inflammatory molecules. These data suggest
that through its ability to bind modified proteins and lipids such as -amyloid and oxidized LDL,
CD36 may play a role in chronic inflammatory conditions such as Alzheimer’s disease and
atherosclerosis through induction of a pro-inflammatory signaling cascade.
Comparison of Thromboelastography and Aggregometry in Monitoring
Glycoprotein IIb/IIIa Inhibition
Ramin Artang, Gitte W Jurgensen, Niels J Frandsen, Ulrik Abildgaard, Jorn D Nielsen.
Gentofte University Hospital, Hellerup, Denmark; Hilleroed Hospital, Hilleroed, Denmark
P180
Objectives: In patients undergoing percutaneous coronary interventions (PCI), there is evidence
of fewer coronary adverse events if platelets are inhibited more than 80% with Glycoprotein(GP)IIb/IIIa
inhibitors. Maintaining an optimal level of inhibition raises the need for a rapid and
reliable bedside monitoring technique. Thrombelastography (TEG), is a bedside method for
evaluation of hemostasis. The aim of this study was, for the first time, to compare TEG with
aggregometry for monitoring platelet inhibition. Methods: After obtaining informed consent, 10
consecutive patients who underwent PCI and received abciximab were included. Blood was
drawn at baseline, and at 2, 8, 24, and 48 hours following bolus and infusion of abciximab
(0.25mg/kg, 0.125 g/kg/min for 12 hours). All patients received heparin in cath. lab. and
continued on aspirin and clopidogrel. Citrated whole blood was analyzed on modified TEG
model 5000 by for guest determination on April of maximum 4, 2013amplitude.
Platelet rich plasma was analyzed by

a-32 Arterioscler Thromb Vasc Biol. Vol 22, No 5
turbidimetric aggregometer using 20 M ADP. Levels of inhibition at intervals mentioned above
with both methods, were correlated using Spearmans correlation coefficient. Findings: See
figure. Conclusion: Percent inhibition of platelets determined by TEG is strongly correlated to
the inhibition determined by standard aggregometry. TEG could therefore be useful in rapidly
monitoring the effect of GPIIb/IIIa inhibitors. This method is far less time and labor consuming.
Larger trials are needed for correlating optimal inhibition as determined by TEG to clinical
outcome.
P181
Association of Risk Factors of Coronary Artery Disease and Clot Strength
Ramin Artang, Esther Jensen, Flemming Pedersen, Niels J Frandsen, Jorn D Nielsen.
Gentofte University Hospital, Hellerup, Denmark; Hilleroed Hospital, Hilleroed, Denmark
Objectives: The clot strength (the elastic shear modulus of clotting blood) is increased in
patients with coronary artery disease. Elevated plasma levels of fibrinogen, C-reactive protein
(CRP), homocysteine and cholesterol are also associated with increased risk of coronary artery
disease. The aim of our study was to investigate the association of plasma concentrations of
these risk markers with clot strength. Methods: After obtaining informed consent, 81 subjects
were included: 20 healthy volunteers, 23 with stable angina, 19 with unstable angina (UA) and
19 with myocardial infarction (MI). Upon admission, blood was drawn from the UA and MI
patients before antithrombotic treatment. Clot strength in dyne/cm was measured on citrated
whole blood by thrombelastography. Plasma concentrations of fibrinogen, CRP, homocysteine
and total cholesterol were correlated to clot strength using Spearmans correlation coefficient.
Findings: A highly significant correlation was observed between fibrinogen concentration and
clot strength (r 0.82, p 0.0001). Clot strength was also logarithmically associated with the
CRP level (r 0.54, p 0.0001). There was no correlation between cholesterol level and clot
strength (r -0.12, p ns), or homocysteine level and clot strength (r 0.1, p ns). Conclusion:
Clot strength is directly correlated to the level of plasma fibrinogen, the structural component
of the blood clot. The association between clot strength and CRP level follows a logarithmic
pattern. The clinical application of the latter finding requires further investigation. Increased
plasma levels of homocysteine and total cholesterol have no direct effect on clot strength.
P182
Overexpression of RISC Attenuates Smooth Muscle Cell Growth Responses
Jiyuan Chen, Joseph M Miano. Center for Cardiovascular Research, University of Rochester
Medical Center, Rochester, NY
We and others have shown that all-trans retinoic acid (atRA), a natural retinoid, is effective in
suppressing smooth muscle cell (SMC) growth in vitro and inhibiting neointimal formation after
vascular injury in vivo. The underlying mechanisms are not clear, but likely involve
retinoid-mediated changes in gene expression. Accordingly, we used the Suppression
Subtractive Hybridization technique to clone a cluster of retinoid-inducible genes from rat aortic
SMC, including a new retinoid-response gene we call retinoid-inducible serine carboxypeptidase
(RISC). We have published the work on the cloning, chromosomal mapping and expression
profile of RISC. To date, we have been unable to document RISC activity in vitro using well
known substrates of other serine carboxypeptidases, suggesting that RISC may catalyze the
C-terminal cleavage of distinct proteins. To begin understanding the role of RISC in mediating
atRA s biological effects, we generated several SMC lines stably expressing rat RISC under
control of the CMV promoter. Of 5 stable cell lines confirmed by Northern blotting to
overexpress RISC, 3 were selected for subsequent study. Growth curves revealed that RISC
overexpression inhibits SMC growth in vitro. To explore RISC function in cellular signal
transduction pathways associated with SMC growth, we examined the effects of RISC
overexpression on PDGF-induced MAPK. The results demonstrate that constitutively expressed
RISC can attenuate phosphorylation of Erk activated by PDGF in PAC1 SMC. These results
provide new insight into RISC function and suggest that this novel retinoid-inducible gene
participates in retinoid-mediated events associated with the inhibition of SMC growth.
P183
Persistence of the Nitric Oxide Pathway and VEGF-Induced Angiogenesis in
Apolipoprotein E-Deficient Mice
Nancy Fournier, Ana Fortuno, Nicole Villeneuve, Marie Sauvage, Christine Breugnot,
Jean-Pierre Iliou, Nathalie Carpentier, Paul M Vanhoutte, Jean-Paul Vilaine. Institut de
Recherches Servier, Suresnes, France
Endothelium-derived NO plays role in the regulation of angiogenesis whereas hypercholesterolemia
impairs NO release. These two parameters were compared in apolipoprotein E deficient
(ApoE KO) and control (C57Bl/6J) mice, 35 weeks old and fed a regular chow. The markers of
the bioavailability of NO (relaxation to acetylcholine, Downloaded production of cyclic from
GMP) in the thoracic
aorta were not different in ApoE KO mice, in comparison with control mice, despite severe
hypercholesterolemia, the presence of atherosclerotic lesions, upregulation of MCP-1 expression
and increased NADPH oxidase activity in the vascular wall. The VEGF-induced angiogenesis
in a sponge implant model was also similar in control and ApoE KO mice. To test if an
increased capacity of NO production in these mice could explain the preservation of vascular
function and angiogenesis, levels of NOS (type III and II) expression were compared in ApoE KO
and control mice using Western blot and immunohistochemistry. The immunodetection signal
for NOS III had the same intensity in the two groups of mice. Staining for NOS II was only
detected in ApoE KO mice and was mainly localized in the lesion areas. The NOS II detected
did not modify vascular reactivity as N-iminoethyl-L-lysine, a specific inhibitor of this isoform,
had no effect. The preserved vascular bioavailability of NO and the persistence of the
angiogenic capability in ApoE KO mice, which contrast with other models of atherosclerosis or
vascular dysfunction, could not be explained by an increased expression of NOS III. An
increased activity of NOS III or upregulation of enzymes involved in redox status such as
superoxide dismutase, catalase or gluthation peroxidase are alternative explanations to the
paradoxical results obtained in this model of atherosclerosis.
P184
High Flow Induced Arterial Remodeling Is Associated with Early Expression
of TGF-1 and MMPs
Eiketsu Sho, Tej M Singh, Mien Sho, Chengpei Xu, Chang Shu, Hirotake Masuda,
Christopher K Zarins. Stanford University School of Medicine, Stanford, CA; Akita University
School of Medicine, Akita, Japan
Arterial adaptation to high flow requires endothelial cell (EC) proliferation, EC migration and
internal elastic lamina (IEL) degradation. Recently, transforming growth factor beta 1 (TGF-1)
and matrix metalloproteinases (MMPs) have been identified as reg ulators of arterial wall
extracellular matrix. We developed a model of acute high flow to identify the time course
expression of TGF-1 and MMPs along with arterial remodeling. Arterio-venous fistulae (AVF)
were created in Japanese male rabbit s between th e left common carotid artery and the
external jugular vein. Animals were sacrificed after 1, 2, and 3 days. Left carotid artery
segments were harvested for either RT-PCR with appropriate rabbit primers for TGF-1 and
MMP-2, 9 or morphologic examination. Differences between groups were analyzed by ANOVE
with Bonferroni correction. On e day after fistula creation, flow increased 5-fold and remained
elevated at all time points. Morphologically, high flow resulted in early IEL degradation and
fragmentation prior to arterial dilatation. Transcription of TGF-1 a nd MMP-2, 9 was increased
as early as one day after flow increase and reduced after 3 days prior to arterial enlargement.
We conclude that arterial adaptation to increased blood flow requires IEL degradation and
fragmentation prior to enlargement. The ea rly expression of TGF-1 and MMP-2, 9 may
provide the initial stimulus to initiate arterial remodeling. Early presence of TGF-1 may initiate
eventual expression of MMPs to induce the required IEL degradation.
P185
Unsaturated Fatty Acids Inhibit Cholesterol Efflux from Macrophages by
Increasing Degradation of ATP-Binding Cassette Transporter A1
Yutong Wang, John F Oram. University of Washington, Seattle, WA
Abnormal high-density lipoprotein (HDL) metabolism may contribute to the increased atherosclerosis
associated with diabetes and insulin resistance. The ATP-binding cassette transporter
ABCA1 is the key mediator of cholesterol transport from macrophages to HDL apolipoproteins.
Because fatty acids are elevated in diabetes, we examined the effects of fatty acids on ABCA1
activity in cultured macrophages. Results showed that unsaturated fatty acids markedly
inhibited ABCA1-mediated cholesterol and phospholipid efflux from macrophages when ABCA1
was induced by a cAMP analog. This was accompanied by a reduction in the membrane
content of ABCA1 and a decrease in apoA-I binding to whole cells and to ABCA1. In contrast,
saturated fatty acids were ineffective in these processes. However, when 22(R)hydroxycholesterol
was used to induce ABCA1, saturated fatty acids palmitate and stearate
inhibited ABCA1-mediated cholesterol efflux, suggesting fatty acid desaturation was also
stimulated. Fatty acids did not alter ABCA1 mRNA abundance or incorporation of methionine
into ABCA1, indicating that decreased ABCA1 transcription, enhanced mRNA decay, or impaired
translation efficiency did not account for these inhibitory effects. Unsaturated fatty acids,
however, increased ABCA1 turnover when protein synthesis was blocked by cycloheximide. We
conclude that unsaturated fatty acids reduce the macrophage ABCA1 content by enhancing its
degradation rate. These findings raise the possibility that an increased supply of unsaturated
fatty acids in the artery wall promotes atherogenesis by impairing the ABCA1 cholesterol
secretory pathway in macrophages.
P186
Hypoxia/Reoxygenation Promotes Myocardial Angiogenesis via NFkB in a
Rat Model of Chronic Myocardial Infarction
Nilanjana Maulik, Dipak K Das, Shoji Fukuda. University of Connecticut Medical Center,
Farmington, CT
We examined a novel method of stimulating myocardial angiogenesis by hypoxic precondi-
http://atvb.ahajournals.org/ tioning at by bothguest capillaryon andApril arteriolar 4, 2013 levels, and the potential role of NFkB in mediating such

a response. We also investigate the functional relevance of such treatment by assessing
whether the induced neovascularization can help preserve left ventricular contractile functional
reserve in the setting of developing heart failure secondary to myocardial infarction. Rats were
divided into 8 groups (n12): normoxia sham surgery (NS), normoxiapermanent left
anterior descending coronary artery (LAD) occlusion (NMI), hypoxic preconditioningsham
surgery (HS), hypoxic preconditioningpermanent LAD occlusion (HMI), PDTC (NFkB
inhibitor)hypoxic preconditioningLAD occlusion (PHMI), PDTCnormoxiaLAD occlusion
(PNMI), PDTChypoxic preconditioning sham surgery (PHS) and PDTC normoxia sham
surgery (PNS). Rats in the preconditioned groups were subjected to systemic hypoxemic
hypoxic exposure for 4 hrs followed by a 24 hr period of normoxic reoxygenation prior to
undergoing LAD. Rats in the normoxia groups were time matched with the preconditioned
group and maintained under normoxic conditions. The HMI group displayed increase in capillary
as well as arteriolar density after 7,14 and 21 days post -operation compared to the NMI. Prior
PDTC administration prevented such increases in the PHMI group and effectively abolished the
proangiogenic effect of hypoxic preconditioning (HP). One, two and three weeks after sham
surgery or LAD, rats underwent a pharmacological stress test which revealed significantly
elevated values of dp/dtmax at each dose point in the HMI group compared to the NMI or PHMI
groups. The results suggest that HP stimulates myocardial angiogenesis via redox regulated
transcription factor, NFkB dependent pathway to an extent sufficient to exert significant
cardioprotection.
P187
Vascular Calcification is Associated with Expression of the Transcription
Factor Cbfa1 in Uremia
Neal X Chen, Kalisha D O’Neill, Danxia Duan, Sharon M Moe. Indiana University School of
Medicine, Indianapolis, IN
Dialysis patients have extensive vascular calcification. We recently demonstrated that
calcification in arteries from renal transplant patients correlates with expression of the bone
proteins such as alkaline phosphatase and osteopontin. In osteoblasts, the expression of these
proteins and the differentiation of osteoblasts is regulated in part by a transcription factor
cbfa1. To test the hypothesis that uremia induces the expression of cbfa1 with subsequent
upregulation of bone proteins, we examined 1)sections of inferior epigastric arteries from renal
transplant patients, and 2)bovine vascular smooth muscle cells (BVSMC)incubated with 10%
sera from pooled uremic serum from patients with low (LP) versus high serum phosphorus (HP)
and from pooled healthy control sera. Cbfa1 and osteopontin expression were assessed by in
situ hybridization, immunohistochemistry, RT-PCR, and Western blotting. The results demonstrate
that the expression of cbfa1 was located in both the media and intima of arteries. The
expression of cbfa1 was in areas also positive by immunostaining for type I collagen and
osteopontin. We also demonstrated that uremic serum increased cbfa1 expression compared
to incubation with control human serum in BVSMC. Changes in cell morphology occurred with
expression of cbfa1. BVSMC cultured with uremic sera also had increased alkaline phosphatase
(ALP) activity and osteopontin expression. Furthermore, blocking sodium dependent phosphate
(Na/Pi) cotransportor with foscarnet only partially inhibited uremic serum-induced osteopontin
expression, indicating that other non-Na/Pi cotransport dependent mechanisms are also
involved. In conclusion, we have demonstrated that cbfa1 is upregulated in areas of vascular
calcification in arteries obtained from uremic individuals. Uremic serum induces cbfa1 and
osteopontin expression in BVSMC. The effect is, at least partially mediated,through ALP activity
and Na/Pi cotransportor dependent mechanisms. The results support the hypothesis that the
accelerated vascular calcification observed in dialysis patients is due, in part, to upregulation
of the transcription factor cbfa1.
Examination of Apolipoprotein A-I Central Domain Conformation in
Lipid-Free and Lipid-Bound States
P188 WITHDRAWN
P189
Michael N Oda, John C Voss, Trudy M Forte, Robert O Ryan. Children’s Hospital of Oakland,
Oakland, AA; UC Davis, Davis, CA; Lawrence Berkeley National Laboratory, Berkeley, CA;
Children’s Hospital of Oakland, Oakland, CA
Apolipoprotein A-I (apoA-I) serves as the primary protein component of HDL, playing a key role
in defining HDL’s structure and biological activity. For a better understanding of apoA-I’s
functional contribution to HDL, we examined apoA-I’s structural organization and defined the
conformational changes that accompany lipid interaction. We focused on the central domain of
apoA-I (residues 80 - 170) in lipid-free and lipid-bound states, applying site directed electron
paramagnetic resonance spectroscopy (EPR) in combination with fluorescence resonance
energy transfer (FRET). A battery of apoA-I mutants were generated: for EPR, 40 individual cys
substitution mutants; for FRET, a series of single trp apoA-I in which three of apoA-I’s four trp
residues were substituted with phe (fluorescence donors), and a set of trp null mutants (all trp
residues were phe substituted) bearing single cys substitutions (fluorescence acceptors).
Recombinant apoA-I were expressed in E. coli, purified, Downloaded cys mutantsfrom used in EPR analysis were
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Poster Presentations a-33
derivatized with the sulfhydryl selective stable nitroxide radical, methanethiosulfonate, and trp
null - cys mutants used in FRET analysis were labeled with AEDANS. From EPR spectral
analysis, solvent accessibility, local flexibility, secondary structure and inter-molecular
distances between spin label(s) were assessed. EPR analysis identified the following elements
in apoA-I secondary structure: residues 80 to 129 are alpha helical, residues 130 to 138 form
a loop, residues 139 to 170 are alpha helical. In addition to secondary structural information,
quaternary structure was observed by inter-molecular spin - spin interactions between paired
monomers. At concentrations 0.1 mg/ml, apoA-I is a dimer with a foci of anti-parallel
interaction at residue 163. EPR examination of nascent HDL revealed that the secondary
structure of the central domain of apoA-I transitions to alpha helix. Coincident with this
conformational transition is repositioning of the foci of inter-molecular alignment from residue
163 to the vicinity of residue 131. These findings were confirmed by FRET analysis of nascent
discs bearing donor, acceptor pairs.
P190
Macrophage Depletion Alters Vein Graft Intimal Hyperplasia and Cytokine
Expression
Jeffrey J Tomas, Randall Wolff, Masaaki Ryomoto, John Hoch. University of Wisconsin
Medical School, Madison, WI
Objectives: We have previously shown that treatment with liposomally encapsulated dichloromethylene
bisphosphonate (L-Cl 2MBP) reduces intimal hyperplasia (IH) development and
macrophage accumulation in a rat epigastric vein to femoral artery model. Our objective in this
study was to determine the effect of L-Cl 2MBP on the expression of two cytokines essential to
neointimal development, transforming growth factor-beta1 (TGF-) and monocyte chemotactic
protein-1 (MCP-1). Methods: We injected rats both 2 days pre and 2 weeks post operatively
with L-Cl 2MBP, liposomally encapsulated phosphate buffered saline (L-PBS), or PBS, and
harvested the grafts at 1 and 4 weeks (n5 per group/time point). Grafts were examined
histologically to determine total and neointimal area, and total and neointimal cellularity. TGF-
and MCP-1 expression was determined by immunohistochemistry and reported as the area
stained as a percentage of total or neointimal area. Results: At 1 week and 4 weeks there was
a decrease in neointimal area in the L-Cl 2MBP group compared with L-PBS or PBS (p.005).
There was also a decrease in neointimal cellularity at 1 week (L-PBS p.0067; PBS
p.0163), with no significant differences observed at 4 weeks. MCP-1 and TGF- total
and neointimal area stained decreased in the L-Cl 2MBP treated group at 1 week when
compared to PBS or L-PBS (Table 1). At 4 weeks there were no significant differences in
total or neointimal area stained for MCP-1 or TGF-. Conclusion: We conclude that
macrophage depletion inhibits neointimal hyperplasia at 1 and 4 weeks, while the major
effect of L-Cl 2MBP treatment on TGF- and MCP-1 protein expression is observed at 1
week. The involvement of TGF- and MCP-1 in the development of IH provides valuable
insight into a significant clinical problem that is responsible for the failure of more than
20% of vein grafts after the first postsurgical year.
P191
High Plasma Level of Asymmetric Dimethylarginine is Associated with
Matrix Metalloproteinase-9 in Patients with Acute Coronary Syndrome
Euy-Myoung Jeong, Jeong-Euy Park. Samsung Medical Center and Samsung Biological
Research Institute, Seoul, Korea
PURPOSE Asymmetric dimethylarginine (ADMA) has been known as an atherogenic molecule
that inhibits nitric oxide synthase. Matrix metalloproteinase (MMP)-9 secreted by macrophages
potentially contributes to plaque rupture and tissue inhibitor of metalloproteinases (TIMP)-1. In
this study, we examine the relationship between plasma ADMA levels and MMP-9 in the
pathogenesis of atherosclerosis. METHODS Plasma samples were obtained from patients with
acute myocardial infarction (AMI, n12), unstable angina (n11), stable angina (n18),
variant angina (n10) and normal healthy subjects (control, n46) who were sex- and agematched.
Plasma ADMA levels were measured by high-performance liquid chromatography.
Plasma total MMP-9 and TIMP-1 levels were determined by ELISA. RESULT The plasma levels
of ADMA and MMP-9 were significantly higher in the patient groups with AMI and unstable
angina compared with control subjects, however not significantly higher in stable angina. In
variant angina by guest group, plasma on April ADMA4, levels 2013 were significantly higher than control subjects, but

a-34 Arterioscler Thromb Vasc Biol. Vol 22, No 5
MMP-9 levels were not changed statistically. Plasma TIMP-1 levels were not changed in any
group of patients. In patients with ACS, plasma ADMA levels were correlated with plasma
MMP-9 (r 0.272, p0.05). CONCLUSION Our results suggest that elevation of plasma ADMA
levels were significantly associated with MMP-9 in the patients ACS. These findings provide an
insight into the relationship between endothelial dysfunction and plaque destabilization.
P192
Gene Expression in Macrophage-Rich Shoulder Area of Atherosclerotic
Lesions Studied by Laser Microdissection and DNA Arrays
Tiina T Tuomisto, Mervi Riekkinen, Anna Korkeela, Juha Rutanen, Jan H Braesen, Konrad
Kolble, Seppo Yla-Herttuala. A.I.Virtanen Institute, University of Kuopio, Kuopio, Finland;
Franz Volhard Clinic, Charite University Hospital, Berlin, Germany; Max-Delbruck-Centrum
fur Molekulare Medizin, Institut fur Pathologie, Universitatsklinikum Charite, Berlin, Germany
Objective During atherogenesis, monocytes adhere to endothelium and are transformed to
macrophages and foam cells. Macrophages are mainly localized in the shoulder area of an
atherosclerotic plaque. Macrophages secrete many growth regulatory molecules and chemoattractans,
which affect atherosclerotic process. Methods Human arterial samples (n5) were
obtained from organ donors. Macrophage-rich shoulder areas of lesions and normal intimas
were dissected using laser microdissection (Leica). RNA was extracted and amplified using
T7-RNA-polymerase-based method. LifeGrid filters that contain 8 400 genes were used in DNA
array analyses. RNA from three different lesions was compared to pooled RNA from normal
intimas. Intensity ratios were calculated. Results were confirmed using RT-PCR. Results
Several macrophage specific genes e.g. interleukin 4 and genes affecting lipid metabolism e.g.
lipase related protein 2 were upregulated in the shoulder area. In addition, upregulation of
many novel genes or genes having unknown function were detected. Downregulation of several
genes, such as E2F transcription factor 4 and farnesul transferase were also detected.
Conclusions DNA array method combined with laser microdissection enables efficient
screening of gene expression in distinct areas of atherosclerotic lesion.
P193
Phospholipid Transfer Protein and Hepatic Lipase Are Associated with
Distinct LDL Subfractions
Susan J Murdoch, Molly C Carr, Amir F Ayyobi, Hal Kennedy, John D Brunzell, John J
Albers. University of Washington, Seattle, WA
Phospholipid transfer protein (PLTP) has been reported to play a significant role in HDL
metabolism but the effect on the apo B-containing lipoproteins has not been established. Due
to our previous observation of a positive correlation of PLTP activity with plasma apo B and LDL
cholesterol, the relationship of PLTP with LDL subfractions was investigated. Plasma
lipoproteins from 50 pre-menopausal women were fractionated by density gradient ultracentrifugation.
Spearman rank order correlations were calculated between the cholesterol
concentration of each of the 38 fractions and the plasma PLTP activity, hepatic lipase (HL)
activity, lipoprotein lipase (LPL) activity and cholesterol ester transfer protein (CETP) mass.
Activities were determined by a radiolabeled phospholipid vesicle/HDL assay (PLTP) or
radiolabeled triolein emulsion assay (LPL and HL). CETP was determined by ELISA. Plasma
PLTP activity was highly, positively correlated with the cholesterol concentration of all buoyant
LDL fractions (r0.47–0.57, p0.001) as well as dense IDL fractions (r0.29–0.41,
p0.003–0.04), but demonstrated no association with dense LDL. In contrast, HL activity
showed no association with buoyant LDL but was positively correlated with dense LDL fractions
(r0.38–0.43, p0.01). LPL activity was positively correlated with 3 buoyant LDL fractions
(r0.30–0.36, p0.01–0.034) and CETP was correlated with 2 dense IDL/buoyant LDL
fractions (r0.32–0.33, p0.02). The positive correlations of PLTP activity with the buoyant
LDL fractions were stronger than those observed for LPL and CETP and remained solely
associated in multivariate analysis. HDL fractions were also positively correlated with PLTP and
LPL activity whereas correlations with HL activity were consistently negative. CETP showed no
associations. In conclusion, the results suggest that PLTP and HL are important determinants
of LDL subpopulation density distributions exhibiting distinct relationships with buoyant and
dense LDL, respectively.
Hypoxia Stimulates Mitogen-Mediated Vascular Smooth Muscle Cell
Proliferation: Role of Heterotrimeric G Proteins
P194
Carita M Lanner, Rodney A Rhoades. Indiana University School of Medicine, Indianapolis, IN
Objectives: Smooth muscle proliferation is an important component in hypoxia-induced
pulmonary hypertension. However, little information is available regarding the effect of hypoxia
on G proteins and specific mitogens. The objectives of this investigation were to determine
if: 1. Hypoxia-induced pulmonary arterial smooth muscle (PASM) proliferation is mitogen
specific. 2. Gi and/or G12/13 proteins play a role in hypoxia-induced PASM cell proliferation.
Methods:. Porcine PASM cells (passages 1–4) were cultured in 5% fetal bovine serum (FBS)
with 5 ng/ml insulin. In some experiments, cells were supplemented with rhPDGF- (10
ng/ml) or hEGF (0.5 ng/ml) and h-bFGF (2 ng/ml). Pertussis toxin (PTX), an inhibitor of Gi proteins, was used at 100 ng/ml. Toxin B, an inhibitor of the small GTPase RhoA, was used at
10 ng/ml, to investigate the role of G12/13 proteins. PASM cells were exposed to 3% O2-5% CO2 (PO2 21–22 mm Hg) at 37°C in a humidified hypoxia chamber (Ruskinn Technology,
LTD; model 200). Proliferation was measured as an increase in cell number, which were
counted on days 1–4 using a Coulter counter (Model ZM). Findings: 1. PASM cells cultured in
EGF-bFGF showed the greatest hypoxic-induced stimulation (60% increase, P0.05, n3)
after 96 hr exposure. Cells cultured in PDGF showed a significant 40% increase (P0.05, n3)
and cells cultured in 5% FBS showed a 15% increase (P0.05, n3). 2. Proliferation of PASM
cells cultured PDGF or EGF-bFGF, showed a synergistic effect with hypoxia by day 4 (P0.05,
n3). 3. The hypoxia-induced proliferation of PASM cells cultured in PDGF or EGF-bFGF was
significantly reduced by PTX (P0.05, n3). Proliferation Downloaded of PTXfrom treated cells returned to
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normoxic levels. The hypoxia-induced cell proliferation was not affected in cell cultures treated
with toxin B. Conclusions: Hypoxia-induced stimulation of PASM cells showed a synergistic
effect when supplemented with autocrine growth factors, but was not mitogen specific. The
data strongly suggest that G i proteins play an important role in mediating hypoxia-induced
PASMC proliferation.
P195
In Vivo High-Resolution MR Imaging of Carotid Artery: Evaluation of Lesion
Type
Jianming Cai, Thomas S Hatsukami, Marina S Ferguson, Chun Yuan. University of
Washington, Seattle, WA; Veterans Affairs Puget Sound Health Care System, Seattle
Division, University of Washington, Seattle, WA; Marina Ferguson Inc, Seattle, WA
Purpose The purpose of this study was to determine whether in vivo high-resolution MR
imaging could accurately classify the human carotid atherosclerotic plaque according to
standard American Heart Association classifications. Methods and Materials Sixty consecutive
patients scheduled for carotid endarterectomy were imaged with a 1.5T scanner. A
standardized protocol was used to obtain 4 different contrast-weighted images (TOF, T1W,
PDW, T2W) of the carotid arteries. Best voxel size was 0.25x0.25x1mm. Carotid plaques were
removed intact and processed for histological examination. Seven patients were excluded
because of a poor image quality. One endarterectomy specimen was not suitable for histology
analysis. In order to decrease dependence, images were selected at 4mm distances. The
remaining 52 patients provided 252 locations. Classification was modified as: Type I-II—
normal wall thickness, no calcification; Type III—diffuse intimal thickening or small eccentric
plaque with no calcification; Type IV-V—plaque with a lipid or necrotic core surrounded by
fibrous tissue with possible calcification; Type VI—complex plaque with possible surface
defect, hemorrhage, or thrombosis; Type VII—calcified plaque; Type VIII—fibrotic plaque with
no lipid core and possible small calcifications. Both MR images and histological sections were
independently reviewed and categorized. Sensitivity and specificity were calculated. Cohen’s
Kappa value was computed to quantify the agreement between the MRI findings and histology.
Results Overall, the classification obtained by MR imaging and the modified AHA classifications
showed good agreement, with kappa 0.74. Compared to the histological gold standard, the
preliminary results of sensitivity and specificity of MR Imaging classification were: Type I-II,
67% and 100%; Type III, 81%and 98%; Type IV-V, 84% and 90%; Type VI, 82% and 91%; Type
VII, 80% and 94%; Type VIII, 56% and 100%. Conclusion High-resolution MRI is capable of
classifying intermediate to advanced atherosclerotic lesions in the human carotid artery, and
of distinguishing early and intermediate plaque from advanced lesions.
P196
Naringenin Inhibits Macrophage Foam Cell Formation Induced by
Cholesteryl Ester-Rich VLDL through Inhibition of Lipoprotein Uptake and
Enhanced Cholesterol Efflux
Carmen A Argmann, Nica M Borradaile, Kyle Geraghty, Robert A Hegele, Murray W Huff.
The John P Robarts Research Institute, London, ON, Canada
The citrus flavonoid, naringenin, has plasma cholesterol lowering and anti-inflammatory
properties. In this study, we assessed whether naringenin could modulate macrophage foam
cell formation induced by native lipoproteins. Macrophage foam cell formation can be induced
by cholesteryl ester (CE)-rich VLDL, which requires initial lipolysis by lipoprotein lipase (LPL)
and subsequent uptake of the remnant by the LDL receptor (LDLr). Incubation of J774A.1
murine macrophages with human VLDL (16h, 50g cholesterol/mL) increased cellular CE and
triglyceride (TG) mass by 12- and 55-fold respectively (both p0.05). Pre-incubation of
macrophages with naringenin (24h) dose dependently decreased CE mass with a maximum
inhibition of 60% at 200M (5.70/13.30 g CE/mg cell protein (PN), p0.05). Consistent with
a decreased CE mass, naringenin (200M) also reduced VLDL-induced oleate incorporation
into CE by 40% (3.30/5.50 nmole/mg PN, p0.05). However, TG mass induced by VLDL was
unaltered by naringenin. Consistent with this observation, naringenin did not affect macrophage
LPL mRNA expression. To address the mechanism for the naringenin-induced reduction in CE,
LDLr activity and the efflux of intracellular free cholesterol to an acceptor in the medium was
determined. Naringenin significantly decreased the total cell association and degradation of 125 I
LDL at 37°C by 57% (31.80/74.80 ng/mg PN) and 44% (156.90/280.70 ng/mg PN),
respectively (both p0.05). Naringenin significantly increased macrophage cholesterol efflux.
Addition of naringenin (200M, 16h) to cells, which had been pre-loaded with cholesterol ( 3 H
cholesterol, Acetylated LDL, 24h) enhanced cholesterol efflux to apolipoprotein AI (12h) by
1.70-fold (p0.05). Furthermore, naringenin increased macrophage ABCA1 mRNA abundance
by 1.50-fold (p0.05). We conclude that naringenin inhibits macrophage foam cell formation
through a two-step mechanism; inhibition of receptor mediated VLDL-remnant uptake and
enhanced cholesterol efflux. Our results suggest that this flavonoid may have anti-atherogenic
effects in vivo.
VASP: A Potential Mediator of the Inhibitory Effect of NO in Vascular
Smooth Muscle Cells
Lihua Chen, Günter Daum, Scott Coats, Daniel Bowen-Pope, Ulrich Walter, Alexander W
Clowes. University of Washington, Seattle, WA; Institut für Klinische Biochemie und
Pathobiochemie, Klinikum der Universität Würzburg, Würzburg, Germany
P197
Smooth muscle cell (SMC) proliferation plays an important role in developing atherosclerosis
and restenosis after angioplasty. Many in vitro and in vivo studies suggested that nitric oxide
(NO), a potent vasodilator, might be an inhibitor of SMC proliferation. The mechanism how NO
inhibits SMC growth is unclear. Several experimental results indicate this inhibitory effect of NO
may be through one of its intracellular signaling pathway: the activation of soluble guanylyl
cyclase, followed by guest by the on increase April of 4, cGMP 2013 level and in turn the activation of cGMP-dependent

protein kinase (PKG). One of the substrates of PKG is vasodilator stimulated-phosphoprotein
(VASP). In SMCs, VASP is mainly localized in focal adhesion sites and can be phosphorylated
upon the stimulation of NO or other vasodilators. This study is to investigate a potential role of
VASP in mediating the growth inhibitory effect by NO in SMCs with a focus on the role of the
PKG-preferred phosphorylation site on VASP: serine239. Methods: Mutant VASP cDNA was
constructed in which Ser239 was replaced by a non-phosphorylatable amino acid, Ala. Cultured
Fischer rat vascular SMCs were transformed with this mutant VASP cDNA as well as wild type
(wt) VASP cDNA using a tetracycline (tet)-regulatable system. Western blot was performed to
examine the expression of VASP in the transformants. The cell proliferation and the inhibition
by NO were assessed by 3 H-thymidine incorporation upon the stimulation with 10% fetal bovine
serum (FBS) in the absence or presence of a NO donor, S-nitroso-N-acetylpenicillamine (SNAP).
Results: Expression of both mutant VASP and wt-VASP in SMCs increase the 3 H-thymidine
incorporation upon FBS stimulation. However, the cells expressing mutant VASP (mt-VASP) is
less sensitive to the inhibition of 3 H-thymidine incorporation upon FBS stimulation by SNAP
when compared to the cells expressing wt-VASP. Conclusion: The phosphorylation site Ser239
of VASP might be involved in mediating the inhibitory effect of NO on SMC proliferation.
Electrical Impulses Prevent Development of Atherosclerosis in
Cholesterol-Fed Rabbits
Valeri S Chekanov, Mohammad Mortada, David Krum, Guennady V Tchekanov, Ruben
Eisenstein, Masood Akhtar. Sinai/St. Luke’s Medical Centers, University of
Wisconsin-Milwaukee Clinical Campus, Milwaukee, WI
P198
Objectives. This investigation studied whether low frequency electrical impulses (EI) induce or
prevent development of new atherosclerotic plaque in previously diseased vessels. Methods. In
all rabbits, an electrode was sutured to the left psoas major muscle close to the abdominal
aorta (AA), and a pacemaker was implanted on the opposite side of the AA. Group 1 received
a high cholesterol diet (HCD) to induce atherosclerosis but no EI (control). Euthanasia followed
after 3 weeks (series I), 8 weeks (series II) and 11 weeks (series III) of HCD. Group 2 received
HCD alone for 3 weeks then EI was added to the HCD for another 8 weeks (weeks 4–11) at
a rate of 30 impulses per minute (IPM) at 3V (series IV), 2V (series V), 1V (series VI) and at a
rate of 60 IPM at 3V (series VII), 2V (series VIII), and 1V (series IX). Euthanasia followed after
11 weeks. Atherosclerotic AA thickness grades were assigned using a 0 (low) to 4 (high)
grading system, and the surface area involved in disease was calculated. Results. In control
rabbits, after 11 weeks of HCD atherosclerotic thickness grade was 2.750.5. In rabbits
exposed to EI (30 IPM at 3V) this grade was 0.50.4 (p0.05). The involved surface area was
only 53% (series IV) vs. 329% in control (p0.05). Results were poorer than in series IV
with the EI regimen used in series V-IX. Conclusion. When applied near the AA, electrical
impulses (30 IPM at 3V) decrease atherosclerotic deposits despite continuation of a high
cholesterol diet.
P199
Vascular Endothelial Growth Factor-Mediated Regulation of Hemostatic
Genes in Endothelial Cells
Zarin Kachra, William C Aird. Beth Israel Deaconess Medical Center, Boston, MA
The endothelium plays an important role in mediating hemostatic balance. During angiogenesis,
hemostasis must be tightly regulated to prevent inappropriate bleeding and/or clotting at
the sites of new blood vessel formation. In the present study we have investigated the role of
vascular endothelial growth factor (VEGF), a potent angiogenic factor, on the regulation of
hemostatic gene expression in human umbilical vein endothelial cells (HUVEC). In Northern blot
analyses, 40 ng/ml VEGF was shown to induce mRNA expression of tissue factor (TF),
plasminogen activator inhibitor (PAI)-1, von Willebrand factor (vWF), urokinase-type plasminogen
activator (uPA), uPA receptor (uPAR), tissue-type plasminogen activator (t-PA) and
thrombomodulin (TM) in a time- and dose-dependent manner. TF was stimulated by 3.26 /-
0.15 fold (maximal at 3h), PAI-1 by 1.43 /- 0.25 fold (3h), vWF by 1.8 /- 0.20 fold (24h),
uPA by 2.22 /- 0.18 fold (12h), uPAR by 2.20 /- 0.27 fold (12h), t-PA by 2.35 /- 0.20 fold
(12h) and TM by 2.72 /- 0.15 fold (24h) compared with untreated controls. In contrast, VEGF
did not alter the expression of tissue factor pathway inhibitor or endothelial protein C receptor.
VEGF-mediated induction of the above genes was blocked by pre-treatment of HUVEC with
actinomycin D or SU1498, a KDR/Flk-1 antagonist. To characterize the signaling pathways
involved, HUVEC were pretreated with specific inhibitors of PKC (bisindolylmaleimide I, BIM and
Gö6983), ERK1/2 (PD98059), PI-3 kinase (LY294002), or P38 MAP kinase (SB203580). BIM and
Göl6983 partially inhibited VEGF stimulation of TF, PAI-1, uPA, uPAR, and TM. PD98059
inhibited the effect of VEGF on TF and vWF. SB203580 inhibited VEGF stimulation of TF and
increased the effect of VEGF on TM. The addition of LY294002 reduced VEGF-mediated
induction of TF, uPA and uPAR and resulted in a superinduction of TM. Taken together, these
latter results suggest that VEGF induces the expression of hemostatic genes via diverse
downstream signaling pathways. In conclusion, we propose that VEGF may play an important
role in regulating the local hemostatic balance of the endothelium.
Inhibition of Hepatocyte ApoB Secretion by the Grapefruit Flavonoid,
Naringenin, Involves Activation of Phosphoinositide 3-Kinase
Nica M Borradaile, Linda E De Dreu, Murray W Huff. The John P. Robarts Research
Institute, London, ON, Canada
P200
We recently reported that naringenin inhibits HepG2 cell apoB secretion via inhibition of MTP
expression and activity, and enhanced LDL receptor (LDLr) expression and activity. We
postulated that the increase in LDLr activity would enhance re-uptake of newly secreted
apoB-containing lipoproteins, thus limiting apoB accumulation in the media. Since activation of
phosphoinositide 3-kinase (PI3-K) by insulin inhibits hepatocyte apoB secretion, we determined
whether this pathway was involved in naringenin-induced Downloaded inhibition from
of apoB secretion and
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Poster Presentations a-35
upregulation of the LDLr. HepG2 cells were pre-incubated with or without the PI3-K inhibitor,
wortmannin (1M), followed by 24h with either increasing doses of naringenin or insulin
(100nM). Naringenin decreased apoB accumulation in the media by up to 89% (200M,
0.03/0.285 OD units, p0.05). This reduction was attenuated by 20% (0.09/0.285, p0.05)
in cells pretreated with wortmannin. Insulin inhibited apoB accumulation by only 44%
(0.16/0.285, p0.01). This effect, however, was completely blocked by wortmannin. To
determine if naringenin-induced upregulation of the LDLr was mediated by PI3-K activation,
cells pre-incubated with either wortmannin or LY294002 (50M) were treated with either
naringenin or insulin for 6h. Both naringenin and insulin increased LDLr mRNA abundance by
1.8 fold (p0.05), effects completely blocked by either PI3-K inhibitor. To determine if
upregulation of the LDLr was mediated via PI3-K-induced increase in SREBP1, cells were
incubated for 6h with naringenin or insulin, and cellular SREBP1 (125KDa) mass was
determined. Naringenin and insulin increased SREBP1 by 2.2 fold and 1.5 fold, respectively
(p0.02), effects again completely blocked by either PI3-K inhibitor. We conclude that
naringenin increases LDLr expression in HepG2 cells via PI3-K-mediated upregulation of
SREBP1, thus enhancing re-uptake of apoB from the media. This mechanism, which is shared
by insulin, contributes to the ability of naringenin to decrease net secretion of apoB from
hepatocytes.
PPAR Gamma Ligands 9-HODE and Troglitazone Stimulate Matrix
Metalloproteinase-1 Expression in Vascular Endothelial Cells via
Extracellular Signal-Regulated Kinase
P201
Bryan A Game, Maria F Lopes-Virella, Yan Huang. Ralph H. Johnson VA Medical Center and
Medical University of South Carolina, Charleston, SC
Matrix metalloproteinase-1 (MMP-1) secreted by vascular endothelial cells plays an essential
role in neovessel formation in atherosclerotic plaques. A well-developed neovascular network
is associated with plaque destabilization and plaque hemorrhage. Since our recent studies have
shown that oxidized LDL stimulates MMP-1 expression in vascular endothelial cells and others
have shown that certain lipid components in oxidized LDL such as 9- and 13hydroxyoctadecadienoic
acid (9- and 13-HODE) activate peroxisome proliferator-activated
receptor gamma (PPAR-), we investigated whether 9- and 13-HODE as well as other PPAR-
agonists are capable of stimulating MMP-1 expression in endothelial cells. Our Northern blot
showed that 9-HODE or troglitazone stimulated MMP-1 mRNA expression in a concentrationdependent
manner, and 10 M of 9-HODE and 10 M of troglitazone stimulated MMP-1 mRNA
expression by 3 folds. Other PPAR- ligands such as 13-HODE and 15-deoxy-12,14-PGJ2
(15-d PGJ2) also stimulated MMP-1 mRNA expression, but to a lower extent. To determine
whether PPAR- is involved in the stimulation, cells were treated with 9-HODE or troglitazone
in the presence or absence of PPAR- antagonist prostaglandin F2alpha (PGF2). Northern
analysis showed that PGF2 (250–1000 M) had no effect on 9-HODE or troglitazone-induced
MMP-1 mRNA expression. Furthermore, overexpression of PPAR- by cells after transfection
with the PPAR- expression plasmid pCMX-P PAR- did not potentiate 9-HODE or troglitazonestimulated
MMP-1 expression. Thus, our results suggest that these PPAR- agonists may
stimulate MMP-1 expression through a PPAR--independent mechanism. Finally, our data
showed that both 9-HODE and troglitazone were able to stimulate ERK1/2 phosphorylation, and
10 M of PD98059 inhibited 90% of 9-HODE or troglitazone-stimulated MMP-1 expression. In
summary, this study demonstrates for the first time that PPAR- ligands 9-HODE and
troglitazone stimulate MMP-1 expression in vascular endothelial cells and the stimulation is
dependent of ERK pathway, but not PPAR-.
The Balance Between PGD Synthase and PGE Synthase is a Major
Determinant of Atherosclerotic Plaque Instability in Humans
P202
Francesco Cipollone, Matteo Marini, Maria Fazia, Annalisa Iezzi, Domenico De Cesare,
Barbara Pini, Sante Ucchino, Francesco Spigonardo, Guido Baiocchi, Cesaria Prontera,
Francesco Chiarelli, Franco Cuccurullo, Andrea Mezzetti. University “G. D’Annunzio”, Chieti,
Italy
Cyclooxygenases (COX) catalyze the conversion of arachidonic acid to prostaglandin (PG)H2, the
first step in PG synthesis. COX-2 is a pro-inflammatory enzyme, and we have described a role
for inducible COX2 and PGE synthase (mPGES) in metalloproteinase (MMP) biosynthesis leading
to plaque rupture. However, COX-2 may be also anti-inflammatory during high PGD synthase
(PGDS) activity. In fact, the PGDS end-product 15-deoxy-12,14-PGJ2 (15d-PGJ2) inhibits
multiple steps in the NF-B pathway, possibly contributing to plaque stabilization. Thus, we
studied whether a shift in the isomerase activity in the sense of a PGES-oriented metabolism
could lead to reduced 15d-PGJ2 and enhanced PGE2biosynthesis, inflammatory activation and
MMP production in rupture-prone plaques. Plaques were obtained from 60 patients undergoing
carotid endarterectomy and divided in symptomatic and asymptomatic according to the
presence of TIA or stroke in the last 120 days. Plaques were analyzed for COX-1, COX-2,
L-PGDS, mPGES, IB, PPAR, NF-B, MMP-2 and MMP-9 by immunocytochemistry,
fluorescence and Western blot, whereas zymography was used to detect MMP activity.
Immunocytochemistry was also used to identify CD68 macrophages, CD3 T lymphocytes
and HLA-DR cells. Inflammatory infiltration and mPGES expression was significantly higher in
symptomatic plaques. In contrast, L-PGDS was the prevalent isomerase in asymptomatic
plaques. Both isomerases were coupled with COX-2 in macrophages. The PGDS-prevalent
profile was associated with enhanced PPAR and IkB and reduced NF-B and MMP
expression and activity. COX-2 inhibition by NS-398 in vitro during minimal 15d-PGJ2 synthesis
was accompanied by reduced NF-B and MMP activity that was reverted by PGE2. In contrast,
NS-398 exacerbated inflammation in the presence of high L-PGDS activity, and this effect was
reverted by replacement of 15d-PGJ2. Thus, these results are consistent with the pro- and the
anti-inflammatory nature of COX-2 in human plaques, and suggest that the shift between PGDS
and PGES in macrophages is associated with ischemic syndromes, possibly through
MMP-induced by guest plaque rupture. on April 4, 2013

a-36 Arterioscler Thromb Vasc Biol. Vol 22, No 5
P203
Thrombin Stimulation of the Vascular Cell Adhesion Molecule-1 Promoter
in Primary Human Endothelial Cells is Mediated by Tandem NF-B and
GATA Motifs
Takashi Minami, Jie Zhang, William C Aird. Beth Israel Deaconess Medical Center, Boston,
MA
Vascular adhesion molecule-1 (VCAM-1) is a 110-kDa cell surface glycoprotein expressed by
cytokine-activated endothelial cells. The goal of this study was to delineate the transcriptional
mechanisms underlying thrombin-mediated induction of VCAM-1. The addition of thrombin to
human umbilical vein endothelial cells (HUVEC) resulted in increased VCAM-1 mRNA levels and
VCAM-1 promoter activity. Thrombin-mediated induction of VCAM-1 mRNA expression and
promoter activity was blocked by inhibitors of PKC (bisindolylmaleimide I, BIM), PI-3 kinase
(LY294002), or P38 MAP kinase (SB203580), but not p42/44 MAP kinase (PD98059). The
upstream promoter region of VCAM-1 contains a thrombin-response element (TRE), two NF-B
motifs and a tandem GATA motif. In transient transfection assays, mutation of the TRE had no
effect on thrombin induction. In contrast, mutation of either NF-B site resulted in a complete
loss of induction, while a mutation of the two GATA motifs resulted in a significant reduction
in thrombin stimulation. In electrophoretic mobility shift assays, nuclear extracts from
thrombin-treated endothelial cells displayed markedly increased binding to the tandem NF-kB
and GATA motifs. The NF-B complex was supershifted with anti-p65 antibodies, but not with
antibodies to RelB, c-Rel, p50 or p52. The GATA complex was supershifted with antibodies to
GATA-2, but not GATA-3 or GATA-6. A construct containing tandem copies of the VCAM-1 GATA
motifs linked to a minimal TK promoter was induced 2.4-fold by thrombin. Thrombin treatment
did not alter GATA-2 mRNA levels, as measured by ribonuclease protection assays. In
immunoprecipitation assays, there was no detectable change in total serine or threonine
phosphorylation of GATA-2 in response to thrombin. Taken together, these results suggest that
1) thrombin stimulation of VCAM-1 in endothelial cells is mediated by tandem NF-B and GATA
motifs, 2) thrombin stimulation of GATA-2 DNA binding activity is mediated at a posttranscriptional
level and 3) thrombin induces VCAM-1 mRNA and promoter activity via a PI3K,
PKC and p38 MAP kinase-dependent pathway.
P204
Hepatic VLDL-ApoB Secretion is Directly Linked to Expression of the ER60
Protease and Microsomal Triglyceride Transfer Protein: Implications in
Insulin-Resistant States
Wei Qiu, Changiz Taghibiglou, Taryne M Chong, Denny Trinh, Khosrow Adeli. University of
Toronto, Toronto, ON, Canada
Objective: A fructose-fed hamster model of insulin resistance has been recently reported by our
laboratory to exhibit hepatic overproduction of VLDL partly due to enhanced free fatty acid flux
and downregulation of hepatic insulin signaling. VLDL-apoB overproduction in this model was
accompanied by enhanced levels of microsomal triglyceride transfer protein (MTP), and
suppressed expression of ER60, a cysteine protease postulated to play a role in intracellular
apoB degradation. Here we investigated the expression and regulation of ER60 and MTP and
their role in controlling VLDL overproduction. Results: First we constructed adenoviral
expression vectors carrying the human or rat ER60 gene. We found a dosage dependent
overexpression of human and rat ER60 protein in HepG2 and HEK 293 cultured cells by
infection of recombinant adenoviral vectors. Overexpression of human ER60 protein in HepG2
cells caused significant downregulation of both cellular and secreted apoB by 50% in
pulse-chase experiments. We also hypothesized that enhanced free fatty acid flux as observed
in insulin resistance may be responsible for upregulation of MTP and VLDL-apoB secretion. To
test this hypothesis, we investigated fatty acid-mediated regulation of MTP gene expression by
using a luciferase reporter construct under the control of the promoter region of the MTP gene.
The MTP promoter activity was significantly stimulated by oleic acid in a dosage and time
dependent manner in HepG2 cells. A 1.85 fold (P0.0032) increase was observed in the
presence of 360 M oleic acid after 1 hour stimulation. Conclusions: Expression of ER60
protein is directly linked to the assembly and secretion of apoB. Fatty acids can regulate MTP
expression, implicating the enhanced free fatty acid flux observed in insulin resistant states
with increased levels of MTP and thus enhanced assembly and secretion of VLDL-apoB.
P205
Overexpression of 12-Lipoxygenase Increases Vascular Smooth Muscle Cell
Proliferation
Pushpa-Rekha R Thimmalapura, Konkal-Matt R Prasad, Rama Natarajan, Jerry L Nadler.
University of Virginia Medical Center, Charlottesville, VA; City of Hope National Medical
Center, Duarte, CA
Vascular smooth muscle cell (SMC) proliferation is a key event in the development of
atherosclerosis and restenosis following vascular injury. Several lines of evidence implicate
12-lipoxygenase (12-LO) pathway to be important for SMC growth. 12-LO expression is
increased in baloon injured rat carotid arteries and inhibition of 12-LO with a specific ribozyme
inhibits neointima formation. Recent evidence shows that ApoE and 12-LO double knockout
mice develop fewer atherosclerotic lesions compared to control ApoE knockout mice. Present
studies were undertaken to determine the direct effect of overexpression of 12-LO on SMC
growth. The mouse leukocyte type 12-LO cDNA (12LO-A10) or the vector pcDNA (control) were
stably overexpressed in A10 cells, a rat smooth muscle cell line. Immunoblotting and
immunohistochemical staining of cells with antibodies against 12-LO showed overexpression
of 12-LO protein in 12LO-A10 cells compared to control cells. 12-LO mRNA was 26 fold
increased in 12LO-A10 cells as determined by reverse transcription and real-time PCR.
12LO-A10 cells showed elevated levels of the 12-LO product 12-hydroxyeicosatetraenoic acid
(12-HETE) in the cell pellet (213.2 Vs 28.15 pg/105 cells) as well as in the media (17.3 Vs 8.6
pg/105 cells) compared to control cells. To examine the effect of 12-LO overexpression on SMC
growth, cell proliferation assay was performed. Cells Downloaded were trypsinized from
and counted in a coulter
http://atvb.ahajournals.org/
counter. Results demonstrate that the 12LO-A10 cells proliferate significantly more compared
to control cells (6628 452 Vs 4433 21 cells at day 5, P0.01). In order to get insight into
the mechanism by which 12-LO increases proliferation, activation of MAP kinases was
determined by immunoblotting with antibodies to phosphorylated ERK1/2 and p38. Results
show that pERK1/2 and p38 are increased in 12LO-A10 cells compared to control cells. This
is consistent with our earlier report that showed that 12-HETE activates ERK1/2 and p38. In
conclusion, these results provide direct evidence for the role of 12-LO in SMC proliferation and
suggests that 12-LO could be a target to reduce abnormal hyperplasia of SMC.
P206
Role of Cyclin-Dependent Kinase Inhibitor p21/Cip1 in Glucose-Induced
Proliferation of Vascular Smooth Muscle Cells
Pushpa-Rekha R Thimmalapura, Jerry L Nadler, Coleen A McNamara. University of Virginia
Medical Center, Charlottesville, VA
Vascular smooth muscle cells (SMC) cultured in high glucose (HG, 25 mM) proliferate
significantly more compared to those in normal glucose (NG, 5.5 mM) conditions. However, the
effects of glucose on the components that drive the cell cycle are poorly understood. The cyclin
dependent kinase inhibitor, p21/Cip1, is an important cell cycle regulatory protein. Although it
is widely recognized as a cell cycle inhibitor, recent studies provide evidence that p21 at low
levels is required for growth factor induced SMC proliferation (Weiss et al, J. Biol. Chem. 275:
10285, 2000). The present study was under taken to determine if glucose regulates the
expression of p21. Expression of p21 in response to Angiotensin II (Ang II) was also examined.
Rat aortic SMC were cultured in NG or HG in regular growth conditions. L-Glucose was used
as an osmolality control. After 7–10 days in different glucose conditions, cells were serum
starved and then stimulated with serum or Ang II in respective glucose conditions. Cells were
harvested 24 h following stimulation and p21 protein expression was examined by immonoblotting.
Results demonstrate that SMC cultured in HG had a significant increase in p21 protein
levels relative to cells cultured in NG in serum starved condition (n4, 1.4 fold, P0.05).
Similar results were obtained for HG cultured cells when stimulated with serum (1.6 fold,
P0.01) or Ang II (1.53 fold, P0.03). This effect was not mimicked by L-glucose. To examine
if the increased expression of p21 was due to increased transcription, SMC were transfected
with a p21 promoter-luciferase reporter construct. Transfected SMC were exposed to HG,
harvested 24 h later and luciferase activity was measured. Results indicate that p21 promoter
activity was 40 % increased in HG compared to NG condition (n3, P 0.003). The modest
increase in p21 expression is consistent with its function as a growth promoting factor rather
than as a growth inhibitor as seen in situations where p21 is overexpressed. Taken together,
these results suggest that increased p21 expression in HG probably contributes to enhanced
SMC proliferation due to HG.
P207
Versican Synthesized in the Presence of Troglitazone is Smaller and Has
Reduced LDL Binding
Lisa R Tannock, Peter J Little, P Hugh R Barrett, Thomas N Wight, Alan Chait. University of
Washington, Seattle, WA; Baker Medical Research Institute, Melbourne, Australia; University
of Western Australia, Perth, Australia; Hope Heart Institute, Seattle, WA
Troglitazone has been shown to reduce development of atherosclerosis in two mouse models,
and to reduce mesangial proteoglycan synthesis. Proteoglycans play a key role in atherogenesis
due to their ability to bind and trap atherogenic lipoproteins in the artery wall. Modifications
of proteoglycans that decrease their ability to bind lipoproteins may be atheroprotective. To
determine if troglitazone modified vascular proteoglycan synthesis, aortic smooth muscle cells
were exposed to troglitazone (10mM) for 24 hours. Secreted proteoglycans synthesized in the
presence of troglitazone were smaller, and sulfate incorporation was reduced to 80.1 5.3%
of control (p0.01). Proteoglycans were separated into peak 1 (versican) and peak 2 (dermatan
sulfate proteoglycans) by molecular sieve chromatography. LDL binding affinity was assessed
using a gel mobility shift assay. Versican synthesized in the presence of troglitazone had lower
binding affinity than control (Kd 47 5 versus 184 26 g/ml LDL for versican synthesized
in the absence or presence of troglitazone, p0.01). However, the binding of dermatan sulfate
proteoglycans synthesized in the presence of troglitazone did not differ from control.
Glycosaminoglycan chains cleaved from versican synthesized in the presence of troglitazone
also demonstrated reduced binding affinity for LDL compared to control glycosaminoglycans
(Kd 345 70 versus 577 7 g/ml LDL for control and troglitazone glycosaminoglycans,
p0.02), whereas there was no difference in binding for glycosaminoglycan chains cleaved
from peak 2 proteoglycans. Thus, troglitazone appears to have a specific effect on
glycosaminoglycan chains on versican, reducing the binding affinity for LDL. This may
contribute to the reduced atherosclerosis seen in animal models.
Movement of PDGF Beta Receptor in Migrating Endothelial Cells on
Vascular Thrombus
Shu Q Liu, Lin Zhong, Jeremy Goldman. Northwestern University, Evanston, IL
P208
The movement of cell membrane receptors has been implicated in the regulation of cell
function and is possibly mediated by cytoskeleton. Here we demonstrate that the motility of
platelet-derived growth factor beta receptor (PDGFR-beta) aggregates depends on the integrity
of actin filaments in migrating endothelial cells (ECs) on vascular thrombus developed on a rat
venous polymeric implant. The movement of PDGFR-beta aggregates was mainly found in
peripheral migrating ECs with a slightly larger degree at the leading edge (0.23/-0.06 and
0.22/-0.05 microns/sec at day 5 and 7, respectively) than at the trailing edge (0.21/-0.04
and 0.19/-0.06 microns/sec at day 5 and 7, respectively). The force required to move a
PDGFR-beta aggregate was 1.94x10 -3/-6.58x10 -4 piconewtons. While the short-term
movement of PDGFR-beta aggregates (within 1 second) appeared random, the long-term
movement by (within guest 30 seconds) on April was4, consistent 2013 with the direction of EC migration. A treatment

with cytochalasin D reduced the movement of PDGFR-beta aggregates. The motility of
PDGFR-beta aggregates also depended on the activity of PDGFR-beta tyrosine kinase, Rho
kinase, and myosin light chain kinase. This study suggests that the movement of PDGFR-beta
aggregates may facilitate cell membrane extension and thereby cell migration.
Is apo B 48 Important for Chylomicron Metabolism?
Cam Phan, Trevor Redgrave, Shuqin Zheng, Shrikant Anant, Nicholas O Davidson, David Y
Hui, Patrick Tso. University of Cincinnati, Cincinnati, OH; University of Western Australia,
Perth, WA, Australia; Washington University, St. Louis, MO
P209
Apolipoprotein B 48 (apo B 48) is an intestinally derived apolipoprotein associated with chylomicrons.
Chylomicrons are rapidly metabolized to form remnant particles before removal by the
liver. In contrast, hepatic derived apo B 100 containing VLDL are converted to LDL. Is the
difference in chylomicron and VLDL metabolism a result of the presence of apo B 48 versus apo
B 100? The apobec-1 knockout mouse allows us the opportunity to test this possibility since the
apoB mRNA editing enzyme, apobec-1 is not present and only apo B 100 containing chylomicrons
are produced. In this study, chylomicrons were collected from C57BL/6 and from apobec-1
knockout mice and the plasma clearance of the different chylomicrons were studied in C57BL/6
mice. Lymph was collected from donor lymph fistula mice (apobec-1 or C57BL/6) infused
intra-duodenally with an Intralipid/taurocholate mixture labeled with 14 C-cholesterol and
3 H-triolein. Chylomicrons were isolated by density gradient ultra-centrifugation and injected, via
tail vein into recipient C57BL/6 mice. Blood samples were taken over 20 mins and plasma
counted to determine the disappearance of both labels over time. Liver and spleen were
removed at the end of 20 mins to determine label uptake. Chylomicron lipolysis was
determined by the plasma disappearance of 3 H-triolein while chylomicron remnant removal
was determined by the disappearance of 14 C-cholesterol. Plasma chylomicron-triolein and
cholesterol disappearance curves were not different for chylomicrons produced from apobec-1
mice compared with those from C57BL/6. Within 3 mins post-injection of both types of
chylomicrons, approximately 70% of the triolein label (chylomicron hydrolysis) disappeared
from the circulation and 90% by 5 mins. About 35–40% of the cholesterol label (remnant
removal) was removed by 3 mins and 90% by 20 min. Organ uptakes of both labels are also
similar between wild type and apobec-1 knockout chylomicrons. We conclude: 1) the
substitution of apo B 100 in place of apo B 48 in chylomicron particles had minimal effects on the
plasma clearance of triglyceride and cholesterol; 2) the difference in plasma clearance between
chylomicron and hepatic VLDL is not due to the type of apo B present.
P210
Retinoids Activate Cyclic-AMP Response Element Binding Protein (CREB) in
Vascular SMC via Protein Kinase A
Jeffrey W Streb, Mary A Georger, Joseph M Miano. University of Rochester Medical Center,
Rochester, NY
Retinoids have been shown to inhibit smooth muscle cell (SMC) growth in vitro and in vivo.
While the mechanism of this inhibition remains unclear, retinoids are known to act through the
retinoid receptors, a group of ligand-activated transcription factors, to modulate gene
expression. One such gene is the tumor suppressor SSeCKS, a multivalent scaffold protein that
binds and modulates the activity of Protein Kinase A (PKA) and Protein Kinase C (PKC). A
downstream target of both of these kinases is CREB, a multifarious transcription factor that also
acts as a co-factor for the retinoid receptors. We therefore examined the possible role of CREB
in mediating retinoid effects in SMC. Agonists that stimulate the cAMP-PKA-CREB pathway
inhibited serum-induced SMC growth to a similar degree as retinoids. While forskolin raised
global cAMP levels, retinoid treatment had little or no observed effect. However, levels of the
phosphorylated, active form of CREB (pCREB) did increase following retinoid treatment. Further,
retinoid treatment led to a dose-dependent increase in CREB-mediated transcription. In
contrast to agonists that lead to a rapid, short-term elevation of pCREB and CREB-assisted
transcription, the retinoid stimulated increase in pCREB and CREB-mediated transcription was
protracted. The increase in pCREB was not due to an increase in total CREB as assessed by
Western blotting. Since CREB can be activated via PKA- and PKC-mediated phosphorylation, we
examined the role of these kinases in retinoid-induced CREB activation. Only the PKA inhibitor
H-89 blocked forskolin-and retinoid-induced activation of CREB. These results indicate that
retinoid-induced activation of CREB involves PKA, but is independent of global increases in
cAMP. Since CREB can inhibit SMC growth, our results suggest a role for this transcription
factor in retinoid-induced SMC growth inhibition. Further studies are underway to better define
this pathway.
FGF2 Overexpression Prevents Ceramide-Mediated Akt Deactivation in
Endothelial Cells Exposed to Oxidized LDL
Jonathan Lu, Shinichi Suzuki, Hazim J Safi, Wei Jiang, Philip D Henry, Chao-Yuh Yang,
Chu-Huang Chen. Baylor College of Medicine, Houston, TX; University of Texas-Houston
Medical School, Houston, TX
P211
Apoptosis of vascular endothelial cells (EC) plays a critical role in atherothrombosis and
modified lipoproteins are considered proapoptotic. We recently demonstrated that copperoxidized
LDL (oxLDL) induces EC apoptosis in part by downregulating fibroblast growth factor
2 (FGF2). FGF2 is a primary activator of the phosphatidylinositol-3-kinase (PI3K) that activates
Akt/protein kinase B by means of phosphorylation. Akt inhibits apoptosis by deactivating
downstream apoptotic mediators. Because our primary experiments showed that oxLDL
increased turnover of ceramide, an intracellular lipid mediator, we investigated the interaction
between the FGF2-PI3K-Akt axis and ceramide in the signaling pathways triggered by oxLDL.
In bovine aortic EC (BAEC) cultures, oxLDL (50 g/mL) induced marked apoptosis at 24 hours,
as assessed by epi-fluorescence microscopy, DNA laddering, and flow cytometry. The
apoptosis was accompanied by Akt deactivation, Downloaded as evaluated byfrom reduced phosphorylation.
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Poster Presentations a-37
Wortmannin (50 nM), a PI3K inhibitor, also deactivated Akt and provoked oxLDL-like apoptosis.
Fumonisin B-1 (400 nM), a ceramide synthase inhibitor, effectively attenuated oxLDL-induced
Akt deactivation and the associated apoptosis, suggesting a participating role of newly
synthesized ceramide. Both membrane permeable C2- and C6-ceramide (50 M) inhibited Akt
phosphorylation and induced apoptosis. Cells transfected with adenoviral vectors containing an
FGF2 antisense oligonucleotide completely abolished FGF2 expression and Akt phosphorylation.
In contrast, overexpressing FGF2 in BAEC by transfecting the cells with FGF2 sense-containing
vectors prevented oxLDL or ceramide-induced Akt deactivation and the associated apoptosis.
Thus, oxLDL acts in part by stimulating production of ceramide, which participates in Akt
deactivation. Concomitant FGF2 downregulation deprives the cells of an important defensive
mechanism that relies on the integrity of the FGF2-PI3K-Akt axis. FGF2 overexpression renders
the cells resistant to ceramide-mediated Akt deactivation in EC exposed to modified
lipoproteins, such as oxLDL.
P212
Topology and Functional Analysis of the Large Extracellular Aminoterminal
and Regulatory Domains
Michael L Fitzgerald, Lorna P Andersson, Sarah Rhee, Armando J Mendez, Mason W
Freeman. Mass. Gen Hospital/Harvard Medical School, Boston, MA; University of Miami
Medical School, Miami, FL
Mutations in ABCA1 can result in a loss of cholesterol efflux from cultured fibroblasts and are
responsible for causing Tangier disease. Hydrophobicity plots of ABCA1 predict more
hydrophobic domains than the canonical 12 which would be expected in a full ABC transporter.
Thus, the topological orientation of the ABCA1 polypeptide chain remains uncertain. Previously
we have shown that the first hydrophobic domain of the protein (AA 25–42) acts as a signal
anchor sequence that results in the translocation of the downstream 590 amino acids to the
extracellular space. Here we report that the central highly hydrophobic domain (AA 1351–1372,
or H8) also traverses the plasma membrane leading to the extracellular positioning of residues
1352–1649. Using the full length transporter with a FLAG tag epitope placed between AA 1396
and 1397 (Flag2), we found comparable antibody binding to this tag compared to the same tag
placed in the amino terminal loop previously demonstrated to be extracellular (Flag1).
Constructs expressing the H8 domain in the context of the entire central regulatory region (AA
843-1650), or a fragment of that region (AA 1311–1650), resulted in the production of a
glycosylated protein, indicating that the H8 hydrophobic residues are capable of directing
translocation across the lipid bilayer as opposed to serving as a hairpin insertion into the
membranes as has been previously proposed. In 293 cells transfected with wild type ABCA1
or Flag1or Flag2, similar amounts of cellular cholesterol was effluxed to ApoA-I (wt 4.2 1.1
% v.s. Flag1, 4.6 0.6 % & Flag2 4.3 1.1 %). Cellular binding of ApoA-I was also similar
(ng of specific ApoA-I bound/mg of cellular protein, wt, 9.31 0.63 v.s. Flag1, 9.1 1.4 &
Flag2, 5.2 0.98). Both Flag proteins could be cross-linked to A-1 and the apoprotein
immunoprecipitated with anti-ABCA1 antibody. These studies indicate that ABCA1 has
extracellular aminoterminal and central regulatory loops and that the insertion of Flag tags in
these loops is structurally compatible with retained ligand binding and efflux function of the
transporter, making such tags useful in future cell biological investigations.
P213
The In Vivo Performance of Vascular Grafts Cryopreserved by Vitrification
K G M Brockbank, Y C Song, M J Taylor. Organ Recovery Systems, Inc., Charleston, SC
The primary objective of our research program is the development of storage methods for living
natural and tissue engineered arterial bypass grafts. It is well known that a principal problem
in attempting to cryopreserve tissues and organs is formation of extracellular ice which
destroys both tissue structure and function. Vitrification is an alternative approach to
preservation that either completely avoids freezing or reduces ice crystallization to a tolerable
minimum. Here we report the results from our 28 day in vivo studies employing autologous and
allogeneic, vitrified, rabbit jugular veins. Patency was equivalent in the experimental and
control groups. The in vivo response was assessed after placing the veins as bypass grafts in
carotid arteries, followed by histopathology and smooth muscle physiology studies of elective
explants. Our results demonstrate that the integrity of a multi-cellular system can be preserved
in the vitreous state without the inherent problems associated with crystallization and so-called
“solution effects injury” that arise in cells due to the removal of water during ice formation.
Glass stability and cell viability of vitrified samples were retained during vapor phase liquid
nitrogen storage for at least four months. Histopathology of fresh and vitrified veins
demonstrated that the proliferative and remodeling responses of the vitrified grafts were
normal. In addition, both endothelial cell and smooth muscle cell functions of vitrified
specimens were well preserved post-transplantation relative to untreated control grafts. These
studies suggest that the vitrification process had no discernible adverse effects on tissues
assessed by transplantation in the rabbit.
Fibrin-Associated Characteristics of Atheroma-Targeted Echogenic
Immunoliposomes
Melvin E Klegerman, Andrew J Hamilton, Susan D Tiukinhoy, Amer A Khan, Shao-Ling
Huang, Robert C MacDonald, David D McPherson. EchoDynamics, Inc, College Park, MD;
Northwestern University, Chicago, IL; Northwestern University, Evanston, IL
P214
Antibody (Ab)-targeted echogenic immunoliposomes (EGIL) have been developed as novel
ultrasound contrast agents for evaluation of vasoactive and pathological endothelium and
atherosclerosis components, particularly surface-accessible fibrin in late-stage and vulnerable
atheroma. In order to monitor the antibody targeting aspect of EGIL, methods have been
developed to assess the immunoglobulin (Ig)-liposome (lp) conjugation (conj) efficiency
routinely, but a reproducible, convenient method for quantitative assessment of EGIL binding
to target antigens by guest is required. on April Previously, 4, 2013a
radioimmunoassay (RIA) protocol was used to

a-38 Arterioscler Thromb Vasc Biol. Vol 22, No 5
establish that a minimum avidity vs. soluble human fibrinogen (fgn) of 1x10 12 M -1 /lp for EGIL
conj to a polyclonal anti-human fgn/fibrin Ab (PAb) was required for optimal EGIL binding to
fibrin in a physiologic flow simulator. Our objective was to develop a more convenient
enzyme-linked immunosorbent assay (ELISA) procedure for routine assessment of EGIL
targeting efficiency and to compare the results with the RIA data. Wells of polystyrene microtiter
plates were coated with human fgn, half of which were then converted to fibrin with thrombin
in order to evaluate the fibrin specificity of PAb-EGIL. Primary incubation of samples diluted in
detergent-free buffer allowed determination of dissociation constants (K D) for intact EGIL.
Secondary incubation with anti-IgG conj to alkaline phosphatase was followed by incubation
with p-nitrophenylphosphate and measurement of absorbance at 405 nm (A 405). The average
K D for PAb-EGIL binding to fgn at 37 o C, determined by RIA, was 1.77 nM, while the analogous
ELISA value was 1.80 nM. The ELISA-determined K D for binding to fibrin was 1.98 nM,
establishing that the PAb has nearly equal affinity for human fgn and fibrin. An avidity of
1x10 10 M -1 /amole lp lipid was found to correspond to the optimal targeting efficiency
determined by RIA analysis. We conclude that the ELISA can be employed for routine
assessment of EGIL targeting efficiency prior to in vitro and in vivo evaluation.
P215
Cyclic AMP-Independent Activation of PKA by Endothelin-1 and Angiotensin
II in Vascular Smooth Muscle Cells
Nickolai O Dulin, Amanda Davis, Jiaxin Niu, Sergei Orlov. University of Chicago, Chicago, IL;
University of Illinois at Chicago, Chicago, IL; University of Montreal Centre de Recherche,
CHUM, Montreal, Canada
INTRODUCTION. Endothelin-1 (ET1) and angiotensin II (AII) are the vasoactive peptides
implicated in cardiovascular pathophysiology. In vascular smooth muscle cells, ET1 and AII
stimulate diverse signaling pathways leading to the cell contraction and hypertrophy. RESULTS
AND METHODS. Employing an in vivo assay for the activity of the protein kinase A (PKA) in intact
cells, we have found that ET1 and AII induce a transient activation of PKA in cultured vascular
smooth muscle cells (VSMC) from rat aorta. PKA activity was assessed by phosphorylation of
the PKA substrate, vasodilator-stimulated phosphoprotein (VASP), and the specificity of the
assay was confirmed by mutation of the PKA phosphorylation site (S153A) of VASP. ET1 and
AII did not stimulate cAMP production in VSMC, suggesting a novel cAMP-independent
mechanism for PKA activation. The ET1- and AII-induced activation of PKA was found
dependent on the inhibitor of kappaB (IB), which is commonly involved in the regulation of the
transcription factor NFB. Employing the co-immunoprecipitation technique, we found that a
certain pool of IB exists in a complex with the catalytic subunit of PKA, cPKA. Moreover, we
demonstrate for the first time that ET1 and AII stimulate a rapid dissociation of the IB/cPKA
complex in VSMC. CONCLUSIONS. These data are consistent with the model wherein a distinct
pool of IB binds and inhibits the activity of cPKA, whereas the stimulation of VSMC with ET1
or AII results in a release of free (active) cPKA, providing a mechanism for the cAMPindependent
activation of PKA.
P216
A Heparin-Binding Deficient Mutant of Endothelial Lipase Has Markedly
Reduced Catalytic and Lipoprotein Bridging Activities
Karen O Badellino, Dawn H Marchadier, Ken Kitajima, Ilia V Fuki, Nadine Blanchard, Jane M
Glick, Daniel J Rader. University of Pennsylvania, Philadelphia, PA
Interaction with cell-surface heparan sulfate proteoglycans (HSPGs) is important to the catalytic
and lipoprotein bridging activities of the triglyceride lipases. The relatively new member of this
family, endothelial lipase (EL), contains three conserved putative heparin-binding motifs, two in
the 40 kDa amino-terminal portion (Lys 294 to Arg 297 and Lys 306 to Arg 312) and one in
the carboxyl terminus (Arg 429 to Lys 434) of the 68 kDa protein. To assess the role of the
carboxyl terminal motif, we used site-directed mutagenesis to substitute the 4 basic amino
acids with asparagine. A similar mutation in lipoprotein lipase resulted in significantly
decreased heparin binding affinity and impaired lipid targeting properties while no changes in
the enzymatic activity was observed [Lutz et al, JCI 107:1183–1192(2001)]. Upon transient
transfection of 293 cells, the mutant EL protein was produced at similar levels as the wild-type
EL protein and exhibited a substantial decrease in heparin binding ability. Interestingly, this
heparin binding defective EL mutant had markedly reduced catalytic activity for both
triglycerides and phospholipids. Whereas wild-type EL is very effective at bridging LDL to
HSPGs on the cell surface, this heparin binding defective EL mutant had virtually no bridging
activity. The loss of bridging activity is unlikely to be related to the loss of catalytic activity since
heparin-binding competent catalytically inactive EL showed no loss of bridging activity. In
summary, the putative carboxyl terminal heparin binding motif in EL is critical for heparin
binding, activity, and lipoprotein bridging.
Nitric Oxide Inhibits Sustained JNK Activation by Hsp70 Induction in
Pulmonary Arterial Hypertension in Rats
Shinichi Kanno, Yi-Jen L Wu, Chien Ho, Timothy R Billiar, Larry L Shears II. University of
Pittsburgh, Pittsburgh, PA; Carnegie Mellon University, Pittsburgh, PA
P217
Background: Pulmonary arterial hypertension (PAH) is associated with dramatic structural
changes in the pulmonary vasculature. We have previously shown that nitric oxide (NO) plays
a crucial role in this process, however, how NO modulates pulmonary vascular remodeling is
not fully elucidated. Heat shock protein 70 (Hsp70), a major stress protein, is induced by NO
in many cell types. We postulated that NO modulates vascular remodeling through the induction
of Hsp 70 expression in the vessel wall. Methods and Results: Monocrotaline (MCT) was
administered to Sprague-Dawley (SD) rats to induce PAH with or without enalapril (ACEI)
treatment. Isolated pulmonary perfusion showed ACEI treatment significantly upregulated NO
production as measured by nitrite levels. MCT treatment resulted in increased c-Jun N-terminal
kinase1/2 (JNK1/2) activation even 5 weeks Downloaded after the treatment. from
The addition of ACEI
http://atvb.ahajournals.org/
suppressed JNK1/2 activation in association with increased expression of Hsp 70. Coimmunoprecipitation
experiments using lysates from the pulmonary vessels revealed that
Hsp70 interacts with JNK1/2. Immunohistochemistry demonstrated sustained activation of
JNK1/2 in vascular smooth muscle cells (VSMCs) in MCT-treated lungs, whereas Hsp70 was
observed in VSMCs in ACEI-treated lungs. Experiments using isolated perfused lungs
demonstrated that ACEI treatment was associated with increased NO production while the
inclusion of an NO synthase inhibitor induced JNK1/2 activation in the pulmonary vasculature.
The capacity of NO to induce Hsp 70 was confirmed using cultured VSMCs exposed to an NO
donor or transduced with using an adenoviral vector expressing iNOS. Conclusions: These
observations suggest that the mechanism by which ACEI suppresses the development of PAH
involves the suppression of JNK1/2 activation with upregulation of Hsp70 induced by nitric
oxide. Hsp70 may physically interact with JNK, inhibiting its activation.
P218
Artery Calcification and Calciphylaxis are Both Inhibited by Doses of the
Amino Bisphosphonate Ibandronate that Inhibit Bone Resorption
Paul A Price, Nika Omid, Truclinh Nguyen, Matthew K Williamson. University of California,
San Diego, La Jolla, CA
The present experiments were carried out to test the hypothesis that there is a common
underlying biochemical mechanism that accounts for the different kinds of soft tissue
calcification observed in animals that are treated with toxic doses of vitamin D. In previous
studies we showed that lethal doses of vitamin D cause extensive calcification of arteries,
lungs, kidneys, and cartilage, and that doses of the amino bisphosphonate ibandronate that
inhibit bone resorption completely inhibit each of these soft tissue calcifications and prevent
death [ATVB(2001)21:817–824]. In the present experiments we have examined the effect of
ibandronate on an entirely different type of calcification, the calciphylaxis induced by
administration of a challenger to rats previously treated with sub-lethal doses of vitamin D.
These studies show that ibandronate doses that inhibit bone resorption completely prevent
artery calcification as well as, in the same rat, the Alizarin red-staining for calcification at sites
of calciphylactic challenge by either subcutaneous injection of 300g FeCl 3 or intrascapular
epilation. Chemical analysis further showed that the level of calcium was increased by 40-fold
at the FeCl 3 -induced calciphylaxis site and by 100-fold at the skin epilation-induced
calciphylaxis site, and that concurrent treatment with ibandronate reduced calcium to control
levels at both sites. Since vitamin D treatment dramatically increased levels of bone resorption,
and concurrent treatment with ibandronate normalized resorption, these results support the
hypothesis that all soft tissue calcifications in the vitamin D treated rat may be linked to bone
resorption. The ability of ibandronate to inhibit vitamin D-associated calcifications in the rat
cannot be explained by an effect of ibandronate on serum calcium, since serum calcium
remained 50% above control levels in the vitamin D-treated animals that also received
ibandronate.
Coronary Artery Lipid Accumulation in Diabetic Dyslipidemic Pigs
Compared to Normoglycemic Hyperlipidemic Pigs
Elzbieta Wysocka, Michael Sturek, Joseph L Dixon. University of Missouri, Columbia, MO
P219
We tested the hypothesis that coronary arteries of diabetic pigs fed a high fat (21%)/cholesterol
(2%) diet (HFC) would accumulate higher amounts of cholesteryl ester (CE), free cholesterol
(FC), and triglyceride (TG) during atherosclerosis than non-diabetic pigs fed the HFC diet.
Experimental Design: Five groups of Yucatan miniature pigs (n4–9 pigs/group) were treated
for 20 weeks: low fat fed control (C), pigs fed HFC diet (F), low fat fed with alloxan induced
diabetes (D), D fed HFC (DF), and mildly diabetic fed HFC (MDF). Methods: Coronary
atherosclerosis defined as percent of artery area with raised endothelium was evaluated by
intravascular ultrasound (IVUS). Left antherior descending coronary artery (LAD) CE, FC, and TG
were measured by HPLC. ANOVA, Student-t, and Mann-Whitney tests were used for statistical
analysis. Results: LAD CE, FC, and TG in the unbranched segments were 0.178/-0.043,
33.32/-3.76, and 331.3/-134.7 ug/mg total tissue protein in C pigs. LAD CE concentration
increased in F, DF, and MDF 295 (52.6/0.178), 71, and 201-fold, respectively, compared to C
(all p0.01). Average LAD FC concentration only increased in F (2.3-fold compared to C,
p0.01). Only small increases in LAD TG were observed in HFC fed pigs. Lipid profiles were
similar in unbranched and branched LAD segments. Although LAD CE was much greater in F
versus DF, IVUS indicated similar sized lesions. Conclusions: 1. CE accumulation in LAD was
more accentuated in non-diabetic HFC pigs compared to diabetic HFC pigs. 2. LAD TG was not
a marker for HFC or diabetes. 3. Non-diabetic HFC fed pigs develop more CE engorged coronary
artery lesions whereas lesions in diabetic pigs may be more proliferative, i.e. cellul
Utility of Abnormal 3-Methylglutaconic Aciduria (3MGA) in Diagnosing
Statin-Associated Myopathy
Paul S Phillips, Richard H Haas, Bruce Barshop, Sergei Bannykh, Darius Amjadi. Scripps
Mercy Hospital, San Diego, CA; University of California, San Diego, San Diego, CA
P220
HMG CoA Reductase inhibitors (statins) are effective in reducing the incidence of cardiovascular
events with a very low rate of toxicity. Generally, patients receiving statins who have muscle
symptoms and a normal creatine kinase level (CK) are believed to have symptoms unrelated
to this therapy. Yet muscle biopsies confirm myopathy in some patients with normal CPK on
statins. We compared the incidence of urine 3-methylglutaconic acid (3MGA) elevation in
patients with normal CK and biopsy-proven statin associated myopathy with the incidence in
patients with muscle symptoms whose muscle biopsies were normal and with patients on
statins without symptoms of myopathy in order to assess the usefulness of this test in
diagnosing statin associated myopathy. Methods: We measured 3MGA by standard mass
spectroscopy techniques in 8 consecutive patients with biopsy-proven statin associated
myopathy, by in 3guest consecutive on April patients 4, with 2013 muscle complaints on statins and negative biopsies,

and also in 6 patients on statins with no muscle complaints. Abnormal muscle biopsies were
attributed to statin toxicity if they demonstrated pathology consistent with mitochondrial
dysfunction such as ragged red fibers, COX-negative staining fibers, or abnormal accumulations
of lipid. 3MGA levels were considered abnormal if they were greater than 2 standard
deviations from the mean values in our laboratory. Results: 7/8 patients with myopathy on
biopsy had abnormal 3MGA 0/3 patients with no myopathy on biopsy had abnormal 3MGA 0/6
patients on statins without muscle complaints had abnormal 3MGA Conclusion: In a small
series, using abnormal muscle biopsies as the gold standard, 3MGA appears 87.5 % sensitive
and 100% specific in confirming statin associated myopathy with normal CK.
P221
A Novel Gas Chromatograph/Mass Spectrometric Method (GC/MS) to
Measure Vascular Smooth Muscle Cell Proliferation, In Vivo, Using 2H2O Alice Chu, Eric T Ordonez, Marc K Hellerstein. University of California at Berkeley, Berkeley,
CA
VSMC proliferation plays a pathogenic role in atherosclerosis and other hyperplastic diseases
of the vessel wall. We have developed a method to measure VSMC turnover using 2 H 2O (heavy
water) to label the deoxyribose (dR) moiety of DNA. The method is based on the exchange of
deuterium into dR in the formation of deoxyribonucleosides (dN) used for DNA replication. Mice
are injected with 100% 2 H 2O to reach a body water enrichment of 2% and maintained on 4%
2 H2O in drinking water for three weeks. Upon sacrifice, the aorta and bone marrow (BM) are
removed. Aorta is subjected to collagenase treatment to remove adventitial and endothelial
layers. DNA is extracted and enzymatically hydrolyzed to dN. Deoxyadenosine is isolated, mild
acid-hydrolyzed to dN to remove adenine, and derivitized to dR-pentane tetraacetate for
analysis by GC/MS (m/z 245,246). Calculation of f(%new cells)(Enrichment of VSMC/
Enrichment of BM)*100 and k (%new cells/day){-ln(1-f)/t}*100. We initially tested the method
in young (age 3–6 weeks) and adult (age 15–21 weeks) C57Bl/6J mice. Significantly higher f
(5.73.5% vs 2.41.5%) and k (0.270.16% vs 0.120.08%) were observed in the 9 week
vs 21 week old mice (P0.05). A delabeling study was also conducted. Mice were labeled for
10 weeks then switched to non-labeled drinking water and sacrificed every 2 weeks. VSMC
enrichments remained nearly constant, confirming slow turnover. VSMC proliferation was
compared in apoE-/- and C57Bl/6J mice fed low-fat or high-fat diet from age 5 weeks. AFter
9 weeks of diet, f of apoE-/- high-fat increased and remained significantly higher than all other
groups (263% vs 1–8%, P0.01). The increased VSMC proliferation preceded and ultimately
correlated with histologic changes in the aortic wall. In conclusion, the method allows the
measurement of VSMC turnover and proliferation in vivo, confirms the normally slow turnover
rate of VSMC in rodents, and demonstrates early changes during the progression of
atherosclerosis; thereby representing a potentially sensitive measure of atherogenesis.
P222
Hyperhomocysteinemia Induces Endothelial Apoptosis: Mechanism and
Implications for Atherogenesis
Ganesh Raveendran, Kalpna Gupta, Anna Solevey, Liming Cheng, Robert Hebbel. University
of Minnesota, Minneapolis, MN
Background: Hyperhomocysteinemia is an independent risk factor for cardiovascular mortality.
Mechanism of action of hyperhomocysteinemia leading to increased cardiovascular morbidity
and mortality is unknown. Endothelial cell (EC) injury, and perhaps apoptosis, are the key
events in the pathogenesis of atherosclerosis and represent an initial step in atherogenesis.
Methods: We hypothesized that endothelial cells from patient with homozygote state for
cystathionine--synthase (CBS) deficiency will have higher apoptosis and higher expression of
adhesion molecule and tissue factor. BOECs were cultured from blood of one homozygote with
CBS deficiency and two normal controls. BOECs were treated with regular EC medium, serum
free medium and escalating concentration of methionine and homocysteine (Hcy). Apoptosis
was estimated by ELISA using optical density measurements and by tunnel assay after 48
hours. Each experiment was performed in duplicate with 8 samples for each test. Results were
analyzed using independent Students T test. Results: Our preliminary results by ELISA show
BOECs from CBS deficient patient had statistically significant difference in apoptosis compared
to the normal controls. Serum free medium induced apoptosis in both group but did have any
significant difference between the groups. Similar comparable results were observed in tunnel
assay. Methionine loading in the culture medium did not have an effect on the measured Hcy
level from the supernatant.
Poster Presentations a-39
effects, Ang II is a potent stimulus for adrenal production of aldosterone, which may also cause
myocardial and vascular injuries. In other studies of ApoE-deficient mice we found that
mineralocorticoid excess produced by infusion of deoxycorticosterone acetate (DOCA-50 mg
pellet implanted for 28 days starting at 8 weeks age) and salt loading increased lesion area of
the descending aorta from 0.40.1% in controls to 549% in treated animals (p0.0001),
while having little effect at the aortic root. Objectives: To determine the role of mineralocorticoid
excess in mediating the effects of Ang II on atherogenesis we measured plasma aldosterone
levels in Ang II-infused mice and then reproduced these levels by direct infusion of aldosterone
via osmotic minipump. Methods: Plasma aldosterone levels measured 4 weeks after Ang II
infusion via osmotic minipump (1,000 ng/kg/min) increased from 185 to19837 ng/dl (n
4, p0.003). Osmotic minipumps were implanted to deliver aldosterone to achieve similar
levels. The final infusion rates were 200 and 400 /kg/day. Findings: After 28 days infusion,
these infusion rates produced plasma aldosterone levels of 13513 and 29857 (n6 for
each group), which spanned the plasma levels observed in the Ang II-infused mice. Systolic
blood pressure, measured by tail cuff increased significantly in both groups by 13 mmHg
compared to sham operated controls, as did kidney weight/body weight ratio. However, in both
aldosterone infusion groups there was no increase in atherosclerosis at either the aortic root
or in the descending aorta. Conclusion: These studies suggest that mineralocorticoid excess
only increases atherosclerosis under the pharmacological conditions of DOCA/salt treatment. If
aldosterone is involved in mediating atherosclerosis in response to Ang II, then plasma Ang II
levels must also be elevated for aldosterone to exert its effects.
Alterations in Apo A-I Conformation Accompanying the Conversion of
Discoidal to Spherical High Density Lipoproteins
Hui-Hua Li, Douglas S Lyles, Michael J Thomas, Wei Pan, Eric Alexander, Michael Samuel,
Mary G Sorci-Thomas. Wake Forest University School of Medicine, Winston-Salem, NC
P224
Apolipoprotein A-I (apo A-I) readily binds and organizes phospholipid bilayers to form discoidal
HDL particles. In the lipid-bound form, apo A-I activates lecithin:cholesterol acyltransferase
(LCAT), which generates cholesterol ester (CE) from free cholesterol (FC). As esterification
proceeds discoidal HDL transforms into a spherical HDL with accumulation of cholesterol esters
within the hydrophobic core of the bilayer. A typical 96Ã. . . diameter discoidal HDL particle
contains two molecules of apo A-I and is oriented in an antiparallel “belt” conformation
surrounding a phospholipid bilayer. However, the conformational changes in apo A-I structure
that take place as the discoidal HDL conforms to the presence of a hydrophobic core are
currently unknown. In order to characterize the changes in apo A-I structure as it adapts to a
sphere, we measured the inter-molecular distance between apo A-I molecules using
fluorescence resonance energy transfer (FRET). Selected domains on each of 2 lipid-bound apo
A-I molecules were studied in both the discoidal and the spherical HDL form. These data
showed that as discoidal particles were converted to spherical HDL through the action of LCAT,
compositional changes included a 35% decrease in phospholipid, 70% decrease in FC, and a
94% increase in CE mass. In addition, after the conversion, spherical HDL particles were found
to have obtained a third molecule of apo A-I, as determined by crosslinking analysis. FRET
analysis showed that the inter-molecular distance between domains corresponding to repeat
5 (amino acids 121–142) showed very little change, while the domain corresponding to repeat
6 (amino acids 143–164) showed a much larger inter-molecular shift as CE accumulated.
These data suggest that the favored “belt conformation” found on discoidal HDL is significantly
altered after the particle accumulates CE in the core.
P225
Selective Cyclooxygenase-2 Inhibition Decreases Monocyte
Chemoattractant Protein-1 Expression and Neointimal Hyperplasia in the
Rabbit Atherosclerotic Balloon Injury Model
Kai Wang, Zhongmin Zhou, Khaldoun Tarakji, A Michael Lincoff, Eric J Topol, Marc S Penn.
The Cleveland Clinic Foundation, Cleveland, OH
Angiotensin II Infusion Increases Plasma Aldosterone Levels and
P223
The inflammation in response to vascular injury is becoming increasingly recognized as a
potential contributor to restenosis. Cyclooxygenase-2 (COX-2) has been shown to be involved
in the proinflammatory response of vascular tissue, potentially through regulation of monocyte
chemoattractant protein-1 expression (MCP-1). We hypothesized that specific COX-2 antagonism
would lead to decreased MCP-1 expression, inhibiting inflammatory response following
vascular injury, and ultimately reducing neointimal formation after vascular injury. Methods and
Results: Bilateral focal femoral artery lesions were induced by air desiccation in New Zealand
White rabbits followed by high cholesterol diet feeding for 28 days. Balloon injury was then
induced at the pre-injured vessel segments by three balloon inflations. Initial studies using
immunostaining (n4 animals) showed that uninjured vessel segments stained positive only
for COX-1 but not for COX-2. Injured vessel segments showed, in addition to COX-1, significant
positive staining for COX-2 in both endothelium and macrophages, demonstrating upregulation
of expression of this pro-inflammatory enzyme in response to injury. In the efficacy study,
celecoxib (75mg/kg/day) was administered orally beginning 3 hours before balloon injury on
Accelerates Aortic Atherosclerosis in ApoE-Deficient Mice, but Aldosterone day 28 and then daily for 21 days. MCP-1 expression was quantified in arterial extracts 4 days
Infusion Alone Has No Effect
after balloon injury by western blot. Morphometric analysis and immunostaining for macrophages
was performed 21 days after balloon injury. Celecoxib treatment significantly decreased
Ying Zhou, Rong Chen, Yuexian Shi, Lufei Hu, Jean E Sealey, John H Laragh, Hayes M
MCP-1 activity (5.3Â1.9 VS 29.0Â4.6 arbitrary units, p0.01), and neointimal hyperplasia
Dansky, Daniel F Catanzaro. Weill Medical College, Cornell University, New York, NY; Mount by more than 30% (0.49Â0.20 vs 0.70Â0.35 mm2, p0.05), accompanied by reduced
Sinai School of Medicine, New York, NY
macrophage infiltration. Conclusions: Selective antagonism of COX-2 decreases the inflammatory
response and intimal hyperplasia following vascular injury, possibly due to the early
Background: Recent studies show that Ang II infusion increases atherosclerosis at both the inhibition of MCP-1 expression, implying a pivotal role of inflammation in the pathogenesis of
aortic root and in the descending aorta of ApoE-deficient Downloaded mice. In addition from
tohttp://atvb.ahajournals.org/ its vasoconstrictor restenosis. by guest on April 4, 2013

a-40 Arterioscler Thromb Vasc Biol. Vol 22, No 5
P226
Small, Dense LDL, Unstable Carotid Plaque and Cerebrovascular Events in
Patients Undergoing Carotid Endarterectomy
Alberto Zambon, Elisabetta Faggin, Sandra Bertocco, Nicola Vitturi, Massimo Puato, Gaetano
Crepaldi, Paolo Pauletto. University of Padova School of Medicine, Padova, Italy
Low-density lipoprotein (LDL) size and density contribute to LDL particle atherogenicity. The
presence of small, dense LDL particles is associated with: 1) a three to six fold increased risk
of coronary artery disease, and 2) with an increased carotid intima-media thickness, a marker
of early atherosclerosis, in normolipidemic subjects. We investigated the possible association
between LDL density, cell composition of carotid plaques (unstable plaques) and the
occurrence of ischemic cerebrovascular events (ICVE). Sixty-eight patients undergoing carotid
endarterectomy, with a carotid stenosis greater than 70% of the lumen, were studied. Plasma
LDL density was evaluated by density gradient ultracentrifugation. Endarterectomy specimens
were examined by immunocytochemistry using the following monoclonal antibodies: i) the
SM-E7 identifying smooth muscle cells (SMCs), ii) the anti-macrophage HAM 56, and iii) the
anti-lymphocyte CD45R0. Fifty patients had at least one episode of ICVE prior the endarterectomy,
while 18 had no ICVE. Mean blood pressure, gender, BMI, total, LDL and HDL
cholesterol, TG, Lp(a), apo AI and apo B levels, smoke habits, glucose, fibrinogen, plasminogen
and homocysteine levels, were not significantly different in those with previous ICVE as
compared with patients without ICVE. Patients with previous ICVE had significantly denser LDL
particles (p0.01) and more macrophages (p0.01) than those without ICVE. LDL density was
significanlty associated with both the number of macrophages (r 0.59, p0.01) and SMCs
(r -0.50, p0.01) in the plaque. In a multivariate analysis including clinical and lipid
variables, LDL density was the only parameter significantly predicting the number of
macrophages in the plaque (p0.01). In a logistic regression analysis the number of
macrophages was the strongest predictor of ICVE (p0.01). The presence of dense LDL
particles is associated with an abundance of inflammatory cells in the carotid plaque (unstable
plaque) and excess prevalence of ICVE in subjects with carotid stenosis.
P227
Delineation of the Role of Pre 1-HDL in Cholesterol Efflux Using Isolated
Pre 1-HDL
Dmitri Sviridov, Osamu Miyazaki, Kally Theodore, Anh Hoang, Isamu Fukamachi, Paul
Nestel. Baker Medical Research Institute, Melbourne, Australia; Diagnostic Research
Laboratories, Daiichi Pure Chemicals Ltd, Tokyo, Japan
The role of pre 1-HDL in cholesterol efflux was investigated by separating human plasma into
purified pre 1-HDL and pre 1-HDL -deficient plasma using a monoclonal antibody specifically
reacting with pre 1-HDL. When compared to whole plasma, pre 1-HDL-deficient plasma was
equally efficient in promoting cholesterol efflux from human skin fibroblasts and THP-1 human
macrophage cells. When added at the same apoA-I concentration pre 1-HDL was less effective
than whole plasma in promoting cholesterol efflux from fibroblasts but equally effective with
THP-1 cells. However, pre 1-HDL-deficient plasma reconstituted with 16% pre 1-HDL was more
active than whole plasma demonstrating that pre 1-HDL does promote cholesterol efflux actively.
The amount of cellular cholesterol present in re-isolated pre 1-HDL was 1.5–2 -fold greater
following incubation of cells with whole plasma, than with pre 1-HDL-deficient plasma or plasma
treated with the antibody. Time-course efflux experiments showed that isolated pre 1-HDL was not
significantly different to whole plasma in promoting efflux from “fast” cholesterol pool, but less
active in recruiting cholesterol from a “slow” pool. Anti pre 1-HDL antibody did not inhibit
cholesterol efflux to whole plasma, pre 1-HDL-deficient plasma or isolated pre 1-HDL and the
epitope of this antibody is located between residues 140–210, i.e. some distance from lipid-binding
domains of apolipoprotein A-I. We conclude that whereas pre 1-HDL are capable of taking up
cellular cholesterol, their presence in plasma is not essential for cholesterol efflux, at least in vitro.
Instead pre 1-HDL may be the first product of apoA-I lipidation during formation of HDL, but might
not play a major role in transferring cellular cholesterol to HDL
Cilostazol Effects on Neointimal Growth in the Carotid Artery of Rats
Following Balloon Injury
Dadong Li, Laurine Bow, Xin Yang, Charles Nightingale, Ronald Langner. Hartford Hospital,
Hartford, CT; University of Connecticut, Storrs, CT
P228
Restenosis of blood vessels following percutaneous coronary angioplasty (PTCA) is a significant
medical problem. Platelet aggregation and the subsequent release of platelet cytokines is
thought to play an important role in promoting blood vessel restenosis. The purpose of this
study was to determine the ability of cilostazol, a phosphodiesterase type III inhibitor, to inhibit
platelet aggregation and to inhibit restenosis in rats. The endothelial lining of the left carotid
artery of 16 male Sprague-Dawley rats was injured by inflating a balloon catheter and moving
it back and forth four times. The 16 rats were randomly divided into two groups. One group was
treated orally with cilostazol, 135mg/kg, twice a day while the other group was treated with
vehicle. Drug and vehicle administration began one day before balloon-injury and was
continued for 15 days. Cilostazol significantly inhibited platelet aggregation after 7 days of
treatment and remained constant through the remainder of the 15 days treatment period. The
extent of neointimal growth in the carotid arteries was estimated by measuring the areas of
intima and media with a computerized morphometric analyzer. The balloon-injured carotid
arteries from vehicle treated rats had a significantly thickened intimal layer which was
characterized by increased proliferation of smooth muscle cells and deposition of connective
tissue compared to its uninjured right carotid artery. In the cilostazol treated rats the intimal
layer of the injured carotid artery was also significantly thicker compared to the uninjured right
carotid artery, however the degree of intimal proliferation in these animals was significantly
decreased compared to that of injured arteries from vehicle treated rats. The data suggests that
treatment with cilostazol may have a beneficial effect in retarding the extent of restenosis in
blood vessels following PCTA.
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Modulation of Arterial Healing in Heterozygous Watanabe Rabbits by
Aspirin and Verapamil
P229
Kenneth J Woodside, Howard H Hayashi, Linda Talken, John A Daller, Charlie Luo, Glenn C
Hunter. University of Texas Medical Branch, Galveston, TX; University of California at Davis,
Sacramento, CA
There is increasing evidence that modulating the components of complex atherosclerotic
plaque may reduce the frequency of symptoms and delay progression of disease. This study
evaluates the effects of aspirin (ASA) and verapamil on the nonlipid components of complex
plaque induced in heterozygous Watanabe Heritable Hyperlipidemic (HWHHL) rabbits by balloon
catheter injury. METHODS: Balloon catheter aortic injury was induced in 27 HWHHL rabbits: 9
control, 9 received 5 mg/kg of aspirin, and 9 receiving 0.15 g/kg verapamil orally daily. Serum
and tissue cholestrol and drugs levels were assayed. Three animals each were sacrificed at 1,
3, and 6 months. The aortas were examined histologically for evidence of neointimal
hyperplasia (NFH), thrombus formation, and calcification. RESULTS: Two animals died during
the study period. Lipid alterations and NFH at the later timepoints are shown in the Table.
CONCLUSIONS: Aortic balloon catheter injury in this model can reproduce all the components
of complex atherosclerotic plaque and allows pharmacologic modulation of each specific
component. Although verapamil appeared to be more advantageous than ASA in altering NFH,
thrombus, and calcification, it seems likely that multiple agent therapy may be required in order
to reduce plaque instability.
Effects of Preprandial Ethanol on Cholesteryl Ester Transfer in
Normotriglyceridemic and Hypertriglyceridemic Subjects
John W Gaubatz, Lynne W Scott, Kay T Kimball, Lisa Wu, Christie M Ballantyne, Henry J
Pownall. Baylor College of Medicine and The Methodist Hospital, Houston, TX
P230
The effects of standard acute doses of alcohol, saturated fat (SF), and alcohol plus SF on
plasma cholesteryl ester transfer activity (CETA) and concentrations of cholesterol, nonesterified
fatty acids (NEFA), and triglyceride (TG) were determined in 9 normotriglyceridemic (NTG)
and 4 moderately hypertriglyceridemic (HTG) subjects (respective fasting TG 1.19 0.19
mmol/L and 4.93 1.20 mmol/L, mean SEM). Over the 10 hours following the ingestion
of alcohol, CETA, cholesterol, and TG values did not significantly change in either group,
whereas NEFA transiently decreased recovering to 100% and 150% of the baseline value at 10
hours in the NTG and HTG groups. In both groups, the fat challenge was followed by an increase
in CETA, NEFA, and TG; cholesterol was not significantly affected. Alcohol plus SF did not affect
cholesterol but, compared with SF alone, increased CETA and the magnitude of lipemia and
suppressed the increase in NEFA in both groups. The fractional increases in CETA and TG with
addition of alcohol were positively correlated in both the NTG (r 2 0.91) and HTG (r 2 0.80)
groups; the increase in CETA with TG was 3.5 times greater in HTG than in NTG. However, a
single line of regression was obtained for the correlation of CETA and TG (r 2 0.62; P
0.0001). This suggests that CETA is a direct function of TG concentration in both NTG and
moderately HTG subjects, and that intestinally derived lipoproteins are the major acceptors of
HDL-CE, particularly in subjects consuming fat with alcohol. Thus, CETA may be involved in the
cardioprotection conferred by regular, moderate consumption of alcohol. This protection could
occur through the increased diversion of HDL cholesterol to intestinally derived lipoproteins,
which are destined for hepatic uptake.
P231
Adventitia-Dependent Intima Hyperplasia and Vasoconstriction of Carotid
Arteries in Rabbits on Cholesterol-Rich Diet
Zhiming Zhu, Huaming Mu. Department of Hypertension and Endocrinology, Daping Hospital,
Third Military Medical University, Chongqing, China
Background: Adventitial cells may play an important role for the neointima formation and
modulation of vascular smooth muscle cell phenotype. The objective of the present study was
to test whether adventitial cells in hypercholesterolemic rabbits on a cholesterol-rich diet
directly affect neointima formation and phenotype of carotid artery. Methods: In 8 rabbits
adventitia was excised from one carotid artery before receiving a cholesterol-rich (2%) diet for
6 weeks. Eight rabbits receiving standard chow were sham-operated. After 1 week and 6
weeks carotid arteries were isolated for histological examination, and vasoconstriction was
measured in carotid rings. Results: After 1 week rabbits on a cholesterol-rich diet showed a
severe hyperplastic intimal lesion after removal of the adventitia compared to sham-operated
rabbits. Only collagen type I were observed in carotid arteries of rabbits on a cholesterol-rich
diet without adventita, whereas collagen type I and type III could be observed in sham-operated
rabbits. KCl-induced vasoconstriction was significantly reduced (1.57/-0.16 g vs 0.57/-
0.08 g, p0.01), whereas acetylcholine (Ach)-induced relaxation was significantly enhanced in
carotid arteries of rabbits on a cholesterol-rich diet without adventitia compared to shamoperated
rabbits by guest ( 0.14/-0.09 on April g4, vs 2013 0.25/-0.08 g, p0.05). After 6 weeks rabbits on a

cholesterol-rich diet without and with adventitia showed similar hyperplastic intima lesions and
distribution of collagen. KCl-induced vasoconstriction was significantly reduced in carotid
arteries without adventitia ( 0.59/-0.08 g vs 0.25 /-0.06 g, p0.05). Ach-induced
relaxation was unchanged. Conclusion: Histological and functional properties of carotid arteries
in rabbits on a cholesterol-rich diet are influenced by adventitial cells (Supported by NSFC grant
39725013 ).
Cell Cycle Protein Expression in Stenosed Arteriovenous Fistulas for
Hemodialysis
Ruben Dammers, Rick De Graaf, Tryfon Vainas, Jan H Tordoir, Peter J Kitslaar, Arnold P
Hoeks. University Hospital Maastricht, Maastricht, Netherlands; University of Maastricht,
Maastricht, Netherlands
P232
Objectives. Renal failure (RF) patients rely on arteriovenous fistulas (AVFs) for hemodialysis
vascular access. Intimal hyperplasia (IH) results in frequent interventions and/or failure of the
AVF. The extent of IH depends on the interplay between cyclins and cyclin dependent kinases
(p.e. CDK2), positively regulating cell cycle progression. CDK activity is negatively modulated
by the interaction with CDK inhibitory proteins, such as p21 and p27. Little is known about the
expression of these proteins in the development of IH in AVFs. Methods. Of 18 failed AVFs
(patency duration: 765 160 days) of 16 RF patients (8 male) stenotic segments were
compared to 5 healthy left-over veins and arteries. The nuclear proteins p21, p27, and CDK2
were immunohistochemically stained on paraffin embedded 5 m tissue sections. To compare
the percentages of positively stained cells an ANOVA with Bonferroni correction was used.
Correlation coefficients were calculated, corrected for age. Findings. Results are summarized
in the table. The percentage of p21-positive cells was significantly less in AVFs compared to
healthy segments (p0.001). CDK2 activity was more pronounced in AVFs (p0.001).
Between healthy veins and AVFs the percentage of p27-positive cells was not different, but in
AVFs it was higher compared to healthy arteries (p0.05). AVF patency correlated with the
number of p27-positive cells (r-0.612, p0.026). Conclusion. p21 is downregulated in
stenotic segments of AVFs and is thus correlated to the development of IH in AVFs.
Furthermore, p27 inversely correlated with the patency, suggesting p27 to have a more
regulatory function in IH development. Future research is needed to decide whether p21 is an
appropriate target for therapeutical intervention to prevent IH in AVFs.
Consequences of Chronically Elevated Plasma Fibrinogen Levels on
Thrombosis in Genetically Hypercoagulable Mice
P233
Bryce A Kerlin, Brian Cooley, Susan Lord, Hartmut Weiler. Medical College of Wisconsin,
Milwaukee, WI; University of North Carolina, Chapel Hill, NC; Blood Research Institute of The
Blood Center, Milwaukee, WI
Elevated plasma fibrinogen (hyperfibrinogenemia) is associated with cardiovascular disease
and may increase the probability of developing arterial vascular disease. Epidemiological
studies remain inconclusive with respect to the question whether elevated plasma fibrinogen
is only associated with disease, or constitutes indeed a causative risk factor for disease
initiation and progression. In order to investigate the cause-effect relationship between
hyperfibrinogenemia and thrombosis, we examined a transgenic mouse model of familial
hyperfibrinogenemia in which plasma fibrinogen levels are approximately 2-fold higher than in
normal mice secondary to the presence of an extra copy of the genetic locus carrying all three
fibrinogen genes (HiFib-mice). HiFib-mice maintained under standard husbandry conditions did
not exhibit an increased propensity for microvascular thrombosis and/or fibrin deposition, yet
showed augmented fibrin degradation products (D-Dimer). Experimentally induced permanent
stasis of blood flow in the carotid artery did not cause formation of fibrin-platelet thrombi in
excess of those formed in wild type mice. Platelet thrombus formation after arterial injury
induced by ferric chloride was indistinguishable from that observed in normal mice. HiFib-mice
were then crossed with a second genetically engineered mouse strain that exhibits a
hypercoagulable state due to reduced thrombomodulin function. Superimposed hyperfibrinogenemia
did not modify the prothrombotic phenotype of thrombomodulin-deficient mice. In
conclusion, in mice, pre-existing hyperfibrinogenemia does not induce a marked prothrombotic
state, and does not appear to alter the incidence/severity of experimentally induced thrombosis.
Identification of the Proteoglycan-Binding Site of Apolipoprotein
B48-Containing Lipoproteins
Maria J Gustafsson, Christofer Flood, Jan Boren. Wallenberg Laboratory, Göteborg, Sweden
P234
The retention of lipoproteins is a key step in atherogenesis. The binding of LDL to proteoglycans
to the extracellular matrix (ECM) is mediate through residues 3359–3369 in apoB100. Despite
the fact that apoB48 lacks this binding site, lipoproteins containing apoB48 have been shown
to bind to proteoglycans in vitro. To identify the principal glycosaminoglycan binding site of
apoB48, we made a series of mutations in the human apoB gene, expressed the mutated genes
in rat McArdle 7777 cells, isolated the recombinant human LDL, and determined their abilities
to bind to the smooth muscle cell proteoglycans, decorin and biglycan. We found that when
mutating all of the basic amino acids in residues 18 –24 or 225–227 of apoB, the assembly of
lipoproteins was affected and there was no secretion Downloaded of human apoB. from
However, recombinant
http://atvb.ahajournals.org/
Poster Presentations a-41
apoB48 with residues 87, 88 and 90 mutated were secreted, but the interaction to the
proteoglycans was abolished. We also generated transgenic mice expressing apoB 80, with or
without residues 3359–3369 mutated. Characterization of wild-type apoB80 and RK3359 –
3369SA apoB80 showed that apoB80-containing LDL had a higher affinity for ECM than
apoB100, and that RK3359–3369SA apoB80 displayed the same proteoglycan binding affinity
as apoB48. Immunoaffinity studies indicated that the proteoglycan-binding site of apoB48 is
masked in apoB100. These results indicate that residues 87, 88 and 90 in apoB48 interacts
with artery wall proteoglycans, and that this binding site is nonfunctional in apoB100.
P235
Protective Effect of Apolipoprotein E2 on Angiographically Determined
Heart Disease in African Americans is Mediated through Serum Lipoprotein
Cholesterol
Lars Berglund, Roberta G Reed, Jill Rubin, Steve Holleran, Rajasekhar Ramakrishnan,
Thomas A Pearson. Columbia University, New York, NY; Bassett Research Institute,
Cooperstown, NY; University of Rochester, Rochester, NY
We studied the relationship of apolipoprotein E (apoE) isoforms to coronary heart disease (CHD)
in 226 African Americans (AA) and 334 Caucasians (C) undergoing diagnostic coronary
angiography. Presence of CHD was defined as 50% stenosis in at least one artery. Allele
frequencies were 12% E2, 62% E3, 26% E4 in AA, and 8% E2, 78% E3, 14% E4 in C. ApoE2/4
heterozygotes were excluded from analysis. Among AA, CHD was present in 9 of 34 E2 carriers,
significantly smaller (P0.05) in proportion compared to 39 of 82 E3/3 and 43 of 93 E4
carriers, suggesting a protective effect of the E2 allele. Among C, the CHD proportions were 23
of 39 E2 carriers, 121 of 205 E3/3 and 40 of 75 E4 carriers, not significantly different from one
another. Case-control differences were significant in both ethnic groups (P0.02 for AA and
P0.01 for C) for LDL cholesterol with cases having higher LDL cholesterol levels than
controls. In AA, HDL cholesterol was lower among cases than controls (P0.03). In AA but not
in C, LDL in E2 carriers was lower than in non-E2 carriers (P0.005). Since lipid levels were
predictive of CHD in both ethnic groups and E2 carriers had more favorable lipid levels, a
multiple logistic regression was done to determine if E2 had a separate protective effect beyond
its effect on lipid levels. After adjusting for lipid levels, the association between apoE2 and CHD
was no longer significant (the P value in AA went from 0.04 to 0.18). Thus, the protective effect
of apoE2 seen in AA could be explained by the favorable lipid profile in E2 carriers, while in C,
the absence of such an effect could be due to the lack of effect of apoE2 on the lipid profile
Differential Effects of Atorvastatin and Fenofibrate on HDL Kinetics in
Overweight Subjects
P236
Hugh R Barrett, Anthony G Johnson, June Ji, Kevin D Croft, Adrian Serone, Gerald F Watts.
University of Western Australia, Perth, Australia; GlaxoSmithKline, King of Prussia, PA;
GlaxoSmithKline, Sydney, Australia
Overweight (OW) and obesity are associated with increased risk for cardiovascular disease
(CVS) and this may be related to low plasma concentrations of high-density lipoproteins (HDL),
and hence to depressed reverse cholesterol transport (RCT). Statins and fibrates are commonly
used in OW, dyslipidemic patients and have been shown to reduce CVS risk in clinical trials.
However, the precise mechanisms of action involved and the specific effects on HDL
metabolism of these agents have not yet been fully established. Therefore, the aim of this study
was to examine the effect of Atorvastatin (AT) and Fenofibrate (FF) on HDL kinetics. We studied
12 dyslipidemic OW men (BMI27 kg/m 2 , plasma cholesterol (C) 6.03/-0.23 mmol/L, HDL
cholesterol (HDLC) 0.96/-0.05 mmol/L, triglyceride (TG) 2.29/-0.29 mmol/L, apoB 1.13/-
0.03 mmol/L, apoAI 1.14/-0.04 mmol/L) in a double-blind, randomized, placebo controlled,
cross-over trial. Subjects were treated once daily with micronised FF (200mg), AT (40mg) or
placebo (P) for 6-week periods with 2-week washouts. HDL apoAI turnover was measured
following iv administration of d 3-leucine. HDL apoAI was isolated using PAGE and isotopic
enrichment measured with GCMS. Metabolic parameters were estimated by compartment
modeling. Compared with reference data, HDL apoAI fractional catabolic rate (FCR) was higher
in the OW subjects, but apoAI production rate (PR) was normal. Compared with P, AT reduced
plasma C 41% (p0.001), TG 35% (p0.002) and apoB 42% (p0.001). FF treatment
reduced plasma TG 28% (p0.004), apoB 12% (p0.015) and increased HDLC 7%
(p0.005). Compared with P, FF increased HDL apoAI concentration 5% (p0.046) and apoAI
PR 14% (p0.037). HDL apoAI FCR did not differ between P, AT or FF. Although AT and FF both
reduced plasma TG concentrations, only AT significantly lowered plasma C. In contrast to AT,
FF was associated with increased plasma HDLC and apoAI concentrations; this effect was
related to increased PR of apoAI and may be due to increased apoAI transcription with FF. This
mechanism of action may account for the favourable effect of FF on RCT and a specific CVS
benefit of PPAR- agonists compared with statins.
P237
Plasma ApoA-I Kinetics in Complete Hepatic Lipase Deficiency in Humans
Isabelle Ruel, Patrick Couture, Jeffrey Cohn, Benoit Lamarche. Nutraceuticals and Functional
Foods Institute, Sainte-Foy, QC, Canada; Lipid Research Center, CHUL, Sainte-Foy, QC,
Canada; Hyperlipidemia and Atherosclerosis Research Group, Montreal, QC, Canada
Complete hepatic lipase (HL) deficiency in humans is a rare monogenic disorder associated
with increased plasma apoA-I and HDL cholesterol levels, and with a marked triglyceride (TG)
enrichment of HDL particles. It has been documented that in the presence of active
intravascular HL, the TG-enrichment of HDL is associated with an enhanced metabolic
clearance of HDL apoA-I. The aim of the study was to test the hypothesis that the catabolic rate
of plasma apoA-I is reduced in complete HL deficiency. The female proband of a kindred with
HL deficiency and 2 of her younger brothers were found to have undetectable HL activity, which
was accompanied by the presence of -VLDL and elevated plasma apoA-I and HDL-TG
concentrations. by guest All 3 subjects on April were4, E32013 homozygotes. The in vivo kinetic of apoA-I was studied

a-42 Arterioscler Thromb Vasc Biol. Vol 22, No 5
in the 3 patients with complete HL deficiency and in 6 male and female control subjects using
a primed-constant infusion of L-[5,5,5-D3]leucine for 12 h in fasting state. The apoA-I (d1.25
g/L) tracer/tracee enrichment curve over time was determined by gas chromatography-mass
spectrometry and fitted to a monoexponential function to determine the rate of apoA-I
production and catabolism. The mean fractional catabolic rate of plasma apoA-I was reduced
in HL deficient patients compared to control subjects (0.118 0.007 vs. 0.173 0.054
pools/day, P0.06). As a result, the residence time of plasma apoA-I was 34 % greater among
HL deficient patients compared to controls. On the other hand, the production rate of plasma
apoA-I was similar between the two groups (9.70 3.66 vs. 9.56 2.63 mg/kg/day in HL
deficient and control subjects respectively, P0.9). Similar trends were observed when men
and women were analyzed separately. These data confirm that the accumulation of
TG-enriched HDL particles in patients with complete HL deficiency is largely due to a reduction
in the catabolic rate of apoA-I
Niacin Non-Competitively Inhibits Hepatocyte Diacylglycerol
Acyltransferase, a Key Enzyme for Triglyceride Synthesis
Shobha H Ganji, S Tavintharan, Daming Zhu, Vaijinath S Kamanna, Moti L Kashyap. VA
Healthcare System, Long Beach, CA; University of California at Irvine, Irvine, CA
P238
Background: Niacin is a widely used lipid regulating agent with a broad spectrum beneficial
effect on lipoproteins in dyslipidemic patients. Previously, we have shown that it increases the
degradation of hepatic apolipoprotein B (apo B, the major apolipoprotein of atherogenic
lipoproteins) by inhibiting triacylglycerol (TG) synthesis from fatty acids and glycerol. In this
study, we hypothesized that niacin inhibits diacylglycerol acyltransferase (DGAT), a key enzyme
for TG synthesis. Methods: Human hepatoma cells (Hep G2) grown in DMEM containing 10%
FBS were used to prepare microsomes by ultracentrifugation at 100,000 g. Microsomal DGAT
activity in the absence or presence of niacin was measured by assessing the formation of TG
using 14C-oleoyl-CoA and dioleoylglycerol as substrates. Enzyme kinetic parameters, including
Km, Vmax, Lineweaver and Burk (LB) plot for competitive/non-competitive inhibition, and IC-50
were determined. Results: DGAT activity measurement at various concentrations of oleoyl-CoA
showed a Km value of 8.3 M . Addition of niacin (0–3 mM) to the assay reaction mixture (at
30 min, 25 C) significantly reduced DGAT activity by 40–50%. Dose-response studies with
niacin showed an IC-50 of 0.3 mM. To assess the competitive or non-competitive type of
inhibition, the rate of reaction of DGAT was measured at various concentrations of substrate
(oleoyl-CoA; 2.5–20 M) in the absence or presence of niacin. LB plot of DGAT inhibition by
niacin showed a non-competitive pattern of inhibition. There was a decrease in Vmax values
with niacin while the Km remained constant in control and in presence of niacin. Conclusions:
These data define DGAT as a major target for the mechanism of action of niacin. We suggest
that the direct inhibition of DGAT by niacin results in the observed increased apo B degradation
leading to a reduction of hepatic atherogenic lipoprotein secretion. DGAT inhibitors therefore
may represent a new class of drugs that regulate atherogenic lipoproteins by this mechanism.
P239
Combination of a Novel Inhibitor of the Apical Sodium-Bile Acid
Cotransporter, SC-435, and Atorvastatin Reduces LDL-Cholesterol through
Decreased Secretion and Enhanced Clearance of LDL ApoB
Dawn E Telford, John R Burnett, P Hugh R Barrett, Bradley T Keller, Murray W Huff. The
John P Robarts Research Institute, London, ON, Canada; University of Western Australia,
Perth, WA, Australia; University of Western Australia, Perth, Australia; Pharmacia Corp, St.
Louis, MO
Discovery of the ileal apical sodium-bile acid cotransporter (ASBT) permitted development of
specific inhibitors of bile acid reabsorption, potentially, a new class of cholesterol lowering
agents. Recently, we showed that SC-435, a novel ASBT inhibitor, significantly reduced
LDL-cholesterol (C) in miniature pigs. In the present study we tested the hypothesis that
combining SC-435 with the HMG-CoA reductase inhibitor, atorvastatin, would potentiate LDL-C
reduction and the regulation of apoB metabolism. ApoB kinetic studies were performed in
miniature pigs after 21 days treatment with SC-435 (5 mg/kg/d) and atorvastatin (3
mg/kg/d)(SC-435 A) or placebo (n6/group). Pigs were fed a diet containing fat (35% of
energy) and C (400 mg/d). SC-435 A decreased plasma total C by 23% (2.22 vs 2.89
mmol/L, p0.004) and LDL-C by 40% (0.86 vs 1.44, p0.0004). Other plasma lipids were
unchanged. Autologous 131I-VLDL, 125I-LDL and 3H-leucine were injected simultaneously and
apoB kinetic parameters determined by multicompartmental analysis (SAAM II). SC-435 A
had little effect on VLDL apoB kinetics. LDL apoB decreased by 35% (6.6 vs 10.1 mg/kg,
p0.004), due to a 45% (0.060 vs 0.044 h-1, p0.05) increase in the LDL apoB fractional
catabolic rate (FCR). Furthermore, LDL direct synthesis was decreased by 17% (0.26 vs 0.32
mg/kg/h, p0.05), whereas conversion of VLDL apoB to LDL was unchanged. SC-435 A
significantly decreased hepatic concentrations of free C (-12%; 2.3 vs 2.0 mg/g) and esterified
C (-47%; 0.18 vs 0.35), increased hepatic 7-alpha OHase activity 2.3-fold (p0.03) and
increased hepatic LDL receptor mRNA 1.8-fold (p0.05). As monotherapy, SC-435 (10
mg/kg/d) decreased LDL apoB by 10% (8.7 vs 9.8; p0.035), due entirely to a 17% (0.052 vs
0.044; p0.032) increase in LDL apoB FCR. Atorvastatin monotherapy (3 mg/kg/d) decreased
LDL apoB by 29% (4.7 vs 6.8; p0.0004), due primarily to a 22% (0.23 vs 0.30; p0.004)
reduction in LDL apoB production. We conclude that SC-435 A potentiates the reduction of
LDL-C and LDL apoB due to their complementary mechanisms of action.
P240
Female Swine Develop Greater Carotid Atherosclerosis than do Male Swine
Care and Use Committee of the University of Missouri approved all procedures. Sexually mature
female (n16) and male (n16) Sinclair miniature swine between the ages of 9 and 12
months were housed in a temperature-controlled room (20 to 22 C) with a 12-hour light/dark
cycle. Pigs were fed once daily for 12 weeks a high fat, high cholesterol diet consisting of
Purina Minipig chow supplemented to contain 2% cholesterol, 17.1% coconut oil, 2.3% corn
oil, and 0.7% sodium cholate. Total, low-density, and high-density lipoprotein (HDL) cholesterol
were analyzed in plasma obtained from blood samples taken following an overnight fast on
days 0 and 84. On day 84 pigs were anesthetized intramuscularly with atropine, ketamine, and
xylazine and the chest was opened to achieve euthanasia. The common carotid arteries were
harvested 5 mm above and below their origin from the brachiocephalic artery and fixed in
neutral buffered 10% formalin. Arterial samples were opened longitudinally, stained with Sudan
IV, flattened under a glass plate, and photographed digitally. The area of positive Sudan IV
staining was calculated as a percent of total luminal area (percent Sudanophilia) utilizing
ImagePro Plus. Cross sections of the arterial wall were taken from the most intensely
Sudanophilic portion of each sample and processed routinely through paraffin embedment. Five
micron sections were stained with Verhoeff’s method for elastin and the maximal intimal
thickness was measured by digital light microscopy. Results and Conclusions: The percent
Sudanophilia was greater in female (37.7 /- 28.4) than male (10.5 /- 9.6, P 0.002) pigs.
The maximal intimal thickness was greater in female (384 /- 333 microns) than male (192
/- 148 microns, P 0.05) pigs. There was no difference in HDL on day 0, however HDL on
day 84 was lower in female (76 /- 14) than male (89/- 15, P 0.02) pigs and correlated
inversely with percent Sudanophilia (R -0.56) and maximal intimal thickness (R-0.39)
Differential Lipid Binding of the N-terminal Domain of ApoE Isoforms
Paul M Weers, Robert O Ryan. Children’s Hospital Oakland Research Institute, Oakland, CA
P241
Apolipoprotein E (apoE) is a 34 kDa protein that is involved in plasma lipid homeostasis through
its interaction with the low density lipoprotein receptor family. The protein contains two
independently folded and functional domains, a 22-kDa N-terminal (NT) and a 10-kDa
C-terminal domain. The NT domain (residues 1–183) undergoes a conformational reorganization
of its four helices upon binding to a lipid surface. Three common isoforms exist (apoE2,
apoE3 and apoE4), differing in cysteine and arginine content at position 112 and 158. They also
differ in biophysical properties, which may be related to isoform-specific correlations with heart
disease and Alzheimer’s disease. We therefore examined the lipid binding properties of the NT
domain of apoE2, apoE3 and apoE4 using model lipid surfaces. The ability of the protein to
interact with large unilamellar phospholipid vesicles composed of anionic dimyristoylphosphatidylglycerol
(DMPG) or zwitterionic dimyristoylphosphatidylcholine (DMPC) was investigated.
Incubation of apoE-NT with vesicle dispersions results in their transformation into
discoidal complexes. Based on the rate of DMPG vesicle transformation, the interaction of
apoE4-NT was much stronger than apoE3-NT, while apoE2-NT showed the weakest interaction.
The transformation of DMPC vesicles by apoE-NT was poor at pH 7.2 but transformation rates
increased dramatically at pH 3. However, no differences in the interaction of apoE-NT isoforms
with DMPC vesicles were observed under these conditions. Fluorescent dye experiments with
8-anilino-1-naphthalene sulfonate revealed increased exposure of the hydrophobic interior at
pH 3 for all isoforms, indicating adoption of a molten globule-like state. In this state,
isoform-specific differences in tertiary structure may be diminished. This study shows that
differences in the biophysical properties of apoE-NT isoforms may result into distinct
interactions with various lipid substrates.
P242
Lipoprotein Lipase Is One of the Important Determinant Factors for LDL
Particle Sizes and Triglycerides and HDL Metabolism, but not Cholesterol
Ester Transfer Protein in Healthy Japanese
Hiroshi Mokuno, Haruyo Yamashita, Kazunori Shimada, Eriko Seki, Yoshitaka Iwama,
Testurou Miyazaki, Yoshirou Watanabe, Hiroyuki Daida. Juntendo University, Tokyo, Japan
Production of small, dense LDL, high levels of triglycerides (TG) and low levels of HDL
cholesterol (Chol) have been reported for atherogenic lipid profile in insulin resistant
metabolism. To determine the lipid metabolic enzyme attributing to this lipid profile, we
measured fasting serum lipoprotein lipase (LPL) and cholesterol ester transfer protein (CETP)
by ELISA in 237 healthy Japanese subjects. LDL size was determined by non-denature gradient
PAGE. Correlation analysis of LPL mass (mean S.D. : 43.713 ng/ml) and CETP mass
(1.920.57 mg/ml) was performed with age (489 yo), BMI (23.062.9), LDL Chol (11632
mg/dl), TG (10887 mg/dl), HDL Chol (6618 mg/dl), glucose (9413 mg/dl) and LDL size
(26.30.9 nm). In result, LPL had positive correlation with HDL Chol (r0.48) and LDL size
(r0.26) and negative correlation with BMI (r-0.26), TG (r-0.34) and glucose (r-0.24)
significantly (p0.01, respectively). In contrast, CETP positively correlated with only LDL Chol
(r0.25, p0.01). These results indicated that LPL is associated with not only TG and HDL
metabolism and also LDL size and glucose metabolism. LPL may be important lipid enzyme
determining atherogenic lipid profile in insulin resistance, compared with CETP.
P243
Atorvastatin Reduces VLDL ApoE and Total Plasma ApoE Production in
Patients with Combined Hyperlipidemia
Jeffrey S Cohn, Michel Tremblay, Rami Batal, Helene Jacques, Lyne Veilleux, Claudia
Rodriguez, P Hugh R Barrett, Lise Bernier, Orval Mamer, Jean Davignon. Clinical Research
Institute of Montreal, Montreal, QC, Canada
James Turk, Tom Thomas, Michael Sturek, M Harold Laughlin. University of Missouri,
Atorvastatin, a synthetic HMG-CoA reductase inhibitor, significantly lowers plasma cholesterol
Columbia, MO
and low-density lipoprotein (LDL) cholesterol levels and reduces total plasma triglyceride and
apoE concentrations. In view of the direct involvement of apoE in the pathogenesis of
Objective: The objective of this study was to examine the influence of gender upon carotid atherosclerosis, we have investigated the effect of atorvastatin treatment (40 mg/day) on in vivo
atherosclerosis induced by an atherogenic diet inDownloaded swine. Materialsfrom and Methods: http://atvb.ahajournals.org/
The Animal rates of plasma by guest apoE production on April and 4, catabolism 2013 in 6 patients with combined hyperlipidemia,

using a primed constant infusion of deuterated leucine. Atorvastatin treatment resulted in a
significant (30–37%) decrease in levels of total triglyceride, cholesterol, LDL cholesterol and
apoB in all 6 patients. Total plasma apoE concentration was reduced from 7.4 0.9 to 4.3
0.2 mg/dl (-38 8%, P 0.05), predominantly due to a decrease in VLDL apoE (3.4 0.8
vs 1.7 0.2 mg/dl; -42 11%) and IDL/LDL apoE levels (1.9 0.3 vs 0.8 0.1 mg/dl;
-57 6%). VLDL apoE production was reduced from 3.82 0.67 to 2.26 0.42 mg/kg/day
(-36 10%, P 0.057) and total plasma apoE production was significantly reduced from
4.67 0.39 to 3.04 0.51 mg/kg/day (-34 10%, P 0.05). VLDL and total plasma apoE
residence times, and HDL apoE kinetic parameters were not significantly affected by drug
treatment. Percentage decreases in VLDL apoE concentration and VLDL apoE production were
significantly correlated with drug-induced reductions in VLDL triglyceride concentration (r
0.99, P 0.001; r 0.88, P 0.05, respectively, n 6). Our results demonstrate that
atorvastatin causes a pronounced decrease in VLDL apoE and total plasma apoE concentrations
and a significant decrease in VLDL apoE and total plasma apoE rates of production in patients
with combined hyperlipidemia.
P244
Validation of 1 H MRS in the Measurement of Hepatic Triglyceride Content
in Mice In Vivo
Xiaobo Lin, Nobuhiro Sakata, Joel Garbow, Zhouji Chen, Gustav Schonfeld. Washington
University in St. Louis, St. Louis, MO
Magnetic resonance spectroscopy (MRS), a noninvasive modality, may be useful for repeated
measurements of hepatic lipids over time, in vivo. The methylene proton ( 1 H) signals in 1 H MRS
originate mainly from the mobile fatty acyl chains of tissue triglycerides. Respiratory gating is
essential for correct placement of voxels. Hepatic lipid contents were measured (n6) both in
vivo using MRS and biochemically by lipid extraction followed by enzymatic assay. MRS results
were significantly correlated with triglyceride levels obtained by biochemical analysis (correlation
coefficient, r 0.97, P 0.001). MRS was used to investigate diurnal changes of
hepatic triglyceride levels in the apoB38.9 truncation-bearing mice (n6), generated by
conventional gene-targeting in embryonic stem cells and the Cre-loxP system. These mice have
a reduced capacity to export hepatic triglycerides. Preliminary MRS data indicate that the mean
hepatic triglyceride level of heterozygous apoB38.9 mice was 1.5-fold higher than wild type
controls (n6) (P 0.07). Food intake for the wild types and the heterozygotes was similar,
with 61% (10.2 g of 17 g) consumed in the dark cycle but only 39% (6.8 g of 17 g) in the light
cycle. Heterozygotes, but not wild types, tended to accumulate more hepatic triglycerides at
22:00 hours and 6:00 hours than at 12:00 hours. Thus, MRS appears to be a useful tool for
measuring hepatic triglyceride levels in mice, in vivo.
Structure and Function Studies of Human Apolipoprotein A-V
P245
Robert O Ryan, Michael N Oda. Children’s Hospital Oakland Research Institute, Oakland, CA
Studies have been conducted to characterize a newly discovered plasma apolipoprotein, human
apolipoprotein A-V (apoA-V). Recent studies reveal an important role of apoA-V in maintenance
of plasma triacylglycerol (TG) levels. In a murine setting, apoA-V transgenic animals display a
threefold decrease in plasma TG while apoA-V null mice have a fourfold increase in plasma TG
levels. The mechanism whereby apoA-V achieves this effect has not been determined although
it is known to associate with high-density lipoproteins in rodent plasma. We have examined the
structure and function of isolated recombinant human apoA-V expressed in E. coli. Spectroscopic
studies and secondary structure analysis reveals that apoA-I possesses a high degree
of alpha helix secondary structure. Lipid binding studies conducted with recombinant apoA-V
reveal a functional ability to transform phospholipid bilayer vesicles into reconstituted high
density lipoprotein complexes. Studies of apoA-V structural and lipid binding properties may
provide insight into the molecular mechanism whereby this protein influences plasma
triacylglycerol levels.
Measuring Blood Viscosity: A Key Factor in Shear Stress and
Atherogenesis
Kenneth R Kensey, Anders Boss. Rheologics, Inc, Exton, PA
P246
Background: Shear stress is a key regulator of endothelial function. Shear stress depends on
blood viscosity, flow velocity and arterial diameter. Of these three variables, blood viscosity is
the most susceptible to influence by exogenous factors such as smoking, lipid levels and
diabetes and is a likely mediator of several well-recognized risk factors. In a normal population,
blood viscosity in the upper quartile is associated with a 50% increased risk of cardiovascular
event, increasing to a 300% in individuals with pre-existing vascular disease. Method: We used
a new device, the Kensey Rheolog, to study blood viscosity in 58 healthy subjects at the AHA
Scientific Sessions 2001. The Kensey Rheolog provides blood viscosity data over a physiological
representative range of flow rate (shear rate) without the need for anticoagulants. We also
studied the effect of regular blood donation in a group of nine healthy volunteers. Results: The
measurements showed little variation at high shear. More individual variations occurred at low
shear rate. Women had on average a lower blood viscosity than men (po .00009). We saw
a change in blood viscosity at low shear in individuals with postprandial hypercholesterolemia,
as well as a difference between the individuals with high and low HDL. The nine individuals who
gave blood regularly over a five-week period demonstrated a progressive drop in blood
viscosity. Conclusions: Blood viscosity varies considerably depending on flow rate with a much
higher viscosity at low flow rates. Cholesterol, HDL levels and HTC clearly influence blood
viscosity. Given the critical role of blood viscosity in determining shear stress and its
relationship to recognized risk factors, blood viscosity is a key determinant of cardiovascular
risk and its study with appropriate technology willDownloaded increase our understanding from
of atherogenesis.
http://atvb.ahajournals.org/
Poster Presentations a-43
P247 WITHDRAWN
P248
The Second Window of Antiarrhythmic Effect Mediated by Adenosine A1
Receptor Agonist R-PIA on Ischemia-Induced Ventricular Tachyarrhythmias
in Rats
Guo Jin-Ai, Jing Chen, Dai Cheng-Xiang. No. 466 Hospital of People’s Liberation Army,
Beijing, China
Objective The aim of this study was to determine whether pretreatment with adenosine A 1
receptor agonist R-PIA 24 hours previous to coronary ligation has antiarrhythmic effects on
ischemia-induced ventricular tachyarrhythmias in rat. Methods Eighty male Wistar rats were
randomly divided into five groups(n16).Group A :normal saline 0.2ml i.c each rat; Group B :
R-PIA 0.03mg.kg -1 i.c each rat; Group C: R-PIA 0.03mg.kg -1 i.c and DPCPX 0.2mg. kg -1 i.c each
rat; GroupD: R-PIA 0.03mg.kg -1 i.c and L-NNA5mg.kg -1 i.p each rat; GroupE: R-PIA 0.03mg.kg -1
i.c each rat and 20min previous to coronary ligation, glibenclamide 0. 3mg.kg -1 i.v each rat.
Sixteen rats in separate groups, 24 hours after pretreatment as above, were opened chest and
subjected to 30 min of regional myocardial ischemia by ligation of the left anterior descending
coronary artery followed by 120 min of reperfusion, and continually monitored and analysed left
ventricular pressure curve and the rhythm and frequency of heartbeat by Cardio2 soft ware.
Combining left ventricular pressure curve with electrocardiography, analysed and educed the
frequency, onset time ,duration and total onset number of ventricular tachycardia and
fibrillation. The experiment data were processed by SPSS, followed by the Fisher protected
least significant difference test (q test) and Chi-square test. The null hypothesis was rejected
at P0.05. Results One-way analysis of variance (ANOVA) express no significant difference in
parameters of left ventricular function, the rate and rhythm of heartbeat between groups before
myocardial ischemia (P0.05). In 30 minutes suffered ischemic insult, the frequency and
duration of ventricular tachycardia and fibrillation in group R-PIA, compared with group normal
saline , significantly reduced (P 0.01);Adenosine A1 receptor antagonist DPCPX and
ATP-sensitive potassium channels antagonist glibenclamide could abrogate complately and
partly respectively antiarrhythmic effect mediated by adenosine A 1 receptor agonist R-PIA
pretreatment on ischemia-induced ventricular tachyarrhythmias, but nitric oxide synthesis
inhibitor L-NNA couldn’t weaken the role of R-PIA. Conclusion This study suggested that
adenosine A 1 receptor agonist R-PIA pretreatment could bring on antiarrhythmic effect on
ischemia-induced ventricular tachyarrhythmias, and the role of R-PIA was partly resulted from
ATP-sensitive potassium channels open through post-receptor mechanism. Nitric oxide didn’t
appear to participate in triggering mechanism of above the role.
P249
Exacerbated Atherosclerosis of Double-Mutant Lyst beige , LDLr-/- Mice Is Not
Due Solely to Altered Macrophage Function
Natalie K Schiller, Audrey S Black, Nobuhiko Kubo, William A Boisvert, Linda K Curtiss. The
Scripps Research Institute, La Jolla, CA
In previous studies, we found that LDL receptor-deficient mice (LDLr-/-) crossed with
lysosomal-defective Lyst beige mice (Bg,LDLr-/-) had increased atherosclerosis compared to
control. Further, it was found that macrophage (M) staining in Bg,LDLr-/- aortic root lesions
was decreased and M distribution was limited to the lesion lumenal surface. In this study,
we tested whether altered M function in Bg,LDLr-/- mice contributed to their exacerbated
atherosclerosis. First, Bg,LDLr-/- M were evaluated in vitro for cellular migration, lipid
accumulation and apoE production. Splenocytes isolated from Bg,LDLr-/- mice migrated better
than LDLr-/- splenocytes through a Matrigel-coated membrane in response to MCP-1 (p
0.007). Bone marrow-derived M were cultured for 24 hours with or without 50 g/mL
acetylated LDL (acLDL) to assess the accumulation of cholesteryl ester and free cholesterol as
well as to quantitate apoE production. Lipid accumulation was similar between Bg,LDLr-/- and
LDLr-/- M. ApoE production was significantly increased in the Bg,LDLr-/- M cultures (p
0.007), however this difference disappeared upon addition of acLDL. To determine whether
these in vitro differences in M function affected atherosclerosis, 24 male LDLr-/- mice (6–8
weeks) underwent total body irradiation (10 gy) and reconstitution with either Lyst beige (Bg) or
control bone marrow. The mice were fed a high fat diet for 16 weeks beginning 4 weeks after
irradiation and reconstitution. Surprisingly, no differences were observed in aortic root lesion
area or lipid accumulation within the aorta. Further, both MOMA-2-positive M staining and
distribution in the aortic root lesions were similar between mice reconstituted with Bg or control
bone marrow. Although functional differences in Bg,LDLr-/- M were observed in vitro, in vivo
manifestations of these differences as assessed by bone marrow transplantation were
unremarkable. This suggests that defective M function is not solely responsible for the
exacerbated atherosclerosis of the Bg,LDLr-/- mice and that Lyst beige defects in other cell types
such as smooth muscle cells may also be involved.
P250
Apolipoprotein B, Small Dense LDL and Body Mass Index as Diagnostic
Criteria for Familial Combined Hyperlipidemia: Results of a 5-Year
Follow-Up Study
Mario Veerkamp, Jacqueline De Graaf, Jan Hendriks, Pierre Demacker, Anton Stalenhoef.
UMC Nijmegen, Nijmegen, Netherlands
Introduction: Familial Combined Hyperlipidemia (FCH) is diagnosed by total cholesterol (TC)
and/or triglyceride (TG) concentration above the 90th percentile. We have shown in 299
subjects of 32 families that the diagnosis FCH was inconsistent in 26% of the subjects over a
five years period (1994–1999). These results emphasize the need for re-evaluation of the
diagnostic criteria for FCH. Aim of the study: To evaluate whether apolipoprotein B (apo B)
levels, small dense LDL and body-mass-index (BMI) improve diagnostic criteria for FCH.
Methods: In by total guest 32 families, on April including 4, 2013 299 subjects, were studied in both 1994 and 1999. Apo

a-44 Arterioscler Thromb Vasc Biol. Vol 22, No 5
B (immunonephelometry, mg/l) and LDL subfractions (density-gradient-ultracentrifugation)
were determined in both 1994 and 1999. LDL subfractions were described by a quantitative
K-value in which a negative value of K reflects small dense LDL. A subject was defined ’truly’-
FCH when diagnosed FCH in 1994 and/or 1999. Logistic discriminant analysis (under condition
of equal assessment of misclassification with maximal sensitivity and specificity) was used to
find the optimal cut-off point for apo B, K-value and BMI for prediction of ’truly’- FCH. Likelihood
ratio tests were used to select between models. Results: In total 40% (n121) of the subjects
were ’truly’-FCH. The ’truly’ affected status of a subject was best predicted by apo B levels
1200 mg/l, a K-value of -0.10 and BMI 25.0 kg/m2 . Both sensitivity and specificity
were satisfactionally high. Sens. ranges from 65% to 77% while the spec. ranges from 75%
to 80%. Multiple logistic regression analysis showed that apo B, K-value and BMI (all p0.05)
independently contribute to better diagnostic criteria for ’truly’- FCH than compared to FCH
based on TC and/or TG alone. Stepwise selection procedures showed that K-value and BMI
were sufficient to predict ’truly’- FCH (adjusted odds ratios 0.24 (95% CI: 0.09–0.60) and 3.8
(95% CI: 1.6–9.9), respectively). Conclusion: Our results show that apo B, small dense LDL and
BMI significantly improve the established diagnostic criteria for FCH, in which K-value and BMI
are most predictive.
P251
Cytotoxicity in J774 Macrophage Foam Cells Induced by Intracellular
Cholesterol Accumulation Is Attenuated by Incubation with Apolipoprotein
AI
Ginny L Kellner-Weibel, Steven J Luke, George H Rothblat. The Children’s Hospital of
Philadelphia, Philadelphia, PA
Excess intracellular free cholesterol (FC) is cytotoxic. The present study examines prevention
of FC-induced cytotoxicity in J774 macrophage foam cells by incubation with apolipoprotein AI
(apoAI). J774 macrophage cells were enriched with esterified cholesterol (EC) by exposure to
acetylated low-density lipoprotein and FC/phospholipid dispersions, thus creating a model foam
cell. Treatment of these foam cells with an acyl coenzyme-A:cholesterol acyltransferase (ACAT)
inhibitor, CP-113,818 (2g/ml), in the absence of an extracellular cholesterol acceptor,
resulted in hydrolysis of stored EC and subsequently FC-induced cytotoxicity. Incubation of
foam cells with the ACAT inhibitor plus apoAI (50g protein/ml) resulted in FC efflux (0.22
0.02% / hour) along with a reduction in cytotoxicity (14.6 1.3% reduction), measured by
adenine release. Small unilamellar vesicles (SUV, 100g phospholipid/ml) caused greater FC
efflux (0.29 0.02% / hour), although only a modest reduction in cytotoxicity (4.7 3.0%
reduction) was measured. Previously we have shown that the intracellular cholesterol transport
inhibitor, U18666A abolishes FC-induced cytotoxicity when macrophage foam cells are
incubated with the ACAT inhibitor by preventing movement of FC to the plasma membrane. In
the present study, co-incubation of the ACAT inhibitor plus U18666A (2g/ml) reduced efflux
to apoAI (40.6 1.31% reduction), but did not result in a reduction of efflux to SUV.
Pre-treatment of the J774 mouse macrophage foam cells with CPT-cAMP (0.3mM for 12 hours)
upregulates hormone sensitive lipase and ABCA1. In experiments using mouse serum as a
cholesterol acceptor, CPT-cAMP caused greater protection against FC-induced cytotoxicity
compared to cells without the pre-treatment, suggesting a role of ABCA1 in removal of cytotoxic
FC. We conclude that in this system, a cytotoxic pool of FC is located in the plasma membrane,
this FC is readily available for efflux to apoAI, and removal of excess cytotoxic cholesterol may
involve the ABCA1 transporter.
P252
Extracellular Superoxide Dismutase Gene Polymorphism in Mouse Models
of Atherosclerosis
Anson P Pierce, Jason Whitlark, Ladislav Dory. University of North Texas Health Science
Center at Fort Worth, Fort Worth, TX
We report for the first time a polymorphism in the mouse extracellular superoxide dismutase
(ecSOD) gene. Two amplicons corresponding to the 3’ untranslated region of the ecSOD gene
were detected in liver and macrophage cDNA from B6.129 ApoE and LDLR double KO mice by
RT-PCR. Sequence analysis revealed that the two amplicons, reproduced by PCR from genomic
DNA, differ by a 10 bp deletion in the 3’ untranslated region (bp 758–767). Restriction enzyme
analysis revealed an additional single nucleotide polymorphism (SNP) site, an A-G change at
bp 61, which always accompanies the 10bp deletion. This SNP leads to an Asn-Asp
substitution that resides in the putative signal sequence of the protein. Since the B6.129 mouse
is an F2 hybrid of the 129Sv/J and C57Bl/6J mouse strains, the 129P3/J (parental strain) and
C57Bl/6J mice were genotyped, by a procedure developed in our lab, for the 10 bp deletion and
the SNP. The atherosclerosis-resistant 129P3/J mouse is homozygous for the ecSOD gene
containing the A-G change and the 10bp deletion (short gene), while the atherosclerosissusceptible
C57Bl/6J mouse is homozygous for the longer variant of the ecSOD gene. This
variation appears to influence the extent of ecSOD expression/activity. Thus, analyses of ecSOD
activities show that age-matched female 129P3/J mice (athero-resistant) have 2-fold higher
circulating ecSOD in plasma (p0.0042), 1.3-fold higher heparin releasable ecSOD
(p0.0002), and a higher level of ecSOD activity bound to the arterial wall when compared to
C57Bl/6J females (athero-susceptible). Age- and sex-matched B6.129 apoE and LDLR double
KO mice homozygous for the long ecSOD gene have 2.6-fold lower ecSOD activity than
heterozygous sex-matched littermates. A failure to recognize this polymorphism has led some
investigators to incorrectly characterize the pattern of ecSOD expression during the development
of atherosclerosis in mice. Therefore, studies using genetically altered, but insufficiently
backcrossed, mouse models of disease should be Downloaded interpreted withfrom caution.
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P253
Integrin-Dependent Adhesion Targets the Protein Synthetic Machinery of
Platelets to Distinct Intracellular Regions: Evidence for the Development of
a “Translational Core”
Stephan Lindemann, Neal D Tolley, Guy A Zimmerman, Andrew S Weyrich. Human
Molecular Biology and Genetics, Salt Lake City, UT
Here we demonstrate that adherence to purified collagen, fibrinogen, and laminin markedly
increases protein synthesis by platelets. As measured by incorporation of [ 35 S]-labeled proteins,
proteins are differentially synthesized on each matrix, with fibrinogen yielding the most robust
response. We next determined if the protein synthetic machinery is redistributed in fibrinogenadherent
platelets and whether spatial targeting of this machinery is required for efficient
translation of mRNAs. As expected, fibrinogen-adherent platelets form actin stress cables and
vinculin-rich focal adhesion complexes. Platelet RNAs, ribosomes, and eukaryotic translation
factors such as eIF4E (eukaryotic Initiation Factor 4E) and eIF2 (eukaryotic Initiation Factor
2) are redistributed to the middle of the cell, forming a nucleus-like complex that is
surrounded by the actin stress cables, referred to here as the Translational Core. This
translational core also contains 3-integrins, suggesting that integrins target the protein
synthetic machinery to distinct regions within the cell. Inhibition of cellular adherence by IIb 3
inhibitors or cell spreading by cytochalasin D prevented the formation of the translational core
and subsequent protein synthesis. Biochemical separation of translational components and
subsequent cDNA array analysis revealed that adherence to fibrinogen markedly increases the
number of transcripts bound to the mRNA-binding protein eIF4E compared to resting platelets.
Two of these eIF4E-bound mRNAs included Bcl-3 and IL-1, proteins that are synthesized in
an integrin-dependent fashion. Lastly, we demonstrate that 3 integrins and eIF4E are
redistributed from the monosomes of quiescent cells to the polysome-rich fractions of
fibrinogen-ligated platelets. Neutralization of IIb 3-integrins blocks this response. These data
indicate that integrins target the protein synthetic machinery to the translational core of
platelets and thereby regulate protein synthetic responses.
P254
Baboon Lp(a) Does Not Bind to Lysine and Fibrin despite the Presence of a
Strong Lysine Binding Site in Apo(a) Kringle IV Type 10
Janet Ho, Andrea Belczewski, Michael B Boffa, Marlys L Koschinsky. Queen’s University,
Kingston, ON, Canada
Insight into the role of lipoprotein(a) (Lp(a)) in fibrinolysis may be gained through comparative
analysis of the structure of the distinguishing protein component, apolipoprotein(a) (apo(a)),
from humans and other species. Human apo(a) contains ten types of plasminogen kringle (K)
IV-like sequences, followed by sequences which are similar to the kringle V and protease
domains of plasminogen. Human apo(a) KIV10 contains a strong lysine-binding site (LBS) which
has been proposed to mediate the lysine-dependent interaction of apo(a) with biological
substrates such as fibrin. Mutations in amino acids which form the LBS in KIV10 are present
in both rhesus monkey (Trp72/Arg) and chimpanzee apo(a) (Asp57/Asn) and have been
proposed to explain the lack of ability of the corresponding Lp(a) species to bind to lysine and
fibrin. The objective of our study was to characterize the lysine-binding properties of Lp(a) in
the baboon in order to further the comparative analyses with the other primate species. We
sequenced a segment of baboon liver cDNA spanning from KIV9 to the protease domain. The
organization of this region is similar to the rhesus monkey in that baboon apo(a) also lacks a
KV domain. Interestingly, we found that the baboon KIV10 sequence contained all of the
canonical residues identified in formation of the LBS. We then characterized the apo(a) KIV10
sequence from an additional 10 unrelated baboons; the sequence was identical to the initial
clone except that one allele from each of two animals encoded Arg at position 72 rather than
Trp. Based on the amino acid sequence of baboon KIV10, we were surprised to find that none
of the corresponding Lp(a) species bound to lysine-Sepharose even upon partial purification.
Additionally, we found that the Lp(a) bound very poorly if at all to plasmin-modified fibrin
compared to human Lp(a). Expression and purification of baboon and human KIV10 in bacteria
allowed us to verify that these domains bind comparably to lysine and lysine analogues. We
conclude that elements of apo(a) structure besides KIV10 sequence dictate the lack of lysine
binding of baboon Lp(a), and perhaps that of other non-human primates.
P255
Changes in Microvascular Structure and Growth Factor Expression in an
Angiogenesis Model
Eric M Brey, Larry V McIntire, Carol Johnston, Charles W Patrick Jr.. Rice University,
Houston, TX; M.D. Anderson Cancer Center, Houston, TX
Understanding the microarchitecture and expression of growth factors during microvascular
development is an important aspect of both normal tissue physiology and the evolution of many
pathologies. We previously reported the development of imaging methods that allow
high-resolution three-dimensional (3D) imaging of vascular microstructure and automated
analysis of comparative growth factor levels from immunohistochemistry. These methods were
employed to quantify the spatial and temporal aspects of angiogenesis in a fibrin gel, a model
of both wound healing and tissue engineering. Gels were implanted on a uniform muscle
surface within Lewis rats. Explants were removed at 7 and 14 days and snap frozen. Forty
serial 6-micron sections were cut on a cryostat, stained for CD31, digitally imaged, processed,
and volume rendered for 3D images of vascular microstructure. Vessel parameters were
calculated for the networks at each time point. Additional 6 mm sections were immunostained
for specific growth factors: VEGF, bFGF, TGF-b1, PDGF-B, and Ang-2, and for H&E’s. These
were selected due to their reported importance in the initial phase of angiogenesis and the
subsequent maturation of vessels through the regulation of stability and recruitment of support
cells. Growth factor levels were determined from the optical density of the samples. Statistical
analysis was performed using ANOVA with Tukey-Kramer post test. Data are presented as
mean /-by SEM. guest P0.05 on was April considered 4, 2013 statistically significant. Angiogenesis occurs rapidly

into fibrin gels by day 7, characterized by dense (242 /- 6 vessels/mm2) networks of small
vessels (diameter 10.7 /- 0.4 microns) expressing high levels of VEGF, TGF-b1, bFGF, and
Ang-1 throughout the tissue. By 14 days the networks are more dense (326 /- 28
vessels/mm2) with a broader range of vessel sizes (10.7 /- 1.4 microns). VEGF and TGF-b1
levels remain high, while bFGF, and Ang-2 are decreased. Spatially, 14 day TGF-b1 expression
localizes to larger vessels in the fibrovascular tissue near the muscle interface. Ang-2 and bFGF
expression decreased at the interface but remained high deeper in the gel.
ADAM-TS and Plasmin-Mediated Degradation of Versican during
Flow-Induced Intimal Atrophy in Baboon Polytetrafluorethylene (PTFE)
Grafts
P256
Richard D Kenagy, Jens Fischer, Mark Davies, Scott Berceli, John Sandy, Suzanne Hawkins,
Thomas Wight, Alexander Clowes. University of Washington, Seattle, WA; University of South
Florida, Shriners Hospital, Tampa, FL; Hope Heart Institute, Seattle, WA
In a bilateral aorto-iliac PTFE graft model of intimal atrophy in baboons, high blood flow caused
by placement of a downstream arterio-venous fistula causes intimal atrophy in the upstream
graft. We have investigated whether matrix degrading proteinases are altered in this model of
atrophy using the normal flow side (no fistula) as a control. After four days of high flow intimal
urokinase was increased (5.03.1 fold of normal flow; p.05; n5) and plasminogen
activator inhibitor-1 was decreased (0.430.15 fold of normal; p.05; n5). After 7 days
plasminogen, proMMP2, and proMMP9 were increased 3.31.2, 1.230.06, and 1.440.18
fold of normal, respectively (p.05; n5). Extracts of 4 day high flow intimas degraded more
35 S-methionine labeled versican (the major proteoglycan of the intimal matrix) compared to
normal flow intimal extracts (114% vs 328% of versican remaining, respectively; p.05;
n5). Degradation was inhibited by the serine proteinase inhibitor, AEBSF, and a blocking
antibody to plasmin , but not by the MMP inhibitor, BB94. Increased amounts of a 70 kD
N-terminal fragment of versican V1 cleaved at E 441 -A 442 were detected in the intima by western
analysis (7.24.9 fold of normal flow; p.03; n6) and immunostaining using a neoepitope
specific antibody (Sandy et al J Biol Chem 276:13372, 2001). This neoepitope is specific for
ADAM-TS enzymes. ADAM-TS4 and ADAM-TS5, but not ADAM-TS1 (TS4 and TS1 are known
to cleave versican at E 441 -A 442 ), protein were detected in intimal extracts. While ADAM-TS4
content was not markedly changed, ADAM-TS5 was decreased at 4 days by high flow
suggesting other ADAM-TSs mediate 70 kD DPE production. It is also possible that activated
ADAM-TSs lose C-terminal TS domains, which mediate binding to the ECM, and are lost from
the tissue. In summary, these data suggest that plasmin and ADAM-TS enzymes mediate
versican removal during flow-induced intimal atrophy in the baboon PTFE graft.
P257
Rho Regulates Extracellular Matrix-Mediated Activation of Arterial Smooth
Muscle Cells
Joy Roy, Bimma Henderson, Johan Thyberg, Ulf Hedin. Karolinska Hospital, Stockholm,
Sweden; Karolinska Institutet, Stockholm, Sweden
The activation of vascular smooth muscle cells (SMCs) from a contractile to a synthetic
phenotype is a prerequisite for the migration and proliferation of these cells after arterial injury.
This process is dependent on changes in the extracellular matrix composition and includes
major changes in SMC structure and function. However, the molecular mechanisms involved
are not known. We have previously shown that integrin-linked tyrosine kinase activity and
ERK1/2 activation are necessary for phenotypic modulation. The rho GTPases have been shown
to regulate signaling pathways that mediate cytoskeletal reorganization and focal adhesion
formation. 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are known to
block rho activation by inhibiting the synthesis of mevalonate and its derivatives that are
involved in protein prenylation. In this study, we investigated the effect of mevinolin and
simvastatin, two lipophilic statins, and the specific rho activation inhibitor, C3 exoenzyme, on
phenotypic modulation of SMCs during primary culture of freshly isolated rat aortic SMCs on
a substrate of fibronectin under serum-free conditions. Treatment with C3 exoenzyme or statins
blocked focal adhesion formation, cell spreading, induction of cyclin D1, and phenotypic
modulation as judged by electron microscopy and by western blotting of SMC -actin. Addition
of mevalonate and geranyl-geranyl pyrophosphate but not cholesterol or farnesyl pyrophosphate
could reverse the effects of mevinolin. In addition, we detected that a smaller fraction
of rho was translocated to the cell membrane in statin-treated cells as compared to control
cells. These results supported the idea that the geranyl-geranylated proteins such as rho, rac
and cdc42 were involved. Our results suggest that rho activation is necessary for SMC
phenotypic modulation and that statins inhibit this process by preventing prenylation of rho
proteins.
Does Manganese Deficiency Reduce Arginase Activity to an Extent
Whereby Vascular Function is Altered?
Fatima A Tenorio, Jodi L Ensunsa, Carl L Keen, J D Symons. University of California Davis,
Davis, CA
P258
Arginase is a manganese-containing enzyme that catalyzes the hydrolysis of arginine to form
ornithine and urea. Nitric oxide synthase is an enzyme that catalyzes the hydrolysis of arginine
to form nitric oxide and citrulline. Animals lacking dietary manganese have reduced arginase
activity. We tested the hypothesis that manganese deficiency decreases arginase activity to an
extent whereby arginine is diverted through the nitric oxide synthase pathway, resulting in
enhanced endothelium-dependent relaxation. Female rats consumed a manganese (Mn)
sufficient (45 mg Mn / kg of diet; n6) or Mn deficient diet (0.5 mg Mn / kg of diet, n6)
for 632 days. After anesthetizing each animal, sections of liver, abdominal aorta, and the
entire heart were excised. Liver Mn (3.40.8 Downloaded vs 41.24.4 nmol/g) from
and arginase activity
http://atvb.ahajournals.org/
Poster Presentations a-45
(0.70.2 vs 1.90.4 U/mg) were lower (p0.05) in Mn-deficient vs Mn-sufficient animals,
respectively. Aortic segments (640 m, internal diameter) and coronary microvessels (118
m, i.d.) were mounted on wire myographs and endothelium-dependent and independent
functions were evaluated using acetylcholine (ACh) and sodium nitroprusside (SNP), respectively.
In aortic segments that were precontracted with norepinephrine, maximal ACh-evoked
relaxation was greater (p0.05) in Mn-deficient (797%) vs Mn-sufficient (549%) animals,
while SNP-evoked relaxation was similar between groups (1012% vs 1083%, respectively).
In coronary vessels precontracted using endothelin-1, maximal ACh-evoked relaxation
(5516% vs 529%) and SNP-evoked relaxation (10521% vs 10310%) were similar in
Mn-deficient vs Mn-sufficient rats, respectively. These data indicate that manganese deficiency
enhances endothelium-dependent relaxation in aortic but not coronary vessels. Therefore, the
vascular effects of reduced arginase activity resulting from manganese deficiency appear to be
heterogeneous throughout the vasculature.
Role of the Plasminogen System in the Inflammatory Response to
Biomaterial Implants
P259
Steven J Busuttil, Victoria A Ploplis, Edward F Plow. Case Western Reserve University
Cleveland VAMC, Cleveland, OH; W M Keck Center for Transgene Research, University of
Notre Dame, Notre Dame, IL; Joseph J Jacobs Center for Thrombosis and Vascular Biology
and the Cleveland Clinic Foundation, Cleveland, OH
Recent evidence speculates that the plasminogen (Plg) system may play a profound role in
mediating cellular recruitment during the inflammatory response. Utilizing a peritoneal
biomaterial implant model in mice deficient in components of the plasminogen system, we
found that both neutrophil (Neut) and macrophage (M) recruitment was significantly
attenuated in the absence of Plg. The present studies were undertaken to elucidate the
molecular mechanism by which the Plg system influences the inflammatory response to
biomaterial implants. This model involves surgical placement of polyethylene terephthalate
disks into the peritoneum of mice. Leukocytes are recovered from peritoneal lavage after 18
hrs and M and Neut populations are distinguished by enzyme activity assays. In mice treated
parenterally with tranexamic acid, an antagonist of the lysine binding sites of Plg, there was
a significant decrease in both M and Neut recruitment (p0.001). Mice treated subcutaneously
with aprotinin, an active site inhibitor of plasmin activity, showed no attenuation in
leukocyte recruitment. In addition, mice treated with intraperitoneal aprotinin showed no
significant change in M recruitment and only a non-significant decrease in Neut recruitment,
although the doses of aprotinin were sufficient to suppress plasmin activity. M and Neut
recovered from the peritoneal lavage were used in an in-vitro fibrin degradation assay. M
from both wild-type and plasminogen-deficient mice displayed a low baseline ability to degrade
fibrin (8.9% and 9.2%, respectively). The fibrinolytic activity of the M increased in the cells
of both strains of mice (24.9% and 19.2%, respectively) after the addition of exogenous
plasminogen, indicating a similarity in the capacity of the cells to form active plasmin. In
conclusion, these studies provide in-vivo verification that the plasminogen system plays an
integral role in inflammatory cell recruitment and indicates that the lysine binding sites of
plasminogen are critical in this response. Manipulation of the function of the lysine binding sites
of plasminogen may provide a means for controlling the inflammatory response to biomaterials.
P260
A Dominant-Negative Variant of Nuclear Receptor TR3 Aggravates Vascular
Lesion Formation
Karin E Arkenbout, Vivian De Waard, Maaike Van Bragt, Tanja A Van Achterberg, Bruno
Pichon, Hans Pannekoek, Carlie J De Vries. Academic Medical Center, Amsterdam,
Netherlands; IRBHHN, Gosselies, Belgium
Vascular smooth muscle cells (SMCs) can undergo relatively rapid and reversible changes in
phenotype in response to local environmental alterations, which occur during atherogenesis.
Transcription factors are key regulators in phenotypic changes of SMCs. Upon activation of
human SMCs with an atherogenic stimulus we found that the mRNA expression of all three
members of the Nerve Growth Factor Induced gene-B (NGFI-B) subfamily of nuclear hormone
receptors is induced rapidly and transiently. This subfamily comprises TR3 orphan receptor
(TR3), Nuclear receptor Of T-cells (NOT) and Mitogen Induced Nuclear Orphan Receptor
(MINOR). In addition, NOT is expressed in differentiating THP-1 cells, whereas MINOR mRNA is
induced both in THP-1 and MonoMac6 cells. In agreement with the expression profiles of these
genes in vitro, TR3, NOT and MINOR mRNA is absent in normal vascular tissue, whereas each
of these genes is expressed in neointimal SMCs in human atherosclerotic lesions derived from
thirteen different individuals. Especially NOT and MINOR are also expressed in lesion
macrophages. To reveal the function of these nuclear receptors in atherogenesis, we applied
a variant of TR3 that lacks the transactivation domain (TA) but exhibits normal DNA binding
and consequently functions as a dominant-negative inhibitor of all three subfamily members.
Adenoviral overexpression of TA in primary SMCs resulted in enhanced DNA synthesis as
illustrated by a 10-fold increase in 3H-thymidine incorporation. Homozygous, transgenic mice
were generated overexpressing TA under control of the SM22-promoter, resulting in arterial
SMC-specific expression. Three founders were bred and show no obvious phenotype and aorta
and carotid arteries exhibit normal morphology. Challenge of TA transgenic mice in the
carotid artery ligation model results in a 4-fold increase in neointima/media ratio. Based on
these data we hypothesize that NGFI-B subfamily members inhibit SMC proliferation in
atherogenesis and that the ligands of these orphan receptors may serve as powerful
therapeuticby tools guest in vascular on April disease 4, involving 2013 excessive SMC proliferation.

a-46 Arterioscler Thromb Vasc Biol. Vol 22, No 5
Cleaved Fibrinogen ’ Carboxyl Terminus Has Heparin-Like Activity
Rehana S Lovely, Lynn K Boshkov, David H Farrell. Oregon Health and Science University,
Portland, OR
P261
Fibrinogen contains three polypeptide chains, , , and , arranged as a dimer, (, , ) 2.
Approximately 10% of fibrinogen molecules contain a highly anionic 20 amino acid sequence
in one chain, termed ’, substituted for the carboxyl terminal four amino acids in the more
common A chain. Previous studies have shown that the ’ chain binds thrombin, and that the
carboxyl terminal 18 amino acids of the ’ chain are cleaved by plasmin during fibrinolysis.
Using fluorescence anisotropy, we found that peptides corresponding to the ’ carboxyl
terminus bound to thrombin anion-binding exosite II. Exosite II ligands, including heparin, a DNA
aptamer directed against exosite II, and a monoclonal antibody directed against exosite II, all
inhibited binding of the ’ peptide. In contrast, two hirudin peptides, known exosite I ligands,
failed to displace the ’ peptide. We next investigated the enzymatic effects on thrombin of the
cleaved 18 amino acid ’ carboxyl terminus. The free ’ peptide had no direct effect on
thrombin’s amidolytic activity towards a peptidyl substrate. However, the ’ peptide accelerated
the inhibition of thrombin by antithrombin III and heparin cofactor II in a dose-dependent
manner. In whole plasma, 500 M concentrations of the ’ peptide prolonged the activated
partial thromboplastin time from 29 seconds to 47 seconds. These results indicate that the
cleaved ’ peptide has heparin-like activity. Our current hypothesis based on these findings is
that the ’ chain provides a high affinity binding site for thrombin during coagulation, in which
the active site remains exposed and can continue to convert more fibrinogen to fibrin. This
bound thrombin is resistant to heparin inhibition since exosite II is blocked by the ’ chain.
However, when fibrinolysis is activated, plasmin cleaves the ’ carboxyl terminus, removing
this high affinity thrombin binding site. In addition, the released ’ 18 amino acid peptide
inhibits thrombin in the presence of antithrombin III or heparin cofactor II. The ’ peptide may
thus provide crosstalk between coagulation and fibrinolysis to prevent further clotting after
fibrinolysis is initiated.
Gene Transfer of a Novel Chimeric Natriuretic Peptide
Shuchong Pan, Laurel S Kleppe, Cheryl S Mueske, Tyra A Witt, Amir Lerman, Robert D
Simari. Mayo Clinic and Foundation, Rochester, MN
P262
DNP is a recently described member of the natriuretic peptide family produced by the green
mamba snake (dendroaspis). Recent studies have suggested unique and potent cardiovascular
and renal properties of DNP compared with other family members. Although the amino acid
sequence of the mature peptide in the snake is known, neither immature forms of the protein
nor the gene encoding for this hormone have been cloned in any species. To develop a system
to efficiently express DNP in heterologous systems, we employed preferred human codons for
the mature snake peptide cloned downstream of the entire pre-pro sequence of the brain
natriuretic peptide (pre-pro BDNP) or the leader sequence of BNP (BDNP) without the
prohormone sequences. Transfections in multiple cells (3T3, 293, HepG2) with vectors
expressing pre-pro BDNP resulted in nonprocessed (18 kD) forms within cell lysates and a
mature form (3kD) in conditioned media. Transfections with vectors expressing BDNP resulted
in mature forms within both cell lysates and conditioned media. Functional studies demonstrated
the ability of both forms of BDNP to stimulate cGMP production in HUVECs in an
endocrine manner. In addition, both forms completely inhibited serum-stimulated (10% FCS)
VSMC proliferation. This stimulation of cGMP and inhibition of proliferation in vascular cells is
greater than that seen with BNP expressed in a similar manner. When exposed to normal
porcine arterial rings BDNP caused significant vasorelaxation (70%) as compared to control
media. Intravenous infusion of an adenoviral vector expressing pre-pro BDNP in normal mice
resulted in systemic expression of BDNP of 40,000 –70,000 pg/ml for up to 3 weeks compared
with undetectable levels in mice infused with a control virus (Ad-LacZ). Expression of BDNP was
associated with a decrease in systemic blood pressure as compared to control virus infection.
Conclusion: Expression of pre-pro BDNP results in a processed, mature form of BDNP that is
able to stimulate cGMP in vascular cells and has potent antiproliferative and vasoactive
properties. Adenoviral delivery results in high levels of circulating BDNP in normal mice and
decreased systemic blood pressure.
P263
Six-Month Patency of ePTFE Grafts Lined with Endothelial Cells Derived
from Peripheral Blood Stem Cells Equals Those Harvested from Native Vein
Laurace E Townsend, Ahmed A Meguid, John F Knipfer, Phillip J Bendick, John L Glover,
Gerald B Zelenock. Wm. Beaumont Hospital Research Institute, Royal Oak, MI
Introduction: A prosthetic vascular graft with hemodynamic characteristics suitable for
implantation is needed clinically. We compare the outcome of ePTFE grafts seeded with
endothelial cells (EC) derived from peripheral blood stem cells (PBEC) versus EC obtained from
native jugular vein (JVEC). Methods: Thirteen dogs had EC harvested from excised jugular veins.
Stem cells were obtained from peripheral blood by phlebotomy followed by gradient
centrifugation. EC developed from stem cells in selective culture. Two 4-mm ePTFE grafts were
prepared for each dog; one graft seeded with autologous JVEC and the other with autologous
PBEC. The grafts were explanted at 6 mo. and evaluated for thrombus free surface area (TFSA)
and for anastomotic intimal hyperplasia. Results: Seven of thirteen animals (54%) maintained
at least one patent graft to the six-month end-point. Of fourteen grafts explanted from these
animals, 64.3% (9 of 14) were patent; 55.6% (5 of 9) PBEC lined, and 44.4% (4 of 9) JVEC
lined, p1.00 by Fishers exact test. There were no significant differences in seeded cell
retention, pre-implant confluence, or TFSA. Samples taken from the center of patent grafts
stained positively for markers of EC. Comparison of proximal versus distal vessel lumen
diameter showed no significant difference (p0.66, exact inference test) indicating that intimal
hyperplasia was not a contributing factor to vessel occlusion. Disparity between graft and
carotid artery lumen diameter contributed to occlusion (p0.05); graft and native vessel were
approximately the same size in 13 of 18 patent anastomoses (72%) and the graft was larger
than native vessel at only 1 patent anastomosis. All occluded graft lumens (9 of 9) were larger
than native vessel lumen. Conclusions: Considering the highly coagulable canine model used
in our study, excellent graft patency results were achieved. Closely matching graft size to
vessel size contributes to patency and is a consideration in future clinical trials. Endothelial
cells derived from stem cells of peripheral blood are as effective in enhancing graft patency as
EC derived from autologous veins.
Flow-Induced Actin Dynamics in Living Endothelial Cells
Brian P Helmke. Univ. of Virginia, Charlottesville, VA
P264
Engineering of artificial vascular grafts requires the establishment of a patent endothelium at
the blood-graft interface. Although the mechanisms by which endothelial cells adapt to
hemodynamic forces remain unclear, maintenance of endothelial function after graft implant
may depend on initial mechanochemical signaling events in response to sudden changes in
mechanical environment. Recent measurements of shear stress-induced cytoskeletal displacement
in living endothelial cells indicate that intracellular deformation is concentrated at discrete
locations. This suggests that force is transmitted via the cytoskeleton to molecular complexes
such as focal adhesion sites or intercellular junctions that may integrate mechanochemical
signals. Actin microfilaments and stress fibers may play an especially important role due to
their structural association with these sites. In the present study, endothelial cells expressing
GFP-actin were exposed to changes in unidirectional laminar shear stress in a parallel plate
flow chamber. High-resolution optical sectioning microscopy acquired simultaneous fluorescence
and DIC 3-D time-lapse images. Fluorescence data was analyzed to demonstrate
displacement and dynamics of GFP-actin, and DIC images demonstrated movement of cellular
structures such as the nucleus, cell boundaries, and intracellular organelles. After onset of
shear stress, ruffles containing F-actin were rapidly projected in an upstream direction, and
existing microfilaments were often displaced or bent. In both GFP-actin and DIC images, active
remodeling of cell edges was induced within minutes after onset of flow. These results indicate
that an increase in shear stress initiates a rapid dynamic response in the actin cytoskeleton that
is capable of transmitting force to sites where mechanotransduction occurs.
P265
Human Smooth Muscle Calponin Protein is Tightly Restricted to Smooth
Muscle Cell Lineages in Transgenic Mice
Mary A Georger, Joseph M Miano. University of Rochester Medical Center, Rochester, NY
Smooth muscle calponin (SM-Calp) is a highly restricted marker for smooth muscle cells (SMC)
whose encoded protein carries out many functions in vitro. Its restricted expression to SMC
makes SM-Calp an ideal model gene to dissect the transcriptional circuitry governing SMC
differentiation. To date, neither the function nor the transcriptional regulation of SM-Calp has
been clearly elucidated in vivo. Accordingly, we generated transgenic mice with a bacterial
artificial chromosome harboring the human SM-Calp (hSM-Calp) gene in an effort to
understand its transcriptional regulation and begin addressing its function in vivo. The purpose
of this study was to optimize conditions for appraising, unambiguously, the expression of
hSM-Calp protein in both transgenic embryos as well as postnatal tissues. We took advantage
of the fact that the SM-Calp antisera (Dako) used was raised to the human antigen and thus
would be expected to have a greater affinity for hSM-Calp than the endogenous mouse
SM-Calp protein. We found that the hSM-Calp protein is present in the embryonic heart at 9.5
days and persists throughout development up to 17.5 days, consistent with in situ mRNA
hybridization studies. All SMC lineages of the developing embryo show clear hSM-Calp protein
with little or no staining of the endogenous SM-Calp protein in non-transgenic embryos. In
postnatal tissues, coronary arteries, aorta, and various microvascular beds (brain particularly)
show exclusive expression of the hSM-Calp transgene in SMC of these tissues. Notably,
staining was also observed in pericytes of the choroid plexus as well as mesangial cells of the
kidney. Visceral tissues showing SMC-restricted staining of hSM-Calp include bladder,
bronchioles of the lung, as well as the SMC investing the alimentary canal. To date, we have
not observed any overt phenotype in these mice. These results demonstrate our ability to
clearly discriminate hSM-Calp protein from the endogenous protein using a variety of antigen
enhancing protocols. The humanized mice we have generated represent powerful tools to
unlock the transcriptional regulation and function of SM-Calp.
3-Dimensional Ultrasound of Extracranial Carotid Arteries: Additional
Information?
Jörg Nossen, Thomas Vierzigmann, Erich Lang. Waldkrankenhaus St. Marien, Erlangen,
Germany
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P266
Background: External vascular ultrasound is an established method for examination of
extracranial carotid arteries. Aim of this study was the evaluation of additional information
about carotid stenoses by 3-dimensional sonography. Methods: Patients with suspected carotid
stenosis underwent B-mode, doppler and color doppler ultrasound of common carotid (CCA),
internal carotid (ICA) and external carotid artery (ECA). Vascular sonography was analyzed for
localisation and severity of stenosis. Second step was evaluation of carotid arteries and
stenoses by 3-dimensional ultrasound (3-scape power doppler, Siemens Elegra). Afterwards all
patients underwent selective angiography of carotid arteries, which was considered as gold
standard. Results: 63 carotid segments (CCA, ICA, ECA left and right) of 11 patients were
analyzed. 2 segments of one patient could not be 3-dimensionally analyzed because of severe
calcifications. Conclusions: Concerning luminal stenoses 80% 3-dimensional ultrasound was

as precise as traditional color doppler ultrasound. In case of severe calcifications 3-dimensional
ultrasound may be impossible.
Endogenous Smooth Muscle Cell PAI-1: Role in Flow-Induced Smooth
Muscle Cell Migration and Regulation of MMP-2 Activity
John P Cullen, Eileen M Redmond, Suzanne M Nicholl, Shariq Sayeed, James V Sitzmann,
S S Okada. University of Rochester Medical Center, Rochester, NY
P267
Several studies have provided compelling evidence for a role of proteases such as
urokinase-type plasminogen activators (uPA) and plasminogen activator inhibitor (PAI-1) in
regulating smooth muscle cell (SMC) migration. We have previously shown that endothelial cell
(EC) PAI-1 inhibits flow-induced SMC migration. In this study we determined the role of SMC
derived PAI-1 in the flow-induced migratory process. Wild type (wt) or PAI-1 knockout SMC
were cultured in the absence or presence of murine EC under pulsatile flow conditions in a
perfused transcapillary culture system. SMC migration was then assessed using a Transwell
migration assay. In the absence, but not in the presence of EC, pulsatile flow significantly
increased the migration of wt SMC (237 11%, n17, p0.05) when compared to wt SMC
cultured under static conditions. While there was no difference in the migration of PAI-1 -/-
SMC compared to wt SMC under static conditions, PAI-1 -/- SMC migration was significantly
increased under pulsatile flow conditions as compared to wild type controls (334 22% vs
237 11%, n6, p0.05). This flow-induced migration was significantly attenuated, but not
completely inhibited, when PAI-1 -/- SMC were cultured in the presence of EC (147 13%,
n6, p0.05). uPA activity in PAI-1 -/- SMC exposed to flow increased 354% (137094 vs
30198) in the absence of EC, and 16% (78086 vs 90693) in the presence of EC. However,
MMP-2 activity in these cells increased 391% (125920 vs 25623) in the absence of EC and
283% (124642 vs 32504) in the presence of EC. These results suggest that, in the presence
of EC, endogenous SMC PAI-1 is required for complete inhibition of flow-induced migration.
Furthermore, gene deletion of SMC PAI-1 causes upregulation of MMP-2 activity under flow
conditions which may contribute to the flow-induced PAI-1 -/- SMC migratory response
observed in the presence of EC.
Modification of the Extracellular Matrix by -Irradiation Increases
Adhesion and Decreases Migration of Vascular Smooth Muscle Cells
P268
Alexandra Kadl, Johannes Breuss, Sophie Ziegler, Florian Gruber, Yuri Koshelnick, Bernd R
Binder. Department of Vascular Biology and Thrombosis Research, Vienna, Austria; Klinische
Abteilung für Angiologie, Vienna, Austria
The limiting factor of percutaneus transluminal angioplasty (PTA) is restenosis, occurring within
6 months in up to 70% of patients with arterial occlusive disease. Restenosisafter PTA is mainly
due to migration of smooth muscle cells (SMC) from the adventitia and media into the intima
and local proliferation. Partial prevention of restenosis has been achieved by combination of
irradiation and PTA. The mechanism by which irradiation prevents restenosis is only partially
known and limitation of proliferation of irradiated cells cannot completely explain the beneficial
effects. Besides cells, also the matrix is a target for irradiation during the combined PTA and
radiotherapy. It was therefore the aim of this study to analyze possible effects of irradiation of
the matrix in the absence of cells on SMCs seeded later on such irradiated matrix, specifically
on adhesion and migration of these cells. Tissue culture supports coated with vitronectin were
-irradiated with 8 Gray. When human arterial SMCs were seeded onto such treated plates,
adhesion was significantly increased and migration was decreased compared to cells seeded
onto not irradiated plates. Western blots of these cells revealed that on irradiated matrix, the
MAP Kinase Erk1/2 phosphorylation was prolonged (3 hrs) as compared to cells seeded onto
not irradiated matrix. Such Erk 1/2 activation was due to increased phosphorylation of the focal
adhesion kinase indicating activation of intergins. This is consistent with the fact that the
increased adhesion on irraditated matrix was RGD dependent. When the effects of irradiation
on vitronectin was analyzed further, it was found that irradiation of the protein resulted in a loss
of tryptophanes as seen also by oxidation and in fact all irradiation effects were abolished in
the presence of an antioxidant. From these data we conclude that -irradiation results in
oxidative modification of matrix proteins and in turn increased adhesion of the cells onto such
matrix and prolonged activation of the Erk1/2 pathway. Oxidation of extracellular matrix might
also contribute to the inhibition of restenosis after radiotherapy.
P269
Measuring Unadulterated Whole Blood Viscosity in Patients with Familial
Hypercholesterolemia on Long-Term LDL Apheresis
Patrick M Moriarty, Cheryl A Gibson, Kenneth R Kensey, William N Hogenauer. University of
Kansas School of Medicine, Kansas City, KS; Rheologics, Inc, Exton, PA
Background. Recent literature indicates that whole blood viscosity and its major determinants
are associated with cardiovascular events and the early stages of atherosclerosis. Major
determinants of whole blood viscosity are red blood cell deformability, hematocrit, red blood
cell aggregation and plasma viscosity. We undertook this study to evaluate whole blood
viscosity changes associated with LDL apheresis (B. Braun, Melsungen, Germany). Unlike
conventional viscometers, we were able to measure unadulterated whole blood over a wide
shear rate, using new technology that does not require anticoagulants (Rheologics, Inc.), which
can severely influence viscosity results. Method. Downloaded We evaluated whole from
blood viscosity over a
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Poster Presentations a-47
wide shear range, and lipid profiles immediately before and after LDL apheresis among patients
who were being treated bi-weekly. Results. We assessed five non-obese patients (4 F; 1 M)
mean age 57.8 years, who have cardiovascular disease and severe hypercholesterolemia
(LDL 200 mg/dl) resistant to diet and pharmacotherapy. Table 1 displays the results for the
pre/post comparison of whole blood viscosity and lipid changes associated with LDL apheresis.
Conclusion. LDL apheresis resulted in significant reductions in whole blood viscosity at both low
and high shear rates. Because elevated blood viscosity may be an important risk factor in
atherogenesis, reductions in shear forces by LDL apheresis therapy may play an essential role
in both acute and chronic cardiovascular disease.
P270
Effect of Intron 4 on eNOS Transcription: Functional Significance of the
27bp Repeats
Jian Wang, Donald J Dudley, Xing Li Wang. Southwest Foundation for Biomedical Research,
San Antonio, TX; University of Texas Health Sciences Center at San Antonio, San Antonio,
TX
Endothelial nitric oxide synthase (eNOS) plays an essential role in vascular NO production. We
have shown that smoker who had a rare 4 repeat of the 27bp in eNOS intron 4 had increased
CAD risk, and associated with a decreased eNOS mRNA and protein levels from placenta
tissues. In the present study, we explored the possible functional significance of the 27bp
repeat in eNOS gene in transcription efficiency. We inserted 4 or 527bp repeat fragments at
either the promoter or the enhancer region of the pGL3 luciferase reporter gene. The constructs
with inserts were then transfected to endothelial cells and assessed for transcription efficiency
by luciferase activity. We found that the 27bp fragment had a promoter effect when inserted
in the promoter region (24.02.1 and 17.00.6 fold for 5 and 4 repeats respectively), which
is more efficient than the eNOS promoter fragment. Cigarette smoke-extract significantly
up-regulated the promoter efficiency (2.4 and 1.5 folds for the 5 and 4 repeats, respectively).
The 27bp repeats also acted as a transcriptional enhancer when inserted at the 3’ region
together with the 1.6kb eNOS promoter fragment at the 5’ of the luciferase gene. Vectors
containing 5 repeats at the enhancer region had a 8.22.1 fold increase in transcription
efficiency as compared to only 3.40.3 fold for the 4 repeats (P0.05). The enhancerless
eNOS promoter alone produced a transcription efficiency about 2.80.2 fold of the basic
control pGL3 vector. In a mobility shift assay, we observed binding of the 27bp fragment with
transcriptional factor(s) from the crude nuclear protein extract of endothelial cells. Our study
provides the first evidence that the 27bp repeat in eNOS intron 4 plays a significant role in
transcriptional efficiency. Further elucidation of transcriptional effect of the 27bp repeat on
eNOS gene, a possible concerted allele-specific effects with eNOS promoter and factors may
influence such effect will have significant implications on regulation and protection of
endothelial function, hence vascular disease.
P271
Matrix Gla Protein (MGP) Expressed in Calcified Lesions of the Aorta Is
Inactive as a Binding Protein for Bone Morphogenetic Protein-2 (BMP-2)
David C Sane, Andrew Sweatt, Reidar Wallin. Wake Forest University School of Medicine,
Winston-Salem, NC
Matrix Gla protein (MGP) has an important role as an inhibitor of arterial calcification. MGP is
a vitamin K dependent protein that is synthesized by vascular smooth muscle cells and is highly
up-regulated in calcified arterial lesions. The mechanism by which MGP works as a calcification
inhibitor has been disclosed by our laboratory. MGP is a binding protein for bone morphogenetic
protein -2 (BMP-2) and possibly other members of the TGF- superfamily of growth factors.
Ligand blotting and affinity chromatography experiments have provided conclusive evidence
that the vitamin K modified, -carboxyglutamic acid residues (Gla residues) in MGP are
essential for binding of BMP-2. We synthesized a peptide covering the Gla region of MGP and
another peptide covering the same region but with Glu residues substituted for Gla residues.
Circular dichroism spectra of the Gla peptide in the presence of Ca or EDTA showed that
the Gla peptide underwent a Ca - dependent conformational change similar to that
demonstrated for the F1 fragment of prothrombin. Both peptides were used to prepare
antibodies. The Gla peptide produced conformational specific (“anti-Gla”) antibodies that only
recognized the Ca induced conformer of mature and functional MGP. The Glu-peptide
produced antibodies (“anti-Glu”), that recognized only immature MGP in which the Glu residues
had not been converted to Gla residues. The antibodies were used to investigate MGP in aortas
containing calcified lesions. MGP was found to be highly up-regulated in calcified regions of the
plaque, but was expressed in a nonfunctional state, since it was detected with anti-Glu but not
with anti-Gla antibodies. In contrast, MGP in cartilage was detected exclusively with anti-Gla.
The production of Gla-less MGP reveals that there is a functional vitamin K deficiency or a
defect in the -carboxylation system in the arterial wall. In conclusion, MGP in calcified arterial
lesions lacks the Gla modification and is therefore a nonfunctional inhibitor, being unable to
bind to orby inhibit guest the initiation on April of calcification 4, 2013 by BMP-2.

a-48 Arterioscler Thromb Vasc Biol. Vol 22, No 5
Significant Improvement in the Positive Predictive Value of Nuclear
Exercise Stress Test by the Presence of Proteinuria
P272
Nour M Juratli, Sambit Mondal, Chizor Iwuchukwu, Pejman Shamekh, Sham Maghout, Soad
Bekheit, Sheldon Breitbart, Louis Salciccioli, Luther T Clark. SUNY-Downstate Medical
Center and Brooklyn Hospital Center, Brooklyn, NY
Background: Nuclear exercise stress test (NES) has suboptimal positive predictive value for the
presence of coronary artery disease (CAD). Proteinuria is an emerging risk factor for CAD
especially in diabetic patients. We intended to study the presence of proteinuria prior to NES
to improve its predictive value. Methods: Between 1/99 and 12/00 all patients (pts) who had
NES followed by cardiac catheterization (CC) were included in the analysis if they had urinalysis
prior to NES. Exercise test database, CC reports, urine chemistry were studied to determine the
presence of CAD and proteinuria. Positive NES was defined by the presence of any defect in
nuclear images. CAD was defined by the presence of any degree of stenosis during CC.
Significant CAD was defined as 50% stenosis in one or more vessels. Severe CAD was
defined as 50% stenosis in left main or 70% stenosis in any other coronary artery.
Proteinuria was defined as positive if it was present in routine urinalysis at any time prior to
NES. Results: Out of 2362 pts who were screened 284 pts had all 3 tests, after excluding pts
with known CAD 192 were eligible for analysis. The mean age was 59.4 11.8 yrs. History
of diabetes (DM) was present in 54 (28%) pts, hypertension in 117 (61%) pts, and
hyperlipidemia in 89 (46.4%) pts. All pts had positive NES. Proteinuria was positive in 55
(28.7%) pts. In all pts NES positive predictive value (PPV) for any CAD had increased from
47.4% to 65.5% (p0.002) by the presence of proteinuria. The PPV for significant CAD had
increased from 36% to 47.3% (p0.016). In pts with DM the PPV of NES had increased from
68.5% to 87% (p0.017) for the presence of any CAD. The PPV of NES with the presence of
proteinuria did not increase significantly for the presence of severe CAD in diabetic and
non-diabetic pts. Logistic regression analysis showed that the presence of proteinuria is an
independent predictor of CAD in DM pts (p0.015). Conclusions: Proteinuria is a powerful tool
to improve the positive predictive value of nuclear exercise stress for the presence of coronary
artery disease. However, it was not associated with angiographic severity of coronary disease.
P273
An MRI Technique for the Quantification of Atherosclerotic Lesions in the
ApoE Knockout Mouse
Stuart M Grieve, David A Priestman, Juergen E Schneider, Fran M Platt, Raymond Dwek,
Stefan Neubauer, Kieran Clarke. Oxford Cardiac Research Group Magnetic Resonance Unit,
Oxford University, Oxford, UK; Oxford Glycobiology Institute, Oxford University, Oxford, UK;
John Radcliffe Hospital, Oxford University, Oxford, UK; BHF Molecular Cardiology Group,
Oxford University, Oxford, UK
OBJECTIVES: Apolipoprotein-E (ApoE) knockout mice spontaneously develop atherosclerotic
lesions and are widely used as a model for human vascular atherosclerotic disease. Standard
methods for quantification of atherosclerotic lesions involve serial sectioning of fixed hearts.
Lesion areas are typically determined by analysis of digitised images of these sections. We
present an alternative method to image excised hearts using MRI after polymer perfusion.
METHODS: Mouse hearts were perfused in situ first with heparanised saline, then with a
solution of MICROFIL MV-122 (Flow Tech, Carver, Mass). Hearts were dissected out after
polymerisation of the perfusate, then embedded in 1% agarose doped with 2 mM Gd-DTPA.
MRI experiments were performed using a Bruker Avance spectrometer at 11.7 T. A 512 x 512
x 256 data matrix was acquired using a 3D frequency selective spin-echo method to give a
resolution of 31 x 31 x 62 Ã,Âm. RESULTS: Figure A shows a cross-section of the aortic root
extracted from a 3D dataset of the whole heart in an Apo-E mouse. The lesion (marked with
arrow) can be clearly identified, both from the lumen of the aorta and also from the endothelial
wall. Figure B shows a similar section from a wild-type mouse. The wild-type mouse shows
a similarly clear definition of the endothelial wall but without the presence of any
atherosclerotic lesions. CONCLUSIONS: These initial results clearly demonstrate the potential of
combining MICROFIL perfusion and MRI as a tool for accurate and rapid quantification of
atherosclerotic lesions in mouse models of human atherosclerotic disease.
P274
Simvastatin Inhibits Chlamydia pneumoniae Induced Nuclear Binding of
Egr-1 in Mouse Macrophages
Florian Bea, Mirja H Puolakkainen, Monica I Shelley, C C Kuo, Lee Ann Campbell, Michael E
Rosenfeld. University of Washington, Seattle, WA
Activation and nuclear binding of the transcription factor early growth response-1 (Egr-1) is an
important modulator of multiple pro-inflammatory genes that are involved in the pathogenesis
of vascular disease. HMG CoA reductase inhibitors have anti-inflammatory and anti-thrombotic
effects that are independent of their capacity to lower plasma lipids and may involve inhibition
of nuclear binding of factors such as Egr-1. To test this possibility, we have investigated
whether infection with Chlamydia pneumoniae, an independent risk factor for cardiovascular
disease, results in increased binding of Egr-1Downloaded and if this induction from
can be inhibited by
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simvastatin. RAW 264.7 mouse macrophages were infected with Chlamydia pneumoniae or
treated with lipopolysaccharide (LPS). Binding of nuclear proteins to Egr-1 consensus
oligonucleotide sequences was evaluated by electrophoretic mobility shift assays. Increased
binding of nuclear proteins occurred 1–4 hours after infection with Chlamydia pneumoniae or
treatment with LPS. Supershift experiments with an Egr-1 specific antibody confirmed binding
of Egr-1 to the Egr-1 binding site. Pretreatment of the cells with simvastatin (1M) for 24 hours
inhibited the DNA binding of Egr-1 induced by Chlamydia pneumoniae or LPS. These data
suggest that infection with Chlamydia pneumoniae may contribute to the development of
atherosclerosis via activation of the binding of pro-inflammatory factors such as Egr-1 and that
the anti-atherogenic effects of the statins may in part be due to their anti-inflammatory
properties.
P275
Endothelial Activation and Myocardial Cell Damage Lead to Transplant
Coronary Artery Disease and Cardiac Graft Failure
Carlos A Labarrere, David R Nelson. Methodist Research Institute at Clarian Health,
Indianapolis, IN; Cleveland Clinic Foundation, Cleveland, OH
Background. Arterial endothelial activation and microvascular fibrin with myocardial cell
damage are independent risk factors for transplant coronary artery disease (CAD) and graft
failure. However, little is known about the temporal association between endothelial activation
and myocardial cell damage or how these changes relate to outcome. Methods. We evaluated
105 heart transplant recipients 46.6 2.5 months after transplantation for CAD (3.7 0.2
angiograms/patient). All patients survived at least 1 year and had at least a first-year
angiogram. Serum soluble intercellular adhesion molecule-1 (sICAM-1) and cardiac troponin I
(TnI) were used as markers of endothelial activation and myocardial cell damage, respectively,
and were studied using an enzyme-linked immunosorbent assay. Serial samples obtained
during the first 3 months after transplantation were evaluated for sICAM-1 while those obtained
between 3 months and 1 year after transplantation were evaluated for cardiac TnI. Results.
Patients with low serum sICAM-1 (308 ng/ml) during the first 3 months post-transplant and
without detectable TnI during months 3–12 after transplantation had significantly less
transplant CAD (p0.001) and graft failure (p0.02) compared to patients with elevated
sICAM-1 (308 ng/ml) and detectable TnI (table). Conclusions. These data suggest that early
activation of the cardiac allograft microvasculature leads to persistent myocardial cell damage
and increased transplant CAD and graft failure.
The Effects of Isometric Exercise on Haemostasis in Ischaemic Heart
Disease
Eileen E Mackie, Anna Beattie, Mandy Dawson, Allison McKenzie, Stewart W Hillis.
University of Glasgow, Glasgow, UK; Western Infirmary, Glasgow, UK
P276
Strenuous dynamic exercise affects haemostasis and may be a trigger for myocardial
infarction.However there is minimal knowledge of the effects of isometric exercise in ischaemic
heart disease (IHD). This study aimed to examine the effects of isometric exercise on
haemostasis in stable angina. Methods: 11 patients with IHD prior to elective angioplasty and
8 healthy controls undertook sustained isometric contraction of their dominant arm to 50% of
their maximal voluntary contraction. Haematological parameters were measured before, after
and 2 hours following exercise. Flow cytometry using the CD62(p-selectin) antibody was used
to assess platelet activation. Results:Expressed as mean(SEM).Paired and 2-sample t-tests
were performed. Platelet count increased post exercise in both groups. CD62 was increased
only in patients post exercise (2.129(SEM 0.609),1.299(0.319)p 0.05) and remained elevated
at 2hr post exercise (2.187(SEM 0.662),1.299(0.319)p0.096).Prothrombin time(PT) shortened
in patients post-exercise (12.636(SEM 0.799),12.273(0.856),p0.038) Fibrinogen levels were
not significantly different at baseline between the groups (2.83(SEM 0.6),3.015(0.14)
p0.846), however there was a reduction at 2hrs in the control group
(2.83(0.6),2.62(0.588)p0.049) not seen in the patient group (2.863(0.13),2.986(0.141)
p0.178).Tissue Plasminogen Activator (tPA) was reduced in patients at baseline
(0.53(0.34),2.98(0.83),p0.023). tPA increased in both groups with exercise(0.53(0.34),2.27
(1.87) p0.377 and 2.98(0.83),3.113(0.975)p0.699). Baseline WCC was significantly higher
in patients than in controls (6.88( 0.22),4.96(0.39) p0.001)and increased only in controls with
exercise(4.96(0.207),5.328(0.245)p0.002) CONCLUSION: Isometric exercise may be prothrombotic
in patients with stable angina relative to controls with increased platelet activation
and stimulation of coagulation. Risk stratification using dynamic exercise is well established
however isometric exercise induces haemostatic imbalance and is essentially ignored in
exercise recommendations by guest on April and risk4, stratification 2013 in IHD.

Species Differences in Endothelial Cell Injury Due to X-rays
Linda Hiebert, Pat Thomas, Tilly Ping, Mark Wickstrom, Bliss Tracy. University of
Saskatchewan, Saskatoon, Canada; Health Canada, Ottawa, Canada
P277 WITHDRAWN
P278
Our previous studies have demonstrated a marked species difference in response of cultured
endothelial cells to free radical injury. Bovine aortic endothelial cells were much more resistant
to injury from hydrogen peroxide (H 2O 2) and xanthine/xanthine oxidase than porcine aortic
endothelial cells (6 vs 0.25 mM H 2O 2 for fifty percent viable cells for bovine vs. porcine cells).
To determine if this same species difference could be demonstrated in response to radiation
injury, porcine and bovine aortic endothelial cells were exposed to increasing doses of x-rays.
Twenty-four hours later, death of irradiated cells was assessed by measuring percent viable
cells and total live cell number by trypan blue exclusion and the release of lactate
dehydrogenase (LDH) into cell medium. Clonogenic survival was determined by measuring the
survival fraction, i.e., the percentage of survivors forming colonies after 14 days. Surprisingly,
bovine cells were more sensitive than porcine cells to x-ray injury when slopes of the
dose-response curves were compared. The decrease in percent viable cells and live cell
number was 2.3–3.4 times greater for bovine vs. porcine cells and increase in LDH release was
8 times greater for bovine vs. porcine cells. Clonogenic survival (25%) was similar at a dose
of 4 Gy with porcine cells being more sensitive at lower doses and less sensitive at higher
doses than the bovine cells. These results suggest that these species differ in their ability to
resist and repair different types of free radical damage; bovine cells may have enhanced
amounts of cytoplasmic free radical scavengers, giving them resistance to damage from
diffusible H 2O 2compared to porcine cells. However damage from x-rays is concentrated along
specific tracks, to which porcine cells appear more resistant. While clonogenic survival curves
are somewhat similar, a species difference still exists, which may reflect differing abilities to
repair DNA damage. These results demonstrate that response to free radical injury depends on
the mode of free radical delivery and cannot be extrapolated between species.
P279
Inhibition of Neointima Formation by Co-Expression of Antisense Thrombin
Receptor Gene and p21waf1
Liguom Mi, Xianmin Meng, Xiuwen Zhao, Dongqing Liu, Jinfeng Ding, Runlin Gao. Molecular
Medicine Center for Cardiovascular Diseases, Cardiovascular Institute & Fu Wai Hospital,
Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China
Associated-adenovirus (AAV) mediated-gene transfer of antisense thrombin receptor gene
(ATR) or p21waf1 has shown to inhibit the proliferation of vascular smooth muscle cells
(VSMCs) and neointima formation in balloon-injured porcine coronary arteries. It is suggested
that co-expression of ATR and p21waf1 (AP) would enhance the effect by adjusting the
expression of thrombin receptor gene and p21waf1 in VSMCs. Here we constructed and
packaged the AAV vectors of expressing ATR (rAAV/ATR), p21waf1 (rAAV/p21), and coexpressing
ATR and p21waf1 (rAAV/AP). The cultured human aorta smooth muscle cells
(ASMCs) were infected with the rAAVs or control vector (rAAV/GFP) respectively, and the effects
of the genes were evaluated by MTT assay and flow cytometry. The rate of survival cells of
co-expression of ATR and p21waf1 was decreased (68.5% vs. 85.6% and 82.0%, AP/GFP vs.
ATR/GFP and p21/GFP), and the apoptosis cells were increased (8.24% vs. 4.84% and 5.17%,
AP vs. ATR and p21) at 48 hours after the gene transfer. In the balloon-injured rat carotid artery
model, both neointima formation and VSMCs of media were significantly inhibited by
co-expressing ATR and p21waf1 as compared with the expression of GFP (control) on days14
and 28 after the gene transfer (table). These data show that co-expression of ATR and p21waf1
can enhance the inhibitory effect of ATR or p21waf1 on proliferation of VSMCs and neointima
formation. This may have implications for developing strategies of gene therapy for restenosis.
P280
Reversal of Tissue Kallikrein Up-Regulation by Revascularization of Lower
Limb Ischemia
Costanza Emanueli, Paolo Porcu, Paolo Madeddu. Cardiovascular Medicine and Gene
Therapy Section National Laboratory INBB, Osilo, Italy; Vascular Surgery, University of
Sassari, Sassari, Italy
BACKGROUND: Tissue kallikrein (tK) and vascular endothelial growth factor (VEGF) are potent
angiogenic factors. Up-regulation of tK or VEGF was documented in animal models of acute
ischemia and impairment in endothelial growth factor surge has been report ed in animal
models of atherosclerosis. Yet, it remains unknown whether these endothelial cell mitogens are
modulated in patients with chronic peripheral vascular insufficiency and whether the levels of
expression correlate with clinical grading, collateral development, and revascularization. AIM:
To evaluate the expression of endothelial growth factors in the context of peripheral ischemia.
METHODS AND RESULTS: Circulating tK and VEGF were measured in 36 patients with
symptomatic peripheral vascular disease beforeDownloaded and after surgical from
revascularization. In 6
Poster Presentations a-49
patients without symptoms at rest, tK was assayed following exercise stress test. VEGF levels
fell within the normal range in all patients (9611 vs. 10913 pg/mL in healthy controls,
PN.S.) and remained unchanged after revascularization. In contrast, tK expression was
upregulated in 34 out 36 patients (1,107203 vs. 8510 pg/mL in controls, P0.05), with
no further increase after exercise. No correlation was found between growth factor expression
and clinical grading. However, TK levels in the venous effluent of ischemic limbs positively
correlated with the number of angiographically recognizable collateral vessels (r0.72,
P0.001). Follow-up studies documented reversal of tK upregulation following revascularization
(P0.01), whereas no change was observed in venous samples from untouched legs.
CONCLUSIONS: Induction of tK could represent a compensatory response to chronic arterial
insufficiency, attempting to maintain an adequate tissue perfusion. Potentiation of this
mechanism may have important implications to therapeutic angiogenesis in limb ischemia.
Effect of Factor VIII on Tissue Factor-Initiated Spatial Clot Growth
Mikhail V Ovanesov, Euguene L Saenko, Fazoil I Ataullakhanov. National Research Center
for Hematology, Moscow, Russia; American Red Cross, Rockville, MD
P281
Intrinsic coagulation factor VIII (fVIII) is an essential component of blood coagulation cascade.
Inherited low levels of fVIII in plasma (Haemophilia A) are associated with bleeding tendency
whereas elevated levels of fVIII are likely to be an independent risk factor for venous
thrombosis. To gain an insight into the role of fVIII in clot formation, we used the novel in vitro
experimental system modelling the physiological spatial conditions in proximity of the damaged
blood vessel wall. Clotting was studied in recalcified plasma freshly drawn from normal donors
(N12) or patients with severe haemophilia A (N23) and supplemented with purified human
fVIII. The light scattering produced by TF-initiated clots formed on fibroblasts was recorded in
a thin layer of nonstirred plasma using time-lapse video microscopy, which allows to monitor
temporal evolution of the clot shape and to determine the clot growth rate. The initial kinetics
of clotting in a 0.2 mm-area near the activator surface was similar in both normal and
haemophiliac plasmas. However, the rate of spatial clot growth (from 0.2 to 1.7 mm width) was
approximately three times slower in haemophiliac plasmas than in normal ones. The clot
growth rate in haemophiliac plasmas increased from 16.11.7 up to 44.92.5 m/min
(normal value) with increasing fVIII concentration from 0.0001 to 0.1 UI. Remarkably, normal
clot formation was restored by addition of fVIII to 5 - 10 %, which is the threshold level defining
requirement for preventive treatment of Haemophilia A. At higher concentrations, fVIII had a
thrombogenic effect, since haemophilic plasmas tended to spontaneously coagulate. These
results support the hypothesis that the intrinsic, fVIII-dependent pathway is responsible for
expansion of the clotting area under conditions when TF remains cell membrane bound but not
present in plasma in its free form. Our experimental approach provides direct evidence of the
critical role of the intrinsic pathway in progression of spatial clot growth
P282
Functional Interactions between Hormone Nuclear Receptors Bound to a
Newly Identified HRE on the Hepatic Control Region-1 and an HRE on the
Proximal ApoC-II Promoter Account for the Transactivation of the ApoC-II
Promoter by HNF-4 and RXR/FXR Heterodimers in Response to Bile
Acids
Dimitris Kardassis, Anastasia Roussou, Paraskevi Papakosta, Costas Boulias, Iannis
Talianidis, Vassilis I Zannis. Department of Basic Sciences, University of Crete Medical
School, Heraklion, Crete, Greece; Institute of Molecular Biology and Biotechnology, FORTH,
Heraklion, Crete, Greece
We have shown previously that the hepatic control region-1 (HCR-1) enhanced the activity of
the human apoC-II promoter in HepG2 cells. This enhancement was mediated by two hormone
response elements B (-102/-81) and C (-156/-116) present in the apoC-II promoter that bind
specifically HNF-4 and RXR/T3R heterodimers respectively. We now report that HCR-1
mediates induction of the apoC-II promoter by chenodeoxycholic acid (CDCA), a natural ligand
of farnesoid X receptor (FXR). In addition, treatment of HepG2 cells with CDCA resulted in
significant induction (3.5-fold) in the steady state apoC-II mRNA levels. In contrast, HCR-1 could
not mediate induction of the apoE promoter by CDCA. Activation of the HCR-1/apoC-II promoter
by CDCA required the binding of RXR/FXR heterodimers to a novel hormone response
element present in HCR-1 which also binds the orphan nuclear receptors HNF-4 and ARP-1.
RXR/FXR heterodimers in the presence of CDCA as well as HNF-4 strongly transactivated the
HCR-1/apoC-II promoter whereas overexpression of ARP-1, which also binds to the element C,
abolished this transactivation. RXR/FXR heterodimers in the presence of CDCA also
transactivated an apoC-II promoter fused with the novel HRE of the HCR-1, whereas mutations
in this HRE which abolished binding of RXR/FXR heterodimers also abolished transactivation
by these nuclear receptors in the presence of CDCA. Finally, the transactivation of the
HCR-1/apoC-II promoter by RXR/FXR in the presence of CDCA was abolished specifically by
mutations in the hormone response element C of the apoC-II promoter. The findings a) establish
functional interactions between hormone nuclear receptors bound to element C of the apoC-II
promoter and the HRE of the HCR-1, and b) suggest that the HCR-1, in addition to its role as
a strong hepatic transcriptional enhancer of genes of the apoE/C-I/C-IV/C-II cluster, also serves
as a mediator of expression of the apoC-II gene in response to bile acids.
P283
The Region Comprising Kringle V and the Protease Domain is Critical for
the Binding of Human Apolipoprotein(a) to Fibronectin
Celina Edelstein, Olga Klezovitch, Angelo M Scanu. University of Chicago, Chicago, IL
In previous studies we have shown that the C-terminal domain (F2) of apolipoprotein(a),
apo(a),but not its N-terminus (F1), binds in vitro to fibrinogen and to the proteoglycans, decorin
http://atvb.ahajournals.org/ and biglycan, by both guest in their on April glycated4, and 2013 non-glycated forms. Fibronectin is also a component

a-50 Arterioscler Thromb Vasc Biol. Vol 22, No 5
of the vascular extracellular matrix (ECM) that may contribute to the atherosclerotic process by
favoring the retention of Lp(a) in the intima of the vessel wall. To this effect, we studied the
binding of Lp(a), apo(a)-derived apo(a) and proteolytic derivatives obtained by elastase
digestion, to fibronectin immobilized onto microtiter plates. The binding was established by
formal kinetic analyses. Apo(a) bound to fibronectin more avidly than parent Lp(a) (Bmax,
92.0 32.0 fmol, n 5, and 1.8 0.5 fmol, n 5, respectively). Of the two apo(a)
fragments, F1 and F2, only the latter bound to fibronectin (Bmax, 162 40 fmol, n 5).
Moreover, of the F2 sub-fragments obtained by more extensive elastase digestion, binding was
only exhibited by the fragment comprising kringle V and the protease domain, KV-PD (Bmax,
349 65 fmol, n 4). This site assignment was corroborated by the fact that the binding was
unaffected by 6-aminohexanoic acid known to act on lysine binding sites not present in KV-PD.
Of interest, this microdomain is the one that we recently reported to be critical for the
pro-inflammatory action of apo(a) in human THP-1 macrophages in culture. We conclude that
fibronectin is one of the ECM components that can contribute to the retention of Lp(a)/apo(a)
in the artery wall and that the effector site in apo(a) lies outside its lysine binding domains.
P284
Apoprotein C-III (ApoCIII) Protein Concentrations and Gene Polymorphisms
in Type 1 Diabetes
Richard L Klein, Steven J Hunter, Alicia J Jenkins, Deyi Zheng, Ngoc-Anh Le, Virgil Brown,
Timothy Garvey, Dcct/edic Research Group. Medical University of South Carolina,
Charleston, SC; Emory University, Atlanta, GA; NDIC/DCCT, Bethesda, MD
ApoCIII concentration and gene polymorphisms have been shown to be risk factors for
cardiovascular disease; however, the underlying mechanisms remain unclear. In addition, no
studies have been performed that address these issues in Type 1 diabetic patients. Fasting
blood samples were obtained in 423 sequential patients in the DCCT/EDIC cohort of patients
with Type 1 Diabetes. Higher ApoCIII concentrations were associated (p0.0001) with
increased triglycerides (r0.75), total (r0.60) and LDL (r0.39) cholesterol, apoAI
(r0.27), and apoB (r0.46), and these relationships persisted after controlling for age,
gender, BMI, and HbA1c. NMR lipoprotein subclass analyses demonstrated that ApoCIII
concentration was correlated with an increase in VLDL subclasses (p0.0001), and that there
were important effects on LDL including decreased LDL size (r-0.29; p0.0001) and an
increase in LDL particle concentration (r0.45; p0.0001) due primarily to an augmentation
in the small L1 subclass (r0.38; p0.0001). ApoCIII concentration was significantly higher
in the group of patients with macroalbuminuria (AER300 mg/24h) compared to the group with
microalbuminuria (AER 40–299 mg/24h) (MANOVA p0.05) or normoalbuminuria (AER40
mg/24h) (MANOVA p0.01). Neither a Sac1 nor the T -455 3C polymorphisms in the ApoCIII
gene were associated with any alterations in circulating ApoCIII concentrations, serum lipids,
apolipoprotein concentrations, or parameters measured by NMR lipoprotein subclass analyses.
Increased ApoCIII concentration may confer increased risk for cardiovascular disease through
its effects on LDL subfraction distribution and particle concentration.
P285
MDA-LDL Immunization of Normal or Hypercholesterolemic Mice Induces
an Inherent Th2 Biased Response
Christoph J Binder, Mi-Kyung Chang, Brian M Crain, Maripat Corr, Joseph L Witztum.
University of California San Diego, La Jolla, CA
Immunization with MDA-LDL significantly reduces the development of atherosclerosis in mice.
However, the mechanism(s) of this protective effect are unknown. To investigate the specific
properties of the cellular immune response following immunization with MDA-LDL, normocholesterolemic
C57BL/6 mice (n4) were immunized three times with homologous MDA-LDL
emulsified in Freunds adjuvant (FA) and the antigen specific IgG isotype responses measured
by ELISA. The antigen specific cytokine secretion patterns of splenocyte cultures from
immunized mice were measured by ELISA and the frequencies of antigen specific IFNg and IL-5
secreting T-cells quantified by ELISpot. In all mice, IgG1 titers to MDA-LDL were significantly
higher (between 4 to 12 fold) than IgG2a titers. Cultures of splenocytes from immunized mice
stimulated for 72 hours with MDA-LDL contained significant amounts of IL-5 (5324 pg/ml)
and IL-13 (2810 pg/ml), but only low amounts of IFNg (15 pg/ml). In the spleens of
immunized mice the frequency of MDA-LDL specific IL-5 secreting T-cells (26.38.4 / 2x10 6
splenocytes) was more than 15 fold higher than the frequency of IFNg secreting T-cells
(1.71.4). This was specific for the immunization with MDA-LDL because native LDL did not
induce any cytokine responses in vitro and splenocytes from naive C57BL/6 mice responded
neither to native nor MDA-LDL. The same Th2 bias was observed in hypercholesterolemic
LDLR -/- mice immunized with MDA-LDL (n4), and in their atherosclerotic lesions there was
a 6 fold increase in T-cells and a 14 fold increase in IL-4 mRNA when compared to PBS / FA
immunized LDLR -/- controls. These data indicate that immunization with homologous MDA-LDL
induces a Th2 biased response independent of hypercholesterolemia and despite conditions
that normally favor a Th1 response (complete FA and C57BL/6 mice). The Th2 biased properties
of antigen specific T-cells in atherosclerotic plaques induced by immunization could well
influence the inflammatory state of the lesion. Therefore, the enhanced Th2 biased response,
which is thought to be anti-inflammatory, may be one mechanism by which immunization with
MDA-LDL is protective.
Effect of BAPN on Coronary Restenosis
Diem T Nguyen, Carl C Danielsen, Henning R Andersen. B-Research, Skejby University
Hospital, Aarhus, Denmark; Inst. of Anatomy, Aarhus University, Aarhus, Denmark
P286
Background: Constrictive vascular remodeling (CVR) is the major mechanism of coronary
restenosis in non-stented vessels, whereas neointima formation is the major mechanism of
in-stent restenosis. Beta-Amino-Propio-Nitrile (BAPN) reduces CVR in non-stented vessels. We
hypothesized that increasing tension in the tissue Downloaded surrounding a stent from
caused by CVR may
http://atvb.ahajournals.org/
trigger cell proliferation and matrix synthesis inside the stent. Therefore, BAPN treatment may
suppress in-stent neointima formation by reducing tissue tension outside the stent. Methods:
24 pigs were randomized in 2 groups treated with oral BAPN or placebo for 28 days. PTCA was
performed at 2 sites in RCA, and at 2 sites with stent in LAD. Angiograms were obtained before
PTCA, immediately after and at follow-up. Postmortem examination included biomechanical
testing of isolated artery rings from media/neointima, adventitia and entire vessel, and the
specimens were evaluated by histology. Results: In RCA, BAPN increased lumen and decreased
tensile strength without affecting neointima formation. Thus, BAPN caused a larger lumen by
reducing CVR. In stented LAD, lumen and neointima were similar in both groups, whereas
tensile strength was reduced in BAPN group. There was no difference in collagen content
between the 2 groups in LAD and RCA. Conclusion: Although BAPN reduced tensile strength
and CVR it did not reduce in-stent neointima formation.
P287
The Presence of Acyl-Coenzyme A:Cholesterol Acyltransferase 2 (ACAT2) in
Human and Mouse Macrophages: Evidence In Vivo and In Vitro
Naomi Sakashita, Akira Miyazaki, Catherine C Chang, Ta-Yuan Chang, Osamu Nakamura,
Emi Kiyota, Maki Satoh, Masaharu Hori, Seikoh Horiuchi, Motohiro Takeya. Kumamoto
University School of Medicine, Kumamoto, Japan; Kumamoto University School of Medicine,
Kumammoto, Japan; Dartmouth Medical School, Hanover, NH; Second Department of
Pathology, Kumamoto University School of Medicine, Kumamoto, Japan
Two distinct ACAT isozymes, ACAT1 and ACAT2, have been identified. In human, we have
previously shown that ACAT1 is ubiquitously expressed in a variety of tissues and cells,
including macrophages, adrenals, hepatocytes, enterocytes, neurons, and skin cells, whereas
ACAT2 is mainly expressed in enterocytes and fetal hepatocytes. The presence of ACAT2 in
macrophages has not been examined carefully. Using high-titer polyclonal antibodies against
ACAT2, we now demonstrate the presence of ACAT2 in certain subpopulations of human and
mouse macrophages. In human, ACAT2 signals are detectable immunohistochemically in
exudate macrophages under various pathological conditions, including sarcoidosis, epitheloid
granuloma, and atherosclerosis, but not detectable in resident tissue macrophages such as
Kupffer cells. In human atherosclerotic aorta, double immunohistochemical staining indicated
that while 100% of infiltrated macrophages express ACAT1, 60–80% of them also express
ACAT2. The expression of ACAT2-positive macrophages correlated positively with lipid
accumulation in the lesion. Immunoblot analysis using the human blood-borne monocyte/
macrophage cell culture system revealed that mature macrophages express both ACAT1 and
ACAT2. Using the mouse model, immunostaining demonstrated that ACAT2 is also present in
atheromatous plaques isolated from the ApoE knockout mice. In addition, immunoblot analysis
revealed that ACAT2 is detectable in thioglycolate-elicited exudate peritoneal macrophages, but
not in resident macrophages. Our results suggest that in macrophages, ACAT2 play an
augmenting role to ACAT1 in the process of cellular cholesterol esterification and storage.
Reactive Carbonyls from Tobacco Smoke Increase Arterial Endothelial
Layer Injury
Adam E Mullick, James M McDonald, Goar Melkonian, Prudence Talbot, Kent E Pinkerton,
John C Rutledge. University of California, Davis, Davis, CA; University of California,
Riverside, Riverside, CA
P288
Environmental tobacco smoke (ETS) has been strongly linked with the development of
atherosclerosis, however the mechanisms of this injury have yet to be elucidated. The goal of
this project was to determine the actions of ETS, during both acute and chronic exposure, on
the artery wall. Additionally, we wanted to determine if estradiol could be protective against the
actions of ETS exposure. For our chronic studies, ovariectomized rats treated with subcutaneous
placebo or 17- estradiol pellets were exposed to ETS or filtered air for 6 weeks. The
rate of accumulation of fluorescently labeled low-density lipoprotein (LDL) in the carotid artery
wall was determined by quantitative fluorescence microscopy. Exposure of the animals to ETS
increased LDL accumulation over 4-fold compared to filtered air exposure, an effect largely
mediated via increased permeability (4.0 0.7 vs 0.8 0.7 ng protein*min-1 *cm-2 ;p0.05).
No protective effect of estradiol was observed. Acute ETS exposure of a buffer solution
containing LDL resulted in over a 6-fold increase in the highly reactive carbonyl glyoxal.
Perfusion of this buffer solution through rat carotid arteries resulted in a 105% increase in
permeability (p0.05). Attenuation of this response was observed with addition of aminoguanidine.
Moreover, perfusion of glyoxal alone resulted in a 50% increase in carotid artery
permeability (p0.05). Hence, these effects of ETS, mediated specifically by reactive
carbonyls, result in increased arterial permeability and LDL accumulation. This arterial injury
may serve as an initiating event in atheroma formation in individuals exposed to environmental
tobacco smoke. by guest on April 4, 2013

P289
Shear Stress Modulates Mesodermic Gene Expression in Mouse Embryonic
Stem Cells
Barbara Illi, Carlo Gaetano, Maurizio C Capogrossi. Istituto Dermopatico dell’Immacolata,
Roma, Italy
Shear Stress (SS) modulates adult endothelial and smooth muscle cells function, but little is
known about its effect during development. Therefore we used mouse embryonic stem cells
(ES) to study the role of SS in an in vitro model of vascular differentiation. ES were adapted in
colture without feeder layer and, 24–48 hours before experiment, cells were deprived of
Leukemia Inhibitory Factor (LIF). We found that SS (10 dyne/cm 2 /sec -1 ) downregulates
Flk-1/KDR gene expression while Platelet Derived Growth Factor receptor- (PDGFR-) mRNA
was not modulated. To investigate whether this phenomenon occurred at transcriptional level,
before exposure to SS, ES were transfected with two promoter reporter constructs, spanning
nucleotides -975/297 and -442/297 of the Flk-1 promoter. In this experimental setting the
-975/297 construct was repressed from 2 to 4 fold by SS, while the -442/297 construct
was unaffected. Surprisingly, administration of trichostatin A (TSA), a histone deacetylase
inhibitor, fully recovered SS repression enhancing 3 to 5 fold the -975/297 Flk-1/KDR
promoter activity in sheared ES cells exposed to SS indicating a role for histone acetylation in
this process. To further investigate the role of SS during vascular development, we performed
cDNA array screening and found that Angiogenin, Thrombin receptor, Myocyte Enhancer
Factor-2C (MEF-2C) and genes of the frizzled/wnt family, important for smooth and skeletal
muscle formation, were upregulated by SS. In conclusion, these data suggest a role for SS in
determining vascular phenotype in embryonic stem cells.
P290
Biphasic Activation of Rac1 in Smooth Muscle Cells by Fibroblast Growth
Factor-2
Euridicky V Fera, Caroline Van Den Diepstraten, J Geoffrey Pickering. John P Robarts
Research Institute, London, ON, Canada
Fibroblast growth factor-2 (FGF-2) is a potent stimulator of vascular smooth muscle cell (SMC)
migration but the signaling cascades mediating this response are not well elucidated. Using
rapid acquisition digital time-lapse microscopy, we observed that p lasma membrane ruffling
was enhanced by FGF-2, especially on a type I collagen substrate. This suggested that Rho
family GTPases might mediate the migratory effects of FGF-2. To test this hypothesis, we
undertook affinity purification of the activated forms of cdc42 and rac1 by interacting SMC
lysates with a PAK1-GST fusion protein. Activated cdc42 was detected in unstimulated SMCs
and the level was unaffected by FGF-2. In contrast, FGF-2 induced a striking, transient
activation of rac1 within 30 seconds of stimulation. This was followed by a second, prolonged
phase of rac1 activation lasting up to 48h. This secondary wave of rac1 activation was greater
for SMCs on collagen than SMCs on fibronectin. Digital overlay of serial time-lapse cell images
revealed an immediate stimulation of plasma membrane protrusion by FGF-2, as well as
sustained lamellipodial protrusion evident 48h later (9.80.2 vs 1.20.4% of total cell area
protruding/min). Because FGF-2 also induces expression of collagenase-1, the role of collagen
degradation was assessed by plating SMCs on collagenase-resistant collagen derived from
mice with a mutation in the collagen cleavage site. On this substrate the acute activation of
rac1 by FGF-2 was preserved. However, the second phase of rac1 activation was blunted and
at 36h the proportion of activated rac1 was reduced to 0.5 of that in SMCs on native collagen.
Furthermore, lamellipodial protrusion rate on this substrate was reduced to 0.4 of that on
wild-type collagen. (p0.001). Conclusions: FGF-2 stimulates direct and transient activation of
rac1 in human SMCs. This is followed by a second wave of sustained rac1 activation that is
mediated by proteolytic modification of collagen. The associated lamellipodial activation
suggests that continual protrusion of the cell front is driven by sequential presentation of
diverse agonists that activate rac1.
P291
Importance of ADP Receptor (P2Y12) in Platelet Activation and Thrombus
Formation under High Shear Rates
Shinya Goto, Noriko Tamura, Minako Yoshida, Shunnosuke Handa. Tokai University School
of Medicine, Isehara, Japan
Background. The role of the ADP receptor P2Y 12 in von Willebrand factor (VWF)-mediated
platelet activation and thrombus formation was investigated. Materials and Methods. Whole
blood samples and platelet-rich plasma was obtained from eleven adult donors. Shear-induced
platelet aggregation and activation was detected by optically modified cone-plate viscometer,
Downloaded from
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and quantitative flow cytometry detecting P-selectin platelet surface translocation. Platelet
thrombus formation on collagen surface under flow condition was detected by parallel plate
flow chamber equipped with epi-fluorescent video-microscopy. Effects of a selective P2Y 12
antagonist AR-C69931MX were tested. Results. AR-C69931MX inhibited shear-induced platelet
aggregation in a dose dependent manner. Maximum inhibition, from 69.79.6% (meanSD)
to 32.48.2% (p0.00011), was achieved at 100 nM. The inhibiting effects of AR-C69931MX
were reversed by exogenous addition of ADP. Shear-induced platelet activation as evidenced
by P-selectin surface translocation was also inhibited from 5964784/10.000 measured
platelet to 2797718/10,000 measured platelet in the presence of 100 nM AR-C69931MX
(p0.0009). Platelet thrombus formation on collagen surface after perfusing blood for 5
minutes at wall shear rate of 1,500 s -1 , which was previously shown to be mediated by VWF-GP
Ib interaction, was also significantly inhibited by 100 nM AR-C69931MX (as shown in the
figure). Conclusions. We demonstrate here that more than 50% of platelet reactions initiated
by the binding of VWF to GP Ib induced by high a shear rate were inhibited by specific P2Y 12
inhibitor.
Multilineage Potential of Vascular Cells
Poster Presentations a-51
P292 WITHDRAWN
Yin Tintut, Karol E Watson, Farhad Parhami, Linda L Demer, Kristina Bostrom. Cardiology,
UCLA School of Medicine, Los Angeles, CA
P293
Mesenchymal cells with multiple differentiation potentials have been isolated from adult
marrow, cord blood and recently in adipose tissue. It is not known whether similar stem cells
are present in adult vasculature. In this study, we assessed the multipotentiality of vascular
cells isolated from aortic media. A subpopulation of vascular cells express osteoblastic markers
and form mineralized nodules. In addition to their osteoblast potential, these calcifying vascular
cells (CVC) exhibit chondrocytic, adipocytic, smooth muscle, and stromal potentials either
spontaneously or in the presence of induction factors. Chondrogenic potential of CVC was
evidenced by the expression of collagen type II and IX by Western blot analysis and Alcian blue
staining. Adipogenic potential of CVC was evidenced by the accumulation of lipid deposits
staining positively by Oil Red O. Smooth muscle cell potential of CVC was evidenced by
expression of smooth muscle cell markers including calponin, smooth muscle alpha-actin, and
smooth muscle myosin heavy chain. Stromal potential of CVC was evidenced by the ability to
support growth of colony forming units of hematopoietic progenitor cells and the survival of
mouse bone marrow cells in coculture. In summary, these results suggest that CVC are
multipotent mesenchymal cells, and that they may serve as a source of stem cells in the event
of vascular tissue maintenance and repair.
P294
Immuno-Laser Capture Microdissection (LCM) Enriches Macrophage Foam
Cell RNA for the Molecular Analysis of Atherosclerotic Lesions
Eugene Trogan, Robin P Choudhury, Hayes M Dansky, James X Rong, Jan L Breslow,
Edward A Fisher. Mount Sinai School of Medicine, New York, NY; The Rockefeller
University, New York, NY
Background. Macrophage foam cells are critical mediators in atherosclerosis. The analysis of
gene expression in foam cells from arterial lesions is complicated by the cellular heterogeneity
and varying complexity of atherosclerotic plaque. To overcome these limitations, we used
immuno-LCM and real-time quantitative RT-PCR to selectively procure and analyze RNA from
lesional macrophages. Methods and Results. Serial aortic root sections from apoE-/- mice were
immunostained for macrophage-specific CD68 by a rapid protocol. RNA was isolated from
either entire sections (analogous to isolation from whole tissue), or from LCM-selected
CD68-positive areas only. Using real-time quantitative RT-PCR, the transcript levels of the
following genes were measured: macrophage-specific CD68, smooth muscle cell (SMC)specific
-actin, and cyclophilin A as normalizing control. Compared to whole section-extracted
RNA, CD68 levels in the LCM-extracted RNA were significantly higher both when adjusted to
RNA mass or to cyclophilin A (fold enrichment: 33.66.17 or 30.44.7, respectively, P0.05).
-actin was absent from the LCM-derived RNA, attesting to the specificity of the cell selection.
The integrity of the LCM RNA sample was represented by amplifying a long (450 bp) region of
GAPDH mRNA. To test this approach toward the study of regulation of candidate genes in
specific cell types in the arterial wall, LCM-selected foam cell RNA from lesions of apoE-/- mice
with or without stimulation with lipopolysaccharide (LPS) (100 g, IP) were analyzed for
LPS-inducible MCP-1, VCAM-1, and ICAM-1 transcripts. Compared to unstimulated mice, foam
cell RNA from LPS-stimulated mice exhibited clear increases (range of fold induction, MCP-1:
18.6–43.6; by VCAM-1: guest on 7.6–15.1; April 4, ICAM-1: 201319.7–40.5).
Conclusions. 1) Immuno-LCM can

a-52 Arterioscler Thromb Vasc Biol. Vol 22, No 5
reliably enrich cell type-specific RNA from mouse atherosclerotic lesions; 2) transcriptional
regulation of candidate genes can be measured by real-time RT-PCR analysis of RNA from
LCM-selected cells; 3) These methods will significantly enhance the cell-specific analysis of in
vivo gene expression in atherosclerosis.
P295
Inhibition of Hedgehog Signalling Accelerates Atherosclerosis and Affects
Serum Lipids
Esther Lutgens, Li Chun Wang, Linda Burkly, Nicholas Davidson, Victor Koteliansky, Mat
Daemen. University of Maastricht, Maastricht, Netherlands; Biogen Inc, Cambridge, MA;
University of Washington, St Louis, MO
To investigate the role of Hedgehog (HH) in atherosclerosis, a gene known to be involved in
embryonic patterning, anti-HH antibody (5E1) was injected twice a week (200g intraperitoneally)
in ApoE-/- mice. Treatment ranged from age 6–12 wks (n4 HH, n4 ctrl) or from age
6–18 wks (n7 anti-HH, n8 ctrl). Subsequently, the arterial tree was perfused and aortic
arches were analyzed. No effects were observed in the 6-wk treatment group. However, when
treatment was prolonged to 12 wks, inhibition of HH signalling significantly increased plaque
area (241,78431,827m 2 anti-HH vs 131,42222,500m 2 ctrl, p0.05). After anti-HH
treatment, plaques were almost entirely composed of giant macrophages (macrophage
content: 70.13.4% anti-HH vs 46.45.6% in ctrl, p0.05). These giant plaque macrophages
contained multiple nuclei and were 79.9% larger than in ctrl treated mice. Anti-HH treatment
decreased lipid core content (17.02.1% anti-HH vs 29.33.2%, p0.05). CD45 cell
content, as well as collagen and ASMA content did not change. Interestingly, anti-HH treatment
induced medial chondral metaplasia, and an increase in medial thickness. Although plaque
area increased after inhibition of HH-signalling, anti-HH treatment induced a decrease in serum
cholesterol (6 wk treatment: 37762 mg/dL vs 49882 mg/dL ctr, p0.05); 12 wk treatment:
43077 mg/dL vs 52064 mg/dL ctrl, p0.05) and triglyceride (6 wk treatment: 10528
mg/dL vs 16126 mg/dL, p0.05; 12 wk treatment 10528 mg/dL vs 16126 mg/dL ctrl,
p0.05) levels. Lipid profiles (VLDL, IDL, LDL, HDL) did not change. In conclusion, inhibition of
HH-signalling induces larger atherosclerotic plaques that are composed of an increased amount
of giant, lipid filled macrophages, that are associated with a decrease in plasma cholesterol and
triglyceride level.
VLA-1 Deficiency Reduces Atherosclerosis and Induces Chondral
Metaplasia in Plaques
Esther Lutgens, Anthonin D Fougerolles, Andrew Sprague, Victor Koteliansky, Mat Daemen.
University of Maastricht, Maastricht, Netherlands; Biogen Inc, Cambridge, MA
P296
To investigate the role of VLA-1 in atherosclerosis, a major collagen receptor, mice deficient in
VLA-1 and ApoE were generated. VLA1-/-/ApoE-/- (hom, n10), VLA1/-/ApoE-/- (het, n10)
and ApoE-/- (E-/-, n10) mice were sacrificed at 26 wks, and the aortic arch was analyzed.
Total plaque area decreased 34.1% (p0.05) in VLA1-/-/ApoE-/- mice, which was due to the
62.2% decrease in individual advanced plaque area (hom 79,7711,4039 m 2 vs E-/-
127,99920,270 m 2 ,p0.05). Besides the decrease in plaque extent, VLA1 deficiency
induced changes in plaque phenotype. In advanced atherosclerotic plaques, inflammatory cell
content decreased significantly (m content: hom 31.54.2% vs E-/- 47.82.9%, p0.05,
CD3 content hom: 0.30.1% vs E-/- 2.90.3%, p0.05, CD45 content hom: 0.60.1% vs
E-/-: 2.90.2% p0.05). Lipid cores were very small (lipid core content hom: 10.21.8% vs
E-/-: 30.71.8%), and seemed to be replaced by chondral metaplasia (content: hom:
19.84.4% vs E-/-: 8.12.5%, p0.05), and extracellular matrix (collagen content hom:
35.85.4% vs E-/-: 22.93.4%, p0.05, ASMA content hom:2.90.7% vs E-/- 1.40.3%,
p0.05). VLA-1 heterozygous plaques showed an intermediate phenotype, with significant
decreases in total plaque area and inflammatory cell content, and a trend to significant
increases in chondral metaplasia (p0.07) and extracellular matrix (p0.06). Chondroid cells
were positive for bone markers like alcian blue, OPG, OP, ON, BMP2/4. In conclusion: deficiency
in VLA-1 reduces atherosclerosis extent, decreases inflammation and induces chondral
metaplasia and an increase in extracellular matrix in atherosclerotic plaques.
C-Reactive Protein and HDL-C Effects of Statins Trial (CHEST)
P297 WITHDRAWN
P298
Benjamin J Ansell, Karol E Watson, Robert E Weiss, Alan M Fogelman, Gregg C Fonarow.
UCLA School of Medicine, Los Angeles, CA; UCLA School of Public Health, Los Angeles, CA
BACKGROUND: Elevated plasma levels of C-reactive protein (CRP) and decreased levels of
high-density lipoprotein (HDL) cholesterol are associated with increased risk of coronary heart
disease (CHD), while statins appear to favorably impact both of these risk markers. We
investigated whether there was a difference in effects on CRP and HDL levels between patients
treated with three commonly-used statins. METHODS: In a prospective, observational study of
74 dyslipidemic subjects from an internal medicine clinic, we measured CRP and lipid profiles
prior to and after 12 weeks of treatment with atorvastatin (A) 10 mg, simvastatin (S) 20 mg,
or pravastatin (P) 40 mg daily. 21 of these patients also underwent CRP testing after 1 and 4
weeks of therapy to assess the time course of CRP reduction. RESULTS: The three treatment
groups experienced comparable reductions in CRP levels (A: 33%, S: 42%, and P: 30%) and
statistically insignificant changes in HDL-C (A: -4%, S: -3%, and P: 0%). Analysis of the
substudy suggested that the CRP levels continued to decrease at 1, 4, and 12 weeks of therapy.
The change in the log CRP concentration correlated with the change in the log LDL cholesterol
concentration (correlation coefficient0.33, p0.004). Subjects had similar baseline CRP
levels, lipid profiles, and CHD risk factors. CONCLUSIONS: At doses achieving similar reductions
in LDL cholesterol, atorvastatin, simvastatin, and Downloaded pravastatin were from
associated with similar
decreases in CRP levels and no significant change in HDL cholesterol levels. The finding of
correlation between the reductions in CRP and LDL cholesterol levels differs from conclusions
of other published studies, and should prompt further investigation of the mechanism by which
statins reduce CRP.
P299
Peroxisome Proliferator-Activated Receptor Alpha Increases Serum
Insulin-Like Growth Hormone-1 Levels but Decreases Insulin Resistance
Jan Malik, Vojtech Melenovsky, Michal Krsek, Tomas Stulc, Vlasta Justova, Jan Simek,
Richard Ceska. 3rd Department of Medicine, General University Hospital, Prague, Czech
Republic
BACKGROUND: Activators of both alpha and gamma type of peroxisome proliferator-activated
receptor (PPAR) decrease serum levels of insulin-like growth hormone-1 (IGF-1) as well as
insulin resistance. Therapeutic administration of IGF-1 to type 2 diabetic patients leads to
decrease of insulin resistance. We studied the effect of fenofibrate, a pure PPAR-alpha
activator, on serum IGF-1 levels and on insulin resistance. METHODS: Thirty-four non-smoking
otherwise healthy subjects (6 of them females) aged 49.2 8.5 (mean SD) years with
combined hyperlipidemia (total cholesterol 7.63 1.45 mmol/L, triglycerides 4.85 4.12
mmol/L at inclusion) were examined before and 10 weeks after treatment by 200 mg of
micronized fenofibrate o.d. Serum levels of IGF-1and its binding protein IGF-BP-1, insulinaemia,
glycaemia and Homeostasis Model Assessment (HOMA) index were analysed. Wilcoxon
matched pairs test was used for the assessment of the effects of fenofibrate on studied
variables. P-values less than 0.05 were considered as significant, values are expressed as
median SEM. RESULTS: The effects of treatment are shown in the Table. CONCLUSIONS:
Pure PPAR-alpha activators increase serum IGF-1 levels, but decrease insulin resistance.
P300
Risk Factors for Myocardial Infarction Before 41 Years of Age: A Danish
Case-Control Study
Finn Edler V Eyben, Ejvind Mouritsen, Jan Holm, Paulius Montvilas, Inge Helleberg, Lisbeth
Kristensen, Georg Dimcevski, Rie V Eyben, Gabriel Suciu. Center of Tobacco Research,
Odense NV, Denmark; Herning Central Hospital, Herning, Denmark; Tarm Hospital, Tarm,
Denmark; Varde Hospital, Varde, Denmark; University of Los Angeles, Los Angeles, CA; Ohio
State University, Columbus, OH
Background Previous studies suggested that a series of anthropometric and biochemical risk
factors added to the coronary risk of major coronary risk factors for myocardial infarction. The
hypothesis has not been addressed in individuals less than 41 years of age where especially
smoking and cholesterol are significant. Methods A prevalence hospital-based matched
case-control study of 22 cases with non-fatal myocardial infarction before 41 years of age and
24 controls without coronary heart disease matching for age and gender. We measured a series
of newer anthropometric and biochemical coronary risk factors blindly. Findings In conditional
univariate logistic regression analyses, family history, smoking, intraabdominal fat as
percentage of total abdominal fat, systolic blood pressure, cholesterol, LDL cholesterol,
homocysteine, fibrinogen, and glycosylated haemoglobin were statistically significant coronary
risk factors whereas the other anthropometric and biochemical coronary risk factors were not.
In multiple conditional logistic regression analyses, smoking (odds ratio (OR) 1.1 for an
increase in consumption of 1 cigarette per day, p 0.0327), LDL cholesterol (OR 2.4 for
an increase of 1 mmol/L, p 0.0173), and fibrinogen (OR 6.9 for an increase of 1 g/L, p
0.0469) were statistically significant. 10 cases (46%) and none of the 24 controls were
smokers with a LDL cholesterol 4.5 mmol/L and a fibrinogen 3.7 g/L (p 0.0003, Fisher’s
exact test). Interpretation Newer coronary risk factors like fibrinogen and LDL cholesterol may
be used together with a major coronary risk factor, smoking, in estimating the coronary risk at
a young age.
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P301 WITHDRAWN

Patients with Combined Hyperlipidemia Have a Longer Period of
Spontaneous Baroreflex Oscillation than Healthy Subjects
P302
Dan Wichterle, Vojtech Melenovsky, Jan Malik, Jan Simek, Richard Ceska, Marek Malik. St
George’s Hospital Medical School, London, UK; General University Hospital, Prague, Czech
Republic
Background: Cardiac autonomic regulations have not been studied in patients with combined
hyperlipidemia (CH). Methods: This study compared 30 males with non-treated CH and 30
healthy controls (C) - age 477 vs476 years, systolic/diastolic blood pressure (SBP/DBP)
12611/827 vs11813/7410 mmHg, body mass index (BMI) 27.82.7 vs 26.53.1
kg/m 2 , total cholesterol (CH) 7.41.3 vs 5.50.9 mmol/l, triglycerides (TG) 5.44.5 vs
1.50.5 mmol/l, and HDL-cholesterol (HDL-C) 1.30.3 vs 1.20.2 mmol/l, respectively. Each
subject underwent ECG and finger arterial pressure recording in a supine position during 5 min
of spontaneous (SR) and 3 min of controlled respiration (CR) at 0.1 Hz. Frequency-domain
indices of heart rate (HR) and SBP oscillations were assessed by autocorrelation method. The
cross-spectral analysis in the low-frequency band was performed to obtain baroreflex
sensitivity (BRS) and appropriate phase shift (PS). Also the frequency of spontaneous baroreflex
oscillation (F SBO) at a distinct peak of maximum coherence between SBP and HR was
determined. All indices were adjusted for age, BMI, SBP, DBP, and HR (ANCOVA). Results: For
both respiratory regimes the patients with CH had reduced time- and frequency-domain indices
of HR variability (not shown), as well as decreased BRS and prolonged PS. However, the only
independent discriminator was F SBO (see the Table). Conclusion: The independently prolonged
period of spontaneous baroreflex oscillation may provide an index of increased cardiovascular
risk, which is superior to other conventional descriptors of HR variability and baroreflex
mechanism.
P303
Upregulation of MCP-1 and VCAM-1 is Associated with Development of
AngII-Induced Arterial Disease in LDL Receptor -/- Mice
Katsuya Tashiro, Lisa A Cassis, Alan Daugherty. University of Kentucky, Lexington, KY
We have previously demonstrated that AngII infusion leads to development of atherosclerosis
and abdominal aortic aneurysms in hyperlipidemic mice. The vascular pathology is associated
with infiltration of both macrophages and T lymphocytes. Since AngII can stimulate the
NF-kappaB signaling pathway, the vascular pathology could be mediated through elaboration
of monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1
(VCAM-1). To provide evidence of the activation of this system, we placed groups of LDL
receptor -/- mice on a high fat diet and for 1 week, then infused them with Ang II (1000
ng/kg/min) via mini-osmotic pumps for 28 days. The infusion of Ang II increased plasma
concentrations of MCP-1 by 344% (22.4 /- 6.8 vs 77.5 /- 23.5 pg/ml; P 0.03) as
determined by ELISA. Western blot analysis of plasma demonstrated that soluble VCAM-1 was
also increased in plasma (140 %). To determine whether activation of NF-kappaB was
responsible for AngII induced vascular pathology, we administered an inhibitor of this system,
pyrrolidine dithiocarbamate (PDTC), at concentrations used previously to inhibit this pathway
(100 mg/kg/day in drinking water). This dose of PDTC failed to inhibit the formation of AngII
induced atherosclerosis and aneurysm formation. However, it also failed to inhibit the AngII
induced increase in plasma MCP-1 concentrations. Therefore, plasma concentrations of MCP-1
and VCAM-1 are associated with AngII induced vascular pathology, but an effective NF-kappaB
inhibitor is needed to define the direct role in the disease process.
Infusion of Large Unilamellar Vesicles (ETC-588) Mobilize Unesterified
Cholesterol in a Dose-Dependent Fashion in Healthy Volunteers
P304
Daniel J Rader, Wendi V Rodrigueza, Narendra D Lalwani, Thomas R Valiquett. University of
Pennsylvania, Philadelphia, PA; Esperion Therapeutics Inc, Ann Arbor, MI
Background: ETC-588 is a novel, phospholipid liposome solution engineered to sequester
cholesterol and other exchangeable lipids from vascular and peripheral tissue and deliver them
to the liver for processing or excretion. This study assessed the safety and tolerability as well
as mobilization of unesterified-cholesterol of five dose levels of ETC-588 (100, 150, 200, 250,
and 300 mg/kg) infused at 10 mL/min given as four separate dose administrations q3d.
Results: Twenty subjects were randomized andDownloaded 17 provided adequate from
data for PK and PD
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Poster Presentations a-53
evaluation. Following four doses of ETC-588, the mean maximal plasma unesterifiedcholesterol
concentration increased significantly in a dose-dependent fashion, as summarized
below. Conclusion: ETC-588 was safe and well tolerated at all dose levels and significantly
mobilized unesterified-cholesterol at all doses studied. Studies of ETC-588 in patients with
atherosclerosis evaluating vascular functional responses and effects on markers of vascular
disease are ongoing to assess direct effects on vascular physiology of ETC-588-induced
mobilization of unesterified cholesterol from vascular and peripheral tissue.
P305
Influenza Infection Exerts Prominent Inflammatory and Thrombotic Effects
on the Atherosclerotic Plaques of Apo E- Deficient Mice
Mohammad Madjid, Silvio Litovsky, Adeeba Akhtar, Philip R Wyde, Samuel W Casscells III,
Morteza Naghavi. University of Texas-Houston Health Science Center and Texas Heart
Institute, Houston, TX; Baylor College of Medicine, Houston, TX
The role of infection in the development and complications of atherosclerosis has been the
focus of much attention. We have previously reported that influenza vaccination was associated
with reduced risk of recurrent myocardial infarction. Here, we report the effect of influenza A
virus on the Apo E -/- mouse, an animal model of atherosclerosis. Methods: Twenty-four apo
E -/- mice over 24 months old were injected with 1 LD50 (lethal dose 50) of influenza A virus
intranasally. Ten wild-type C57BL/6 infected mice and 11 non-infected age-matched apo E -/mice
served as controls. Animals were sacrificed 3, 5 and 10 days after inoculation. Multiple
aortic sections were studied histologically. Results: The infected mice showed markedly
increased intimal cellularity compared to the non-infected apo E -/- mice. No aortic
abnormalities were seen in infected wild type mice. Ten infected apo E -/- mice had a
prominent subendothelial infiltrate composed of a heterogeneous group of cells that stained
positively for smooth muscle cell actin, F4/80 (macrophages) and CD3(T lymphocytes). Infected
animals with the focal cellular infiltrate had clusters of platelets in the lumen overlying the
infiltrate. One case of subocclusive platelet and fibrin-rich thrombus was seen. Neither the
subendothelial infiltrate nor the large platelet clusters and thrombus were seen in the
non-infected mice. Conclusion: This study shows for the first time that influenza infection
promotes inflammation, growth, and thrombosis of atherosclerotic plaques. This may prove
useful as a model for studying the roles of infection and inflammation in atherogenesis.
Detection of Endothelin 1 Deposits by Inmunofluorescence in Human
Healthy and Diseased Coronary Arteries
Mauricio J Pineda, Luz Maldonado, Ana Uribe. Cardioinfantil Foundation, Bogotá, Colombia
P306
The role of ET1 in the genesis of atherosclerosis is not well known. Davenport published some
works demonstrating a significant rise in plasma levels of ET1, and local deposits into the
plaque in patients with some types of CHD. OBJETIVES: To standardize the IF technic in CA, in
order to detect ET1 deposits in the arterial wall. To demonstrate a significant difference of ET1
deposits in healthy versus diseased CA. MATERIALS AND METHODS. We obtained fragments of
CA from patients died because CHD or fragments from autopsies with casual finding of CHD
(Group I; n17) and fragments from autopsies of patients died because noncardiac gunshots
or traffic accidents and normal CA(Group II,n17) before 3 hours from death and stored in
Normal Saline 0,9% for transportation and then in Liquid Nitrogen for tissue preservation.
Specimens were cut in 3 micron slides and incubated with the antisera and fluoresceine
conjugateby andguest analyzedon under April a Fluorescence 4, 2013 Microscope 4 times in a blind fashion. Exclusion

a-54 Arterioscler Thromb Vasc Biol. Vol 22, No 5
criteria:Any condition with known rise in plasma ET1 levels. We standardize the IF technic using
pig CA segments with and without balloon barotrauma to obtain positive and negative controls
and used different consecutive dilutions of antisera and conjugate to obtain the optimal dilution.
RESULTS: We obtain positive IF in 100% of specimens in Group I ( Diseased arteries) and
negative IF in 100% of specimens in Group II ( Healthy arteries). DISCUSION. This findings
suggest that, different from healthy human CA, which have no detectable deposits of ET1, in
atherosclerotic CA exist deposits of ET1 probably secreted permanently after the strong stimuli
of Oxidized Cholesterol entrance to the arterial wall and the presence of macrophages .ET1
stimulates the cellular hyperplasia and hypersecretory status of the atherosclerotic plaque. In
other words, ET1 could play a physiopathological role in human atherosclerosis.
P307
Oleic and Linoleic Acid Potentiate the Mitogenic Effects of Insulin-Like
Growth Factor I via a Phospholipase D-Dependent Pathway in Arterial
Smooth Muscle Cells
Bardia Askari, Ross G Gerrity, Farah Kramer, Karin E Bornfeldt. University of Washington
School of Medicine, Seattle, WA; Medical College of Georgia, Augusta, GA
In a porcine model of diabetes-accelerated atherosclerosis there are increases in both the
proliferation of smooth muscle cells (SMCs) in lesions and in levels of plasma triglycerides. We
investigated if increased levels of fatty acids could contribute to the proliferation of SMCs seen
in diabetic lesions. Because levels of immunoreactive insulin-like growth factor I (IGF-I) were
found to be higher in lesions from diabetic animals, we stimulated porcine SMCs with IGF-I (1
nM), oleic acid (OA; cis-18:1) and linoleic acid (LA; cis-18:2). SMC proliferation was assessed
by measuring [3-H]-thymidine incorporation into DNA and by analysis of cell number. IGF-I
alone increased [3-H]-thymidine incorporation (590 46% vs. untreated cells, mean S.E.M,
n3). OA and LA stimulated thymidine incorporation a dose-dependent manner (EC50 70
M). However, incubation of IGF-I with OA (70M) or LA (70M) resulted in a further
stimulation of thymidine incorporation (8011 183 vs. 5438 45 cpm, IGF-ILA VS. IGF-I;
10810 696 vs. 5438 45 cpm, IGF-IOA vs. IGF-I, p.05), an increase that was also
reflected in cell number. Pretreatment of SMC with the trans isomer of OA (elaidic acid,
trans-18:1) and with conjugated-linoleic acid (c-LA) did not potentiate the effects of IGF-I.
Pharmacological inhibition of the phospholipase D (PLD) pathway via propranolol (50M), a
phosphatidate phosphohydrolase inhibitor, or tert-butanol (1-butanol, 0.1 % v/v), a direct
inhibitor of PLD activity, abolished the fatty acid-mediated potentiation of thymidine incorporation
in IGF-I treated cells, while inhibition of the PLA2 and PLC pathways was without effect.
Furthermore, increasing DAG levels by inhibiting diacylglycerol (DAG) kinase activity using
R59022 (3 M) potentiated the mitogenic effect of IGF-I more than 2-fold. In conclusion, IGF-I
is present in diabetic lesions of atherosclerosis and OA and LA potentiate the mitogenic effects
of IGF-I in SMCs via a PLD-dependent pathway leading to an increase in DAG. It is possible that
increased levels of fatty acids contribute to the acceleration of atherosclerosis seen in diabetes.
P308
Vasorelaxation to C-Type Natriuretic Peptide (CNP) Is Enhanced in Rabbit
Carotid Arteries with Atheroma-Like Lesions
Melissa Barber, Greg Dusting, Robyn Woods. Howard Florey Institute, Melbourne, Australia
Atrial and C-type natriuretic peptides (ANP and CNP) have antiproliferative effects in cultured
vascular smooth muscle cells and can prevent neointima formation in balloon injured rabbit
carotid arteries. Moreover, we previously demonstrated that both CNP and ANP prevent
dysfunction of the endothelial nitric oxide system accompanying neointima formation induced
by periarterial collars in rabbits. Since the soluble guanylate cyclase is the receptor for
endothelium-derived nitric oxide, we have now explored whether the sensitivity of the
particulate guanylate cyclase system, activated by natriuretic peptides, is altered in this model.
A non-occlusive silastic collar was placed around each carotid artery in the anesthetised rabbit.
After 14 days, animals were euthanased and control and collared sections were taken for
pharmacological and morphological studies. Morphometric measurements showed a neointima
formed in collared arteries. Vascular reactivity studies demonstrated vasoconstrictor supersensitivity
to serotonin in collared artery rings. Vasorelaxation of collared arteries to ANP was
not significantly different from control arteries (maximum relaxation: 77 vs 63 7%,
within-animal SEM, n 5 rabbits). In contrast, vasorelaxation to CNP was greater in collared
arteries than in control arteries (maximum relaxation: 86 vs 50 7%, n 8 rabbits, P
0.01). CNP is thought to be the endogenous ligand for the natriuretic peptide B (NP B) receptor.
The observed supersensitivity to CNP in this model may indicate an upregulation of the NP B
receptor or that its post-receptor signalling is enhanced in atherosclerosis. By contrast, the
natriuretic peptide A (NP A) receptor, primarily responsive to ANP, is either unaltered or
downregulated during the development of atherosclerosis. The selective, increased responsiveness
to CNP in our model indicates an alteration in the endogenous natriuretic peptide
system in early atherosclerosis. Since the NP B receptor mediates anti-atherogenic activity, the
local CNP system may be counteracting other pro-atherogenic factors.
Gene Expression Profiling in Atherosclerotic Coronary Arteries
Astrid B Wietelmann, Stephanie I Hehlgans, Birgit Weiss, Manfred Richter, Annette Zeyer,
René Zimmermann, Dietmar Von der Ahe. Kerckhoff-Klinik, Vascular Genomics, Bad
Nauheim, Germany
P309
Atherosclerosis is a complex and chronic inflammatory disease process involving generally
larger and medium-sized muscular arteries. Damage of the arterial wall is a key factor in the
initiation of atherosclerosis which is potentiated by systemic factors including lipoprotein
disorders, hypertension and diabetis. Although clinically significant complications of atherosclerosis,
such as plaque rupture and thrombosis, are the leading cause of death in western
countries, the underlying pathogenic mechanism(s) of lesion formation and progression of
plaque formation ist not well understood. The elucidation Downloaded of new factors from
and their correspond-
http://atvb.ahajournals.org/
ing genes playing a causative role in atherosclerosis may lead to interventions that delay or
prevent lesion progression. The aim of this study is to use a molecular approach to identify
atheroma-specific genes expressed by vessel wall cells (endothelial cells, smooth muscle cells,
macrophages, T-lymphocytes) and their role in lesion formation. We applied subtractive
hybridization to compare two populations of RNA and obtain clones of genes that are expressed
in one population but not in the other. RNA was isolated from human healthy and
atherosclerotic coronary arteries. Full-length cDNA was amplified by SMART cDNA synthesis
technology and used for subtractive hybridization. Several subtractive libraries have been
established each including forward subtractions in which atherosclerotic tissue serves as the
tester and reverse subtractions in which healthy tissue serves as the tester. PCR products were
cloned and analyzed for differential expression. Northern blot hybridization with randomly
selected, subtracted clones will be time consuming and inefficient. So we decided in a first step
to spot these clones for a secondary screening to minimize false positive. Dot blot arrays of
clones from the subtracted library are hybridized with labeled probes from both RNA
populations. In the first attempt we analyzed about 2000 clones. About 10 % were in our
screening confirmed. Sequencing and homology searches identify several known and unknown
genes, either expressed or repressed in atherosclerotic tissues.
P310
Macrophage-Derived Apolipoprotein E Increases Atherosclerosis in LDL
Receptor-Deficient Mice
Weibin Shi, Aldons J Lusis. University of Virginia, Charlottesville, VA; UCLA, Los Angeles, CA
LDL receptor-deficient (LDLR-/-) mice exhibit severe hyperlipidemia and develop advanced
atherosclerotic lesions when fed a Western diet. To determine the role of macrophage-derived
apoE on hyperlipidemia and advanced atherosclerosis, female LDLR-/- mice were lethally
irradiated and reconstituted with bone marrow from either apoE-/- or apoE/ mice. Four
weeks after transplantation, the mice were fed a Western diet for eight weeks. Surprisingly, we
found that reconstitution of LDLR-/- mice with apoE-/- bone marrow resulted in significant
reductions in atherosclerotic lesions in the proximal aorta and also decreased plasma levels of
LDL/VLDL cholesterol. The deposition of apoE and apoB in the aortic wall was also significantly
reduced. These data clearly indicate that apoE can exhibit pro-atherogenic as well as
anti-atherogenic effects.
Human Endothelial Lipase Activity is Partially Inhibited by ApoA-II
Uli C Broedl, Weijun Jin, Anthony J Secreto, Jane M Glick, Daniel J Rader. University of
Pennsylvania, Philadelphia, PA
P311
Introduction: ApoA-II is the second most abundant HDL-apolipoprotein, but its physiological role
is poorly understood. Previous studies have shown that apoA-II may modulate hepatic lipase
activity. These observations led us to hypothesize that apoA-II might also modulate human
endothelial lipase (hEL), another enzyme of the same triacylglycerol lipase gene family.
Overexpression of hEL in human apoA-I transgenic mice was previously shown to markedly
reduce HDL-C and apoA-I levels. Methods: Five human apoA-I and 5 human apoA-I:A-II
transgenic mice were injected with 3x3 10 particles of an adenovirus encoding full-length hEL
cDNA. Mice were bled before and several times after injection to determine lipid and
apolipoprotein levels and phospholipase activity. Results: Baseline values were 17622 vs.
14917 md/dl for HDL-C (p0.07), 38126 vs. 30316 mg/dl for PL (p0.01) and 34043
vs. 25026 mg/dl for apoA-I (p0.01), apoA-I vs. apoA-I:A-II transgenic mice, respectively.
HEL overexpression markedly increased post-heparin phospholipase activity in both groups but
on day 7 post injection activity levels were significantly less in apoA-I:A-II transgenic mice
(8554 nmol ffa/ml/hr) than in apoA-I transgenic mice (220109 nmol ffa/ml/hr; p0.038).
HEL overexpression reduced HDL-C, PL and apoA-I to similar levels in both groups of mice after
injection. However, apoA-I:A-II transgenic mice had significantly higher HDL-C and PL levels on
day 7 and 28 post injection than apoA-I transgenic mice (HDL-C on day 7: 156% vs. 60.4%
of baseline values (p0.015), day 28: 677% vs. 4015% (p0.01); PL on day 7: 3722%
vs. 111% (p0.03), day 28: 604% vs. 3615% (p0.01)). ApoA-I levels were
significantly higher in apoA-I:A-II transgenic mice than in apoA-I transgenic mice on day 7
(126% vs. 51% of baseline values (p0.036)) but not on day 28 (6113% vs.4318%
(p11.3)). ApoA-II levels in apoA-I:A-II transgenic mice were reduced to 4922% of baseline
(652 mg/dl) on day 7 and could almost completely recover on day 28 (924%). Conclusions:
Our results suggest that apoA-II has an inhibitory effect on endothelial lipase activity. This might
be part of a mechanism whereby apoA-II can modulate HDL-C levels.
P312
Native LDL and Oxidized LDL Modulate Cyclooxygenase-2 Expression in
Human Endothelial Cells through a p38-MAPK Dependent Pathway
Alberico L Catapano, Angela Pirillo, Fabio Pellegatta, Danilo Norata. University of Milan,
Milan, Italy
Low density lipoproteins (n-LDL) and oxidized low density lipoproteins (Ox-LDL) play a central
role in atherogenesis and possess a wide variety of biological properties. We investigated
whether n-LDL or Ox-LDL modulate cyclooxygenase 1 and 2 (Cox-1 and Cox-2) expression in
human endothelial cells. As various growth factors and cytokines induce Cox-2 expression
through the activation of several mitogen-activated protein kinases (MAPKs), we also
investigated the role of these pathways on the modulation of Cox 1 and/or Cox-2 expression
by n-LDL or OxLDL. We report that n-LDL and Ox-LDL (3 to 30 mg/mL) induce Cox-2 expression
in a time- and in a dose-dependent manner. The Cox-2 protein is present in unstimulated
endothelial cells and is strongly induced 2 h after exposure to n-LDL or Ox-LDL; the induction
is maximal after 4 hours and sustained for at least 8 hours. The effect is specific for Cox-2,
as Cox-1 expression is not modulated neither by n-LDL nor by Ox-LDL. N-LDL induce the
phosphorylation of ERK1/2 to a large extent, while Ox-LDL are less effective; both n-LDL and
Ox-LDL induce by guest the phosphorylation on April 4, of 2013 p38 MAPK. The induction of Cox-2 expression is mainly

dependent on the activation of p38 MAPK as the preincubation with the p38 MAPK inhibitor
SB203580 (1mM) strongly reduces n-LDL- or Ox-LDL-induced Cox-2 protein and mRNA
expression; the MEK1 inhibitor, PD98059 (25mM) slightly affects Cox-2 expression. This is the
first observation that n-LDL and Ox-LDL induce Cox-2 expression in HUVECs and that this
regulation is mediated through a MAPK dependent pathway. The finding that n-LDL and Ox-LDL
induce Cox-2 in human endothelial cells through a MAPK dependent pathway suggests a new
level of regulation that could be inhibited to interfere with cellular signals driven by n-LDL and
Ox-LDL.
Characterization of the Glypican-Mediated Pathway for Metabolism of
Atherogenic Lipoproteins
Ilia V Fuki, Nadine Blanchard, Daniel J Rader. University of Pennsylvania, Philadelphia, PA
P313
Cell-surface heparan sulfate proteoglycans (HSPGs) participate in the catabolism of many
physiologically important ligands including lipoproteins. Three major families of cell-surface
HSPGs exist based on the core protein structure and nature of their attachment to the plasma
membrane: perlecan, syndecan, and glypican. Glypicans are a family of HSPGs characterized
by core proteins linked to plasma membrane via a glycosylphosphatidylinositol (GPI) anchor and
relatively short HS chains which are positioned close to the plasma membrane. Upon addition
of lipoprotein lipase (LpL), a molecule that bridges between lipoproteins and cell-surface
HSPGs, cells expressing glypican but no other HSPGs exhibited 5.6-, 2.7-, and 2.0-fold
increased [125I]-LDL surface binding, intracellullar accumulation and degradation, respectively.
Addition of heparin (100ug/ml) to the incubation medium or removal of HS side chains
with heparitinase, resulted in inhibition of LpL-dependent catabolism of [125I]-LDL by more
than 90%, indicating a key role for the HS side chains of glypican in this process. We next
compared metabolism of LpL-enriched [125I]-LDL in cells expressing either glypican or
syndecan alone. Interestingly, although internalization of the surface-bound lipoproteins was
quite similar via two pathways, the proportion of degraded material was about 50% lower
through the glypican-mediated process. Moreover, a tyrosine kinase inhibitor genistein and
microfilament disruptor cytochalasin D were much less effective in inhibiting internalization via
glypican compared to that via syndecan while both inhibitors efficiently blocked degradation via
glypican and syndecan. Finally, preincubation of cells for 18h with lipoprotein-deficient serum
resulted in 1.5 to 3-fold increase of the glypican-mediated ligand catabolism with no effect on
the syndecan-mediated pathway. In conclusion, glypican can mediate efficient processing of
atherogenic lipoproteins with a relatively low rate of ligand degradation in a manner that is
different from the syndecan-mediated pathway.
P314
THP-1 Cells Internalize Cholesteryl Ester from Oxidized LDL by a Selective
Lipid Uptake Process
Debin Lan, Willem J De Villiers, Wolfgang Sattler, Frederick C De Beer, Deneys R Van der
Westhuyzen. University of Kentucky, Lexington, KY; Institut fur Medizinische Biochemie,
Universitat Graz (W.S.), Graz, Austria
The formation of lipid-laden macrophage foam cells is a key event in atherogenesis. We
investigated the uptake of the protein and lipid components of oxidized LDL in THP-1 cells to
determine if a selective lipid uptake process, as described for the uptake of HDL and LDL by
the SR-BI receptor, contributes to oxidized LDL uptake. The uptake of oxidized LDL and HDL,
both double labeled with 125I and [3H]-cholesteryl ether, was assessed in human THP-1 cells.
Cell association of cholesteryl ether from oxidized LDL exceeded the association of protein
when expressed on a particle basis, indicating selective lipid uptake not accounted for by the
uptake of whole particles. Selective uptake accounted for approximately 75% of the total cell
associated cholesteryl ester. Selective uptake of oxidized cholesteryl ester occurred at a
markedly greater rate (6-fold) compared with cholesteryl ester. Lipid uptake from HDL was also
markedly selective. Increased levels of expression in CD36 in PMA-treated cells were not
associated with increased selective lipid uptake from oxidized LDL. Transfected COS-7 cells did
not exhibit selective lipid uptake from oxidized LDL in a CD36 or SR-BI-dependent manner,
indicating that neither of these receptors was responsible for macrophage selective uptake
from oxidized LDL. We conclude that a selective lipid uptake process, independent of whole
particle internalization, contributes to cholesteryl ester uptake in macrophages.
P315
Human Plasma Phospholipid Transfer Protein Is Expressed in Macrophages
and Present in Atherosclerotic Lesions
Catherine M Desrumaux, William A Boisvert, Matti Jauhiainen, Christian Ehnholm, Linda K
Curtiss. The Scripps Research Institute, La Jolla, CA; National Public Health Institute,
Helsinki, Finland
Phospholipid transfer protein (PLTP) in plasma promotes phospholipid transfer from
triglyceride-rich lipoproteins to HDL and also plays a major role in HDL remodeling. Recent in
vivo observations support a key role of PLTP in cholesterol metabolism. The expression of PLTP
by macrophages has not been reported, although human PLTP is found in gonads, thymus,
pancreas, and lungs, and mouse PLTP is found in lungs, heart and brain. We observed PLTP
gene expression in elicited mouse peritoneal macrophages and in cultured Raw264.7 cells.
Using RT-PCR and Western Blot with a monoclonal anti-PLTP antibody (Mab 66), we also
observed PLTP mRNA and protein expression in human macrophages. PLTP gene expression
was initially downregulated when proliferating monocytic THP-1 cells were transformed into
non-dividing macrophages by phorbol-myristate acetate (PMA) treatment. However the high
levels returned over 7 days of culture in the absence of PMA. In adherent peripheral blood
human macrophages, PLTP expression was increased by culturing the cells in the presence of
granulocyte-macrophage colony stimulating factor (GM-CSF). Moreover, incubation of human
macrophages with acetylated-LDL induced a marked increase in PLTP mRNA expression that
paralleled cholesterol loading. Immunohistochemical Downloaded analysis of human from
aortic atherosclerotic
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lesions indicated the presence of immunoreactive PLTP in areas that colocalized with
CD68-positive macrophages, suggesting that PLTP is produced locally by intimal macrophages.
Because PLTP is expressed by macrophages and is present in human atherosclerotic lesions,
macrophage-derived PLTP may play a key role in disease progression.
P316
Simvastatin Induces Apoptosis and Up-Regulates Caveolin-1 Expression in
Mouse Peritoneal Macrophages
Peter Gargalovic, Ladislav Dory. University of North Texas Health Science Center, Fort
Worth, TX
Cell death by apoptosis is recognized as a significant event influencing the development and
stability of the atherosclerotic lesion. Evidence suggests that the predominant cell type
undergoing apoptosis in formed lesions is the macrophage. HMG-CoA reductase inhibitors
(statins) have been previously reported to induce apoptosis in several cell types. Their extensive
use in the treatment of hypercholesterolemia therefore justifies the examination of the effects
of statins on macrophages. Incubation of thioglycollate-elicited mouse peritoneal macrophages
(Tg-MPM) with simvastatin (10 M) leads to an induction of apoptosis, as measured by DNA
fragmentation, cell morphology changes and phosphatidylserine externalization. Simvastatininduced
apoptosis is accompanied by specific induction of caveolin-1 expression, as assessed
by immunoblotting. Simvastatin exposure has no significant effect on caveolin-2 expression.
The statin- induced caveolin-1 expression is dose- and time- dependent, with a 20- fold
induction in macrophages treated with 10 M simvastatin for 72 hrs. Increased caveolin-1
expression is at least partially the result of increased caveolin-1 mRNA levels, as simvastatin
treatment increases caveolin-1 mRNA 2- fold. Addition of mevalonate (100 M) completely
prevents the simvastatin- induced apoptosis as well as increase in caveolin-1. Because the
simvastatin- mediated increase in caveolin-1 expression correlates with the degree of
apoptosis, we also examined caveolin expression in macrophages deprived of glucose or
treated with ethanol. Both treatments induced macrophage apoptosis in a time dependent
manner with a concomitant and specific increase in caveolin-1 expression. In conclusion, our
data suggest that increased caveolin-1 expression in macrophages undergoing apoptosis is not
specific to simvastatin action but rather to induction of apoptosis. This suggests that caveolin
and cholesterol-rich membrane domains such as caveolae/lipid rafts may be of general
importance in the activation of the apoptotic cascade and influence the extent of macrophage
apoptosis in the lesion area.
Role of Macrophage Apoptosis in Atherogenesis
Poster Presentations a-55
P317
Nobuhiko Kubo, Linda K Curtiss, Shigeki Yamada, Hiroshi Kanno, Ikunosuke Sakurabayashi,
Kouichi Itoh, William A Boisvert. Jichi Medical School, Tochigi, Japan; The Scripps Research
Institute, La Jolla, CA; Jichi Medical School, Saitama, Japan; Yokohama City University
School of Medicine, Yokohama, Japan
Fas (CD95)-mediated apoptosis has been postulated to play a role in atherosclerosis. To
examine the connection between apoptosis and lesion morphology as well as stability,
macrophage foam cell apoptosis was investigated by detection of single strand DNA in foam
cells of multiple human carotid atherosclerotic tissue obtained by endatherectomy. Evidence of
cell death was highest in areas adjacent to the lipid core where there was relatively thin layer
of connective tissue, a feature that is characteristic of unstable lesion, than in other areas of
foam cell accumulation. The role of macrophage-specific Fas in atherosclerosis was tested
directly by repopulating bone marrow cells of LDLR-/- mice with marrow from either
Fas-deficient lpr (lpr-BMT) or wild-type (WT-BMT) mice. After confirming the lack of Fas
expression in the peripheral blood of lpr-BMT mice, all mice were given an atherogenic diet for
16 weeks. The extent of atherosclerosis was similar between the 2 groups of mice, however
there was a striking difference in morphology of the lesions. The connective tissue fibrous cap
was much thinner and the lipid core was more prominent in the lesions of lpr-BMT mice.
Furthermore, compared to the WT-BMT mice, lpr-BMT mice had considerably less TUNELpositive
staining throughout the lesion suggesting a defect in their apoptotic mechanism due
to the lack of Fas. Thus, although overall lesion size was not affected, the lack of Fas-mediated
apoptosis in leukocytes led to a morphologically less stable lesion characterized by a thinner
fibrous cap which is more prone to thrombotic events.
P318
Modified Lipoproteins and Acute Phase Reactants Induce Ligand Specific
Clustering of an Innate Immunity Receptor Complex in Macrophage Rafts
Alexandra Pfeiffer, Michael Torzewski, Alfred Boettcher, Evelyn Orso, Wolfgang Drobnik,
Gregor Rothe, Gerd Schmitz. Institute for Clinical Chemistry and Laboratory Medicine,
Regensburg, Germany
Cellular recognition of modified lipoproteins by scavenger receptors has been linked to the role
of these receptors in innate immunity while the precise cooperation of these receptors remains
unclear. We recently showed by fluorescence resonance energy transfer (FRET), that the LPS
receptor CD14, an innate immunity receptor in monocytes, is clustered with integrin associated
protein CD47 and the Fcg-receptors CD32 and CD64. This receptor complex is activated by LPS
as revealed by co-association of CD14 with complement receptor 3 (CD11b) and the scavenger
receptor (SR) CD36. Other ligands to CD14 also induce conformational activation while they
differ in the pattern of receptors targeted into the cluster. Thus only LPS induces co-clustering
with Toll-like receptor 4 and Fcg-RIIIa, whereas the ceramide cluster contains CD47. The
effects of both agonists are modulated by the cholesterol content of the plasma membrane
suggesting that rafts are an important determinant. Interestingly, the same receptor complex
which recognizes LPS or ceramide represents a recognition structure for modified lipoproteins
and acute phase proteins. Thus, CD36 represents the major binding site for enzymatically
modified LDL (E-LDL) and the two Fcg-receptors bind CRP with high affinity. The same FRET
approach revealed by guest thaton in acute April phase 4, 2013 plasma cellular uptake of E-LDL comprises both direct

a-56 Arterioscler Thromb Vasc Biol. Vol 22, No 5
recognition of non-opsonized E-LDL by CD36 and recognition of CRP-opsonized E-LDL by CD32
and CD64. Moreover, CRP accelerates E-LDL induced macrophage foam cell formation. Cellular
binding and uptake of E-LDL was neither competed by ac-LDL nor the class A SR-inhibitor
polyinosinic acid but was partially inhibited by an excess of ox-LDL or an anti-CD36 antibody
suggesting that charge and motif recognition of E-LDL is mediated in part by the class B
scavenger receptor CD36. These data indicate the exogenous as well as endogenous host
defense systems engage similar mechanisms, that are related to an intact raft structure and
the formation of ligand specific multimeric receptor complexes, followed by a ligand specific
integrated signaling response.
P319
Relationship between Cellular Cholesterol Homeostasis and Sphingolipid
Metabolism in Primary Human Monocytes with Potential Impact for Cell
Regulation
Gerhard Liebisch, Michael Kapinsky, Wolfgang Drobnik, Chinh Duong Quoc, Gerd Schmitz.
Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg,
Regensburg, Germany
Sphingolipid molecules are important structural components of cell membranes but also
function as signal transducers in a variety of cellular processes, including proliferation,
differentiation, apoptosis. Cholesterol loading of primary human monocytes with atherogenic
E-LDL resulted in the surface exposition of ceramide and different glycosylated ceramide
species, including lactosylceramide (CDw17), dodecasaccharide ceramide (CDw65) and
globotriaosylceramide (CD77). This effect was reversed by HDL mediated cholesterol depletion
and removal of E-LDL from the culture medium. Moreover electrospray tandem mass
spectrometric measurement revealed an increase of cellular sphingomyelin and phosphatidylcholine
on E-LDL loading. In summary these data show a clear relationship between cholesterol
homeostasis and sphingo-/glycerophospholipid metabolism. Recently, our laboratory identified
ABCA1 as a central regulator of membrane traffic in macrophages and other cell types. Beside
an impaired apo AI mediated cholesterol and phospholipid efflux ABCA1 deficient cells show
increased ceramide concentrations, associated with cell cycle G2/M-phase arrest and in vitro
growth retardation. This indicates that cellular cholesterol traffic and ABC transporter function
are linked to ceramide metabolism. Furthermore, sphingolipids were found as important
regulators of lipid efflux. Thus, sphingosine as well as sphingosine-1-P, a ligand of the
EDG-receptors EDG1, EDG3, EDG5 and EDG6, inhibited apo AI mediated cholesterol and
phospholipid efflux with different concentration kinetics and lipid selectivity. C8-ceramide,
however, showed a concentration dependent effect with stimulation at 3 M and inhibition at
30 M. In summary, these data indicate a tight association between cellular cholesterol and
ceramide metabolism as well as ABC-transporter function, which may be of fundamental
importance for the regulation of diverse cell functions, including differentiation, proliferation
and apoptosis.
P320
Lipoprotein-Associated Phospholipase A2 Is Associated with Coronary
Artery Calcification in Men with a Family History of Premature Coronary
Heart Disease
Megan L Wolfe, Nisha Dada, Keith E Suckling, Nigel K Spurr, David A Campbell, Colin H
Macphee, Daniel J Rader. University of Pennsylvania, Philadelphia, PA; diaDexus, Inc, South
San Francisco, CA; GlaxoSmithKline, Harlow, UK; GlaxoSmithKline, Upper Merion, PA
Lipoprotein-associated phospholipase A2 (Lp-PLA2) circulates in the blood associated with low
density lipoproteins (LDL) and its levels are correlated with LDL cholesterol. Recent studies
have shown increased serum levels of Lp-PLA2 are independently associated with an increased
risk of coronary events. Subjects with a family history of premature coronary artery disease
were recruited to study biochemical and genetic predictors of coronary artery calcification
(CAC) as assessed by electron beam tomography (EBT). Medical history, fasting blood drawn
for standard lipid measurements, blood pressure, height, weight and waist measurements were
collected. Fasting plasma Lp-PLA2 levels were measured using a sandwich ELISA assay
utilizing two monoclonal antibodies with specificity for Lp-PLA2. In 264 men aged 40 –68, the
range in LpPLA2 levels was 0.93–4.33 ug/mL. In multiple variate analysis, only age (odds
ratio 1.2, 95% confidence interval 1.10 – 1.35) and Lp-PLA2 levels (odds ratio 2.15, 95%
confidence interval (1.09 – 4.25) were significantly associated with CAC. There were no
associations between CAC and total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol,
body mass index, or blood pressure. Our results indicate that increased plasma levels of
Lp-PLA2 are associated with coronary plaque burden and suggest that Lp-PLA2 may promote
atherosclerosis.
Effect of Trans Unsaturation on the Susceptibility of Phospholipids to
Oxidation
Robert M Sargis, Papasani V Subbaiah. Rush University, Chicago, IL
P321
Dietary trans fatty acids are known to increase the risk of atherosclerosis. Although previous
studies have shown that trans fatty acids are more slowly metabolized than the more common
cis fatty acids, the mechanisms by which trans fatty acids increase atherogenic risk are
incompletely understood. In this study we sought to determine whether trans fatty acids
modulate the oxidizability of LDL, a process believed to be critical in atherogenesis. We
synthesized 16:0–18:2 PC with either linoleic acid (18:2c,c) or linoelaidic acid (18:2t,t) atsn-2
position, incorporated them into liposomes by extrusion, and determined the rates of oxidation
in presence of either Cu2 or the free radical generator 2,2’-azo-bis(2-amidinopropane)
dihydrochloride (AAPH) by measuring the absorbance at 234 nm. Compared to 16:0 –18:2c,c
PC, the rate of oxidation of 16:0–18:2t,t PC was reduced up to 80% in presence of Cu2 , and
by 50% in presence of AAPH. We also found that Downloaded the trans unsaturated from
phospholipids inhibited
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the oxidation of 16:0–18:2c,c PC , when incorporated into mixed liposomes at 20 mol%. To
investigate whether this inhibition is related modification of bilayer fluidity by the trans fatty
acids, we studied the effects of di16:1t PC and di16:1c PC on the oxidation of 16:0–18:2c,c
PC at increasing temperatures, up to 55° C. The 16:1t PC inhibited the oxidation more than the
cis isomer at all temperatures, although the differences narrowed at higher temperatures,
indicating that the trans double bond inhibits oxidation by other mechanisms in addition to
rigidifying the membrane. These results show that the atherogenic properties of trans fatty
acids are not related to their effects on LDL oxidation. On the contrary, they might inhibit the
oxidation of lipoproteins. These results would also, in part, explain our previously reported
observation that sphingomyelin, which contains a trans double bond in its sphingosine
backbone, inhibits lipid peroxidation in LDL.
P322
Relation between Plasma Concentration of Gamma Tocopherol and Severity
of Coronary Artery Disease Diagnosed by Coronary Angiography
Cyndya A Shibao, Irma Arima, Carlos Grijalva, Ivan Best, Ana C Colarossi, Pedro Salazar.
Universidad Peruana Cayetano Heredia, Lima, Peru; Instituto Nacional del Corazon-
EsSALUD, Lima, Peru; Universidad Peruana San Luis Gonzaga de Ica, Lima, Peru; Hospital
Nacional Cayetano Heredia, Lima, Peru
The oxidation hypothesis of atherosclerosis implies that oxidation of low-density lipoproteins
(LDL) plays an important role in atherogenic process. Antioxidant vitamin E (tocopherol) protects
against coronary artery disease (CAD). Gamma tocopherol, tocopherol ’s isomers, has been
recognized to be more potent than alpha tocopherol on endogenous antioxidant activities
despite it is found in low amounts in plasma. In order to establish whether plasma
concentration of gamma tocopherol was related to the severity of coronary artery disease
inhabitants of Lima, Peru, we measured the lipophilic antioxidant gamma tocopherol in patients
with and without CAD diagnosed by Coronary Angiography (CA) and assesed by a semiquantitative
scoring system which analyzes the number and size of distinct stenotic lesions (
stenosis score) and diffuse atheromatosis lesions (atheromatosis score). We studied 48
patients, both sexes, 35 to 80 years old, with CAD versus two age matched controls subjects,
23 patients without CAD assesed by CA and 37 patients assesed by exercise test. We measured
gamma tocopherol by High Performance Liquid Chromatography (HPLC) and it was analyzed in
plasma as absolut amount and corrected by lipid profile. The analysis showed no inverse
association between plasma levels of gamma tocopherol in patients with and without CAD for
controls assesed by CA (p 0.569) or controls assesed by exercise test (p0.143), in addition
no inverse correlation was found between gamma tocopherol and coronary stenosis score( r
0.237 p 0.105) or atheromatosis stenosis score (r 0.258 p0.076).We concluded that there
is no relationship between gamma tocopherol plasma concentrations and severity of CAD
diagnosed by coronary angiography inhabitants of Lima,Peru .
Role of Egr-1 in LPS Induction of Inflammatory Genes in Monocytes
P323
Mausumee Guha, Carolyn Mackman, Devi Navamani, Ian De Belle, Eileen Adamson, Nigel
Mackman. The Scripps Research Institute, La Jolla, CA; The Burnham Institute, La Jolla, CA
Lipopolysaccharide (LPS) from Gram-negative bacteria induces a number of inflammatory
target genes in monocytes. We have recently demonstrated that LPS induction of Egr-1 in
monocytes and THP-1 cells contributes to the expression of target genes, such as tissue factor
(TF) and TNF-alpha. Currently, we are attempting to identify other Egr-1 target genes in LPS
stimulated monocytic cells. We have employed a technique called chromatin immunoprecipitation
(ChiP) that identifies Egr-1 target genes by directly crosslinking Egr-1 to regulatory
regions followed by immunoprecipitation with an anti-Egr-1 antibody. We prepared PCR
amplified target gene DNA and libraries from both unstimulated and LPS stimulated (90 min)
THP-1 cells. To validate the process, we used PCR primers spanning Egr-1 sites in the TF and
TNF-alpha promoters and successfully amplified a 452bp (TF) and a 167bp (TNF-alpha) product
from the PCR amplified target gene DNA. We observed insert sizes ranging from 150-1800 bp
in the library prepared from LPS stimulated cells. MegaBLAST (nr), pairwise BLAST, exon
prediction and splice site identification were used to analyze the sequences with homologies
in the NCBI database. Direct sequencing of clones identified the VAMP (vesicle-associated
membrane protein)-associated protein B gene and the FK506 binding protein (FKBP2) gene as
potential Egr-1 regulated genes. The VAPB gene has been shown to function in the regulation
of protein trafficking and may play an important role in protein secretion in LPS stimulated
monocytic cells. The FKBP2 represents a direct target of the PI-3K pathway, an important
signaling pathway in LPS stimulated monocytic cells. These and other Egr-1 target genes are
being further characterized. The role of Egr-1 in regulation of these targets will be confirmed
using RNA interference (RNAi) to inhibit Egr-1 expression in human monocytic cells and by
using peritoneal macrophages from Egr-1-/- mice. These studies should allow us to further
elucidate the by role guest of Egr-1 on April in LPS 4, regulation 2013 of gene expression in monocytic cells.

P324
Growth-Factor Induced Gene Expression and Transcription Factor
Activation are Attenuated in Vascular Smooth Muscle Cells Derived from
12/15-Lipoxygenase Knockout Mice
Marpadga A Reddy, Linda Lanting, Rama Natarajan. Beckman Research Institute of the City
of Hope, Duarte, CA
Biochemical and genetic evidence support the involvement of lipoxygenases in the atherogenic
process. We have previously demonstrated that the 12-lipoxygenase (12-LO) pathway plays a
key role in growth factor (GF) induced responses in vascular smooth muscle cells (VSMC). We
also recently reported that VSMC derived from 12/15-LO knock out mice (LOKO) show slower
growth rates and matrix protein production relative to control (WT) mice. Here we demonstrate
that changes at the level of gene expression and transcription factor activation may contribute
to the reduced growth and GF responses in VSMC from LOKO mice. We compared GF (serum
and PDGF)- stimulated activation of MAPKs, key transcription factors involved in cell growth
and FN production, the expression of immediate early genes and matrix protein fibronectin (FN)
in aortic VSMC cultures derived from WT and LOKO mice. Immunoblotting of cell lysates with
phosphospecific-MAPK antibodies revealed that the GF stimulated activation of p38MAPK was
markedly reduced in LOKO cells but p44/42 MAPK activation was similar in both cell types. The
expression of immediate early genes c-fos and c-myc and matrix protein FN mRNA were
examined by RT-PCR using 18S RNA as the internal standard. Results showed that the
expression levels of both c-fos and c-myc mRNAs were greatly reduced in LOKO cells (40% to
50% of WT, n2). Basal FN protein expression was lower in LOKO cells (55% of WT, p0.001).
GF stimulated FN mRNA peaked at 4 hours in both WT and LOKO cells. It was sustained up to
24 hours in WT cells but returned to basal levels after six hours in LOKO cells. Basal DNA
binding activity of AP-1 and CREB transcription factors were 50% lower in LOKO cells
compared to WT cells. GF stimulated AP-1 activation was reduced by 50% and CREB activation
was completely absent in LOKO cells. In contrast DNA binding activity of a constitutively active
transcription factor SP1 was similar in both cells. These results suggest that LO activation may
play a key role in GF induced VSMC proliferation, migration and matrix responses which are
associated with the development of atherosclerosis.
P325
Inducible Transgene Expression in Adult Murine Vascular Endothelium for
the Analysis of TGF- Signal Transduction In Vivo
Peter I Teng, Laszlo G Komuves, Shouchun Liu, James E Tomlinson, Keith Y Abe, Tony
Wyss-Coray, Thomas Quertermous, James N Topper. COR Therapeutics, South San
Francisco, CA; Gladstone Institute of Neurological Disease- UCSF, San Francisco, CA;
Stanford University, Stanford, CA
TGF- is a pleiotropic growth factor that is essential for embryonic development including
vasculogenesis. In the adult, the actions of this growth factor are thought to be important for
vascular homeostasis; and dysregulation of the actions of TGF- have been implicated in
vascular disease pathogenesis. In the mouse, standard manipulation of TGF- signaling
pathways (via transgenic or knock out strategies) are often complicated by developmental
defects which limit their applications to adult vascular disease models. We have developed a
novel transgenic model utilizing the TIE-2 promoter/enhancer elements coupled with the
“tetracycline-on” system to allow regulated and selective expression of transgenes in the adult
murine vascular endothelium. In response to exogenous doxycycline, these mice demonstrate
selective and inducible endothelial expression of a -galactosidase reporter transgene in all
tissues examined. En face analysis of the aorta and its principle branches (e.g., carotid,
femoral, etc.) indicates that the vast majority of the lumenal endothelial cells express the
transgene and this expression is only seen in the endothelium. We have utilized this system to
modulate TGF- signaling in adult murine vascular endothelium via two independent
strategies, over-expression of inhibitory Smad7 or over-expression of a dominant negative form
of TGF- type II receptor. Preliminary analysis of the TIE2-Smad7 and TIE2-TBRIIdn double
transgenic animals reveals no overt adverse effects after up to 3 months of doxycycline
induction. Smad7 transgene induction is demonstrable by immunoblotting in several highly
vascularized organs. In summary, we have developed a novel approach to the study of TGF-
signaling in the adult murine vascular endothelium. This tool can now be utilized to further
understand the role of TGF- and related growth factors, in the modulation of endothelialdependent
process in vivo such as angiogenesis, inflammation and thrombosis.
P326 WITHDRAWN
Oral Administration of Isomers of Higenamine Improves LPS-Induced
Experimental Disseminated Intravascular Coagulation in the Rat
Ki Churl Chang, Hye Sook Yun-Choi. College of Medicine, Gyeongsang National University,
Jinju, Korea; Natural Product Research Institute, Seoul National University, Seoul, Korea
P327
(RS)-higenamine, an active ingredient of Aconite root, was reported to possess anti-platelet and
anti-thrombotic activities, and to attenuate experimental disseminated intravascular coagulation
(DIC) in rats injected by LPS. In general, pharmacological characteristics between isomers
are not identical. In the present study, therefore, two enantiomers of higenamine were
separately synthesized and their effects on some parameters of DIC and anti-platelet
aggregation activity were compared. (S)-higenamine (S-H, IC50 ; 1.6 M) was observed to be
10 times more potent than (R)-higenamine (R-H, IC50 ;14M) against platelet aggregation (rat)
induced by epinephrine. Both enantiomers showed beneficial effects on the experimental DIC
induced by LPS in rats. When administered orally 1h before starting infusion of LPS (through
tail vein, 4 h), both enantiomers inhibited the decrease in platelet count (417 25.9 and
480 29.1 for 10 mg/Kg and 25 mg/Kg of S-H vs 259 19.5 for LPS group) and fibrinogen
level (115 6.2 mg/dl and 257 4.4 mg/dl for Downloaded 10 mg/Kg and 25from mg/Kg of S-H vs 100
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Poster Presentations a-57
11.4 mg/dl for LPS group). The prolonged activated partial thromboplastin time (25.2 1.07
sec and 19.4 1.41 sec for 10 mg/Kg and 25 mg/Kg of S-H vs 38.9 2.84 sec for LPS group)
and prothrombin time (18.0 0.50 sec and 15.4 0.45 sec for 10 mg/Kg and 25 mg/Kg of
S-H vs 25.1 1.23 sec for LPS group) were shortened. The drastic increase in fibrin and
fibrinogen degradation products were also strongly inhibited (53 3.7 g/ml and 68 25.0
g/ml for 10 mg/Kg and 25 mg/Kg of S-H vs 202 41.0 g/ml for LPS group). R-H also
attenuated the experimental DIC conditions, however the effects were much less potent than
S-H. Furthermore, both enantiomers reduced the production of nitric oxide in vascular smooth
muscle cells activated with LPS/IFN-, which was associated with suppression of inducible
nitric oxide synthase (iNOS) expression. The inhibition of iNOS expression by higenamine was
due to inhibition of NF-kB activation. Taken together, it is concluded that both enantiomers of
higenamine are orally effective, S-isomer is better than R-isomer, in experimental DIC
conditions.
P328
Diabetic Lymphocyte Rheology—Evidence for Increased Cortical Tension
Elizabeth J Bray, Cecile M Perrault, Nicolas R Didier, Roger Tran-Son-tay, C K Ozaki.
University of Florida College of Medicine and Malcolm Randall VAMC, Gainesville, FL;
University of Florida Department of Aerospace Engineering, Mechanics and Engineering
Science, Gainesville, FL
Altered physical properties of white blood cells (WBCs) from diabetic patients have been
associated with vascular complications of diabetes. We used micropipette techniques to
examine WBC rheology in the non-obese diabetic (NOD) mouse model of diabetes mellitus. We
hypothesized that lymphocytes from diabetic mice are “stiffer” (higher cortical tension, T o) than
controls. Methods Lymphocytes were isolated by density gradient separation from diabetic
(NOD, glucosuria positive, n18) and control (B6x129, n11; NOD, glucosuria negative, n7)
mice. Individual lymphocytes were manipulated using a micropipette apparatus. Cellular
activation was assessed visually. Recovery length following expulsion from the micropipette
was used to derive the ratio of cortical tension over viscosity (T o/) for each cell. Projection
length into the micropipette during constant pressure aspiration was plotted against time to
generate a two-phase aspiration curve for each cell. Viscosity values were calculated from the
slopes of these curves. Results Forty-two percent (265 of 624) of diabetic lymphocytes
compared to 31% (298 of 967) of controls were spontaneously activated (p0.0001). T o/
values for diabetic cells were significantly higher than controls (35.4 16.5 10 -6 cm/s,
meanSD vs 25.211.5 10 -6 cm/s, p0.003). First phase viscosities for diabetic lymphocytes
were modestly lower than controls (312.65236.32 Pa●s vs 519.96274.38 Pa●s,
p0.0047). Second phase viscosities were similar (1770.771692.19 Pa●s vs
1987.531904.99 Pa●s, pNS). Conclusions Lymphocytes from diabetic mice tend to
spontaneously activate. Diabetic lymphocytes have a larger recovery ratio (T o/) compared to
controls. Aspiration experiments reveal that lymphocyte viscosity is only modestly lower in
diabetic mice. By mathematical implication, the cortical tension (“stiffness”) of diabetic
lymphocytes is greater than controls. Our results provide strong evidence that rheological
properties of lymphocytes are altered in diabetes. Understanding these differences may point
to novel therapeutic strategies for diabetic vasculopathy.
P329
VISTA and PIP Analyses Reveal Unconventional Regulatory Domains in a
Vascular Smooth Muscle Cell-Restricted, Retinoid-Inducible Integrin
Subunit
Chad M Kitchen, Jiyuan Chen, Jeffrey W Streb, Joseph M Miano. University of Rochester
Medical Center, Rochester, NY
In a subtractive screen for retinoid-inducible genes, we discovered that a relatively understudied
alpha integrin subunit (a8) was induced by retinoids in rat aortic smooth muscle cells (SMC).
Its binding partner, beta 1 integrin, is similarly upregulated. Northern analysis shows that unlike
virtually all other SMC-restricted genes, a8 is expressed robustly in the aorta, but barely
detectable in other SMC-containing tissues. These results suggest a unique mode of
transcription, distinct from other SMC markers, which are dependent on serum response factor
(SRF)-binding CArG elements. In an effort to begin understanding the nature of such restricted
gene expression, we have chosen to take a bioinformatic approach. We used PIP (Percent
Identity Plot, http://bio.cse.psu.edu) and VISTA (VISualization Tools for Alignments, http://wwwgsd.lbl.gov/vista)
to solve the a8 gene structure, to assign regions of interspecies homology,
and to direct wet lab experimentation in order to assess the function of conserved regulatory
domains in and around the a8 locus. The assembled mouse a8 cDNA (5,831 bp) comprises 30
exons spread over 180 kb of genomic sequence. The human a8 cDNA is slightly larger in the
untranslated regions and is contained within 200 kb of genomic sequence. The start site of
transcription was determined by RACE and RNAse protection assays, which closely matched in
silico predictions. Comparative sequence analysis found no conserved CArG elements between
mouse and human a8. Moreover, no consensus retinoic acid response elements were identified
around the start site of transcription. The proximal a8 promoter exhibits minimal activity in
PAC1 SMC and is unresponsive to retinoids. Importantly, VISTA reveals two highly conserved
regulatory domains located 4 kb upstream (80% over 400 bp) and 8 kb downstream (93% over
170 bp) of the start site of a8 transcription. These results demonstrate the power of in silico
assays to rapidly solve complex gene structures, define promoter regions, and pinpoint remote,
highly conserved by guest sequences on April in order 4, to 2013 accelerate wet-lab efforts.

a-58 Arterioscler Thromb Vasc Biol. Vol 22, No 5
P330
The Effect of Individual Antioxidant Vitamins in Suppressing the HDL2-C
Response to Lipid Therapy
Andrew C Lee, Josh Morse, Marian Cheung, Xue-Qiao Zhao, Alan Chait, John J Albers, B
Greg Brown. University of Washington School of Medicine, Seattle, WA
Background: Antioxidant (AOx) vitamins E, C, -carotene and selenium in combination have
recently been found to blunt the substantial rise in HDL2-cholesterol (HDL2-C) seen in patients
given simvastatin plus niacin (SN) therapy. In this study, we attempt to identify the individual
antioxidant(s) responsible for this effect. Methods and Results: After completing a 3-year
angiographic trial, 44 patients with coronary disease, low HDL-C (31 mg/dl), and low HDL2-C
(3.9 mg/dl) were treated for 3 additional months with simvastatin 10 mg plus niacin 2.5 gm per
day and randomized to either vitamins EC (n7), -carotene (n12), vitamin A (n7),
selenium (n7), AOx placebo (n5), or the full AOx combination (n6) in dosages identical
to those of the original trial. Samples were obtained at 3 months on-therapy and 2 months
post-therapy. The original trial revealed a mean difference in HDL2-C levels between on- and
off-SN, of 1.88 mg/dl for placebo (n38) and 0.63 for AOx combination (n40; p0.01).
This effect was reproduced, 2.40 vs. 1.17 mg/dl (PNS) in the smaller follow-up study. Those
receiving vitamin EC, -carotene, and vitamin A had comparably blunted HDL2-C responses,
0.86, 1.00, and 0.71 mg/dl (p0.27, 0.26, and 0.28 respectively, vs. placebo). In contrast,
selenium enhanced the HDL2-C response, 3.86 mg/dl (p0.05 vs. placebo, 0.09 vs. vitamin
A, 0.02 vs. -carotene, 0.01 vs. vitamin EC, and 0.001 vs AOx combination). Conclusions:
While this analysis is underpowered to reach compelling conclusions, these results suggest that
no single component of this AOx combination is the culprit, but rather the blunting is a
generalized antioxidant effect. We conclude that treatment with any of the AOx vitamins E, C,
-carotene, or A in the face of SN therapy may be counterproductive. The AOx vitamin may
be down-regulating the expression of genes for the ABCA1 or apoA-I proteins, or up-regulating
cholesterol ester transfer protein (CETP) activity, each of which are important in HDL-C and
particularly HDL2-C metabolism. Surprisingly, selenium enhances the effect of lipid therapy on
HDL2-C, differing significantly from the other AOx vitamins in this regard.
P331
Oxidative Stress Induces the De-Phosphorylation of the Retinoblastoma
Family of Proteins in Endothelial Cells
Fabio Martelli, Lucia Cicchillitti, Pasquale Fasanaro, Giulio Pompilio, Maurizio C Capogrossi.
Istituto Dermopatico dell’Immacolata-IRCCS, Rome, Italy; Centro Cardiologico
Monzino-IRCCS, Milan, Italy
Background: Oxidative stress has been shown to induce cell death and growth arrest of
Endothelial Cells (EC). The retinoblastoma family of proteins is constituted by pRb, p107, and
p130, collectively named pocket proteins. They are the substrate of Cyclin Dependent Kinases
(CDKs) and, in their hypo-phosphorylated forms, bind to selected cellular proteins, playing a key
role in cell growth and apoptosis control in response to certain stress stimuli. We investigated
whether pocket proteins phosphorylation is modulated by oxidant stress. Methods and Results:
Upon exposure of Human Umbelical Vein EC (HUVEC) to H 2O 2 (200 M), pRb, p107 and p130
accumulated in their under-phosphorylated form in less than 30 minutes. Similar results were
obtained in Bovine Aorta EC (BAEC). The following observations helped to characterize the
molecular mechanisms regulating this event: 1. Pocket proteins hypo-phosphorylation preceded
both the down modulation of Cyclins or CDKs and the induction of p53 and p21 waf1 ,an
inhibitor of cyclin/CDKs. 2. Treatment of HUVEC either with 500 nM okadaic acid or with 50 nM
caliculinA, two inhibitors of phosphatases, prevented pocket proteins hypo-phosphorylation.
Moreover, in an in vitro de-phosphorylation assay, pRb accumulated in its underphosphorylated
form, only in cell extracts derived from H 2O 2-treated HUVEC and this event was
blocked by phosphatase inhibitors. These results show that oxidative stress induces a
phosphatase activity targeting pRb. 3. In HUVEC expressing SV40 small t, that binds and inhibits
PP2A phosphatase, H2O2 treatment failed to induce pocket protein de-phosphorylation,
suggesting that the activity of PP2A is necessary for pocket proteins hypo-phosphorylation. 4.
BAEC expressing E1A of adenovirus, that binds and inactivates pRb, p107 and p130, were more
sensitive to H 2O 2-induced cell death, suggesting that pocket proteins play an anti-apoptotic role
in EC response to oxidative stress. Conclusions: Exposure of EC to oxidative stress induced
rapid de-phosphorylation of pocket proteins. That event may be part of a cell survival
mechanism.
P332
Identification of Angiogenic Inhibitors that also Disrupt Mature Endothelial
Capillary Networks
Chumpon Wilasrusmee, Monica S Da Silva, Igor R Yusupov, Bhupender Sing, Michael Sobel,
Dilip S Kittur. SUNY Upstate Medical University, Syracuse, NY; VA Puget Sound HCS, Seattle,
WA
Introduction: Several therapeutic strategies prevent or halt the progression of angiogenesis but
few destroy newly formed capillary networks. Agents that disrupt new blood vessels may be
valuable in the treatment of conditions with established pathological neovasculature, such as
cancer, hyperproliferative retinopathies and rheumatoid arthritis. We report on agents identified
in vitro as having the potential to destroy newly formed blood vessels. Methods: We studied the
effects of potential anti-angiogenic agents in two models of angiogenesis. First, we used an in
vitro model in which human aortic endothelial cells formed capillary tubes on Matrigel; we
studied the effect of cyclosporine A (CyA), heparin, and an antibody against 1 integrins, on
two parameters of endothelial dysfunction: the inhibition of tube formation and the disruption
of mature capillary tubes. All experiments were performed in triplicate and repeated twice. The
most potent of these agents, CyA, was also studied for its capacity to inhibit in vivo
angiogenesis by a Matrigel plug assay. Results: The following agents completely inhibited in
vitro capillary tube formation: CyA (2ng-20g/ml), unfractionated heparin sulfate ( 20g/ml),
and an antibody against 1 integrins (P5D2). Without Downloaded affecting cell from
viability by trypan blue
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assay, high dose cyclosporine A ( 10 g/ml) and the blocking antibody against 1 integrins
(P5D2) were the only agents that disrupted mature capillary tubes. CyA (10 g/ml) also
completely inhibited VEGF and FGF-induced in vivo angiogenesis in the Matrigel plug assay.
Conclusion: Our results demonstrate a novel yet practical approach to the evaluation of
anti-angiogenic agents in vitro. Moreover, we have identified two angiogenic inhibitors that also
destroy mature capillary networks in vitro, high dose cyclosporine A and a 1 integrin blocking
antibody. Agents that disrupt mature capillary tubes have a therapeutic potential to regress
tumor-associated blood vessels and neovasculature of other angiogenesis-related diseases.
P333
Plaque Inflammation in Atherosclerotic Rabbits Can Be Identified by SPIO:
Introducing a Noninvasive Method for Imaging Macrophage Infiltration in
Active or Inflamed Vulnerable Plaque
Maziar Azadpour, Silvio Litovsky, Samuel W Casscells III, James T Willerson, Morteza
Naghavi. Division of Cardiology, University of Texas-Houston Health Science Center and
Texas Heart Institute, Houston, TX
SPIO (super para magnetic iron oxide) nanoparticles are magnetic resonance imaging contrast
agents with a central core of iron oxide coated by polysaccharides which are currently used for
MRI detection of metastatic cancer. These particles are avidly engulfed by circulating
monocytes and tissue macrophages. SPIO creates significant darkening in MR images mainly
due to its T-2 shortening effect. Accumulation of SPIO loaded macrophages in inflammatory foci
enables MR imaging of inflammation . Previously we have shown accumulation of SPIO loaded
macrophages in aortic plaques of Apo E deficient mice. Here we report similar finding in
atherosclerotic plaques of hereditary hypercholestrolemic Watanabe rabbits. Methods: Three
Watanabe rabbits (36 months) were injected IV 1mmol/kg SPIO. The animals were sacrificed
at days 5 and 10 days after injection. Multiple samples from aortic root to abdominal aorta, as
well as heart, lung, kidneys, liver and spleen were taken and stained with H&E, Perl’s and RAM
11. SPIO particles were abundantly found in macrophages in RES liver, spleen and lungs. In the
aorta, the presence of SPIO was mostly restricted to macrophages and foam cells in the
sub-endothelial areas of the atherosclerotic plaques suggesting only newly recruited macrophages.
No difference in the distribution pattern was detected between days 5 and 10. There
was a high correlation between iron-containing cells in Perl’s staining and cell density in H&E
(r0.956). Areas of plaque hemorrhage were minimal in these animals. The cap overlying the
macrophages was always thin. Iron-laden monocytes were also seen in the lumen, suggesting
a relatively easy transport across the vessel endothelium. Conclusion: 1) The SPIO nanoparticles
offer a unique and physiologic opportunity to trace macrophage infiltration into
atherosclerotic plaque. 2) SPIO can be imaged non-invasively by MRI enabling detection of
plaque inflammation, a well-known feature of vulnerable plaques. 3) Since SPIO is clinically
available, human clinical trials are feasible and greatly warranted.
P334
Competitors of Scavenger Receptors Stimulate Macropinocytosis in Pigeon
Macrophages
Nancy L Jones, Marie A Plyler. Wake Forest University School of Medicine, Winston-Salem,
NC
Macropinocytosis, a fluid uptake pathway, occurs constitutively, but is dramatically upregulated
in cultured macrophages by growth factors (ex. macrophage colony stimulating
factor, M-CSF) and phorbol esters. Recently, modified LDLs, AcLDL and OxLDL were shown to
stimulate macropinocytosis and be internalized in part via macropinosomes by cultured pigeon
monocyte-derived macrophages (Jones and Willingham, Anatomical Record, 255:57–68). We
hypothesized that binding of ligands to the scavenger receptors is sufficient to stimulate the
membrane ruffling and macropinosome formation. We tested non-lipoprotein scavenger
receptor ligands for the ability to stimulate membrane ruffling and formation of macropinosomes.
Macropinosomes are classified as phase-bright, fluid-filled organelles forming primarily
at the base of membrane ruffles (diameters 0.2 -5.0 um). Macropinosomes can also be
detected using fluid phase indicators by either light microscopy (dextran-fluorescein 10,000
MW) or electron microscopy (dextran-biotin, followed with avidin-HRP). AcLDL was used as a
positive control for stimulating macropinocytosis and LDL was used as a negative control.
Polyinosinic acid (1.5 ug/ml), dextran sulfate (3 ug/ml) but not fucoidan (10 ug/ml) stimulated
macropinosome formation by 20 minutes. Dextran sulfate, but not fucoidan stimulated
peripheral membrane ruffling and macropinosome formation as detected by video time-lapse
phase microscopy. Dextran sulfate stimulated more numerous macropinosomes of intermediate
size (0.2–0.5 um), while AcLDL stimulated larger macropinosomes( 0.5 um). This
indicates that ligand interaction with the scavenger receptor is sufficient to stimulate
macropinocytosis in pigeon macrophage.
P335
Any Amount of Hyperglycemia Is Predictive of the Presence of Coronary
Atherosclerosis
Stefan Aczel, Werner Benzer, Peter Langer, Christoph Saely, Zeynep Vetter, Heinz Drexel.
VIVIT-Institute, LKH Feldkirch, Feldkirch, Austria
Background:Although diabetes is a strong risk factor for coronary atherosclerosis, the precise
atherogenic role of hyperglycemia is at debate. Materials and Methods:We therefore looked at
the coronary angiograms of 751 consecutive patients (512 men and 239 women) referred to
coronary angiography for the evaluation of chest pain. From angiograms, distinct stenotic
lesions as well as diffuse coronary sclerosis were recorded; in the absence of either the
angiogram was considered normal. With respect to the glycemic status, 4 patient categories
were built: established diabetes (n128), fasting plasma glucose (FPG) 125 mg/dl (n60),
HbA1c 6.1% and FPG 126 mg/dl (n99); and 464 patients fulfilled none of the 3 criteria
and were, by thus, guest considered on April to be normoglycemic. 4, 2013 Results: Hence, 38% of patients referred for

angiography proved hyperglycemic by at least one of the criteria. The rate of normal coronary
angiograms was significantly (p0.024) higher in normoglycemic than in hyperglycemic
patients (22% vs. 16 %). However, among the three hyperglycemic subgroups the rate was
comparable (16 % vs. 15 % vs. 16%). In a logistic model, glycemic status (normoglycemia vs.
hyperglycemia) was significantly (p0.04) predictive of the presence of coronary atherosclerosis.
Upon inclusion into the model of age, gender, alcohol intake, cigarette smoking,
low-density lipoprotein cholesterol, and hypertension, glycemic status remained a significant
predictor of the presence of coronary atherosclerosis. Conclusions: This large coronary
angiographic study thus allows three important conclusions: 1) About two out of five patients
referred to coronary angiography are hyperglycemic. 2) Hyperglycemia is significantly
associated with the presence of coronary atherosclerosis and this holds true for slight
elevations of fasting glucose or HbA1c as much as for established diabetes. 3) Upon inclusion
of traditional risk factors into a logistic model, glycemic status remains independently predictive
of coronary atherosclerosis. From our angiographic approach, the new ADA criteria for diabetes
thus are useful in the setting of diabetic macroangiopathy.
HepG2 Cells Are Capable of Utilizing apo B-100 but not apo B-48 to
Produce Very Low-Density Lipoproteins (VLDL)
Zhouji Chen, Robin L Fitzgerald, Xiaobo Lin. Washington University School of Medicine, St.
Louis, MO
P336
The HepG2 cell has been widely used to study the intracellular degradation and secretion of
human apo B-100 but it is considered to be of limited usefulness for studying VLDL assembly
and secretion because it is generally considered incapable of producing VLDL. However, oleic
acid (OA)-dependent VLDL production by HepG2 cells is evident in the literature. Heparinreleasable
lipase activities have also been shown in these cells. To examine whether the
capability of HepG2 cells for VLDL production is masked by the re-uptake of triglycerides from
the nascent VLDL, we used the receptor-associated protein (RAP), apo CIII, and heparin to block
VLDL re-uptake/modeling. In experiments where RAP and apo CIII were used, the cells were
pre-incubated with these two inhibitors for 30 min. In the presence of OA (0.5 mM), both apo
CIII and heparin doubled the 3 H-labeled triglyceride secretion in a dose dependent manner after
incubation for 3 h, while RAP had no effect (up to 100 g/ml). All three inhibitors slightly
increased (by 20–30%) the accumulation of the labeled apo B-100 in the media but had no
effect on that of albumin and apo AI. Without OA, 5% of the 35 S-labeled apo B-100 secreted
during a 3-h incubation period floated at VLDL densities (d1.009) regardless of the use of
inhibitors. In the presence of OA (0.5mM), 30% of the secreted apo B-100 was in the VLDL
fractions of control cells but VLDL-apo B-100 increased to 60% in the apo CIII (50 g/ml)- and
heparin (5 mg/ml)-treated cells. RAP had no effect. To determine whether HepG2 cells are also
able to use apo B-48 to assemble VLDL, they were stably transfected with a mouse apobec-1
cDNA. These transfectants produced apo B-100 and apo B-48 at 1:1 ratios. Unexpectedly,
secretion rates of apo B-48 were not affected by OA. Apo B-48 was secreted more efficiently
and faster than apo B-100 in the same cell. While the density distribution of the apo B-100
secreted by these cells was similar to that of the untransfected cells, apo B-48 was found in
the HDL fractions regardless of the availability of OA and/or inhibitors. Thus, HepG2 cells utilize
apo B-100 but not apo B-48 to assemble VLDL.
P337
C-Type Natriuretic Peptide Suppresses Plasminogen Activator Inhibitor-1 in
Rabbit Carotid Arteries with Early Atherosclerosis
Robyn Woods, Evette Kairuz, Melissa Barber, Colin Anderson. Howard Florey Institute,
Melbourne, Australia; University of Melbourne, Parkville, Australia
PAI-1 increases in human atherosclerotic blood vessels and this might contribute to decreased
fibrinolysis and progression of atherosclerosis. In vitro, CNP has antiproliferative effects and
inhibits the production of PAI-1 in rat aortic vascular smooth muscle cells and endothelial cells.
Whether CNP can affect PAI-1 in vivo, particularly in the setting of atherosclerosis, has not been
reported. We investigated this possibility using a peri-arterial collar model of early atherosclerosis
in rabbits. The vascular neointima formed with this model shares a number of features
with the early stages of atherosclerosis in humans. Non-occlusive silastic collars were placed
on both carotid arteries of rabbits (n6) under anesthesia. One collar was filled with saline and
the contralateral collar was filled and continuously infused with 10M CNP by osmotic
minipump. After 7 days, the animals were euthanased and from each carotid artery, sections
of normal and collared vessels were taken for morphology and localization of PAI-1 by
immunohistochemistry. In sections of normal vessels, the highest density of PAI-1 immunostaining
was in the endothelium although small amounts were present in the media and
adventitia. Relative densities of PAI-1 immunoreactivity were determined using the normal
sections of artery from the saline-collared side as controls (100%, within-animal design). PAI-1
staining was not different between uncollared, control sections from the two sides. In
saline-collared arteries, PAI-1 increased (P0.05) in the endothelium (1196%) but not in the
adventitia or in the media. PAI-1 was present in the neointima of collared arteries. CNP
treatment reduced PAI-1 (P0.01) in the endothelium (803%) and neointima (by 40%) of
the collared arteries to below the levels seen in control arteries. Thus we have shown for the
first time that CNP attenuates PAI-1, in vivo, in the atherosclerotic artery wall providing
evidence to support previous in vitro observations. These findings have implications for the
local CNP system as a potential therapeutic target in atherosclerosis or restenosis.
P338
Matrix GLA Protein and Bone Morphogenetic Protein-2 Regulate Vascular
Cell Differentiation
replacement of vascular smooth muscle cells (SMC) by cartilage undergoing bone formation.
We have demonstrated that MGP modulates differentiation induced by bone morphogenetic
protein-2 (BMP-2), a strong osteoinductive factor also expressed in calcified plaques and in
vascular endothelium. In studies further clarifying the relationship between MGP and BMP-2,
we showed that MGP co-precipitates with BMP-2 and inhibits BMP-receptor binding and
SMAD1-activation when present in 10-fold excess relative to BMP-2. We also showed that
MGP sequesters MGP in extracellular matrix suggesting this to be part of the inhibitory effect
on BMP-2. To quantitate the effect of MGP on BMP-2 activity, we used a 96-well assay for
alkaline phosphatase, an early osteoblastic marker, and marrow stromal cells sensitive to
BMP-2. Unexpectedly, we found that MGP modulates BMP-2 in a dose-dependent manner. Low
levels (1-fold) of MGP relative to BMP-2 results in mild enhancement of BMP-2’s
osteoinductive effect, intermediate levels (1–15-fold) in the strong inhibition described above,
and high levels (15-fold) in pronounced enhancement. To determine whether MGP affects
differentiation in SMC and calcifying vascular cells (CVC), SMC and CVC were treated with
increasing levels of MGP and showed a significant decrease in expression of SMC-markers
(alpha-actin, calponin, and myosin heavy chain). A significant decrease of SMC-markers was
also seen in SMC and CVC in an in vitro model of the vascular wall, where SMC or CVC are
grown opposite to bovine aortic endothelial cells (BAEC), separated by a membrane. This was
combined with a significant increase in CBFA1 (an osteoblast-specific transcription factor) and
mineralization, likely induced by BMP-expression in BAEC. Our results suggest that both MGP
and BMP are important for vascular cell differentiation depending on the cellular milieu.
P339
Bone Marrow Stem Cells Are a Source of Smooth Muscle and Endothelial
Cells in Atherosclerotic Lesions
Stephen B Huebner, Vladimir R Babaev, Tianli Zhu, Youmin Zhang, Sergio Fazio, Macrae F
Linton. Vanderbilt University School of Medicine, Nashville, TN
Smooth muscle cells (SMCs) and endothelial cells (ECs) are two important cell types in the
atherosclerotic lesion, and both are thought to arise from local sources in the artery wall.
Recent evidence suggests that bone marrow stem cells (BMSCs) can differentiate into ECs and
SMCs. Furthermore, BMSC-derived SMCs have been shown to contribute to the lesion of
transplant atherosclerosis. We sought to test the hypothesis that BMSCs can give rise to SMCs
and ECs in atherosclerotic lesions. Chimeric mice were generated by transplantation of bone
marrow from mice expressing enhanced green fluorescent protein (EGFP). Twenty-one
10-week old male low-density lipoprotein receptor deficient mice were lethally irradiated and
transplanted with bone marrow from EGFP mice (EGFP3LDLR -/- ;n12) or from wild-type
mice (WT3LDLR -/- ; n9). Six weeks after transplantation, 50% to 80% of monocytemacrophages,
B-lymphocytes, and T-lymphocytes in recipient mice were found to express
EGFP by flow cytometry. Mice were challenged with a Western diet containing 21% fat and
0.15% cholesterol for 10, 16, or 24 weeks. No difference in plasma cholesterol levels,
triglyceride levels, or lipoprotein distribution was noted between the two groups. As expected
based on previous studies in our laboratory, macrophages in the atherosclerotic lesions
expressed EGFP. Donor-derived SMCs and ECs expressing EGFP were detected in the
atherosclerotic lesions of EGFP3LDLR -/- but not WT3LDLR -/- mice after 10–24 weeks of diet.
These cells were identified by immunofluorescence using antibodies to -actin (SMCs) and von
Willebrand factor (ECs). The contribution of BMSCs to the population of SMCs and ECs in
atherosclerotic lesions was estimated at less than one percent of the total cell number. These
results indicate that engrafted bone marrow stem cells or their progeny can migrate to the site
of atherosclerotic lesion formation, where they are capable of differentiating into SMCs or ECs
and contributing to the process of atherogenesis.
P340
Aspirin Prevents Fatty Streak Formation without Altering CD4 TH Cell
Phenotype
Sally A Huber, Russell P Tracy. University of Vermont, Burlington, VT
Poster Presentations a-59
Aspirin is a potent non-steroidal anti-inflammatory agent and may have anti-atherosclerotic
effects in humans. We have recently shown that CD4 T lymphocyte expression is a powerful
promoter of fatty streak formation in mice under conditions of mild hypercholesterolemia
through production of interferon-gamma (IFN) at the site of the lesion. Therefore, we
investigated whether aspirin modulates fatty lesion development in mildly hypercholesterolemic
mice, and if so, through effects on T cell and IFN responses. C57Bl/6 male mice were
maintained on 20% fat, 1.5% cholesterol diets with or without 460 mg/kg acetylsalicylic
acid(10.58 mg/mouse/day)for 16 weeks. Fatty lesion size was determined by image analysis
in four aortic sections 80 microns apart in each heart. Average lesion size was 1630 /- 482
m 2 in animals not given aspirin (n 14) and 0 /- 0 m 2 in the group with aspirin (n
7; p 0.01) demonstrating complete protection in mice under conditions of mild hyperlipidemia
(cholesterol: no asiprin group, 170 /- 8 mg/dl; aspirin-treated group, 163 /- 7 mg/dl).
Despite protection by aspirin, there were no significant differences in total % CD4 cells in the
blood (23.9 /- 4.7 no aspirin; 22.5 /- 1.5 with aspirin), %CD4 IFN cells (7.6 /- 1.9
no aspirin; 5.6 /- 0.4 with aspirin) or CD4IL4 cells (0.3 /- 0.1, no aspirin; 0.3 “0.1 with
aspirin). We conclude that while aspirin is a potent anti-atherosclerotic agent in mice, it does
not exert this effect through modulation of T cell expression. Other possible targets include
monocyte/macrophage activation and growth factor contribution by activated plat.
Thioredoxin Inhibits ASK1-Induced Apoptosis by Promoting ASK1
Degradation
Yingmei Liu, Wang Min. University of Rochester, Rochester, NY
Amina F Zebboudj, Kristina I Bostrom. UCLA School of Medicine, Los Angeles, CA
Thioredoxin (Trx) is a cellular redox-sensor protein and plays multiple functions in regulation of
Matrix GLA protein (MGP) has been identified as an inhibitor of vascular calcification. Its cell growth, apoptosis and activation. These functions have been largely attributed to its redox
expression is increased in calcified plaques, Downloaded and absence of from
MGP inhttp://atvb.ahajournals.org/ mice results in activity. Itby hasguest been shown on April that 4, Trx, 2013 in a reduced form, binds to and inhibits apoptosis
P341

a-60 Arterioscler Thromb Vasc Biol. Vol 22, No 5
signal-regulating kinase 1 (ASK1). The oxidized form (intramolecular disulfide between C32 and
C35) or redox-inactive form (mutations at catalytic sites C32 and C35) of Trx does not bind to
ASK1. Apoptotic stimuli such as TNF and reactive oxygen species (ROS) activate ASK1 in part
by oxidizing Trx to release Trx from ASK1. In the present study, we show that the single
mutation of Trx at C32 or C35 (Trx-C32S or Trx-C35S) retains binding activity for ASK1. Unlike
Trx-WT, Trx-C32S and Trx-C35S constitutively bind to ASK1 in an ROS- and TNF-resistant
manner. Consistent with constitutive binding activity, Trx-C32S and Trx-C35S (but not Trx-WT)
inhibited ASK1-mediated apoptosis induced by TNF in endothelial cells by inhibiting TNF/ASK1induced
JNK activity, Bcl-2 degradation and caspase 3 activation. To further examine the
mechanism by which Trx inhibits ASK1 activity, we compared the expression level of Trx and
ASK1 in EC to that in tumor cells in which Trx is overexpressed. We found that EC express lower
Trx but higher ASK1 than tumor cells. Further studies indicate that overexpression of Trx or
activation of Trx by selenium in EC induced ASK1 degradation which was blocked by MG32, an
inhibitor of ubiquitin/proteosome pathway. Our data suggest that Trx inhibits ASK1 apoptotic
activity by promoting ASK1 degradation. The cytokine-resistant inhibition of ASK1 by Trx-C32S
and Trx-C35S suggests a novel therapeutic approach to treat proinflammatory and apoptotic
diseases such as atherosclerosis and myocardial infarction.
The Pathobiological Determinants of Atherosclerosis in Youth Study;
Summary, Recent Findings, and Significance
P342
Arthur W Zieske, Gray T Malcom, Jack P Strong. Louisiana State University Health Sciences
Center, New Orleans, LA
The PDAY Study documented the natural history of atherosclerosis and determined the relation
of cardiovascular risk factors (RFs) to atherosclerosis in young subjects. This project continues
to provide fresh insight into atherogenesis. New observations from PDAY Study include the
associations of RFs with intermediate atherosclerotic lesions, the high prevalence of advanced
coronary artery plaques with qualities indicating vulnerability to rupture, the significant effects
of non-lipid RFs on atherosclerosis even in the presence of favorable lipoprotein profiles, the
importance of obesity in atherosclerosis in youth, and that lipoprotein cholesterol levels using
the new Third Report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood
Cholesterol in Adults guidelines affect atherosclerosis in these young subjects. The PDAY Study
is the most comprehensive source of information in the U.S. about how atherosclerosis
progresses. Pathology laboratories in 15 centers collected coronary arteries, aortas, and other
tissues from over 3,000 subjects aged 15 to 34 who died of trauma between 1987 and 1994.
The extent, prevalence, and topography of arterial lesions were evaluated and RFs were
analyzed in a central laboratory. Postmortem RFs included serum lipoproteins, serum
thiocyanate (smoking), glycohemoglobin (diabetes), thickness of panniculus adiposus and body
mass index (obesity), and changes in small renal arteries (hypertension). The PDAY Study
confirmed that atherosclerosis begins in the teens, and showed that atherosclerosis is
influenced by the same RFs that predict clinically manifest coronary heart disease (CHD) in
adults. The results emphasize the need for control of RFs in young persons for long-range
prevention of CHD. The PDAY Archives enables exploration of less established RFs and
evaluation of mechanisms of atherogenesis utilizing cellular and molecular techniques. The
archive and data library is available for use by researchers. The changes in the medical,
scientific, financial, social, and legal environments over the past decade make it impossible to
duplicate this unique resource.
Differences in Body Fat Distribution and Endocrine and Endothelial
Vasodilator Functions in Postmenopausal Women With or Without
Cardiovascular Disease
Yangsoo Jang, Jong Ho Lee, Oh Yoen Kim, Ha Jung Ryu, Seok Min Kang. Cardiovascular
Genome Center, Yonsei University, Seoul, Korea; College of Human Ecology, Yonsei
University, Seoul, Korea
P343
In addition to simple obesity, accumulation of intra-abdominal visceral fat, and abnormal
endocrine and endothelial vasodilator functions have been shown to be effective markers for
risk of cardiovascular disease (CVD). The purpose of this study was to determine the
differences in body fat distribution, endocrine profiles and endothelial vasodilator function in
postmenopausal women with or without CVD. Thirty-five CVD patients had angiographic
evidence with 50% occlusion of one or more major coronary arteries. Eighty healthy
postmenopausal women with similar age, body mass index and serum lipid profile were
selected for the control group. Adipose tissue areas were calculated from computed
tomography scans made at the L1 and L4 vertebrae, mid-thigh and mid-calf. Brachial artery
endothelium-dependent vasodilation was assessed by flow mediated dilation (FMD) and
endothelium-independent vasodilation by 0.6mg sublingual nitroglycerin (TNG). The brachial
artery was imaged using a 10-MHz linear phased array ultrasound transducer and diameters
measured offline using computer software. The visceral fat area was about 25% higher in CVD
patients than control subjects at both the L1 and L4 vertebrae. The total and subcutaneous fat
area at the L1 vertebra was 15–20% higher in CVD patients than control subjects. CVD patients
had lower serum concentrations of insulin-like growth factor I (-21%), and higher levels of free
androgen index (40%), leptin (34%) and insulin (41%) than the control. Compared to
control subjects, CVD patients showed lower mean of FMD at 1 min (7.3 vs 4.6%, p0.05) and
nitroglycerin induced dilation (11.3 vs 7.8%, p0.05). This study suggests that the presence
of CVD in postmenopausal women is associated with greater negative effect on cardiovascular
risk factors, typically associated with visceral fat accumulation, and abnormal endocrine and
brachial endothelial vasodilator functions. Downloaded from
http://atvb.ahajournals.org/
P344
Combined Deficiency of Fatty Acid-Binding Proteins aP2 and mal1 Lowers
Serum Cholesterol and Triglycerides and Reduces Atherosclerosis in
ApoE-Deficient Mice
Jeffrey B Boord, Kazuhisa Maeda, Vladimir R Babaev, Liza Makowski, Youmin Zhang, Z C
Gorgun, Sergio Fazio, Gokhan S Hotamisligil, Macrae F Linton. Vanderbilt University Medical
Center, Nashville, TN; Harvard School of Public Health, Boston, MA
Fatty acid-binding proteins aP2 and mal1 are both expressed in adipocytes and macrophages.
Deficiency of aP2 increases insulin sensitivity in obese but not lean mice, and macrophage aP2
deficiency protects against atherosclerosis independent of its effects on insulin sensitivity. Mal1
expression is significantly increased in aP2 -/- adipocytes but not in aP2 -/- macrophages. We
examined the effects of combined aP2 and mal1 deficiency on lipid metabolism, insulin
sensitivity, and atherosclerosis in apoE -/- mice. Male and female aP2 -/- mal1 -/- apoE -/- (n15
male/13 female) and age-matched apoE -/- (n15 male/12 female) control mice were placed on
chow diet for 20 weeks. Male aP2 -/- mal1 -/- apoE -/- mice had significantly lower fasting serum
cholesterol (32491 vs 39589; mg/dlSD; p0.048) and triglycerides (8824 vs 13040;
mg/dlSD; p0.009) than apoE -/- controls. Female aP2 -/- mal1 -/- apoE -/- mice also had
significantly lower serum cholesterol (23043 vs 32559; mg/dlSD; p0.0002) and
triglycerides (8128 vs 14045; mg/dlSD; p0.0006) than controls. There was a
significant increase in insulin sensitivity by insulin tolerance testing in both male and female
aP2 -/- mal1 -/- apoE -/- mice compared to controls. Atherosclerotic lesion analysis showed a 53%
reduction in the proximal aorta in the aP2 -/- mal1 -/- apoE -/- males (586316638 vs
12516517332; m 2 /sectionSEM; p0.001) and a 37% reduction in the aP2 -/- mal1 -/apoE
-/- females (12778414522 vs 20328423728; m 2 /sectionSEM; p0.01) compared
to controls. There was a 44% reduction in the en face aorta in aP2 -/- mal1 -/- apoE -/- males
(0.1720.03 vs 0.3110.03; % lesion areaSEM; p0.003) and a 37% reduction in
aP2-/-mal1-/-apoE-/- females (0.2590.03 vs 0.4130.06; % lesion areaSEM; p0.03)
compared to controls. In summary, combined deficiency of aP2 and mal1 in lean apoE -/- mice
increases insulin sensitivity and reduces both serum lipid levels and atherosclerosis. Thus,
mal1 deficiency enhances the effect of aP2 deficiency on insulin sensitivity and atherosclerosis.
Aging-Associated Differential Cardiac Microvascular TNF Epitope
Distribution
Dongqing Cai, Jacquelyne Holm, Jorge R Kizer, Jay M Edelberg. Weill Medical College of
Cornell University, New York, NY
P345
Endothelial function is impaired with aging. We hypothesized that characterization of the
senescent changes in cardiac microvascular endothelial phenotype in vivo would provide novel
insight into the molecular mechanisms that may underlie the increased pathogenesis
associated with cardiovascular disease in older individuals. In order to define the changes in
cardiac endothelial cell surface molecules during aging, we performed in vivo bio-panning with
a cyclic octopeptide phage display library (6 amino acid viable region; 10 7 total complexity) in
young adult (3-month-old) and senescent (18-month-old) C57Bl/6 mice. Analysis of over 100
individual clones isolated after three rounds of cardiac phage isolation and enrichment
demonstrated an age-associated difference in epitope profile. Protein blast analysis (FASTA3.0)
demonstrated that majority of the phage insert peptides in the both the young and aging heart
derived isolates were most homologous (E1) to previously defined soluble and membrane
binding proteins. Peptides from the young, but not older, hearts demonstrated homology to
tumor necrosis factor (TNF) alpha complexes (2/101 young clones vs. 0/100 aging clones;
P0.002, computed using the binomial distribution), indicating that cardiac microvascular
cytokine receptor expression may be altered in the aging heart. Immunostaining revealed
similar levels of TNF receptor 1 in the epicardial microvasculature of the 3 and 18-month-old
mice, but markedly diminished levels in the endocardium of older hearts as compared with
younger hearts. These findings suggested that decreases in the multifunctional hemostatic and
angiogenic roles of the TNF alpha pathway may contribute the impairment in senescent cardiac
vascular function.
Proteomic Analysis of HUVEC Proteins Modulated by High-Density
Lipoproteins
P346
Mehul B Dhinoja, Vaksha Patel, Poonam Tripathi, Robin Wait, Markus Lang, Gillian Cockerill,
Mike Dunn. St Bartholomew’s and the Royal London School of Medicine and Dentistry,
London, UK; Institute of Psychiatry, King’s College, University of London, London, UK;
Kennedy Institute of Rheumatology, Imperial College, University of London, London, UK; ZLB
Bioplasma AG, Bern, Switzerland
Background: High-density lipoproteins (HDLs) have an inverse relationship with the risk of
coronary artery disease (CAD). The mechanisms by which HDLs exert their protective effect are
unclear. The earliest observable event in coronary atherosclerosis is leukocyte adhesion to the
endothelium. HDLs have been shown to inhibit the cytokine-induced expression of adhesion
molecules and chemokines, important in the adhesion and transmigration of leukocytes. HDLs
significantly reduce the transcription of the E-selectin and VCAM-1 genes without any effect on
NF-B translocation. HDLs also synergise with interleukin-1 to increase the level of
cyclo-oxygenase-2, increasing the synthesis of prostacyclin, which has both vasodilatory and
anti-thrombogenic properties. Hypothesis: HDLs maintain an anti-atherogenic endothelial
phenotype, by differentially regulating the expression of endothelial cell genes. HDL effects on
the complete HUVEC proteome: Using proteomic analysis, we have demonstrated HDL
modulation of the basal human umbilical vein endothelial cell (HUVEC) proteome and
characterised 100 landmark proteins. We have demonstrated good reproducibility using
HUVECs derived from different cords. HDL effects on the HUVEC nuclear proteome: By
subcellular fractionation, we have successfully enriched for nuclear proteins. 2D-PAGE and
silver staining by of guest theseon proteins April demonstrated 4, 2013fewer
spots of higher intensity, which were either

absent or only faintly visible in the complete HUVEC proteome. This has been confirmed
subsequently by the identification of nuclear landmark proteins. We have demonstrated HDL
modulation of the basal HUVEC nuclear proteome and identified 10 reproducibly modulated
spots. Their molecular weights (MWs) lie in the range 66.0–21.5 kDa; two spots lie in the pH
range 4–7 and the remaining eight lie in the pH range 6 –9. This represents the correct ranges
of pIs and MWs for several transcription proteins. We are currently characterising these
proteins by MALDI-ToF MS. These studies should provide an insight into the mechanisms by
which endogenous HDLs exert atheroprotective effects, and open up new therapeutic avenues
for the management of CAD.
P347
Regulation of Initial Steps of Blood Coagulation by Tissue Factor Pathway
Inhibitor
Mikhail A Panteleev, Veronica I Zarnitsina, Fazoil I Ataullakhanov. National Research Center
for Hematology, Russian Academy of Medical Sciences, Moscow, Russia
Blood coagulation is initiated upon contact of the integral membrane glycoprotein tissue factor
(TF) with plasma. TF is expressed on the membrane of tissue cells, which are normally not in
contact with blood. After vascular damage TF is exposed to plasma and binds to circulating
factor VIIa greatly enhancing its proteolytic activity. The VIIa-TF complex initiates cascade of
enzymatic reactions of coagulation via activation of factors IX and X. The main regulator of the
VIIa-TF complex activity is tissue factor pathway inhibitor (TFPI), which inhibits VIIa-TF activity
towards factors IX and X in a factor Xa-dependent way. Common two-step mechanism of TFPI
action suggests that TFPI binds factor Xa and then the Xa-TFPI complex inhibits VIIa-TF. This
mechanism is based upon observation that free TFPI, unlike Xa-TFPI, cannot bind VIIa-TF.
However, it was shown recently (J. Biol. Chem., 273 (8): 4378–4386, 1998) that factor Xa
activation in the presence of TFPI is inhibited much more efficiently than this two-step
mechanism predicts. We analyzed several mathematical models, which describe different
mechanisms of inhibition of the VIIa-TF complex by TFPI. This analysis led us to proposition of
a new mechanism of TFPI action. Proposed mechanism describes all experimental data
quantitatively. The experiments to test the hypothesis are suggested.
P348
FXI is Essential for Thrombus Formation Following Ferric Chloride-Induced
Injury of the Carotid Artery in the Mouse
Elliot D Rosen, Julie Roahrig, Francis J Castellino. University of Notre Dame, Notre Dame, IN
Thrombus formation in mice with a FVII or FXI deficiency was monitored in vivo following FeCl 3
or laser-induced injury. FVII-tTA mice, in which FVII expression is regulated by the tTA-Tc
genetic switch, have no (0.05% of normal) detectable FVII in plasma when fed a diet
containing doxycycline. Thrombus formation was similar in wild-type, FVII-tTA-, and FXIdeficient
mice following laser induced injury in veins of the ear. Following FeCl 3-induced injury
to the carotid artery, blood flow began to restrict similarly in FVII-tTA and wild type mice
approximately 8 minutes after injury. Although wild type and FVII-tTA mice initiated
thrombogenesis similarly, the ability of FVII-tTA to form occlusive clots was impaired,
suggesting FVII plays a role in the propagation of thrombi. Interestingly, clot initiation was
impaired in FXI-deficient animals. Unlike wild-type or FVII-tTA animals in which there was a
sharp reduction in flow within 10 minutes of injury, blood flow through arteries in FXI-deficient
decreased slowly for 60 minutes following injury, to approximately 50% of pre-injury flow rates.
The results underscore the fact that the mechanisms of thrombus initiation are varied in
different injury models. While FXI is critical for thrombogenesis in the FeCl 3 injury, it does not
appear to be essential in laser-induced injury. The dramatic consequences of FXI deficiency on
thrombus formation in the FeCl 3 model are somewhat surprising since it is generally accepted
that TF-FVII interactions are the physiologically significant initiators of coagulation in vivo. The
results of the above study underscore the caution that the mechanisms of thrombogenesis in
these disparate injury models might be quite different. Better understandings of how clot
formation is initiated, propagated, and regulated in each of the different injury protocols is
essential before conclusions are drawn about global mechanisms (if any) of thrombogenesis in
vivo.
P349 WITHDRAWN
Eicosapentaenoic Acid, but Docosahexaenoic Acid, Decreases Mean
Platelet Volume in Normal Subjects
Yongsoon Park, William S Harris. Saint Lukes’ Lipid and Diabetes Research
Center/University of Missouri-Kansas City, Kansas City, MO
P350
Background: Mean platelet volume (MPV) may be a risk marker for thrombosis since this
parameter is increased in patients at elevated risk for athero-thrombotic diseases. The purpose
of this study was to determine the effects of individual omega-3 fatty acids on MPV and platelet
count (PLT-CT). Method: Healthy subjects (n 33) Downloaded received olive oilfrom placebo for 4 weeks, and
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Poster Presentations a-61
then were randomly assigned to 4gofeither safflower oil, eicosapentaenoic acid (EPA), or
docosahexaenoic acid (DHA) for 4 weeks. At the end of placebo run-in and treatment periods,
MPV (fL; mean SEM) and PLT-CT (10 3 /L blood) were measured in the basal states and after
stimulation with collagen (10g/mL), cold (ice) and heat exposure (37°C) during the fasting
state and again four hours after consuming a high-fat drink (fed state). Results: EPA
supplementation significantly lowered MPV during the fasting (7.6 0.2 vs. 7.3 0.2) and
the fed states (7.6 0.2 vs. 7.4 0.2), and raised basal PLT-CT (192 18 vs. 211 18)
during the fasting state. Safflower oil and DHA supplementation had no significant effect on
MPV and PLT-CT. Collagen and cold increased MPV whereas heat lowered MPV regardless of
treatments (p 0.05). All stimuli decreased PLT-CT. EPA supplementation significantly
increased platelet EPA (0.2 0.1 vs. 3.3 0.4 %) and docosapentaenoic acid (DPA; 2.2
0.3 vs. 2.9 0.3 %) concentrations, but not DHA. DHA treatment significantly increased DHA
(1.4 0.2 vs. 4.1 0.5 %) and DPA (2.0 0.4 vs. 3.0 0.4 %) concentrations, but not EPA.
In conclusions, EPA, but DHA, reduces platelet activation, an early step in platelet aggregation.
Biomarkers of Atherosclerotic Disease in Asymptomatic Individuals
Identified by Calcium Score
Ngoc-Anh Le, Yadon Arad, Patricia Huey, Nora Ngai, Alan D Guerci, W Virgil Brown. Emory
University and Atlanta VAMC, Atlanta, GA; St Francis Hospital, Roslyn, NY
P351
Coronary calcium score (CS) as assessed by electron beam computed tomography (EBCT) has
been reported as a noninvasive assessment of calcification of the arteries. Prevalence of
biomarkers of lipid abnormalities in asymptomatic individuals with high and low CS is
examined. From a cohort of 1460 aymptomatic men and women, frozen plasma from a random
subset of individuals with CS in the 10th percentile (n50) and CS in the 90th percentile
(n100) were analyzed. Basic lipid profile and apolipoprotein levels, AI, B, CIII, E and Lp(a),
were determined. Composition of triglyceride-rich lipoproteins (TRL), isolated by ultracentrifugation
(d 1.020) was also determined. Apolipoprotein levels were done by immunoturbidometric
methods. Preliminary analysis in 24 subjects with low CS and 28 individuals with high
CS is presented. Tw0-sample analysis with equal variance was used. There was no difference
between the 2 groups with respect to CHOL, HDL, LDL, Lp(a), AI, E and CIII. The difference was
primarily in B-containing lipoproteins. Individuals with high CS had higher plasma TG (178 v.
117 mg/dL, p0.04), higher plasma FFA (0.35 v. 0.29 mg/dL, p0.04) and higher plasma B
(108 v. 88 mg/dL, p0.0002). From the composition of TRL, individuals with high CS had lower
CIII/B ratio (0.07 v. 0.1, p0.04). This is explained by a higher concentration of B in TRL (17
v. 14 mg/dL, p0.05). The present data would support the hypothesis that individuals with
high CS have impaired metabolism of B-containing lipoproteins as suggested by the presence
of increased number of TRL particles of abnormal composition.
P352
Framingham Risk Indexes Underestimate Subclinical Atherosclerosis in an
Asymptomatic Brazilian Population
Raul Santos, Romeu Meneghelo, Fabio Nasri, Tania Martinez, Jairo Hidal. Hospital Israelita
Albert Einstein and Heart Institute University of Sao Paulo Medical School, Sao Paulo, Brazil;
Hospital Israelita Albert Einstein, Sao Paulo, Brazil; Heart Institute University of Sao Paulo
Medical School, Sao Paulo, Brazil
Coronary heart disease (CHD) is a leading cause of death in Brazil. However, the prevalence of
this disease is lower in Brazil than in the United States. Framingham risk indexes (FRI) have
been developed in North American populations to identify patients at risk of CHD events. These
indexes, however were not tested in the Brazilian population which presents a different ethnic
composition (Portuguese, Africans, Spanish and Italians mainly). Coronary artery calcification
(CAC) is a marker of atherosclerotic plaque burden and has also been correlated with CHD
events. Objective: Correlation of FRI with CAC in a cohort of asymptomatic Brazilian subjects.
Methods: We measured FRI based on arterial blood pressure, total and HDL cholesterol, age,
sex and smoking status and CAC by electron beam tomography (EBT) in 626 asymptomatic
subjects (age 45 8 years, 10% females). Calcium scores were determined by the Agatston’s
method. Fasting plasma lipids were determined by enzymatic methods. Results: The
percentage of subjects with positive scores ( 0) was 37 %, the Spearman’ s correlation index
between CAC and FRI was 0.42 , p 0.0001 . Conclusion: In our population the FRI
underestimated by guest sub- clinical on April atherosclerosis 4, 2013diagnosed
by EBT.

a-62 Arterioscler Thromb Vasc Biol. Vol 22, No 5
P353
Apoptotic Cells with Oxidation-Specific Epitopes Are Immunogenic and
Proinflammatory
Mi-Kyung Chang, Christoph J Binder, Yury I Miller, Ganesamoorthy Subbanagounder, Judith
A Berliner, Gregg Silverman, Joseph L Witztum. University of California, San Diego, La Jolla,
CA; University of California, Los Angeles, Los Angeles, CA
Oxidation of LDL (OxLDL) generates a variety of oxidatively modified lipids and lipid-protein
adducts that are immunogenic and proinflammatory, which in turn contribute to atherogenesis.
In addition to OxLDL, cells undergoing apoptosis present oxidation-specific epitopes on their
surface membrane that share identity to those of OxLDL. Therefore, we tested the hypothesis
that apoptotic cells with oxidation-specific epitopes would also be immunogenic and
proinflammatory. To determine the immunogenicity, mice were immunized with syngeneic
apoptotic cells bearing oxidation-specific epitopes, and humoral/cellular immune responses
were determined. Mice immunized with apoptotic thymocytes, induced by dexamethasone,
generated high autoantibody titers to a variety of oxidation-specific epitopes of OxLDL. In
contrast, immunization with viable thymocytes, or with cell lysates generated by freeze-thaw
cycles, or PBS did not yield such titers. Mixed splenocyte cultures from mice immunized with
apoptotic cells exhibited an apparently spontaneous release of significant levels of Th1/Th2
cytokines, whereas mice immunized with viable cells released only low levels. Studies revealed
formation of apoptotic cells in splenocyte cultures, providing the apparent antigen stimulus for
primed T-cells obtained from the mice immunized with apoptotic cells. To investigate
proinflammatory properties of apoptotic cells, we performed monocyte adhesion assays.
Preincubation of apoptotic cells induced monocyte adhesion to endothelial cells, which was
abolished by EO6 antibody to oxidized phospholipid (OxPL), demonstrating that OxPL mediated
this effect. To demonstrate the presence of OxPL in apoptotic cells, mass spectrometry of
cellular lipid extracts was performed. In comparison to viable cells, apoptotic cells showed 2–3
fold increases of bioactive OxPLs including POVPC, PEIPC, and PGPC. These results suggest
that, in analogy to OxLDL, apoptotic cells with oxidation-specific epitopes can be both
immunogenic and proinflammatory, which in turn could contribute to autoimmune and
inflammatory responses during atherogenesis.
Coiled-Coil Helices in C-Terminal Domain of Apolipoprotein E Promote
Oligomerization in Lipid-Free State
Vasanthy Narayanaswami. Children’s Hospital Oakland Research Institute, Oakland, CA
P354
Apolipoprotein E (ApoE) is a critical component of several classes of plasma lipoproteins that
play a crucial role in atherosclerosis and other cardiovascular diseases. It is a 34-kDa
exchangeable apolipoprotein that exists predominantly as a tetramer in lipid-free state. ApoE
is composed of a 22 kDa N-terminal (NT) domain (residues 1–191) housing low density
lipoprotein receptor binding sites and a 10 kDa C-terminal domain (residues 216 –299) bearing
lipoprotein binding and self-association sites. The NT domain is comprised of 4 amphipathic
-helices that form a helix bundle. However, the molecular architecture of the CT domain is
not known. Computer-based sequence algorithms identify segments of sequential heptad
repeats in apoE, with residues 225–261 bearing a high propensity to form coiled-coil helices.
In this study, spectroscopic and hydrodynamic analyses were performed to evaluate the
structural organization of recombinant human apoE with respect to the CT domain. Circular
dichroism spectroscopy of apoE in lipid-free state indicates 68% -helical and 26% disordered
structure. The ratio of molar ellipticities at 222 and 208 nm was 0.97, indicating the presence
of a coiled-coil helical configuration in the protein. In the presence of trifluoroethanol, a
co-solvent that disrupts tertiary and quaternary interactions, an increase in -helicity to 94%
and a decrease in the ratio to 0.86 was noted, suggesting disruption of coiled-coil structures.
Hydrodynamic studies indicate that apoE exists predominantly as a tetramer in aqueous
solutions, and, as a monomer/dimer in the presence of trifluoroethanol. Taken together, the
data indicate that in aqueous state, apoE tetramerization is promoted by intermolecular
coiled-coil formation, mediated by the CT domain. It is postulated that this quaternary
interaction facilitates sequestration of the lipoprotein binding surface at helix-helix contact sites
in lipid-free state, which are replaced by helix-lipid contacts upon lipid association. These
findings are an important contribution towards understanding the structural basis of the role of
apoE in lipoprotein metabolism.
P355
Structural Identification of a Novel Family of Oxidized Phospholipids that
Mediate Foam Cell Formation via the Macrophage Scavenger Receptor
CD36
Eugene A Podrez, Eugenia Batyreva, Zhongzhou Shen, Yijun Deng, Mingjiang Sun, Renliang
Zang, Paula J Finton, Maria Febbraio, David P Hajjar, Roy L Silverstein, Robert G Salomon,
Henry F Hoff, Stanley L Hazen. Cleveland Clinic Foundation, Cleveland, OH; Case Western
Reserve University, Cleveland, OH; Cornell University Weill Medical College, New York, NY
The macrophage scavenger receptor CD36 plays an important role in binding and uptake of
oxidized forms of low-density lipoprotein (LDL), foam cell formation and lesion development
during atherosclerosis. The structural basis of CD36 - lipoprotein ligand recognition is
unknown. We identify here a novel class of oxidized choline glycerophospholipids that serve as
specific high affinity ligands for CD36 on macrophages. We further demonstrate their
enrichment in atherosclerotic lesions and their formation during oxidization of LDL by multiple
distinct pathways. The critical structural elements of these endogenous ligands for CD36 were
defined - a phospholipid with a truncated sn-2 acyl group that incorporates a terminal
-hydroxy(or oxo)-,-unsaturated carbonyl (oxPCCD36). CD36 is the major receptor on
macrophages that accounts for the recognition of oxPCCD36. Incorporation of few molecules
of oxPCCD36 per particle (liposome, vesicle or lipoprotein) is shown to confer binding, uptake
and cholesterol accumulation in macrophages from wild type but not CD36 knock-out mice.
Using a panel of truncated forms of the recombinant Downloaded extracellular binding from
domain for CD36, we
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show that the binding site for oxPCCD36 species (and oxLDL) on CD36 is spatially and
functionally distinct from that of other known CD36 ligands (PS, thrombospondin-1 or long
chain fatty acids). Our data suggest that formation of this novel class of atherogenic
phospholipids plays an important role in CD36-mediated recognition of oxidized lipoproteins
and foam cell formation in vivo.
P356
Probucol Prevents Restenosis by Regulating ERK1/2 Activity and Improving
Vascular Remodeling after Angioplasty in Rabbits
Duan-Fang Liao. Nanhua University, Hengyang, China
Probucol has been shown to prevent effectively against restenosis after PTCA. However, its
mechanism remains unclear. In this research, we investigated the correlation between
preventive effects of probucol on restenosis and its improving vascular remodeling by inhibition
of ERK1/2 activation. New Zealand rabbit thoracic aorta atherosclerosis was induced by 3.5F
balloon catheter injury following a 4-week feeding of high cholesterol diet, and Percutaneous
Translumanal Angioplasty (PTA) was performed by using 3.5F balloon catheter. After two weeks
of PTA, the bore and diameter of aorta, intimal elastic lamina(IEL), extral elastic lamina (EEL),
neointima area (NEA), medial area(MA) and NEA/MA ratio were measured The relaxation and
contraction responses of rabbit aortic rings to acetylcholine, serotonin, norepinephrine and
potassium chloride were measured by bioassay. ERK1/2 activity was determined by western
blot using phospho-ERK1/2 antibody, and the expression of MKP-1 and caveolin-1 were
determined by Western Blot using MKP-1 or caveolin-1 antibody, respectively. Results showed
that probucol treatment for 5 weeks significantly improved the restenosis of aorta as shown by
increasing bore, diameter, lumen area, IEL, EEL of restenotic aorta and decreasing NEA,
NEA/MA. In addition, probucol could protect the relaxation responsibility of restenotic vessel to
acetylcholine and decrease the contraction responsibility to serotonin, norepinephrine and
potassium chloride. Interestingly, the western blot analysis showed that probucol markedly
inhibited the ERK1/2 activity by enhancing the expression of caveolin-1 and MKP-1 of
restenotic vessel wall. Furthermore, probucol also was showed to enhance nitric oxide level of
serum and inhibit expression of c-myc and PCNA. Conclution: 1)Probucol prevented restenosis
by improving vascular remodeling after angioplasty. 2)The regulating effect of probucol on
remodeling is related to enhance expression of caveolin-1 and MKP-1, and therefore to inhibit
ERK1/2 activity. This work was supported by the National Major Basic Research Program of
China(G2000056905)
Altered Endothelial-Derived Fibrinolytic and Antithrombotic Factors:
Implications for Increased Thrombotic Events in Smokers
Rajat S Barua, John A Ambrose, Lesley-Jane Eales-Reynolds, Dhanonjoy C Saha. Saint
Vincents Catholic Medical Centers of New York, New York, NY; The School of Biomedical
and Life Sciences, University of Surrey, Surrey, UK
P357
Background: Data regarding the effects of smoking on thrombo-hemostatic molecules (TF &
TFPI) are limited and on fibrinolytic variables (t-PA & PAI-1) are debatable. The current study
investigates the smoking-related, endothelial cell (EC)-specific responses for these molecules
and their relation to nitric oxide (NO) production in vitro. Methods: To elucidate EC-specific
responses, serum from 8 nonsmokers (NS) and 15 smokers (SM) were incubated with confluent
(85%) human umbilical endothelial cells (HUVECs) in 24-well tissue-culture plates for 12
hours. After the incubation, basal (12 hour) NO, t-PA, PAI-1, TF, TFPI production and substance
P (SP)-stimulated (30 min) NO, t-PA, PAI-1 production were determined in the cell culture
supernatant. NO was measured by a chemiluminesence method, serum levels of cotinine and
all the thrombo-fibriolytic variables were determined by ELISA. Results: HUVECs treated with
SM serum showed lower basal (2.30.3 vs 4.91.1 ng/ml, p0.02) and SP-stimulated (
% baseline: 14.84.3% vs 3512%, p0.06) t-PA production compared to NS but had
similar basal and stimulated PAI-1 production (p0.9 and p0.6). Basal t-PA/PAI-1 molar ratio
was significantly reduced in SM compared to NS group (0.020.01 vs 0.040.01, p0.005).
TFPI production was significantly lower in SM compared to NS group (5.70.4 vs 8.21.3
ng/ml, p0.05). TF levels were not different between both groups (p0.5). Both basal
(1.20.2 vs 3.60.4 M, p0.001) and stimulated ( baseline: 0.100.07 vs 1.10.5 M,
p0.02) NO production were significantly reduced in SM compared to NS. Basal TFPI
correlated positively with basal NO production (r0.42, p0.04), and negatively with serum
cotinine level (r -0.6, p0.01). Conclusions: These results indicate that cigarette smoking is
associated with alterations in EC-derived fibrinolytic (t-PA) and anti-thrombotic (TFPI) molecules.
To our knowledge this is the first demonstration that EC TFPI production is affected by
smoking and endogenous NO or degree of smoke exposure may influence TFPI levels in an EC
milieu.
P358
Proteasome Inhibition Limits Smooth Muscle Cell Growth and Neointimal
Formation after Arterial Injury
Kurt G Barringhaus, Richard A Birnbaum, Martin E Matsumura. University of Virginia Health
Sciences Center, Charlottesville, VA
Background: Cell cycle factors are attractive targets for limiting smooth muscle cell (SMC)
growth and vascular lesion formation. The ubiquitin-proteasome degradation pathway is a
critical regulator of many cellular proteins, including many cell cycle regulatory factors, and
evidence supports an important role for this pathway in neoplastic and other proliferative
disorders. Accordingly, we examined the effect of proteasome inhibition on SMC growth and on
neointimal formation following rat carotid arterial injury. Methods: Cultured rat aortic SMC were
treated with the proteasome inhibitor lactacystin (20 M) or vehicle control and assayed for cell
number, BrdU uptake, and p21 protein levels. To determine the effect of proteasome inhibition
in vivo, balloon carotid arterial injury was performed in the rat followed by a surgical dwell with
either lactacystin by guest (40 M) on April or vehicle. 4, 2013 Carotid arteries were harvested 4 days after injury to

assess p21 protein by immunohistochemistry (IHC) and 14 days after injury to assess
histomorphometry and reendothelialization. Results: Proteasome inhibition resulted in a 60%
and 80% decrease in cell number at day 3 (11098 vs. 4406, p.001) and day 5 (14830 vs.
3022, p.001), respectively. Lactacycstin treatment increased p21 as assessed by Western
blot analysis and decreased BrdU uptake (49% vs. 7% positive cells; 86% reduction, p.005).
Local treatment with lactacystin following rat carotid arterial injury increased p21 early after
injury and resulted in a 59% decrease in neointimal area 14 days after injury (.029mm 2 /-.007
vs. 075mm 2 /-.017, p.05). No differences in EEL or media areas were noted between
groups. Uniform PECAM staining along the luminal surface suggesting reendothelialization was
seen in both groups at 14 days. Conclusions: Proteasome inhibition increases p21 and
decreases SMC S-phase entry and SMC growth. Local administration of a proteasome inhibitor
decreases neointimal growth following arterial injury of the rat carotid artery. These data
support the proteasome as a potential target for limiting SMC growth in vascular proliferative
disorders.
P359
Modulation of the Human APJ Receptor mRNA by Mevastatin in Cultured
Endothelial Cells
Tina M Thorne, Nancy Stagliano. Millennium Pharmaceuticals, Cambridge, MA
The widespread use of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors
(statins) has shown benefit in patients with coronary artery disease. The effects of the statins
are not fully explained by their lipid lowering and appear to directly modify gene expression
within the vascular wall via the interruption of non-steroidal isoprenoid synthesis. In this study,
we used transcriptional profiling to identify genes which may be involved in the antiatherogenic
or pro-angiogenic action of the statins. Millennium nylon cDNA gene arrays were
probed with RNA derived from commercially purchased HUVECs (Clonetics) treated with
Mevastatin (SIGMA, 2 or 20uM) or vehicle. Differentiated genes were further validated using
TaqMan Q-PCR (Applied Biosystems). Although many genes were shown to be regulated by
mevastatin in this experiment, the striking induction of the G-protein coupled receptor for
Apelin (APJ) is the focus of this particular study. TaqMan PCR was unable to detect APJ in the
vehicle-treated HUVECs but showed significant induction with mevastatin. In addition to
regulation in HUVECs, follow up Taqman analysis showed human APJ mRNA tissue distribution
in spinal cord hypothalmus hemangioma heart. fetal tissue skeletal muscle and
adipose. Because reports of possible association of this GPCR with angiogenesis, we further
evaluated expression in human hemangiomas and a mouse angiogenic ovarian model by
rodioactive In Situ Hybridization. Specific signal for APJ was detected in endothelial cells of
multiple hemangiomas. Additionally, TaqMan and ISH using the mouse orthologue of APJ
receptor demonstrated expression in mouse ovaries in association with the vascular support of
developing follicles. To our knowledge, this is the first demonstration of regulation of this GPCR
in statin-treated HUVECs and of its presence in human pro-angiogenic tissues. Our data,
coupled with the observations of others support a role of statins in angiogenesis and highlight
the potential involvement of the APJ receptor pathway in this process.
P360
Functional Evidence for the Existence of Cys560-Cys583 Disulfide Bond
Linking EGF-3 with EGF-4 Domains of the 3 Integrin Subunit
Shaoying Chen, Qi-Hong Sun. Blood Research Institute, The Blood Center of Southeastern
Wisconsin and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee,
WI
The 3 integrin subfamily includes v3 and IIb3. The IIb3 ligand-binding function is
rapidly regulated by integrin activation or inside-out signaling, and activation of certain cells
leads to v3-mediated adhesion. We have shown that a naturally-occurring Cys560Arg
mutation within the 3 cysteine-rich domain from a patient results in constitutively active v3
and IIb3 complexes (Liu CY, et al. Blood 96 Suppl 1: pp40a, 2000; Liu CY, et al. Blood 98:
2432, 2001). We hypothesize that substitution with alanine of the cysteine that is paired with
Cys560 will also activate the 3 integrins. Among the cysteines within the 3 cysteine-rich
domain, Cys567 and Cys583 are particularly interesting because they have been proposed to
be paired with Cys560 (Calvete JJ, et al. Biochem. J. 274: 63, 1991; Xiong JP, et al. Science
294: 339, 2001). The purpose of this study was to identify disulfide bond in the cysteine-rich
domain of the 3 subunit that are involved in activation of the 3 integrins. We found that both
Ala583 and Ala567 isoforms of 3 associated with either v orIIb; and were expressed on
CHO and 293 cells. These surface-expressed isoforms of the v3 orIIb3 bound normally
to a number of monoclonal antibodies (mAbs) specific for 3, and the 3 integrin complexes.
The isoforms of Ala5833 integrins also bound better to several conformationally-sensitive 3
specific Ligand-Induced Binding Site (LIBS) mAbs, as did as Arg5603 integrins. More
importantly, Ala5833-transfected cells, not Ala5673, adhered to immobilized fibrinogen (Fg)
with higher affinity than that mediated by wild type (WT) 3 integrins and in a Mnindependent
manner. Indeed, cell adhesion to immobilized Fg mediated by Ala5673 integrins
was lower than that mediated by WT 3 integrins. Since both Arg560 and Ala583 mutations
in 3, but not Ala567, resulted in similar effects on the adhesive properties of the 3 integrins,
we conclude that Cys560 forms a disulfide bond with Cys583, and Cys560-Cys583 disulfide
bond in the cysteine-rich domain of the 3 subunit participates in the conformational changes
associated with receptor activation. Downloaded from
http://atvb.ahajournals.org/
Poster Presentations a-63
P361
The Mammalian Target of Rapamycin (mTOR) is Synthesized by Activated
Polymorphonuclear Leukocytes
Christian C Yost, Stephan Lindemann, Neal D Tolley, Larry W Kraiss, Thomas M McIntyre,
Andrew S Weyrich, Guy A Zimmerman. Human Molecular Biology and Genetics, Salt Lake
City, UT
The mammalian Target of Rapamycin (mTOR) is critical regulator of translational events. It is
inhibited by rapamycin and neutralization of mTOR activity inhibits overall protein synthesis by
approximately 15% in most cell systems. It is generally believed that mTOR is ubiquitously
expressed in all mammalian cells. Here we demonstrate that primary human lymphocytes,
monocytes, and platelets constitutively express mTOR. However, resting polymorphonuclear
leukocytes (PMNs) do not express mTOR as measured by immunocytochemical or western
analysis. PCR analysis and subsequent sequencing of the cDNA demonstrated that PMNs
contain mRNA for mTOR1, the primary mTOR homologue found in most mammalian cells.
Further examination at the protein level revealed that platelet-activating factor (PAF) induces
the transient expression of mTOR protein in PMNs. Trace levels of mTOR are present in resting
cells. PAF-stimulation increases the expression of mTOR within 30 minutes and maximal
expression is observed by 1 hour. mTOR protein levels are negligible 4 hours after
PAF-activation. Pretreatment of the PMNs with actinomycin D, cycloheximide, or puromycin
prevents the accumulation of mTOR protein indicating that mTOR is being synthesized by
activated PMNs. Downstream targets of mTOR, including 4E-BP1 and p70S6 kinase, exhibit
prolonged phosphorylation that is sensitive to rapamycin. This indicates that newly synthesized
mTOR is functional in PMNs. Since we have previously demonstrated that protein synthesis by
PMNs is primarily controlled at the translational level , the rapid synthesis of mTOR may be a
primary mechanism by which these terminally differentiated cells synthesize proteins in a
regulated fashion.
Cytokines Upregulate Endothelial Lipase Expression and Activity in
Endothelial Cells
Weijun Jin, Gwo-Shing Sun, Uli C Broedl, Dawn H Marchadier, Jane M Glick, Daniel J
Rader. University of Pennsylvania, Philadelphia, PA; Lockheed Martin Space Operations,
Moffett Field, CA
P362
Cytokines such as tumor necrosis factor (TNF) and Interleukin 1 (IL-1) mediate a broad
spectrum of inflammatory responses that in turn alter lipid levels and lipoprotein metabolism.
Two members of the triacylglyceride lipase gene family, lipoprotein lipase (LPL) and hepatic
lipase (HL) are downregulated in the inflammatory state. In this study, the regulation of another
member of this gene family, endothelial lipase (EL) by cytokines is investigated in three types
of primary endothelial cells in vitro. Low levels of both phospholipase and triacylglyceride lipase
activities in human umbilical vein endothelial cells were detected under basal conditions, and
treatment with cytokines significantly increased both activities. Using a polyclonal antibody to
EL, we determined that both activities were due to EL. In addition to the increase in lipolytic
activity, cytokine treatment was demonstrated to substantially upregulate EL protein and EL
mRNA in a time and dose-dependent manner. TNF(10ng/ml) caused a 12 fold increase of EL
protein expression, while IL-1(1ng/ml) resulted a 5 fold increase. Both of these showed a
similar pattern of effect on EL mRNA, increasing at 6 hrs and reaching the maximum level at
24 hrs. TNF induced EL expression with little or no additional effect from IL-1. Similar
results were obtained in two additional microvascular endothelial cell types. We examined the
potential role of NFkB in the regulation of EL expression by cytokines. Overexpression of NFkB
subunit P65 or P100 in endothelial cells increased EL protein expression about 1.5 fold.
Inhibition of endothelial NF kB activation using the NFkB inhibitor SN50 decreased basal EL
expression, but not the effect of cytokines. Dominant inhibitory mutant IkBa and pyrrolidinedithiocarbamate
had no effect on basal and cytokine-induced expression of EL. These results
indicate that NFkB proteins are not major regulators of EL expression by cytokines. In summary,
the upregulation of EL by cytokines has implications for the physiologic role of EL in
inflammatory conditions and its potential role in the modulation of lipoprotein metabolism
during inflammatory conditions, including atherosclerosis.
High Pulse Wave Velocity Associated with Male Gender and Old Age
Predicts the Presence of Coronary Artery Stenosis
Ryo Imanishi, Shinji Seto, Genji Toda, Yuji Koide, Katsusuke Yano. Nagasaki University
School of Medicine, Nagasaki, Japan
P363
Pulse wave velocity (PWV) is used as a noninvasive index of arterial stiffness. The clinical
application of arterial stiffness determined by PWV to distinguish between patients with and
those without coronary artery stenosis was indecisive. This study was conducted to evaluate
whether PWV can be used as an indicator to identify the presence of coronary artery stenosis.
Methods: Brachial-ankle PWV was measured in successive 131 patients (79male, 52female
;63.9/-12.3 years old), who underwent coronary angiographic examination from April 2000
to January 2001. Coronary artery stenosis was defined angiographycally as the presence of
75% or more diameter stenosis. Results: Systolic and diastolic blood pressure and heart rate
were similar between patients with coronary artery stenosis (39M,14F) and those without
coronary artery stenosis (40M,38F), although the age was higher in patients with coronary
artery stenosis (68.6/-2.1 vs 60.6/-1.9 years old, p0.01). Brachial-ankle PWV was
significantly higher in male patients with coronary artery stenosis than in male patients without
those (1835/-51 vs 1516/-51 cm/s, p0.001), however, this difference was not observed
in female patients. Logistic regression analysis showed that higher age and higher PWV were
significantly related to the presence of coronary artery stenosis in male patients, but no
relationship was detected in female patients in term of age, PWV, systolic blood pressure,
diastolic blood pressure and heart rate. Furthermore, in male patients, combinations of high
age ( 65by years) guest and high on April PWV (1850cm/s) 4, 2013 produced the positive predictive value of 83.3%

a-64 Arterioscler Thromb Vasc Biol. Vol 22, No 5
for the presence of coronary artery stenosis, on the contrary, combinations of lower age(65
years)and lower PWV(1500cm/s) produced the negative predictive value of 87.0%. Conclusion:
High PWV value when observed in male patients of 65 years or older can be considered
as one of indicators to identify the presence of coronary artery stenosis.
Influenza Infection Exerts Prominent Inflammatory Effects on the
Atherosclerotic Plaques of Aged Apo E-Deficient Mice: A Comparison
between Intranasal and Intravenous Route of Infection
Silvio Litovsky, Philip Wyde, Mohammad Madjid, Adeeba Akhtar, Samuel W Casscells III,
Morteza Naghavi. Center for Vulnerable Plaque Research at the University of Texas-
Houston, and the Texas Heart Institute, Houston, TX; Baylor College of Medicine, Houston,
TX
P364
We have previously reported that influenza vaccination lowers the risk of recurrent MI.
Recently, we showed that influenza infection administered intranasally (the standard route of
infection) promotes inflammation and thrombosis of atherosclerotic plaques in the apo E -/mouse.
Here, we report the effect of influenza A virus administered intravenously in the same
model. Methods: Nine apo E -/-mice over 24 months old were injected with 1 LD50 (lethal dose
50) of influenza virus IV. 24 apo E -/- mice were inoculated intranasally (IN) and 11 non-infected
age-matched apo E -/- served as controls. Animals were sacrificed 3, 5, 10 and 15 days after
inoculation. Results: Twelve IN but no IV mice died before sacrifice. All animals lost weight
suggesting 100% attack rate. The weight lost in the IN and IV groups were 5.2/-0.5 and
6.1/-0.8 grams respectively (pN/S). The virus titer 5 days after infection were 5.3–5.8 log
10/g and 6.8–7.3 log 10/ g of lung tissue for the IV and IN groups respectively (P0.0001).
Intimal cellularity (plaque infiltration) was 104.7/-40.9 in the intranasal group, 76.9/-13.0
in the IV group and 53.4/-9.7 in the non-infected control group (p0.0004). While 10 IN mice
had a prominent subendothelial plaque infiltration composed of a heterogeneous group of cells
mostly macrophages, T lymphocytes and smooth muscle cells, only one IV mouse showed
similar findings. Conclusion: Infection with influenza A via the IV route produced less systemic
sickness comparing to the usual intranasal route, and promoted significantly less inflammatory
response in the atherosclerotic plaque of apo E -/- mice. Since influenza viremia is a rare
phenomenon, the prominent plaque inflammation and its thrombotic complication in intranasally
infected animals suggests the involvement of inflammatory stimuli from a remote infection
(lung) instead of local plaque infection. Our findings help in elucidating the association between
influenza infection and the increased risk of cardiovascular events in the flu season.
LDL Cholesterol Is Significantly Lower in Diabetic than in Nondiabetic
Coronary Patients
Heinz Drexel, Stefan Aczel, Christoph Saely, Guenther Hoefle, Peter Langer. VIVIT-Institute,
LKH Feldkirch, 6800 Feldkirch, Austria
P365
Background: Epidemiologic evidence infers that diabetes mellitus is atherogenic and induces
a typical dyslipidemia with elevated triglycerides and LDL cholesterol (LDL-C) and decreased
HDL cholesterol (HDL-C). However, studies rarely report on patients affected by both, diabetes
and coronary atherosclerosis. Materials and Methods: We therefore performed an angiographic
study of 767 patients referred to coronary angiography. After exclusion of patients with type 1
diabetes and of those taking lipid-lowering drugs, 532 patients were investigated. According to
the glycemic status 4 groups of patients were built: group 1, established diabetes (14 %); group
2, fasting plasma glucose (FPG) 125 mg/dl (8 %); group 3, HbA1c 6.1 % (14 %); and
group 4: non-diabetic patients fulfilling none of the 3 above criteria (65 %). Serum lipids were
determined enzymatically by standard methods, LDL-C was measured directly by Quantolip,
and serum insulin by radioimmunoassay. Results: Table 1 The major determinant for diabetic
dyslipidemia was elevated fasting blood glucose (present in groups 1 and 2 but absent in
groups 3 and 4). Consistent with current knowledge, diabetic dyslipidemia in our coronary
patients was characterized by elevated insulin and triglyceride levels as well as decreased
HDL-C. Most surprisingly however, hyperglycemic coronary patients had significantly decreased
LDL-C levels. After exclusion of patients with normal coronary arteries the differences
remained statistically significant. Conclusions: We conclude that diabetic coronary patients
exhibit low LDL-C in addition to the high triglyceride/low HDL pattern. Although this finding does
not imply that lowering of LDL-C in diabetic patients with coronary atherosclerosis is not
worthwile it may be preferable to also consider the main abnormalities of these patients as
targets of lipid lowering treatment (e.g. hypertriglyceridemia and low HDL).
A Novel Serine Protease Predominantly Expressed in Macrophage
P366
Cailin Chen, Andrew Darrow, Jian-Shen Qi, Michael D’Andrea, Patricia Andrade-Gordon. The
R.W.Johnson Pharmaceutical Research Institute, Spring House, PA
Serine proteases are members of a conserved multigene family that are involved in the
post-translational processing of many polypeptides and play central roles in the regulation of
a wide variety of physiologic processes including coagulation, fibrinolysis, fertilization,
development, malignancy and inflammation. A homology search program has been engaged to
mine novel serine proteases using bioinformatic databases. Downloaded We have from
identified a novel serine
protease (EOS), which is homologous to tryptase and belongs to the S1 trypsin-like serine
protease family. It also maps within a gene cluster at human chromosome 16p13.3. This
protease showed S1 protease activity cleaving its substrates prior to the arginine residue at an
optimum pH of 8.5–9.5. By immunohistochemistry, EOS is highly expressed in spleen, and
moderately in intestine, colon, lung and brain. The positive staining appears to be of a particular
cell type rather than a broad range of different cells within a given tissue. We confirmed this
expression pattern of EOS by performing in situ hybridization using a digoxigenin-labeled cRNA
probe. The results from both immunohistochemistry, and in situ hybridization indicate that EOS
is associated with macrophage. We corroborated this observation by double immunofluorescence
using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker.
Furthermore, we have detected a dramatic increase in immuno-staining with both CD68 and
EOS antibodies in cultured U937 cells treated with PMA, which represent activated macrophages,
compared to un-induced U937 cells. This up-regulation of EOS gene expression is also
reflected by elevated EOS mRNA in the PMA treated U937 cells, as detected by Northern
blotting. Since macrophages play important roles in various pathological conditions such as
wound healing, artherosclerosis and numerous inflammatory diseases, the localization of this
novel serine protease to active macrophages may further help the elucidation of the roles of
this novel protease in modulating these disorders.
Glucose-6-Phosphate Dehydrogenase Protects Endothelial Cells from
Nitrosative Stress
Frederick L Ruberg, Jane A Leopold, Joseph Loscalzo. Whitaker Cardiovascular Institute,
Boston, MA
P367
Vascular endothelial cells (EC) respond to cellular stress by increasing the activity of enzymes
with antioxidant properties. Glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in
the pentose phosphate pathway, is the principle source of intracellular NADPH. NAPDH, in turn,
serves as a reducing equivalent that maintains cellular glutathione (GSH) stores. Nitric oxide
(NOÂ●) is a potent vasodilator that plays a critical role in EC homeostasis; however, elevated
levels of NOÂ● may deplete GSH stores and result in cytotoxicity. We sought to determine
whether G6PD over-expression and subsequent augmentation of intracellular NADPH and GSH
could enhance EC viability when cells were exposed to cytotoxic nitrosative stress. Bovine
aortic endothelial cells (BAEC) were exposed to an exogenous NO donor, sodium nitroprusside
(SNP, 1mM), for 24 hours. Cell viability decreased over time as determined by increased
concentrations of lactate dehydrogenase (LDH) release at 24 hours (458 /- 89 vs. 2098 /-
35 U/L, p0.0001). Cell death occurred by an apoptotic mechanism as demonstrated by
increased concentrations of histone-associated DNA degradation products (enrichment factor
1.0 /- 0.2 vs 3.3 /- 0.8, p0.05 at 24 hours). Over-expression of G6PD was achieved by
trans(in)fection of BAEC with an adenovirus containing G6PD cDNA (AdG6PD). EC infected with
AdG6PD demonstrated increased G6PD protein by Western analysis, elevated G6PD activity
(108.3 /- 7.0 vs 571.2 /- 30.8 U/6 min/mg protein, p0.0001), and elevated NADPH levels
(0.42 /- 0.03 vs 0.65 /- 0.02 mmol/L/mg protein, p0.009) as compared to control cells.
After exposure to SNP (1mM for 24 hours), AdG6PD EC were protected from the cytotoxic
effects of nitrosative stress as compared to control cells (LDH concentration 1600 vs 2800 U/L
at 24 hours). These studies demonstrate that G6PD protects EC from apoptosis mediated cell
death caused by nitrosative stress.
Iron Loading Accelerates Arterial Thrombosis after Vascular Injury
Sharlene M Day, William P Fay. University of Michigan, Ann Arbor, MI
P368
Objectives: Iron is essential for many cellular processes, but can also damage tissues by
catalyzing the formation of reactive oxygen species. Some studies suggest that increased
stores of total body iron are associated with atherosclerosis and myocardial infarction. This
study tested the hypothesis that systemic iron overload increases the thrombotic response to
arterial injury. Methods: C57BL/6 mice (8–10 week old males) were injected intraperitoneally
with iron dextran (15 mg) or saline in divided doses over 6 weeks. The time required to form
an occlusive thrombus after oxidative vascular injury (green laser light/Rose Bengal model) of
the carotid artery was measured. The effects of iron loading on organ iron content, histology,
and hemostatic parameters were also examined. Statistical analysis was performed using
Student’s t-tests. Findings: Mean occlusion times after carotid injury were 20.4 8.5 min. in
iron loaded mice vs. 54.5 35.5 min. in controls (n10 for both, p0.009). Iron loading
significantly increased the serum transferrin saturation (77% vs. 55%, p0.0001), as well as
the tissue iron content of aortas (4-fold vs. controls; p0.002), livers (60-fold vs. controls;
p0.0001), and spleens (6-fold vs. controls; p0.0001) but did not induce detectable
histologic abnormalities. Mean platelet count, hemoglobin, plasma clotting times (PT and aPTT),
and in vitro platelet aggregation did not significantly differ between groups. Iron loading did not
increase extractable carotid artery tissue factor activity. Conclusion: Moderate iron loading,
comparable to levels seen in human iron storage diseases such as hemochromatosis, markedly
enhances the thrombotic response to arterial injury. This pathologic effect could contribute to
the development of complications of atherosclerosis such as myocardial infarction. Since iron
loading had no apparent effects on plasma clotting, platelet reactivity, or vascular wall tissue
factor activity, we hypothesize that iron accelerates thrombus formation by increasing the
extent of tissue damage after vascular injury.
P369
Apolipoprotein E Accumulation in Carotid Arterial Smooth Muscle Tissue
after Endothelial Denudation in Mice
Zachary W Moore, Binghua Zhu, David G Kuhel, David Y Hui. University of Cincinnati,
Cincinnati, OH
This study was designed to characterize the nature of the effect apoE plays in the vascular
http://atvb.ahajournals.org/ response to by endothelial guest on denudation. April 4, Two 2013 strains of mice have been shown in our laboratory to

have differential neointimal response to carotid artery endothelial denudation; the inbred
FVB/NJ strain has pronounced neointima at 14 days after denudation while the inbred
C57BL/6J strain is resistant to neointimal hyperplasia. A previous study in our laboratory
showed that the apoE genotype affects the response to endothelial denudation in mice.
C57BL/6J mice with apoE-null mutation responded with increased neointima compared to the
C57BL/6J wildtype strain. In order to determine the local presence of apoE in the injured artery,
mice from both wildtype strains were injured and then sacrificed 1, 5, 7, 10, 14, and 28 days
later. Both injured and uninjured carotid arteries were frozen-sectioned and immunostained
using -mouse apoE antibodies. Antibody specificity was determined by staining denuded
artery sections from apoE-KO mice. Staining of sectioned arteries from the wildtype C57BL/6J
timecourse showed increased apoE presence in the injured artery peaking at 5 days after
injury, then gradually reducing to just above levels found in the uninjured artery. Denuded
arteries from the wildtype FVB/NJ mice showed high accumulation of apoE at 14 days after
denudation, comparable to the 5-day peak found in C57BL/6J arteries. These data indicate that
apoE accumulates in arterial smooth muscle cells after endothelial denudation and may directly
inhibit smooth muscle cell migration and proliferation.
P370
Vascular Smooth Muscle -Actin is Expressed in HUVECs and Could Be a
Potential Marker for Circulating Endothelial Cells
Xiaomei Lu, Simon V Boudouin, James I Gillespie. University of Newcastle, Newcastle upon
Tyne, UK
Recent observations suggest that both endothelial cells (EC) and endothelial progenitor cells
(EPC) co-exist in adult circulation. Identification of differences EPC and circulating EC has been
hampered by the facts that (i) both EC and EPC express similar endothelial markers including
vWF, PECAM-1, VE-cadherin, vascular endothelial growth factor receptor-2 (KDR) and eNOS, (ii)
hematopoietic cell subsets express markers similar to those of ECs. Endothelial and vascular
smooth cell (VSMC) may be derived from the same vascular progenitor cell (Nature 408, 92–96
2000) and VSMC -actin is recognised to be an early marker of differentiated smooth muscle
cell. We here examined the possibility that VSMC -actin is expressed in endothelial cells and
might be used to distinguish mature EC from EPC. mRNA was extracted from cultured HUVECs
(human umbilical cord vein endothelial cells) and VSMCs, freshly isolated CD34 and CD133
hematopoietic stem cells, putative EPCs and mononuclear cells (MNC). Human heart mRNA
(purchased from Ambion Inc.) and VSMC mRNA was used as negative and positive control.
RT-PCR was carried out using access RT-PCR system (Promega) by VSMC -actin specific
primers. After RT-PCR amplification we found HUVECs expressed VSMC -actin. This was
confirmed by PCR product sequences. Neither putative EPC nor hematopoietic cell showed
VSMC -actin expression. Our results suggest that VSMC -actin expression could be used to
distinguish circulating mature ECs from EPCs and hematopoietic cells.
Elevated Serum Interleukin-12 Levels in Unstable Angina Pectoris
P371
Juliano L Fernandes, James L Orford, Consilia Garcia, Otavio R Coleho, Andrew P Selwyn,
Maria Heloisa S Blotta. Heart Institute (InCor) - University of Sao Paulo Medical School, Sao
Paulo, Brazil; Brigham and Women’s Hospital, Boston, MA; University of Campinas,
Campinas, Brazil
Background: Acute coronary syndromes are associated with evidence of local and systemic
inflammation as evidenced by elevated highly sensitive C-reactive protein (hsCRP), serum
amyloid A (SAA) protein and interleukin-6. The role played by this cytokine and acute phase
proteins in these syndromes is still unclear. Interleukin-12 (IL-12) and interferon-gama have
been shown to inhibit vascular smooth muscle cell proliferation and collagen gene expression,
suggesting a direct role in plaque instability. Objectives: Document elevated serum levels of
IL-12 and interferon-gamma in patients with unstable angina, and to correlate these
differences with hsCRP and SAA. Methods: We measured serum levels of IL-12 and
interferon-gamma in 15 patients with unstable angina and compared the results with 15
patients with stable angina as controls. We also correlated these data with serum levels of
hsCRP and SAA. Results: Mean levels of IL-12 were 57.1 Â 31.0 pg/ml in the stable angina
group, and 94.5 Â 57.5 pg/mL in the unstable angina group (P0.035). Median level of SAA
was higher in the unstable angina group (8.3 pg/mL; range, 0.7 to 69 pg/mL) compared with
the stable angina group (2.7 pg/mL; range, 0.7 to 2.6 pg/mL; P0.001). There was a
significant correlation between IL-12 and SAA (R0.42, P0.025). hsCRP was higher in the
unstable angina group (median, 4.7; range, 0.8 to 15.2 pg/mL) than the stable angina group
(median, 2.6 pg/mL; range, 0.3 to 8.7 pg/mL; P0.021). hsCRP was correlated with SAA
(R0.56, P0.003), but not with IL-12 (R0.27, P0.14). There was no significant difference
in levels of interferon-gamma between the groups. Discussion: This study demonstrates
elevated levels of IL-12 in patients with unstable angina compared with a control group of
stable angina patients. SAA and hsCRP, established markers of inflammation and predictors of
poor outcome in patients with unstable angina, are also elevated. IL-12 is significantly
correlated with SAA. These findings support the hypothesis that IL-12 is a marker of
inflammatory activity in patients with unstable angina, and suggest that this cytokine may play
an important role in the pathophysiology of the vulnerable plaque.
P372
Perlecan Side Chains Suppress PDGF-BB-Induced Proliferation in Vascular
Smooth Muscle Cells: Potential Role of Basic FGF
Oliver Schmidt, Phan-Kiet Tran, Andreas Kalmes, Guenter Daum, Johan Thyberg, Ulf Hedin,
Alexander Clowes. University of Washington, Seattle, WA; Karolinska Institute, Stockholm,
Sweden
Poster Presentations a-65
thought to reside in the carbohydrate side chains of perlecan. To further investigate the
potential role of perlecan heparan sulfates in regulation of SMC activation, we compared the
proliferative response to PDGF-BB of SMCs derived from wild type mice (control SMCs) with
SMCs from transgenic mice expressing a deletion mutant of perlecan that lacks the N-terminal
glycosylation sites (mutant SMCs). Results: When compared to wild type SMCs, mutant cells
respond to platelet-derived growth factor (PDGF)-BB with increased DNA synthesis (3 fold) and
proliferation (2 fold after 4 days). Addition of heparin has no effect on wild type SMCs but
inhibits both PDGF-BB-induced DNA synthesis and proliferation to 40% in mutant cells. When
mutant SMCs were plated onto matrix produced by wild type cells, they respond to PDGF-BB
like wild type cells. Vice versa, when wild type cells were plated onto matrix produced by
mutant cells, they respond to PDGF-BB like mutant cells. In the presence of neutralizing
antibodies against basic fibroblast growth factor (bFGF), PDGF-BB-induced DNA synthesis is
reduced in both cell types. The extent of inhibition, however, is greater in mutant cells so that
both cell types respond equally to PDGF-BB under these conditions. Conclusion: Our data
demonstrate that perlecan side chains suppress the response of murine SMCs to PDGF-BB. We
suggest that release of bFGF is part of the PDGF-BB response and that bFGF is sequestered by
perlecan located in the extracellular matrix.
P373
Identification of Aldehyde Modified Peptide Sequences in Apolipoprotein B
Which Give Rise to Antiatherogenic Immune Responses
Gunilla Nordin Fredrikson, Bo Hedblad, Ingrid Söderberg, Marie Lindholm, Paul Dimayuga,
Göran Berglund, Jan Nilsson. Malmo University Hospital, Lund University, Malmo, Sweden
Low density lipoprotein (LDL) oxidation is believed to play an important role in the development
of atherosclerosis, and oxidized LDL particles have been shown to become targets for the
immune system. Immunization of animals with oxidized LDL results in r eduction of
atherosclerosis suggesting an athero-protective effect of this immune response. Using a library
of polypeptides covering the complete sequence of apolipoprotein B (apo B-100), the only major
protein of LDL, we have identified over hundred dif f erent human antibodies reacting against
aldehyde-modified apo B-100 sequences. IgM antibody titer levels decreased with age and
were associated with severity of cardiovascular disease in subjects younger than 60 years. In
prospective clinical studies an ti body levels against several aldehyde-modified apo B-100 sites
predicted risk for development of coronary heart disease in this group. Immunization with
peptide sequences, against which high levels of IgG or IgM antibodies were found in healthy
human co ntr ols reduced atherosclerosis in apo E null mice by about 60%. Immunizations with
these peptides were also found to affect plaque stability as assessed by an increased collagen
content of subvalvular lesions. We have identified several specific epitopes within the apo
B-100 component of oxidized LDL those provoke an immune response in humans. Furthermore,
certain identified peptide sequences in oxidized LDL induced immune responses, which
inhibited atherosclerosis in mice. This suggests a way of develop ing an immunization therapy
for coronary heart disease.
P374
Effect of Estrogen on Superoxide Production in Oophorectomized Rats
Maria Florian, Sheldon Magder. MUHC Royal Victoria Hospital, Critical Care Division, McGill
University, Montreal, QC, Canada
Estrogen is asscociated with a reduced incidence of cardiovascular disease in women but the
mechanism is unknown. Oxidant stress contributes to atherosclerosis by affecting intracellular
pathways and decreasing bioavailable nitric oxide (NO). A membrane bound cytochrome b 558
enzyme complex which uses NAD(P)H has been shown to be a major vascular source of
superoxide (O 2 - ). Objective: We hypothesized that estrogen treatment (E): a) decreases O2 -
production by NAD(P)H oxidase and thus increases bioavailable NO, b) decreases expression of
components of the NAD(P)H oxidase. Methods: We studied 8-week old oophorectomized (OVX)
Sprague-Dawley rats 14 days after surgery. We implanted pellets with 0.25 mg of
17--estradiol or placebo, which was released over 21 days. After 21 days the aorta was
removed, cut into segments and used for contractility studies, O 2 - measurement by
chemiluminescence, western analysis of components of NAD(P)H oxidase and nitric oxide
synthase (eNOS). Results: Basal O 2 - production of 0.07 0.02 nmol O2 - / min was lower in E
compared to 0.19 0.03 in controls (C) (P0.5). DPI, an inhibitor of NAD(P)H oxidase,
decreased O 2 - production in C to 0.09 0.02 (P0.5) but did not change the already low O2 -
in E. The NOS inhibitor L-NAME decreased O 2 - generation in C from 0.17 0.04 to 0.12
0.03, but not in E (0.08 0.01 and 0.08 0.02 respectively). This is consistent with higher
O 2 - generation in C than E. Isometric tension induced by phenylephrine (PHE) (10 -10 -10 -4 )M
was similar in endothelium intact and denuded aortic tissue of C and E and so were the
acetylcholine relaxation dose-response curves. The addition of SOD after PHE preconstriction
dilated aortic ring by 34 8% in C and by 19 4% in E, consistent with more O 2 - in C. eNOS
mRNA and protein were similar in E and C. NAD(P)H oxidase components p 22phox ,p 47phox ,p 67phox
and gp 91phox were all similar in aorta, heart, lung. Conclusion: Estrogen replacement decreased
O 2 - production in the aorta of OVX rats and appeared to act by altering activation rather than
expression of NAD(P)H oxidase. The change in O 2 - could affect endothelial cell signaling
mechanism.
P375
Can the Lack of Association between Antiphospholipid Antibodies and
Stroke Recurrence Be Explained by Their Failure to Persist?
Stanley Tuhrim, James H Godbold, Martin E Goldman, Deborah R Horowitz, Jesse M
Weinberger, Xiao-Xuan Wu, Jacob H Rand. Mount Sinai School of Medicine, New York, NY
Objectives: Case-control studies have demonstrated an association between ischemic stroke
Objectives: Perlecan is a component of the basal membrane and a major heparan sulfate and anticardiolipin (aCL) and antiphosphatidyl serine (aPS) antibodies. Prospective cohort
proteoglycan of the extracellular matrix. The molecule is growth inhibitory for vascular smooth studies have generally failed to confirm this finding. Similarly, small prospective studies have
muscle cells (SMCs). Given the inhibitory effectsDownloaded of heparan sulfates from
on SMCs, http://atvb.ahajournals.org/
this activity is not found by positive guest antiphospholipid on April 4, antibody 2013(aPL)
titers at time of initial stroke to be predictive

a-66 Arterioscler Thromb Vasc Biol. Vol 22, No 5
of stroke recurrence. We attempted to replicate this finding in a large cohort of ischemic stroke
patients and, finding no significant association, hypothesized that the apparent paradox
between initial and subsequent stroke risk and between prospective and case-control studies
may be due to variability in aPL antibody titers. We examined this by performing follow-up
testing in a subset of the initial cohort. Methods: We obtained IgG and IgM aCL and aPS
antibody titers in 662 patients entered in the Minorities Risk Factors and Stroke Study within
48 hrs of an acute ischemic stroke and at follow-up (24 12 mos) in a subgroup of patients.
Findings: The presence of any aPL at index stroke did not increase the relative risk of
subsequent stroke (p.05, see Table). Of the 201 patients with repeat titers, 50% (100) were
negative for all aPL antibodies tested; 20% (40) had some positive titer initially and at
follow-up; 13% (26) were positive initially but negative subsequently; and 7% (35) were
negative initially but positive on follow-up, for an over all discordance rate of 30% Individual
aPL antibody discordance rates varied from 5.4% for IgM aPS to 26% for IgG aCL. Conclusion:
aPL antibody status at initial stroke does not appear to affect risk of recurrence. aPL antibody
status frequently changes in the years following stroke. This may explain the failure to
demonstrate an association between the presence of antibodies at time of initial stroke and
stroke recurrence. Furthermore, this finding may help to reconcile the largely disparate results
of previous case-control and prospective cohort studies.
P376
The Associations of Serum Lipid Levels with Bone Mineral Density and
Risk of Fracture in Older Women
Paul D Varosy, Steven R Cummings, Douglas C Bauer, Katie L Stone, Warren S Browner.
University of California, San Francisco, San Francisco, CA; California Pacific Medical Center
Research Institute and the University of California, San Francisco Departments of Medicine
and of Epidemiology and Biostatistics, San Francisco, CA
Background Drugs that inhibit hydroxy methyl-glutaryl coenzyme-A (HMG CoA) reductase
(statins) may increase bone mineral density and reduce the risk of fracture, but whether these
effects result from lipid lowering or other properties is unclear. Methods In 228 women older
than 65 years of age randomly selected from the Study of Osteoporotic Fractures cohort, we
determined whether baseline serum levels of total cholesterol, low-density lipoprotein (LDL)
cholesterol, high-density lipoprotein (HDL) cholesterol or triglycerides were associated with
bone mineral density (BMD) of the hip in linear regression models. In case-cohort analyses (101
hip fractures and 100 vertebral fractures), we then used Cox proportional hazard models to
determine whether serum lipoprotein levels predicted risk of hip fracture, and we used logistic
regression to determine whether serum lipoprotein levels predicted the odds of vertebral
fracture. Results We found weak, but statistically significant unadjusted associations of serum
HDL cholesterol (r -0.14, P 0.005) and triglyceride (r 0.15, P 0.05) with BMD of the
hip. After adjusting for age and body mass index, neither serum HDL (-0.004 g/cm 2 of hip BMD
per 10-mg/dl increase in HDL; 95% confidence interval (CI), -0.014 to 0.007; P 0.48) nor
serum triglycerides (0.0005 g/cm 2 of hip BMD per 10-mg/dl increase in triglycerides; 95% CI,
-0.001 to 0.002; P 0.61) was associated with BMD of the hip. In both unadjusted and
adjusted models, serum lipid levels were not associated with hip or vertebral fracture. For
example, after adjusting for age and body mass index, the risk of hip fracture was 1.00 (hazard
ratio, 1.00; 95% CI, 0.94 - 1.06; P 0.94) for every 10 mg/dl increase in LDL cholesterol.
Conclusions We found no independent associations between lipid levels and either bone density
or fractures. This suggests that the lipid-lowering properties of statins are unlikely to explain
their reported beneficial effects on bone.
P377
Experiment of Nature: Expansive Remodeling Is a Local Response in the
Aortic Bulb of the ApoE-Deficient Mouse
Jacob Bentzon, Gerard Pasterkamp, Erling Falk. Skejby Hospital, Aarhus C, Denmark;
Experimental Cardiology Laboratory, University Medical Center, Utrecht, Netherlands
Atherosclerotic plaque formation is often compensated for by expansive remodeling, thereby
preventing luminal narrowing. The mechanisms underlying this important phenomenon are
unknown, mainly due to the lack of relevant animal models. It may be caused by a homeostatic
response of normal adjacent vessel wall, or digestion of the media beneath atherosclerotic
plaques by proteolytic enzymes, or both. In the present study we 1) validate the apo-E deficient
(apoE-/- ) mouse aortic bulb as a model to study expansive remodeling and 2) examine whether
remodeling is a response of the media underlying plaques or of the adjacent vessel wall in this
model. Methods. Cross-sections of the distal aortic bulb are divided into left coronary (LC),
noncoronary (NC) and right coronary (RC) segments by the commissures of the aortic cusps.
The LC segment is a predilection site for atherosclerosis while the RC and NC segments are
less involved. This constitutes an experiment of nature in which to assess whether expansive
remodeling occurs beneath plaques or in normal/mildly diseased adjacent vessel wall. ApoE-/ mice were terminated at 11 weeks (n13), 6 months (n21) and 13 months (n14). Wild type
C57BL/6J mice were used to control for normal growth (n 6, 3 and 11, respectively). Plaque
area and length of the internal elastic lamina (IEL-length) were measured for each segment at
the level of the commissures of the aortic cusps. Results. Expansive remodeling was evident
in the aortic bulb by 6 months of age and showed the effect of overcompensation by 13 months
of age. When each segment was looked at individually, strong correlations were revealed
between plaque area and IEL length for the LC (r20.84, p0.0001) and for the RC (r20.67, p0.0001). Multiple regression analysis revealed that plaque amount in one segment did not
influence IEL length in adjacent segments (for LC, RC an NC, p0.2). Conclusion. The aortic
bulb of the apoE-/- mouse is a model in which expansive remodeling consistently occurs in
response to plaque formation and is therefore suitable to study its mechanism. In this model
expansive remodeling was a local response of the Downloaded media underlying from
plaque.
http://atvb.ahajournals.org/
P378
Stimulation of Macrophage CXCR2 with KC/Gro-Alpha Promotes MRP14
Expression
Ejaz Ahmed, Linda K Curtiss, William A Boisvert. The Scripps Research Institute, La Jolla,
CA
OBJECTIVE: Macrophage entry into the vessel wall is a key event in the pathogenesis of
atherosclerosis. We have shown previously that the chemokine receptor CXCR2 is strongly
expressed on lesion macrophages and that its expression on macrophages is important for the
accumulation of these cells in lesions. Moreover, leukocyte-specific deficiency of CXCR2 led to
significantly reduced atherosclerosis in the LDLR-/- mice. However, the mechanism by which
CXCR2 affects macrophage biology in atherogenesis has been elusive. The objective of this
study was to determine using the latest gene chip technology the genes that are differentially
regulated upon stimulation of mouse macrophage CXCR2 with its ligand KC/Gro-alpha.
METHODS: Mouse macrophages were cultured from the bone marrow of C57Bl/6 wild type
mice and one batch was stimulated with KC/Gro-alpha and the other was not. The RNA from
these cells were analyzed with Affymetrix gene (murine genome U74Av2) which can detect
approximately 13,000 genes, and the results were analyzed using the Affymetrix GeneChip
algorithm. RESULTS: Amongst the genes that were upregulated by stimulation with KC/Groalpha
the most significantly upregulated gene (13 fold) was macrophage related protein,
MRP14, which is known to be important in adhesion and migration of macrophages among
other roles. To confirm these results, macrophages from bone marrow of CXCR2-/- mice were
cultured and stimulated with KC/Gro-alpha and the RNA was analyzed exactly as above. When
these results were compared with above results from wild type mouse macrophages also
stimulated with KC/Gro-alpha, there was a 14 fold decrease in the expression of MRP14 in the
macrophages from the CXCR2-/- mice. CONCLUSION: These results strongly suggest that
engagement of CXCR2 with its ligand KC/Gro-alpha has downstream effects that may alter the
behavior of macrophages that may in turn influence their participation in atherogenesis. Of
those, the upregulation of MRP14 may indeed play an important role in the participation of
macrophages in the atherosclerotic process.
P379
Hypercholesterolemia Promotes Cuff-Induced Vascular Remodeling Lesions
Associated with Bone Marrow-Derived Cells in Mice
Yang Xu, Masayuki Yokode, Hiroshi Kataoka, Toshinori Murayama, Xin Zhuge, Toru Kita.
Kyoto University, Kyoto, Japan
Background: We have recently reported that bone marrow (BM)-derived cells are involved in
vascular remodeling process initialized by polyethylene cuff placement in mice and that the
progenitor of smooth muscle cell (SMC) and macrophage could have opposing roles in the
lesion formation (Xu et al. Circulation 104 (17): II-299). In the current study, we examined the
effect of hypercholesterolemia on cuff-induced vascular remodeling lesion formation. Methods:
[1] Double-transgenic mice which lack apolipoprotein (apo) E gene and express ubiquitously
green fluorescent protein (GFP) transgene were obtained by crossbreeding apoE-deficient mice
with the GFP-expressing transgenic mice. [2] Either apoE-deficient mice (810 weeks of age)
fed high fat diet containing 0.3% (w/w) cholesterol or control C57/Bl6 mice fed normal chow
were subjected to 9 Gy irradiation and received the BM cells from the double-transgenic mice.
Four weeks later, a nonconstrictive cuff was placed around the right femoral artery of each
mouse. After another 2 weeks, both right and left femoral arteries were harvested and
subjected to histochemical analysis. Results: [1] As compared with C57/Bl6 mice, the
apoE-deficient mice showed a markedly larger number of GFP-positive BM-derived cells
clustering in the cuff-planted artery. In the recipient apoE-deficient mice, the majority of the
GFP-positive BM-derived cells were stained by Oil red O. [2] In accordance with our previous
observation, the distribution manner of the cells recognized by anti-macrophage antibody BM8
were distinct from that of the cells detected with anti-SMC alpha actin antibody. [2] No
GFP-positive cells were observed in the sham-operated left artery in either apoE-deficient or
control mice. Conclusion: These results indicate that hypercholesterolemia promotes accumulation
of BM-derived macrophage and SMC thereby accelerating vascular remodeling lesion
formation.
P380
Hematein Inhibits Atherosclerosis by Inhibition of NF-kB-Dependent Gene
Expression and Reactive Oxygen Generation
Goo Taeg Oh, Tae Sook Jeong, Jae Hoon Choi, Dae Yong Kim, Young Myeong Kim. Korea
Research Institute of Bioscience and Biotechnology, Taejon, Korea; College of Veterinary
Medicine, Seoul National University, Suwon, Korea; College of Medicine, Kangwon National
University, Chucheon, Korea
Hematein, a natural flavonoid, is known as an analgesic and anti-inflammatory agent. Here we
investigated the effects of this compound on atherosclerosis in low density lipoprotein receptor
deficient (Ldlr-/-) fed high cholesterol diet alone or in combination with hematein for 8 weeks.
Mice fed high cholesterol diet alone develop the fatty streak lesion in the aortic sinus, whereas
this lesion was significantly reduced by hematein treatment. Infiltrated macrophages, iNOS
protein, tumor necrotic factor-alpha, interleukin-6, interleukin-1b and nitrotyrosine expression
were localized in the atherosclerotic lesion. Hematein treatment reduced serum levels of nitrite
and nitrate, TNF-a, IL-1b, and lipid peroxide, but not changed the plasma cholesterol level,
compared with the levels of control mice. In addition, hematein inhibited NF-kB activation and
the activity of transient tranfected NF-kB and iNOS promoters in peritoneal macrophages from
Ldlr-/- mice. This compound also suppressed NO production and iNOS expression in
LPSINFg-stimulated peritoneal macrophages from Ldlr-/- mice. Hematein inhibited the
production of NF-kB-dependent cytokines TNF-a, IL-1b, IL-6 in LPSINFg-stimulated peritoneal
macrophages. Hematein also inhibited superoxide generation in LPS-stimulated peritoneal
macrophages. by guest These results on April suggest 4, that 2013 hematein inhibited atheroscleotic lesion formation,

possibly by inhibiting NO production, lipid peroxidation, and pro-inflammatory cytokine
expression through the inhibition of NF-kB activation and reactive oxygen production.
P381
Oxidized Phospholipid-Induced Activation of Endothelial 1 Integrins Is
Mediated by an R-Ras/PI3-Kinase Pathway
Amy L Cole, Srirupa Mukhopadhyaya, Devendra K Vora. University of California at Los
Angeles, Los Angeles, CA
Monocyte recruitment to the sub-endothelium may be important for the initiation and
progression of atherosclerosis. We have previously reported that oxidized 1-palmitoyl-2arachidonyl-sn-glycero-3-phosphorylcholine
(OxPAPC) and its component 1-palmitoyl-2-(5oxyvaleroyl)-sn-glycero-3-phosphorylcholine
(POVPC), induce monocyte-specific binding to
human aortic endothelial cells (HAEC) by increasing levels of cAMP and by activating apical
surface 1 integrins in HAEC. We also demonstrated activated 1 integrins in human
atherosclerotic lesions. To further elucidate the signaling pathway involved in OxPAPC-induced
activation of 1 integrins, we treated HAEC with OxPAPC for varying periods and assessed for
activated Ras-like G-proteins by using the Ras-binding domain of Raf to affinity-purify
GTP-bound (activated) Ras-like molecules. We found that OxPAPC activated endothelial R-Ras,
but not H-Ras, by 3–4 fold. Since PI3-kinase (PI3K) is a downstream mediator of R-Ras action,
we treated HAEC with the PI3K inhibitor LY-294002 and then exposed them to OxPAPC. We
found that OxPAPC-induced 1 integrin activation and monocyte binding to HAEC were
significantly reduced by LY-294002. In addition, treatment of HAEC with pertussis toxin (Gi
inhibitor demonstrated to elevate intracellular cAMP levels) activated R-Ras. Furthermore,
expression of a constitutively active R-Ras construct was able to mimic some effects of OxPAPC
in HeLa cells. Taken together, these data suggest that elevated cAMP levels and the R-Ras/PI3K
signal transduction pathway are important mediators of OxPAPC action that may contribute to
1 integrin activation and atherosclerosis.
Identification of a Feed-Forward Mechanism of NF-B Signaling in
Vascular Endothelial Cells Using Transcriptional Profiling
Xixi Yin, Paul Krikorian, Tom Logan. Millennium Pharmaceuticals, Cambridge, MA
P382
Inflammatory cytokines play a key role in the vascular inflammation that is associated with
atherosclerosis. Recently it has been demonstrated that activators of the nuclear receptor
PPARalpha inhibit the effect of inflammatory cytokines by interfering with the activation of
NF-kB. To better understand the mechanisms mediating this inhibition of cytokine signaling,
transcriptional profiling was utilized. This approach allows for the simultaneous analysis of
thousands of genes in a biologically relevant model. In this study, transcriptional profiling was
used to monitor the effect of PPARalpha agonist pre-treatment on the cytokine response in
vascular endothelial cells. Human aortic endothelial cells were pre-treated with vehicle or the
PPARalpha selective agonist WY-14643 (125 M) for 24 hrs., followed by treatment with
TNFalpha (10 ng/ml) for 5, 12 and 24 hrs. Cluster analysis was used to identify specific gene
expression patterns at individual time points. Expression of the pro-atherosclerostic chemokine
MCP-1 was shown to be up-regulated at each time point following cytokine stimulation.
However, cytokine induction of MCP-1 gene expression was significantly repressed when cells
were pre-treated with WY-14643. RIP2 is a member of a family of caspase recruitment domain
(CARD) proteins that have been implicated in mediating inflammatory signaling via activation
of NF-kB. Cluster analysis identified RIP2 as exhibiting a similar pattern to that of MCP-1. In
addition to induction of RIP2 mRNA, Western blot analysis shows that TNFalpha also
up-regulates RIP2 protein levels in vascular endothelial cells. Gel shift analysis indicates that
WY-14643 mediated repression of RIP2 induction correlates temporally with decreased NF-kB
DNA binding activity. Taken together, this data indicates that RIP2 is up-regulated by TNFalpha
via NF-kB. Because RIP2 has previously been shown to activate NF-kB through interaction with
IKKgamma, these observations represent a novel, transcriptionally mediated feed-forward
mechanism of NF-kB signaling.
P383
Elevated Plasma Interleukine-6 Levels Are Associated with an Increased
Risk of Ischemic Heart Disease in Men
Benoit Lamarche, Annie C St-Pierre, Bernard Cantin, Jean Bergeron, Gilles R Dagenais,
Jean-Pierre Despres. Nutraceuticals and Functional Foods Institute, Ste-Foy, QC, Canada;
Quebec Heart Institute, Ste-Foy, QC, Canada; Lipid Research Center, Ste-Foy, QC, Canada
There is increasing evidence to suggest that plasma C-reactive protein (CRP), a non-specific
acute phase reactant whose secretion is induced by interleukine (IL)-6, predicts the risk of
ischemic heart disease (IHD) in healthy individuals. However, whether elevated plasma IL-6
levels also predict an increased risk of IHD remains to be clearly established. The purpose of
the present study was to investigate the relationship between plasma IL-6 levels and incident
IHD in a sample of 2008 men (mean age (SD) 56 7 yrs) from the Quebec Cardiovascular
Study. All men were initially free of IHD at baseline and were followed for a period of 5 years,
during which 95 first IHD events were recorded. Plasma IL-6 levels were measured in the entire
cohort of men on baseline samples using a commercially available ELISA. Plasma IL-6 levels
were significantly (P0.01) but weakly correlated with age (r0.24), body mass index
(r0.08), smoking (median IL-6 levels 1.20 mg/l in cigarette smokers vs. 0.89 mg/l in others)
and with plasma triglyceride (TG) (r0.08) and HDL cholesterol concentrations (r-0.13).
There was also a strong positive association between plasma CRP and IL-6 levels (r0.54,
P0.001). Plasma IL-6 concentrations were virtually identical between incident IHD cases and
non cases (median levels 1.0 mg/l vs. 0.94 mg/l respectively, P0.70). However, there was
a significant interaction between plasma IL-6 levels and age at baseline in modulating IHD risk.
Indeed, increased plasma IL-6 levels (0.94 mg/L, the median in the cohort) predicted an
increased risk of IHD in young men (age55 yrs, RR2.4, P0.01) but not in older men
(age55 yrs, RR1.3, P0.5). When considering Downloaded the interactionfrom between IL-6 and age in
http://atvb.ahajournals.org/
Poster Presentations a-67
multivariate modeling of IHD risk, elevated plasma IL-6 levels remained a strong and
independent risk factor (RR7.6 in men with elevated vs. low IL-6 levels, P0.007) after
adjustment for non lipid and lipid risk variables as well as for plasma CRP concentrations.
Results from this large, population-based cohort of men provide strong evidence that elevated
plasma IL-6 levels may be associated with an increased risk of IHD, particularly in young men.
P384
Safer NSAID Therapy: A Review of the Literature on Platelet Effect from
Human Randomized Controlled Trials of Celecoxib, Rofecoxib and
Meloxicam
Alan D Bell, Simon Huang, John F Chiu, Lydia Hatcher, Algis V Jovaisas, Sheilah Lamb,
Jean-Marc Noiseux, Proton Rahman, Kevin Saunders, David Morgan. University of Toronto,
Toronto, ON, Canada; University of British Columbia, Vancouver, BC, Canada; University of
Alberta, Edmonton, AL, Canada; Memorial University of Newfoundland, St. John’s, NF,
Canada; University of Ottawa, Ottawa, ON, Canada; McMaster University, Hamilton, ON,
Canada; Centre Hospitalier Pierre-Boucher, Longueuil, PQ, Canada; Wellness Institute of
Seven Oaks General Hospital, Winnipeg, MB, Canada
BACKGROUND Inhibition of platelet function is well-documented with traditional non-steroidal
anti-inflammatory drugs (NSAIDs), a phenomenon not likely to occur with agents that spare
COX-1. This premise was evaluated by reviewing the literature on platelet effects in human
randomized controlled trials of apparent COX-1 sparing NSAIDs. METHODS A MEDLINE
literature search for human trials of meloxicam, celecoxib and rofecoxib was conducted.
Studies of platelet function were selected based on the following criteria: randomized controlled
trial (RCT) or meta-analysis, human ex vivo, at least 10 human subjects and at least 5 days’
duration. Papers were evaluated individually and consensus reached on the criteria following
in Table 1. Criteria for each drug for complete lack of platelet effect was at least one leve l“A”
paper and no level “X”, “Y” or “Z” papers; for modest platelet sparing was more than one “B”
level studies, AND no “X” or “Y” level papers; for significant platelet effect was no level “A”
papers and at least one level “X” or more than one “Y” level studies. RESULTS A total of 465
abstracts were obtained from which 73 papers were reviewed, eight of which related to platelet
effects. Table 2 summarizes the findings of the evaluation. Celecoxib and rofecoxib fulfilled the
pre-specified criteria for platelet sparing. Meloxicam fit the criteria for modest effect on
platelets due mainly to inhibition of thromboxane B production. CONCLUSION In contrast to the
coxibs, meloxicam demonstrated no convincing platelet-sparing effect. This has implications
for GI safety, surgical and non-surgical bleeding and cardiovascular issues.
P385
1-Adrenoceptor (AR)-Mediated Vascular Wall Growth: Systemic 1A-AR
Antagonist Inhibits Restenosis of Rat Carotid
Cauveh Erami, John C Teeters, Hua Zhang, James E Faber. University of North Carolina,
Chapel Hill, NC
In previous cell and organ culture and chronic local perivascular drug-delivery in vivo studies
(see Zhang and Faber, Circ Res 89(8), 2001) we have shown that: 1) norepinephrine (NE) has
direct trophic effects on smooth muscle cells and adventitial fibroblasts of rat aorta and carotid;
2) the trophic effect of endogenous NE is strongly augmented after balloon injury to contribute
significantly to neointimal and adventitial hypertrophy, lumen loss, and inward remodeling; 3)
antagonist studies suggest these effects are mediated by 1A- & 1B- but not 1D-, 2D/A
or -ARs that are also expressed in both media and adventitia. Herein, our purpose was to test
the feasibility of chronic intravenous 1A-AR antagonist KMD-3213 for inhibition of adrenergic
growth. In an initial anesthetized-rat experiment, “selective” low (L) & 2.5 fold higher (H) doses
were identified: L and H had no effect on MAP and baseline renal (RVR; 1A-dominant bed) and
hindquarters resistances (HVR; 1A- & 1D-dominant bed). KMD caused dose-dependent
decreases in the iv phenylephrine dose-response curve for MAP (10 –30% inhibition) and RVR
& HVR (greater inhibition of RVR than HVR). In a 2nd experiment, rats received carotid balloon
injury, an osmotic pump for iv KMD delivery, and an arterial line for a 2-week study. L&H had
no effect on mean, systolic or diastolic pressures, heart rate or body weight of conscious
unrestrained rats. L&H reduced neointima area by 30 & 46% (p0.01), respectively, and
adventitia area by 14 & 19% (p0.05). These data are consistent with our hypothesis that
overzealous trophic activity of catecholamines is counterproductive and contributes to
hypertrophic by vascular guest disease. on April 4, 2013

a-68 Arterioscler Thromb Vasc Biol. Vol 22, No 5
P386
Macrophage-Expressed Group V Secretory Phospholipase A2 Modifies LDL
and HDL Particles and is Present in Human and Mouse Atherosclerotic
Lesions
Clavia R Wooton-Kee, Munira Nasser, Willem J De Villiers, Frederick C De Beer, Nancy R
Webb. University of Kentucky, Lexington, KY
Secretory phospholipase A2 (sPLA2) enzymes catalyze the hydrolysis of the sn-2 fatty acyl ester
bond of phospholipids to produce a free fatty acid and a lysophospholipid. Several lines of
evidence suggest that sPLA2s may play a role in atherogenesis by modifying lipoprotein
particles. Studies in vitro indicate that LDL modified by sPLA2 is more susceptible to
aggregation, which may lead to enhanced lipid accumulation in the arterial wall. In addition,
hydrolysis of HDL phospholipids by Group IIA sPLA2 leads to accelerated HDL catabolism in
vivo. Recently, a new member of the sPLA2 family, Group V sPLA2, has been identified in
macrophage cell lines. In the present study, we show by immunohistochemical staining that
Group V sPLA2 is detectable in macrophages in both human and mouse atherosclerotic lesions.
To assess Group V sPLA2 activity towards LDL particles, the enzyme was over-expressed in
COS-7 cells by adenoviral vector-mediated gene transfer. Twenty-four hours after adenovirus
treatment, cells were incubated an additional 24 hours with media containing 0.2 mg/ml LDL
(0.4 M). The extent of LDL hydrolysis was assessed by measuring the free fatty acid (FFA)
content of media. This analysis showed that 85 molecules of FFA were generated per particle
of LDL added to Group V sPLA2-expressing cells. Analysis by non-denaturing gel electrophoresis
showed that LDLs incubated with Group V sPLA2-expressing cells migrated as smaller
lipoprotein particles compared to LDLs incubated with control cells. Over-expression of Group
V sPLA2 by adenoviral vector in C57BL/6 mice resulted in a significant reduction in plasma
HDL-cholesterol (17.9 /- 7.0 mg/dl) compared to mice injected with a control adenoviral
vector (61.7 /- 5.2 mg/dl). Taken together, our data indicates that macrophage-expressed
Group V sPLA2 may play a role in atherosclerotic lesion development by modifying LDL particles
in the arterial wall and by altering HDL metabolism.
P387 WITHDRAWN
Reducing Oxidative Damage to Endothelium Obtained from Brain and
Aorta: The Protective Action of the Polyanion Dextran Sulfate
Scott N Schneider, Tilly Ping, Linda Hiebert. University of Saskatchewan, Saskatoon, SK,
Canada
P388
It is widely accepted that endothelial cell dysfunction due to oxidative stress is implicated in the
pathogenesis of numerous vascular diseases. The objective of the present study was to assess
the protective effects of dextran sulfate (molecular weight 8000) in reducing oxidative damage
in cultured endothelial cells obtained from the brain and aorta of two different species (bovine
and porcine). Using hydrogen peroxide (H 2O 2) as a free radical generator, the concentrations
that resulted in an approximate 50% reduction in percent viable cells after a 24 hour exposure
were used. Dextran sulfate was added in concentrations of 0.05 mg/mL, 0.50 mg/mL, 5.0
mg/mL and 50 mg/mL. After a 24 hour exposure, media was changed and replaced with fresh
media, dextran sulfate and H 2O 2. Percent viable cells and lactate dehydrogenase (LDH) release
were assessed following a 24 hour exposure to H 2O 2. Bovine aortic endothelial cells and bovine
brain capillary endothelial cells both required an H 2O 2 concentration of approximately 7.0 mM
to reduce percent viable cells to 50% of control. All concentrations of dextran sulfate examined
offered no significant protection to either bovine cell type. Porcine aortic endothelial cells and
porcine brain capillary endothelial cells required H 2O 2 concentrations of approximately 0.25 mM
and 1.6 mM respectively to reduce percent viable cells to 50% of control. Dextran sulfate at
concentrations of 0.5 mg/mL and 5.0 mg/mL offered significant protection (increased percent
viable cells and reduced LDH release) to both porcine cell types (P0.05, one-way ANOVA).
The lack of protection offered by dextran sulfate to the cells obtained from the bovine species
may be attributed to their higher resistance to oxidative stress and the subsequently higher
concentration of H 2O 2 used. The protection offered to the cells obtained from the aorta and
brain of the porcine species suggests a role for dextran sulfate and possibly other polyanionic
compounds in reducing oxidative damage to endothelial cells. Supported by the Heart & Stroke
Foundation of Saskatchewan.
P389
Acidosis Does Not Directly Stimulate Prostacyclin Release from Venous
Endothelial Cells
Jaehwa Choi, Jerry L Mamaril, Carmen R Overstreet, Robert L Hester. University of
Mississippi Medical Center, Jackson, MS
Local blood flow in a peripheral tissue is regulated by the metabolic demand of that tissue.
During an increase in metabolic activity, arteriolar dilation serves to meet the demand for
additional blood supply in the tissue. Previous studies in our laboratory have indicated that
arachidonic acid metabolites from the venous endothelium play an important role in the dilation
of adjacent arterioles during muscle stimulation. In this study, we investigated the role of
acidosis on prostacyclin release from veins to determine whether exercise-induced acidosis
contributes, directly or indirectly, to the synthesis of vasodilatory substances that diffuse to
adjacent arteries and lead to arteriolar dilation. Small branches of femoral veins were isolated
from male golden hamsters, cannulated and perfused with MOPS-buffered physiological salt
solution at 37oC. Prostacyclin synthesis was determined by enzyme immunoassay of the
metabolite 6-keto-prostaglandin F1a. Veins were perfused with solutions of different pH to
assess the direct role of acidosis on prostacyclin synthesis; perfusion at pH 7.2 (n8) or 6.9
(n6) did not significantly change the basal level of prostacyclin synthesis compared to pH 7.4
(P0.05, repeated measures ANOVA on ranks). We also tested whether ATP-induced
prostacyclin synthesis is modulated by acidosis. Downloaded Recent studies from
have suggested that ATP
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released from erythrocytes during muscle stimulation mediates arteriolar dilation. The EC 50 for
ATP-induced prostacyclin synthesis was 268 mM (meanS.E., n6), 186 mM(n7), and
133 mM(n6) at pH 7.4, 7.2, and 6.9, respectively (P0.05, one-way ANOVA). These
findings suggest that acidosis by itself neither stimulates prostacyclin synthesis in veins nor
increases ATP-induced prostacyclin release. It is possible that acidosis might, instead, regulate
the release of ATP from erythrocytes.
P390
Thrombogenic Properties of the Endothelium of Apolipoprotein-E Deficient
Mice
Marie-Pierre Fournet-Bourguignon, Christine Breugnot, Ana Fortuno, Delphine Saboureau,
Edwige Brule, Nicole Villeneuve, Paul Vanhoutte, Jean-Paul Vilaine. Institut de Recherches
Servier, Suresnes, France
Endothelium-derived NO plays an important role in the regulation of activation of platelets
whereas hypercholesterolemia impairs NO release and enhances the adhesive properties of the
endothelium and platelets. Apolipoprotein E deficient (ApoE KO) mice, a model of atherosclerosis,
early express adhesive proteins like ICAM-1 and VCAM-1 in the neointima and the
smooth muscle cells underlying the lesions indicating an important inflammatory process.
Surprisingly, vascular reactivity studies showed that the markers of the bioavailability of NO
(relaxation to acetylcholine and cyclic GMP level) in the thoracic aorta were not different in ApoE
KO in comparison with control mice. Under inflammatory conditions, the endothelial cells may
develop thrombogenic properties which could be modulated by the production of NO. This
pathological phenomenon was compared in control (C57Bl/6J) and ApoE KO mice, using a
sensitive method quantifying platelet adhesion to the aortic endothelium . Washed platelets
labeled with 111Indium were administered in the caudal vein of conscious mice. Platelets from
control mice were injected because similar aggregations in whole blood induced by ADP and
collagen were observed with platelets from control and ApoE KO mice. Platelet associated
radioactivity in the total aorta was measured after 30 minutes of circulation in the blood.
Platelet adhesion was increased significantly by 21 % and 69 % in 20 and 35 weeks old ApoE
KO in comparison with control mice. A chronic treatment with N-nitro-L-arginine(an inhibitor of
NOS activity) did not increase these responses in ApoE KO mice. In conclusion, in the
inflammatory context of atherogenesis, the preserved NO pathway, resulting in a normal
endothelium-dependent relaxation, is not able to limit the platelet-adhesion to the endothelium
of blood vessels of the ApoE KO mice.
Glycoprotein IIb/IIIa Inhibitors Increase the Number of Fibrinogen
Receptors on Platelets
Ramin Artang, Gitte W Jurgensen, Neils J Frandsen, Jorn D Nielsen. Gentofte University
Hospital, Hellerup, Denmark; Hilleroed Hospital, Hilleroed, Denmark
P391
Objectives: Glycoprotein (GP)IIb/IIIa receptor blockade reduces the risk of thromboembolic
episodes during percutaneous coronary interventions. Currently three intravenous agents are
available: abciximab, eptifibatide and tirofiban. An upregulation of GPIIb/IIIa receptors during
treatment with abciximab has been reported both in vivo and in vitro. The aim of this study was
to investigate whether eptifibatide and tirofiban have the same effect on platelets. Methods:
After obtaining informed consent, blood from 6 healthy male volunteers (mean age /- SD: 42
/- 8 yrs.) was drawn. Citrated whole blood was incubated with abciximab in concentrations
0, 1, 2, 3 and 4 g/ml, epifibatide in concentrations 0, 0.1, 0.5, 1 g/ml and tirofiban in
concentrations 0, 10, 20, 50 and 100 ng/ml, based on previous pharmacokinetic studies of
patients receiving recommended doses of each drug. For evaluation of platelet inhibition and
number of GPIIb/IIIa receptors, platelets were labeled with FITC-conjugated antibodies against
fibrinogen and CD41 (clone SZ22, non-fibrinogen ligand of GPIIb/IIIa receptor), and analyzed by
flowcytometry. Mean fluorescence intensities at baseline and at mentioned concentrations
were compared using Friedmann Repeated Measures ANOVA test. Findings: Inhibition of
platelet fibrinogen binding of 80% was achieved with 2 g/ml abciximab, 0.5 g/ml
eptifibatide and 50 ng/ml tirofiban. At these concentrations, the mean number of GPIIb/IIIa
receptors increased 37 /- 13% (mean /- SD, p0.01) for abciximab, 10 /- 3.4% (ns) for
eptifibatide, and 36 /- 10% for tirofiban (p0.05) compared to baseline. Conclusion: While
all three GPIIb/IIIa agents inhibit platelet fibrinogen binding effectively in the recommended
doses, they have the capability of increasing the number of fibrinogen receptors on platelets
in vitro. We have demonstrated for the first time an increase in the number of fibrinogen
receptors induced by tirofiban. An increase in the number of receptors may be one of the
mechanisms of post-procedural coronary adverse events despite GPIIb/IIIa inhibition. Clinical
relevance of this phenomenon requires further investigation.
Role of Adenosine and Nitric Oxide on the Mechanisms of Actions of
Dipyridamole
P392
Robert Abraham, Terry Ketch, Ginnie Farley, Italo Biaggioni. Vanderbilt University, Nashville,
TN
Dipyridamole (Aggrenox) has recently been shown to be more effective than aspirin alone in the
prevention of secondary stroke. Dipyridamole may act by inhibiting adenosine clearance via
blockade of the nucleoside transporter, thus potentiating its action. It is also an inhibitor of
cGMP-specific phosphodiesterases (PDE5) and, through this mechanism, could potentiate
cGMP-mediated actions of nitric oxide. To define the mechanism of action of dipyridamole, we
studied the local vascular effects of adenosine, acethylcholine (NO-mediated dilation) and
nitroprusside (cGMP-mediated dilation) at baseline, and two hours after treatment with
Aggrenox (250 mg dipyridamole/25 mg aspirin PO) in seven normal volunteers. Drugs were
administered into the brachial artery and forearm blood flow (FBF) was measured by venous
occlusion plethysmography. Adenosine 31.25 ug/min increased FBF from 11.21.5 to 16.82
(52% increase) by guest beforeon Aggrenox, April and 4, 2013 from 9.31 to 28.23 ml/100ml forearm/min (204%

increase) after Aggrenox (p0.005 by ANOVA). In contrast, Aggrenox did not alter the response
to Acetylcholine or to nitroprusside. In conclusion, the vascular actions of Aggrenox can be
explained by potentiation of adenosine mechanisms but not by potentiation of nitric oxide or
other cGMP-mediated actions.
P393
Upregulation of the Cardiac Serine Protease Corin in a Rat Model of Heart
Failure
Katherine Tran, Wei Yan, Qingping Feng, Qingyu Wu. Berlex Biosciences, Richmond, CA;
Univ. of Western Ontario, London, Canada
Corin is a transmembrane serine protease originally cloned from human heart. Our recent
studies indicate that corin is the proteolytic enzyme responsible for processing pro-atrial
natriuretic peptide (pro-ANP) to mature ANP (Yan et al. PNAS 2000;97:8525). Heart failure is
associated with a marked increase in myocardial ANP production. It is not known if corin is
overexpressed under the pathological conditions and contributes to the increased ANP
production in heart failure. In the present study, rat corin cDNA clones were isolated from a
heart library using a PCR-based strategy with oligonucleotides derived from the human and
mouse corin cDNA sequences. The full-length rat corin cDNA is 4,885 bp in length and encodes
a polypeptide of 1,111 amino acids. Rat corin protein shares 82 and 90% sequence homology
with human and mouse corin, respectively. In Northern analysis, corin mRNA was most
abundant in the heart. To examine the potential role of corin in heart failure, we determined
corin mRNA expression in a rat model of heart failure induced by ligation of the left coronary
artery. Eight weeks after coronary artery ligation, non-infarcted LV myocardium mass and LV
end diastolic pressure were increased while myocardial contractility and cardiac output were
decreased compared to sham controls (n8 per group, P0.01). Corin mRNA expression in
the non-infarcted LV myocardium was 2.8-fold higher than that in sham controls as measured
by Northern blotting (P0.01). The elevated corin mRNA expression in the myocardium was
associated with a 5-fold increase of plasma ANP in the heart failure animals (P0.01). Our
results suggest that up-regulated corin expression increased ANP production in rats with heart
failure.
Non NO, Nonprostanoid Dependent Hyperpolarization in the Aorta of
Control and Apolipoprotein E-Deficient Mice
Ana Fortuno, Catherine Thollon, Nicole Villeneuve, Christine Brunel-Jacquemin, Paul M
Vanhoutte, Jean-Paul Vilaine. Institut de Recherches Servier, Suresnes, France
P394
In 35 weeks old apolipoprotein E deficient (ApoE KO) mice no decrease in endotheliumdependent
relaxation to acetylcholine (ACh) was observed despite severe hypercholesterolemia,
the presence of atherosclerotic lesions and increased NADPH oxidase activity. As different
endothelial factors can be released by ACh the potential contribution of NO, prostacyclin (PGI2)
and endothelium-derived hyperpolarizing factor (EDHF) as vasodilators in control and ApoE KO
mice aorta was analyzed. In the two strains, the cyclooxygenase inhibitor indomethacin did not
modify the relaxation to ACh in phenylephrine constricted aortas and N-nitro-L-arginine (LNA,
an inhibitor of NOS activity), or oxyhemoglobin (a scavenger of NO), abolished it, indicating the
exclusive involvement of NO in the response. Relaxation to ACh was partly inhibited in the
presence of 30 mM KCl indicating that NO could Downloaded hyperpolarize this from
tissue. Iberiotoxin, an
http://atvb.ahajournals.org/
Poster Presentations a-69
inhibitor of BKCa produced a similar inhibition of ACh relaxation as KCl while charybdotoxin (an
inhibitor of IKCa) plus apamin (an inhibitor of SKCa) did not reduce it, suggesting that this
relaxation is not attributable to EDHF. Nevertheless, hyperpolarizations of the smooth muscle
cells, in response to ACh, were observed in the non constricted aorta, in the presence of LNA
and indomethacin and were similar in control and ApoE KO mice (22.5 mV and 24.0 mV
respectively). In conclusion, the mechanism underlying the vasodilator action of ACh in mice
aorta is NO dependent. Nonetheless, a potent hyperpolarization, independent of NO and PGI2
was demonstrated with ACh. Similar results were obtained in control and ApoE KO mice
suggesting that a contribution of EDHF or PGI2 can not explain the preserved endotheliumdependent
relaxation in the hypercholesterolemic mice.
P395
Platelet-Derived Growth Factor-A Expression Is Upregulated in Arterial
Remodeling in Response to Elevation of Blood Pressure
Chengpei Xu, Chang Shu, Sheila Lee, Hirotake Masuda, Christopher K Zarins. Stanford
University, Stanford, CA; Akita University, Akita, Japan
Hypertension induces both cellular and extracellular responses in arterial remodeling.
Platelet-derived growth factor (PDGF)-A has been shown to play a role in a variety of
hypertrophic events. To define time course response of PDGF-A to elevated blood pressure, we
assessed the levels of PDGF-A mRNA and protein in the aortic wall of hypertensive rats.
Hypertension was established using mid-thoracic aortic coarctation in rats. The proximal aorta
to the coarctation was studied using RT-PCR, Western blotting and immunohistochemistry at
time points ranging from 1 day to 8 weeks (n5 each) after coarctation. PDGF-A mRNA levels
were standardized with GAPDH expression. ANOVA and Bonferroni test were used for statistical
analysis. Coarctation resulted in significant elevation of blood pressure in the proximal aorta
with 118/-9 mmHg vs. 94/-6 mmHg in controls (P.002). RT-PCR revealed 3-fold increase
in relative mRNA levels of PDGF-A at 1 week, 2 weeks and 4 weeks (P.001) with
normalization at 8 weeks (Figure). Western blot results indicated 1.5 fold increase in PDGF-A
protein at 1 week, 2 weeks, and 4 weeks (P.03). Both smooth muscle cells and endothelial
cells proliferated with a proliferation rate peaked at 1 week. Furthermore, immunohistochemistry
confirmed the localization of PDGF-A mainly in the intima, both inner and outer zones of
the media, and the adventitia, which was associated well with cell proliferation response in
these zones. We conclude that PDGF-A chain plays a key role in the early hypertrophic
response to acute hypertension in the aortic wall.
P396
Macrophage Expression of Group IIA sPLA2 in LDL Receptor-Deficient Mice
Increases Atherosclerotic Lesion Formation in the Absence of Systemic
Effects on Lipoprotein Metabolism
Meredith A Bostrom, Frederick C De Beer, Alan Daugherty, Nancy R Webb. University of
Kentucky, Lexington, KY
Secretory phospholipases A2 (sPLA2) are thought to be responsible for amplifying the
inflammatory component of many disease processes, including atherosclerosis. Transgenic
mice expressing human Group IIA sPLA2 develop significantly larger atherosclerotic lesions
compared to non-transgenic littermates. The mechanism for this pro-atherogenic effect is likely
complex, as HDL-cholesterol is significantly lower and LDL/VLDL cholesterol is slightly higher
in transgenic mice compared to non-transgenic littermates. In the present study, we show that
peritoneal macrophages from transgenic mice express human Group IIA sPLA2. To assess
whether macrophage-expressed Group IIA sPLA2 plays a role in atherogenesis, bone marrow
cells from either sPLA2 transgenic mice or non-transgenic littermates were transplanted into
lethally irradiated LDL receptor -/- mice (n 10 per group). After a 6-week recovery period,
animals were fed a diet enriched with saturated fat (21% w/w) for 12 weeks. Engraftment of
donor cells was confirmed by the presence of the LDL receptor gene in bone marrow cells of
transplanted mice. Expression of human Group IIA sPLA2 in bone marrow-derived cells had no
effect on plasma total cholesterol (1205 /- 340.3 mg/dl and 1293 /- 402.2 mg/dl for
non-transgenic and transgenic, respectively) or HDL cholesterol (66.8 /- 19.7 mg/dl vs 75.6
/- 10.0 mg/dl). Despite a lack of effect on circulating lipoprotein concentrations, the presence
of bone-marrow-derived cells expressing human Group IIA sPLA2 resulted in a significant
increase in the extent of atherosclerosis in the aortic arch compared to control (percent lesion
area 12.75 /- 4.57 vs 7.37 /- 2.75 for transgenic vs non-transgenic, respectively; P
0.005). Taken together, our data suggests that macrophage-expressed Group IIA sPLA2 can
contribute to atherosclerotic lesion development in the absence of effects on systemic
lipoprotein metabolism. This effect may be mediated through Group IIA sPLA2 influencing
lipoproteinby metabolism guest on in the April microenvironment 4, 2013 of a developing lesion in the arterial wall.

a-70 Arterioscler Thromb Vasc Biol. Vol 22, No 5
P397
Differential Expression of Adenosine Receptors in Human Endothelial Cells:
Role of A 2B Receptors in Angiogenic Factor Regulation
Igor Feoktistov, Anna E Goldstein, Sergey Ryzhov, Dewan Zeng, Luiz Belardinelli, Tatyana
Voyno-Yasenetskaya, Italo Biaggioni. Vanderbilt University, Nashville, TN; CV Therapeutics,
Palo Alto, CA; University of Illinois, Chicago, IL
Adenosine has been reported to stimulate or inhibit the release of angiogenic factors depending
on the cell type examined. To test the hypothesis that differential expression of adenosine
receptor subtypes contributes to endothelial cell heterogeneity, we studied microvascular
(HMEC-1) and umbilical vein (HUVEC) human endothelial cells. Based on mRNA level and
stimulation of adenylate cyclase, we found that HUVEC preferentially express A 2A adenosine
receptors and HMEC-1 preferentially express A 2B receptors. Neither cells expressed A 1 or A 3
receptors. The nonselective adenosine agonist 5’-N-ethylcarboxamidoadenosine (NECA) increased
mRNA and protein expression of interleukin-8 (IL-8) and vascular endothelial growth
factor (VEGF) in HMEC-1, but had no effect in HUVEC. In contrast, the selective A 2A agonist
2-p-(2-carboxyethyl)phenethylamino-NECA (CGS 21680) had no effect on protein levels of IL-8
and VEGF. Cotransfection of each type of adenosine receptors with a luciferase reporter in
HMEC-1 showed that A 2B receptors, but not A 1,A 2A or A 3, activated IL-8 and VEGF promoters.
These effects were mimicked by constitutively active G q, G 12 and G 13, but not G s or
G i1–3. Furthermore, stimulation of phospholipase C indicated coupling of A 2B receptors to G q
proteins in HMEC-1. Thus, differential expression of adenosine receptor subtypes contributes
to functional heterogeneity of human endothelial cells. A 2B receptors, predominantly expressed
in human microvascular cells, modulate expression of angiogenic factors via coupling to G q,
and possibly via G 12/13
P398
ASK1-Induced Phosphorylation of Myofilament Proteins Causes Cardiac
Contractile Dysfunction
Xiangrong He, Wang Min. University of Rochester, Rochester, NY
There is increasing support for the idea that excessive production of proinflammatory cytokines
such as TNF and reactive oxygen species (ROS) contribute to the pathogenesis of cardiac
dysfunction. Phosphorylation of myofilament proteins, including troponin (Tn, subunits T, I and
C) and sarcomeric structural proteins plays important roles in the regulation of the contractile
activity. However, the linkage between cytokine / ROS production and phosphorylation of
myofilament proteins has not been established. Given that the MAP kinase kinase kinase, ASK1
(apoptosis signal-regulating kinase 1) is highly expressed in cardiac muscle and that ASK1 is
an important mediator in the signaling pathway induced by TNF, IL-1 and ROS, we examine the
role of ASK1-induced phosphorylation of myofilament proteins in cardiac contractile function.
Using yeast two-hybrid and mammalian expression/immunoprecipitation assays, we show that
ASK1 specifically interacts with cTnT and myomesin in vitro and in vivo via the C-terminal ASK1
domain. ASK1 specifically phosphorylates cTnT and myomesin in vitro and in vivo. Mutations
at an ASK1-phosphorylation consensus sequence (T194/S198) in cTnT molecule significantly
reduced its phosphorylation by ASK1. ROS induces ASK1 activation and contractile dysfunction
in neonatal rat cardiomyocytes. Moreover, overexpression of the constitutively active ASK1
resulted in inhibition of Ca2-stimulated myosin MgATPase activity and cardiac contractile
dysfunction. We conclude that ASK1 plays an important role in regulation of cardiac contractile
function by phosphorylating myofilament proteins and may mediate ROS-induced pathogenesis
such as cardiomyopathy and heart failure.
Structure-Function Analysis of Recombinant Human Triacylglycerol
Hydrolase (hTGH) Expressed in Sf-9 Cells
Mustafa Alam, Dennis E Vance, Richard Lehner. University of Alberta, Edmonton, AB,
Canada
P399
The majority of VLDL-triacylglycerol (TG) is derived from lipolysis of an intracellular TG storage
pool followed by re-esterification of the lipolytic products. We have identified and characterized
a triacylglycerol hydrolase (TGH) that mobilizes intracellular TG for VLDL secretion. Detailed
knowledge of structure-activity relationships will assist in generation of specific TGH inhibitors.
Using site-directed mutagenesis and expression of the mutants in Sf-9 cells we have shown
that the amino acid residues (Ser-221, Glu-354 and His-468) comprise the catalytic triad of
hTGH and that glycosylation of hTGH may be required for optimal activity. Human TGH also
contains within its primary amino acid sequence a putative neutral lipid binding domain (NLBD)
that we hypothesized to play a role in lipolysis. In addition, hTGH also contains a free cysteine
residue (C390) that we hypothesized to play a role in structure/interaction with the endoplasmic
reticulum membrane. The present study utilized a mutagenesis approach to address the
importance of the NLBD and C390. Our results demonstrate that deletion of NLBD yields an
inactive lipase. Chemical modification of C390 has no effect on TGH activity. However,
mutagenesis of C390 to alanine yields an inactive enzyme, suggesting an important structural
role of C390.
P400
The Matrix Metalloproteinase Inhibitor Doxycycline Reduces the Incidence
and Severity of Angiotensin II-Induced Aortic Aneurysms
Michael W Manning, Lisa A Cassis, Alan Daugherty. University of Kentucky, Lexington, KY
We have recently demonstrated that infusion of angiotensin II (AngII) induces abdominal aortic
aneurysms (AAA) and atherosclerosis in hyperlipidemic mice. Specific matrix metalloproteinases
(MMPs) are upregulated by AngII and could mediate the induced vascular disease. To
determine the role of MMPs in AngII-inducedDownloaded vascular pathology, from
we administered the
non-selective MMP inhibitor doxycycline to AngII-infused mice. Male LDL receptor-/- mice were
implanted with mini-osmotic pumps delivering AngII subcutaneously at 1,000 ng/kg/min for 28
days. Doxycycline (30 mg/kg/day) was administered via the drinking water to saline and
AngII-infused mice. Doxycycline did not significantly influence systolic blood pressure, serum
concentrations of cholesterol and triglycerides, or lipoprotein-cholesterol distribution. Doxycycline
decreased AngII induced atherosclerosis as defined by both percent of intimal surface
covered by lesions (7.2 0.6% vs 4.9 0.7%, P 0.05; n 16 and 13, control vs
doxycycline, respectively) and sterol content of arteries (1.0 0.1 vs 0.8 0.1 g/mm 2 ,P
0.05). Doxycycline significantly decreased the incidence of AngII induced AAAs (86% vs 46%;
P 0.04). In addition, doxycycline also markedly reduced the severity of AAA formation. AngII
infusion in the control group led to pronounced tissue hypertrophy and thrombus in the
supra-renal aorta. In contrast, AAA in doxycycline treated animals had limited tissue expansion
and no thrombotic material present. Therefore, inhibition of MMPs induced by AngII
administration resulted in a reduced atherosclerosis and decreased incidence and severity of
AAA. These results infer a role of MMPs in AngII-induced vascular pathology.
P401
Matrix Metalloproteinase-9 Expression Is Associated with the Presence of
Chlamydia pneumoniae in Coronary Atherosclerotic Plaques
Gavin Arno, David A Smith, Aldwin J Haven, Juan Carlos Kaski, Christina Baboonian. St
Georges Hospital Medical School, London, UK
Background: Chronic inflammation plays a role in atherosclerosis. Macrophages, smooth
muscle cells and endothelial cells present in the lesion produce matrix metalloproteinases
(MMPs). MMPs have been shown to play an important role in smooth muscle cell migration,
vascular remodelling, fibrous cap degradation and plaque vulnerability. Active synthesis of
MMP-9 is associated with unstable angina and plaque disruption in coronary and carotid
atherosclerosis. Chlamydia pneumoniae has been strongly associated with atherosclerotic
disease although little is known about the mechanism by which C. pneumoniae can affect the
lesion. Recently C. pneumoniae has been shown to induce MMP-9 expression in mouse
macrophages and human monocyte derived macrophages in vitro. However no substantial
evidence for this has been demonstrated within atherosclerotic plaques. The aim of this study
was to investigate the in situ association between intralesional presence of C. pneumoniae and
MMP-9 expression. Methods: 23 coronary endarterectomy specimens from first time bypass
surgery patients (21 men, mean age 62.1 6.1 years) were studied by immunocytochemistry.
Serial 5m frozen sections were double stained with mouse anti-C. pneumoniae and mouse
anti-CD68 (macrophage marker) or mouse anti-MMP-9 and mouse anti-CD68. Results were
analyzed using Fisher’s exact test. Results: C. pneumoniae immunoreactivity was observed in
15/23 samples (65%) and was frequently associated with intimal macrophages. MMP-9
immunoreactivity was observed in 20/23 samples (86%). 15/15 (100%) C. pneumoniae positive
samples were also positive for MMP-9. MMP-9 immunoreactivity was frequently observed in
macrophage rich, Chlamydia positive regions within the plaque. Detection of MMP-9 was
associated with intralesional presence of C. pneumoniae (p0.03). Conclusions: This is the first
clear evidence that expression of MMP-9 in the atherosclerotic plaque is associated with the
presence of intralesional C. pneumoniae. This data may suggest a possible mechanism by
which the bacteria may contribute to progression of atherosclerotic disease.
P402
Chronic Oxidative Injury of Vascular Smooth Muscle Cells Alters Expression
of Cell Cycle Regulatory Proteins and Activation of MAP Kinase
Sarah A Jones, Jan L Patterson, Kenneth S Ramos, Emily Wilson. TAMUS Health Science
Center, College Station, TX; College of Veterinary Medicine, TAMU, College Station, TX
Chronic oxidative injury by allylamine (AAM) induces phenotypic changes in rat aortic smooth
muscle cells similar to those seen in vascular smooth muscle cells (vSMC) within atherosclerotic
lesions. The molecular mechanisms that mediate altered regulation of vSMC growth and
differentiation following chronic oxidative injury involve alterations in integrin-extracellular
matrix (ECM) signaling. To test this hypothesis, cells were harvested from rats treated in vivo
with AAM or water (control cells), and seeded on different growth matrices to evaluate the
relationship between cell-matrix interactions, MAP kinase activation and cell cycle regulatory
proteins. Western blot analysis using antibodies specific for phosphorylated MAP kinase was
used to monitor MAP kinase activation. Time course studies following serum stimulation of
mitogen restricted cultures showed a 1.4 fold increase and prolonged (90 min) activation of
MAP kinase in AAM cells when compared to control cells. A decrease in peak activation of MAP
kinase was observed in AAM cells compared to controls seeded on collagen. Western blot
analysis also showed that cyclin kinase inhibitors, p21 cip1 and p27 kip1 , were decreased by 2.8
fold and 3.0 fold, respectively, in AAM cells compared to control cells on plastic. Similar results
were observed on collagen. No difference was observed in cyclin D1 or cyclin E expression
between the two cell types. Based on these data we conclude that both the degree and duration
of activation of MAP kinase contributes to the proliferative advantage of AAM cells through its
interaction with cell cycle regulatory proteins and ECM.
P403
Lipid-Lowering and Antiatherogenic Effects of Primary Metabolic Products
of Naringin and Hesperidin in Cholesterol-Fed Rats or Cholesterol-Fed LDLr
Knockout Mice
Tae-Sook Jeong, Goo-Taeg Oh, Song-Hae Bok, Goong-Woo Nam, Myung-Sook Choi,
Mi-Kyung Lee. Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea;
Kyungpook National University, Taegu, Korea
The effects of two primary metabolic products of naringin and hesperidin,
4-hydroxyphenylpropionic acid(p-HPPA) and 3,4-dihydroxyphenylpropionic acid(3,4-DHPPA), in
http://atvb.ahajournals.org/ rats or LDLr by knockout guest mice on April fed a 1% 4, cholesterol 2013 diet. The metabolic products(0.1% wt/wt diet)

or lovastatin (1 mg/kg body wt) supplements were given for 6 weeks. The plasma total
cholesterol and triglyceride levels were significantly lowered by the p-HPPA and 3,4-DHPPA
supplements compared to the control or lovastatin group. The hepatic HMG-CoA reductase
activity was significantly lower in the 3,4-DHPPA group than in the control or lovastatin group.
The acyl coenzyme A: cholesterol O-acyltransferase (ACAT) activities were significantly lower
in the p-HPPA and 3,4-DHPPA groups compared to the control or lovastatin group. As regards
the hepatic antioxidant enzyme system, the catalase activity was significantly lower in the
p-HPPA and 3,4-DHPPA groups compared to the control or lovastatin group. The two metabolic
products resulted in an increased hepatic superoxide dismutase (SOD) and glutathione
peroxidase (GSH-Px) activities, meanwhile all the supplements significantly lowered the hepatic
TBARS content. However, the p-HPPA and 3,4-DHPPA supplements did not alter the neutral
sterol and total fecal sterol. Accordingly, both metabolic products were potent in lipid-lowering
and altering the antioxidative enzyme. Eighteen female Ldlr-/- mice were randomly divided into
three groups (n6 per group). The mice were fed a 1% cholesterol diet alone (control group)
or supplemented with either 1 mg/kg lovastatin, or 0.1% p-HPPA for 8 weeks. The plasma
lipoprotein levels, total cholesterol, triglyceride and high-density lipoprotein were not shown
significant changes in control and experimental groups. The mean lesion area of the aortic
sinus was significantly reduced in the p-HPPA group compared with the control group
(86758.031004.2 um2 vs. 127253.427574.8 um2, P0.001). The metabolite of naringin,
p-HPPA exhibited potent anti-atherogenic effect in Ldlr-/- mice.
Simplified Rabbit Jugular Vein Graft Interposition Model
Zhihua Jiang, Elizabeth J Bray, Zaher S Abouhamze, Scott A Berceli, C K Ozaki. University
of Florida College of Medicine and the Malcolm Randall VAMC, Gainesville, FL
P404
Reconstructions utilizing vein grafts develop neointimal hyperplasia (NIH) and are prone to
occlusive failure. The rabbit vein graft model of NIH involves jugular vein (JV) interposition into
the carotid artery (CA) using hand-sewn anastomoses, a technically complex procedure. We
sought to simplify the rabbit JV graft carotid interposition model to create a more broadly
applicable tool for the study of NIH. Methods New Zealand white rabbits (n10) underwent JV
interposition into the CA using a cuff technique. Polyurethane cuffs, consisting of a body loop
and an extension tab were fashioned from 4-French catheters. Three cm of the right external
JV was excised. Each JV end was passed through a cuff, everted, and fixed to the cuff using
6–0 silk. The right CA lumen was exposed using a2cmarteriotomy and the cuffed, reversed
JV ends inserted. Another 6–0 silk ligature fixed the CA wall around the cuff. After anastomosis
completion, one centimeter of CA back wall was removed, permitting tension-free vein graft
extension. Four weeks post-op graft blood flow was measured and grafts were harvested.
Morphometry, proliferation (BrdU), and smooth muscle cell (SMC) localization were determined
using histochemistry and immunohistochemistry. Results Mean time for graft construction was
123 (SD) minutes. All 10 rabbits survived without complications and grafts were patent at
harvest. Blood flow was 31.58.6 ml/min at harvest. SMC rich neointima progressed to
0.40.18mm 2 at 4 weeks (baseline 0). At 4 weeks there were 6341 BrdU positive cells/mm 2
intimal area (5%). Conclusions The non-suture external cuff technique represents a reliable
and fast method for performing JV graft CA interposition in rabbits. This model requires less
technical skill than the traditional hand-sewn model, reproducibly produces NIH, and provides
a useful tool for the study of vein graft failure.
Dimunition of Advanced Atherosclerosis by Electrical Impulses in the
Rabbit Abdominal Aorta
Valeri S Chekanov, Mohammad Mortada, David Krum, Guennady V Tchekanov, Ruben
Eisenstein, Masood Akhtar. Sinai/St. Luke’s Medical Centers, University of
Wisconsin-Milwaukee Clinical Campus, Milwaukee, WI
P405
Objective: Our objective was to investigate slowed progression or elimination of atherosclerosis
by low-frequency electrical impulses (EI) in abdominal aortas (AA) of rabbits with moderate
atherosclerosis induced by a high cholesterol diet (HCD). Methods: Series I rabbits (control)
were fed HCD for 8 weeks. Series II had HCD for 8 weeks, then were switched to normal diet
for 8 weeks (no EI). Series III had HCD for 8 weeks, then normal diet simultaneously with EI
applied around the AA for 8 weeks (3V, 30 single impulses per min, 24 hrs/day). After
euthanization, atherosclerosis level and percentage of surface area involved in atherosclerosis
were calculated in thoracic and abdominal aortas. Results: Atherosclerosis levels were
significantly different (p0.05) in AA between series III (0.40.2) and the two other groups:
1.50.4 in series I (HCD only), 1.20.3 in series II (HCD then normal diet). In series III, there
was scant evidence of atherosclerosis: few signs of endothelial cell injury, undisturbed elastic
lamina, few fat laden macrophages, no migration of skeletal muscle cells. Gross examination
of percentage of involved surface area revealed significant differences (p0.05) between
series I (control) (30.14.1%), series II (21.33.6%), and series III (5.55.5%). Conclusion:
Our study showed that, when applied near the abdominal aorta, low frequency electrical
impulses decrease atherosclerotic deposition in the Downloaded abdominal aorta. from
http://atvb.ahajournals.org/
Poster Presentations a-71
Lactosylceramide Recruits Phospholipase A 2 to Stimulate PECAM-1
Expression in Human Monocytes and Adhesion to Endothelial Cells
Nanling Gong, Sanaul Haq Chowdhury, Heming Wei, Yean Leng Lim, Subroto Chatterjee.
Johns Hopkins Singapore and National Heart Centre Vascular Biology Centre, Singapore
P406
Although platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) has been implicated
in the adhesion and trans-endothelial migration of monocytes / lymphocytes, little is known
about mechanisms by which this cell adhesion molecule is regulated. In the present study, we
explored the role of a glycosphingolipid, lactosylceramide (LacCer) in modulating PECAM-1
expression and cell adhesion. We observed that in monocytes (differentiated U-937 cells)
LacCer exerted a time and concentration-dependent increase in the expression of PECAM-1.
The stimulation of PECAM-1(but not other cell adhesion molecules) by LacCer was also
observed at the transcriptional level. Western immunoblot assays revealed that maximal
increase in PECAM-1 expression ( 2-fold) occurred within 6 hours of treatment of cells with 100
nM LacCer. And this was accompanied by a significant increase in the adhesion of these cells
to human endothelial cells. This phenotypic change was abrogated by pre-incubation of cells
with staurosporine and antibody against PECAM-1. However,other homologs of LacCer such as
ceramide and glucosylceramide did not alter PECAM-1 expression. Moreover, scavengers of
free oxygen radicals and inhibition of NADPH oxidase did not impair LacCer induced PECAM-1
expression. We observed that within 5 min LacCer stimulated the activity of phospholipase A 2
in monocytes. At this time [ 3 H] LacCer was associated with the cells in a trypsin-sensitive state
and was not internalized/metabolized. PLA 2 inhibitors abrogated LacCer mediated PECAM-1
expression and cell adhesion and this was bypassed by arachidonate but not by a variety of
other fatty acids and other lipid second messengers. In sum, LacCer, by virtue of activating
PLA 2 and producing arachidonic acid, stimulates PECAM-1 expression, cell adhesion and
trans-endothelial migration. Since the level of LacCer is elevated in atherosclerostic plaques,
the findings above may be relevant in explaining its role in the pathophysiology in this disease.
P407 WITHDRAWN
P408
Mycophenolic Acid Inhibits Neointimal Growth in the Balloon-Injured Rat
Carotid Artery
Xin Yang, Andrea Hubbard, Dadong Li, Charles Nightingale, Ronald Langer, Laurine Bow.
Hartford Hospital, Hartford, CT; University of Connecticut, Storrs, CT
We have investigated the effects of mycophenolic acid (MPA) (Roche Laboratories, Inc.) on
neointimal growth in balloon-injured rat carotid arteries. Briefly, male Sprague-Dawley rats
(400–500g) were administered daily 20 mg/kg MPA or vehicle intravenously post injury.
Carotid arteries were collected for morphometric analysis and immunohistochemical staining 3
and 14 days after surgery, to determine early and later effects on intimal growth. MPA
significantly inhibited intimal growth and intima/media ratio in 14-day animals. In animals
terminated at 3 days, there is no intimal layer formation and there is no difference of medial
layer areas between MPA and vehicle treated groups. To evaluate macrophage recruitment into
the intima/media of these arteries, immunohistochemical staining for macrophage marker ED1,
and monocyte chemoattractant protein-1 (MCP-1) was performed. In addition, SMCs were
stained for proliferating cell nuclear antigen (PCNA) and smooth muscle cell (SMC) -actin.
Vehicle treated animals terminated at 3 days demonstrated more ED1 expression as compared
to MPA treated animals, however there was no difference in the percentage of PCNA positive
SMCs between two groups. Animals terminated at 14 days showed no ED1 expression for both
vehicle and MPA treated groups, and showed significantly less PCNA positive SMCs in MPA
treated group than vehicle group. Finally, 14-day MPA treated animals showed significantly
decreased MCP-1 expression in the intima as compared to vehicle group. The effect of MPA
on cell proliferation of SMCs isolated from rat aorta was also investigated. Cells treated with
PDGF, serum and PDGF plus serum stimulated proliferation of SMCs. A dose dependent
inhibition by MPA was determined using 3 H-thymidine incorporation assay. This effect was
reversible and dependent on the amount of MPA bound to SMCs. No toxicity was observed by
MPA washout assay during SMC culture. These data suggest that inhibition of macrophage
recruitment and/or SMC proliferation by MPA may contribute to the decrease of neointimal
growth seen in animals treated with MPA at 14 days.
P409
Atherosclerosis Is Reduced in Cholesterol-Fed ApoE(-/-) Mice Administered
an ASBT Inihibitor or a Selective COX-2 Inhibitor
Elaine S Krul, Nida Napawan, Dustie T Butteiger, Kyle Hayes, Lorrie Krause, Gregory E
Frierdich, Kay O Broschat, Bradley T Keller. Pharmacia Corp., St. Louis, MO
Atherogenesis also involves the stimulation of inflammatory pathways and the relative benefit
of lipid-lowering versus anti-inflammatory therapy on the initiation and progression of
atherosclerosis has not been established. To test this, we administered an ASBT inhibitor or a
selective COX-2 inhibitor to apoE(-/-) mice fed a diet with 0.125% cholesterol to assess the
effects of the compounds on the progression atherosclerosis as monotherapy or in combination.
6 week old mice were placed on the cholesterol diet to initiate atherosclerosis and then
divided into by 6 guest groups: on control April (A), 75 4, mpk 2013 celecoxib (B), 1 mpk ASBTi (C), 10 mpk ASBTi (D),

a-72 Arterioscler Thromb Vasc Biol. Vol 22, No 5
1 mpk ASBTi75 mpk celecoxib (E) or 10 mpk ASBTi75 mpk celecoxib (F). Mice were
maintained on the test diets for 12 weeks. Serum total cholesterol levels were identical during
the study for groups A and B. ASBTi diets C and D reduced serum cholesterol by 42% and 53%
at week 12 compared to control. Triglycerides were increased 2.5-fold by ASBTi treatment,
however, lipid levels were not affected by celecoxib. Serum thromboxane (TxB2) was reduced
in the ASBTi-treated mice by 60–75%, suggesting a reduction of COX-1 activity in the ASBT
inhibitor treated groups. No significant difference in serum TxB2 was seen with celecoxib
treatment alone indicating selective inhibition of COX-2 in this model. Aortic lesion area was
reduced by 76% and 81% in the 1 mpk and 10 mpk ASBTi treatment groups, respectively.
There was a more modest, but significant 22% reduction in atherosclerosis in the celecoxibtreated
mice. However, the combination of ASBTi and celecoxib did not result in a further
decrease in atherosclerosis than that seen with either dose of ASBTi alone (73% and 82%
reduction in the combination groups, respectively). Thus, lipid lowering by an ASBT inhibitor
reduces atherosclerosis progression which may be partly mediated through reduction of
pro-inflammatory prostaglandin production. Administration of a selective COX-2 inhibitor in the
absence of lipid-lowering, resulted in modest reduction in atherosclerotic lesion area and when
combined with the ASBT inhibitor, did not lead to any further reduction in area.
P410
The Proximal Serum Response Element in the Egr-1 Promoter Mediates
Response to Thrombin in Primary Human Endothelial Cells
Sheng-Qian Wu, Takashi Minami, Diana J Donovan, William C Aird. Beth Israel Deaconess
Medical Center, Boston, MA
Thrombin signaling in endothelial cells provides an important link between coagulation and
inflammation. The Egr-1 transcription factor couples short-term changes in the microenvironment
to long-term changes in gene expression. In this study, our goal was to investigate the
effect of thrombin on Egr-1 gene expression in primary human endothelial cells (EC). The
treatment of EC with 1.5 U/ml of thrombin resulted in a 6-fold increase in Egr-1 mRNA
expression and a 3-fold increase in Egr-1 promoter activity. The Egr-1 promoter region contains
5 serum response elements (SREs), arranged in a 5 cluster of three SREs and a 3 cluster of
two SREs. In transient transfection assays, deletion of the 3 cluster resulted in a loss of
thrombin response, whereas deletion of the 5 cluster had no such effect. Mutation analyses
revealed that the 3-most SRE (SRE-1), but not the other SREs, was necessary for mediating
the thrombin response. In electrophoretic mobility shift assays, nuclear extracts from
thrombin-treated EC displayed increased binding of total and phosphorylated serum response
factor (SRF) to SRE-1, compared with untreated controls. Thrombin-mediated induction of the
endogenous Egr-1 gene and the Egr-1 promoter was blocked by inhibitors of MEK1/2, but not
by inhibitors of PKC, PI3K or p38 MAPK. Finally, the treatment of EC with thrombin resulted in
the rapid nuclear translocation of SRF. Taken together, these data suggest that thrombin
induces Egr-1 expression in endothelial cells by a MAPK-dependent mechanism that involves
an interaction between SRF and SRE-1.
SR-BI-Specific Selective Lipid Uptake from HDL Ligands Lacking
Apolipoprotein E
P411
Lei Cai, Frederick C De Beer, Daniel J Rader, Deneys R Van der Westhuyzen. University of
Kentucky, Lexington, KY; Veteran Affairs Medical Center; University of Kentucky, Lexington,
KY; University of Pennsylvania School of Medicine, Philadelphia, PA
Apolipoprotein E (apoE) mediates the clearance of chylomicron remnants, very low density
lipoproteins as well as HDL. The class B scavenger receptor type I (SRBI), plays an important
role in HDL metabolism through its ability to mediate the selective uptake of cholesterol ester
(CE) from HDL. However, the role of apoE in SR-BI-mediated selective CE uptake is poorly
understood. In this study, the influence of apo E on SR-BI-dependent selective CE uptake was
investigated using HDL isolated from normal mice and apoE-deficient mice (E-/-HDL) as well
as mouse HDL depleted of apoE (E(-)HDL) by heparin sepharose chromatography. HDL
preparations were double-labeled with 125I and 3H cholesteryl oleoyl ether. In Chinese hamster
ovary (CHO) cells transfected with murine SR-BI (CHO-SRBI), SR-BI-specific selective CE uptake
from E-/-HDL was significantly reduced (about 2-fold) compared with normal HDL, despite
increased cell association of these particles. HepG2 cells and primary hepatocytes showed a
40% decrease in the uptake of 3H-cholesterol oleoyl ester from E-/-HDL compared to normal
HDL. Mouse and human HDL depleted of apoE also exhibited reduced SR-BI-mediated selective
uptake of CE. However, addition of apoE through the use of adenoviral-mediated over
expression had little or no effect on SR-BI-mediated selective uptake of CE from HDL and
E-/-HDL in CHO-SRBI cells. These findings indicate that the presence or absence of apoE on
HDL does not influence SR-BI-specific selective uptake of CE in CHO-SRBI cells and do not
explain the difference in selective uptake from normal, E-/-HDL or E(-)HDL.
P412
Quantitative Analysis of Stretch-Induced Cytoskeletal Remodeling in the
Human Umbilical Vein Endothelial Cells
Masaaki Yoshigi, Kazushi Yasuda, Edward B Clark, H Joseph Yost. University of Utah, Salt
Lake City, UT; Huntsman Cancer Institute, University of Utah, Salt Lake City, UT
Mechano-sensing of the vascular wall plays a crucial role in normal and abnormal development
of vascular formation. When linearly stretched for several hours, the human umbilical vein
endothelial cells (HUVECs) align cell axis perpendicular to the stretch direction. This
mechano-sensing and remodeling mechanisms remain unclear, partially due to difficulty to
elaborate the directional response. We therefore developed a novel image processing system
that quantifies stretch-induced responses. Methods HUVECs were grown on a rectangular
silicone rubber membrane using standard techniques until 100% confluent. Linear strain of
10% was applied for 0 (control), 0.5, 1, 2, 5, 10, and 20 hours at 0.5 Hz using custom devices.
The cells were fixed, permeabilized and stained Downloaded by rhodamine-phalloidin from
to visualize F-actin.
http://atvb.ahajournals.org/
Fluorescent images were processed by custom software based on Sobel convolution kernel.
Our software automatically calculates average orientation of actin fibers, standard deviation of
actin fiber orientation, local density index of actin fibers, and display fiber arrangement with
colorized manner. ANOVA was employed to analyze ’randomness’ and ’density’ of the actin
fibers, and their correlation to the stretch direction was compared. Results Stretch-induced
cytoskeletal remodeling was visualized by color, which was easy to comprehend. Initial
response after 1 to 2 hours of stretch increased actin fiber density, whereas late response
induced perpendicular alignment of actin fibers. Fiber density index increased from 67.38.2
to 73.59.6 after 1 hour of stretch (p0.01). Standard deviation of fiber orientation decreased
from 49.6 to 19.8 degree after 5 hours of stretch (p0.01). These two parameters clearly
differentiated initial and late response of the stretch-induced cytoskeletal remodeling.
Pre-incubation in cyclohexamide (5M, 30 min) did not alter two parameters after 1 hour of
stretch, indicating stretch-induced cytoskeletal remodeling is post-transcriptional. Thus, image
processing of stretch-induced cytoskeletal remodeling allows us to dissect molecular
mechanisms of mechano-sensing more precisely.
P413
Expression of the Growth Promoting Helix-Loop-Helix (HLH) Transcription
Factor Id3 Is Enhanced in Response to Vascular Injury in the Obese Zucker
Rat Model
Pushpa-Rekha R Thimmalapura, Hong Pei, Feng Li, Kevin Naran, Martin E Matsumara, Jiali
Gu, Jerry L Nadler, Coleen A McNamara. University of Virginia Medical Center,
Charlottesville, VA
Diabetes is an important risk factor for vascular proliferative disorders although the molecular
mechanisms are poorly understood. Previous data from our laboratory has implicated the HLH
factor Id3 in the regulation of vascular smooth muscle cell (SMC) proliferation. To determine if
Id3 is an important mediator of accelerated SMC growth in insulin resistant states, the Zucker
rat model was used. The obese zucker (OZ) rats show an increased intimal SMC proliferation
and restenosis in response to vascular injury. Accordingly we hypothesized that Id3 expression
will be more in OZ relative to lean Zucker (LZ) rats in response to injury. To test this, the left
common carotid arteries of LZ and OZ rats were injured using a 1.8 F PTCA balloon catheter.
3, 7 and 14 days later, injured and uninjured samples were immunostained with Id3 antibody.
Results demonstrate an abundant expression of Id3 protein at 7 and 14 days after injury in OZ
rats relative to LZ rat. To determine if this was due to an increase in Id3 mRNA, Id3 mRNA was
quantitated by reverse transcription and real-time PCR. Results demonstrate that Id3 mRNA
levels increase in both LZ and OZ rats following injury. Further, Id3 mRNA was 2.3 and 6.23
fold more in OZ relative to LZ rats at 7 and 14 days, respectively, following injury. A marked
increase in Id3 expression at 14 day is consistent with the kinetics of SMC proliferation that
demonstrates significant differences in SMC proliferation at 14 and 30 days following baloon
injury in OZ vs LZ rats. To examine the effect of Id3 on SMC growth, Id3 was stably
overexpressed in rat SMC and SMC growth was determined. Results show that SMC
overexpressing Id3 proliferate significantly more compared to control cells. Taken together,
these results implicate Id3 as an important mediator of accelerated SMC proliferation in the OZ
rat model of insulin resistance.
Forkhead Transcription Factors Lie Downstream of VEGF Signaling in
Endothelial Cells
P414
M D Ruhul Abid, Shaodong Guo, Takashi Minami, Katherine C Spokes, William C Aird. Beth
Israel Deaconess Medical Center, Boston, MA
VEGF promotes endothelial cell survival, proliferation and migration. In non-endothelial cells,
PI3/Akt signaling has been reported to induce the phosphorylation and nuclear exclusion of
forkhead transcription factors, FKHRL1 (FOXO 3), FKHR (FOXO 1), and AFX (FOXO 4), an effect
that may result in increased cell survival and proliferation. We hypothesized that VEGF signaling
may be coupled to the activity of forkhead proteins in endothelial cells. Here we show that VEGF
transiently phosphorylates endogenous FKHRL1 (at Ser-253 and Ser-315), FKHR (at Thr-24 and
Ser-256) and AFX (at ser-193) in human coronary artery endothelial cells (HCAEC). In
immunolocalization studies, VEGF treatment resulted in the nuclear exclusion of FKHRL1. In
co-transfection assays, VEGF resulted in a significant (50%) reduction of WT-FKHRL1mediated,
but not triple mutant (TM)-FKHRL1-mediated, transactivation of a plasmid containing
the forkhead-responsive element coupled to luciferase reporter gene. Overexpression of
TM-FKHRL1, but not WT-FKHRL1, inhibited VEGF-mediated migration and in vitro tube
formation of HCAEC. Overexpression of TM-FKHRL1 or WT-FKHRL1 resulted in increased
apoptosis of HCAEC, as assayed by FACS analyses of annexin V staining. Only the
WT-FKHRL1-induced apoptosis was significantly inhibited by VEGF. Finally, VEGF treatment
resulted in a significant reduction of the p27kip1 transcripts in HCAEC. Taken together, these
findings demonstrate that 1) VEGF induces phosphorylation of AFX, FKHR and FKHRL1, 2)
VEGF-mediated phosphorylation of FKHRL1 correlates with nuclear exclusion of the protein, 3)
VEGF-mediated phosphorylation and nuclear exclusion of FKHRL1 is associated with a
reduction in its transcriptional activity, 4) VEGF survival signal is mediated, at least in part, by
the phosphorylation and nuclear exclusion of FKHRL1, 5) the forkhead transcription factor plays
a specific role in mediating the effect of VEGF on endothelial cell migration and tube formation,
and 6) VEGF-mediated induction of endothelial cell survival, migration and angiogenesis may
be mediated, by at guest least in on part, April by a4, forkhead-dependent 2013 inhibition of p27kip1 expression.

Platelet Factor 4 Localization in Carotid Atherosclerotic Plaques:
Correlation with Clinical Parameters
P415
Bruce S Sachais, Stephanie Pitsilos, Jennifer Hunt, Emile R Mohler, Anand M Prabhakar,
Mortimer Poncz, Tigran Z Khalapyan, Megan L Wolfe, Ronald Fairman, Marc Mitchell,
Michael Golden, Douglas B Cines. University of Pennsylvania, Philadelphia, PA; University of
Pittsburgh Medical Center, Pittsburgh, PA
Background:Platelet activation is an important mediator of terminal thrombotic events in
atherosclerosis. Emerging evidence supports an additional role for platelets in the pathogenesis
of atherosclerosis itself. Platelet Factor 4 (PF4), a cationic protein released by activated
platelets, stimulates several pro-atherogenic processes including endothelial activation, smooth
muscle cell proliferation, and vascular recruitment and differentiation of monocytes. We
recently found that PF4, but not NAP-2 (a structurally related platelet protein) inhibits
LDL/LDL-receptor endocytosis and promotes uptake of oxidized LDL by macrophages.
Therefore, we studied the vascular localization of PF4 during the evolution of human
atherosclerotic plaques. Methods: Carotid endarterectomy plaques were collected from 142
patients with critical carotid stenosis, and 6 autopsy specimens with minimal disease.
Specimens were characterized histologically and graded (3–6) according to a standard AHA
system. Immunohistochemical staining for PF4, NAP-2 and cell specific markers was
performed. Clinical, histologic and immunohistologic data were analyzed using a 2 test.
Results: PF4 was observed in the cytoplasm of macrophages and luminal and neovascular
endothelium, as well as in calcifications, adherent clot, intimal smooth muscle and mast cells.
PF4 expression in macrophages and in neovascularization correlated with more severe disease
(p0.001). The presence of PF4 in luminal endothelium and neovascularization correlated with
symptomatic coronary artery disease (CAD:p0.04, 0.019). In early lesions, PF4 was
commonly present in fatty streak macrophages, while NAP-2 was rarely present. Conclusions:
The correlation of PF4 localization with CAD and lesion grade suggests that persistent platelet
activation may contribute to the evolution of vascular lesions. These studies provide a rationale
for the long-term use of anti-platelet therapy in patients at risk for atherosclerosis. Further, the
differences between NAP-2 and PF4 in early lesions suggests that the PF4 noted inside the
lesion may have an etiologic role in the development of lesions.
CETP and LCAT Contribute to Formation of Small Dense LDL In Vitro
Patricia U Huey, Xianzhou Li, Harnish Patel, Mary E Sweeney, Warren Davis, William V
Brown, Ngoc-Anh Le. Atlanta VA Medical Center, Decatur, GA
P416
We examined lipoprotein composition and metabolism in plasma from 11 patients with type 2
diabetes and 19 normal individuals. Whole plasma was subjected to fractionation using fast
protein liquid chromatography (FPLC) and the fractions corresponding to each lipoprotein class
were assayed for total cholesterol (C), unesterified cholesterol (UC) and triglyceride (TG)
content. Diabetic patients with low plasma TG (100 mg/dl) had a higher % of their total low
density lipoprotein (LDL)-C in the small dense LDL (sdLDL) fraction (32.9 vs. 27.4%, p0.003)
than did normal individuals with similar plasma TG levels. Diabetic patients with moderate TG
(100 TG 250 mg/dl) also showed higher % of sdLDL (36.7 vs. 28.8%, p0.0001) than
nondiabetic patients with similar plasma TG. Mean LDL particle size was determined by
nondenaturing gradient gel electrophoresis (GGE) and was found to be negatively correlated
with % sdLDL (r-0.516, p0.005). To examine the origin of sdLDL in these patients, we
incubated whole plasma at 37°C for 6 hr to allow lipoprotein remodeling by the plasma
enzymes cholesteryl ester transfer protein (CETP) and lecithin-cholesterol acyltransferase
(LCAT). Plasma was then fractionated by FPLC and mean LDL particle size was determined by
GGE. LDL particle size decreased upon incubation in both diabetic (from 248 to 246 Å) and
nondiabetic (256 to 253 Å) patients in spite of an increase in LDL-TG as assessed by FPLC. The
decrease in LDL size observed after in vitro remodeling was due to loss of total C. In diabetic
patients with low plasma TG, loss of LDL-C occurred preferentially from sdLDL subfractions; in
nondiabetic individuals, LDL-C loss occurred primarily from larger LDL subfractions. Both
diabetic and nondiabetic patients with moderate hypertriglyceridemia exhibited even greater
loss of LDL-C from the sdLDL fractions. This drop in LDL-C was due in part to a decrease in
LDL-UC, presumably via LCAT activity. These data suggest that a significant portion of
esterified C transferred to high density lipoprotein (HDL) by CETP is derived from LDL-UC via
LCAT and that complementary actions of CETP and LCAT play a role in the formation of sdLDL
in plasma.
P417
ApoA-I Deficiency Results in Increased Atherosclerosis in LDLR Knockout
Mice Fed a Chow Diet
Ryan E Moore, Masa-Aki Kawashiri, Anthony Secreto, Daniel J Rader. University of
Pennsylvania, Philadelphia, PA
INTRO: ApoA-I is the primary protein constituent of HDL. Overexpression of apoA-I in mice
reduces atherosclerosis, but the impact of apoA-I deficiency has been less clear. To investigate
the hypothesis that apoA-I deficiency results in increased atherosclerosis on a chow diet, mice
deficient in both the LDLR and apoA-I (LDLR-/- /apoA-I-/- ) were compared to mice deficient in
either the LDLR (LDLR-/- ) or apoA-I (apoA-I-/- ) alone. METHOD: Mice deficient in apoA-I (n8),
the LDLR (n14), or both (n51), were fed a chow diet. Aortas and plasma were collected at
10 and 15–16 months. Plasma lipids were measured and lipoprotein profiles were characterized
by FPLC, density ultracentrifugation, and lipoprotein electrophoresis. Atherosclerosis was
measured in aortas by Sudan IV staining and en face lesion quantitation. RESULTS: Both LDLR-/ and LDLR-/- /apoA-I-/- mice had a 3-fold elevation of total cholesterol, and 9-fold elevation of
non-HDL-C, relative to wt mice. LDLR-/- /apoA-I-/- mice however had decreased HDL-C levels
relative to both LDLR-/- and wt mice. At 10 months of age, apoA-I-/- mice had no detectable
atherosclerosis, LDLR-/- mice had some lesion (1.560.54 % lesion area), and LDLR-/- /apoA-I-/ mice had significantly greater lesion (4.060.44 % lesion area, p0.006 vs. LDLR-/- Downloaded from
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Poster Presentations a-73
15–16 months of age, LDLR -/- mice did not have significantly increased atherosclerosis
compared to 10 month old LDLR -/- mice (2.471.20 % lesion area at 15–16 months). 15–16
month old LDLR -/- /apoA-I -/- mice however had significantly increased atherosclerosis compared
to 10 month old mice, which was increased nearly 4-fold compared to age matched,15–16
month old LDLR -/- mice (9.521.79 % lesion area at 15–16 months, p0.006 vs. 15–16
month old LDLR -/- ). CONCLUSION: Despite similar levels of total and non-HDL cholesterol,
apoA-I deficiency results in markedly increased atherosclerosis in LDLR -/- /apoA-I -/- mice
compared with LDLR -/- mice. This demonstrates that deficiency of apoA-I is associated with a
loss of protection from the formation of atherosclerosis, resulting in significant time dependent
lesion formation.
P418
Endothelial Specific Molecule-1 (ESM-1) is a VEGF-Responsive Gene That Is
Preferentially Expressed in Tumor Vascular Endothelium
Xianjin Yi, Jo C Tsai, William C Aird. Beth Isreal Deaconess Medical Center, Boston, MA
Angiogenesis is important for tumor growth and metastasis. The identification of genes that are
differentially expressed in tumor endothelium will establish a foundation for developing tumor
selective anti-angiogenesis therapies. In this study, we investigated the distribution of
endothelial specific molecule-1 (ESM-1) in normal mouse tissues and tumor xenografts. In
Northern blot analyses, ESM-1 mRNA was detected in the kidney, spleen and lung of adult
mice. In in situ hybridization assays, ESM-1 transcripts were detected in peritubular cells of the
kidney and a subpopulation of mononuclear cells in the spleen, but were undetectable in the
lung. Northern blot assays revealed ESM-1 mRNA in all tumor xenografts examined, including
human lung carcinoma, human renal cell carcinoma, rat glioma, and mouse breast carcinoma.
Expression of ESM-1 in the tumors was restricted to the vascular endothelium, as determined
by in situ hybridazation studies. In contrast, there was no detectable expression of ESM-1 in
mouse embryos. Under in vitro conditions, ESM-1 expression in human umbilical vein
endothelial cells (HUVEC) was upregulated by tumor cell-conditioned medium. The effect of
tumor cell-conditioned medium on ESM-1 expression was significantly inhibited by the addition
of neutralizing anti-VEGF antibody. Moreover, treatment of HUVEC with VEGF resulted in a doseand
time-dependent increase in ESM-1 mRNA. Induction of ESM-1 in HUVEC by tumor
cell-conditioned medium or VEGF was blocked by inhibitors of PKC, but not by inhibitors of
ERK1/2 MAPK or PI3K signaling pathways. Finally, the addition of conditioned medium from
primary human keratinocyte and human kidney tubular cells to HUVEC inhibited ESM-1
expression, an effect that was prevented by preincubation with neutralizing anti-EGF antibody.
Taken together, these results suggest that ESM-1 is a marker for tumor endothelium and that
its vascular bed-specific expression is regulated by the microenvironment.
P419
The Role of Sphingolipids in Apolipoprotein-Mediated Cholesterol Efflux to
Apolipoprotein-AI
Scott Witting, W Sean Davidson. University of Cincinnati, Cincinnati, OH
Recent evidence indicates that the ATP-binding cassette transporter AI (ABCAI) is critical for the
lipidation of apoA-I. It is thought that apoA-I may first obtain phospholipids and then receive
cholesterol secondarily as these two steps can be pharmacologically uncoupled. However, the
cholesterol pool(s) and the cellular signaling processes utilized by the ABCAI-mediated pathway
remain unclear. One potential factor governing membrane cholesterol availability is membrane
sphingomyelin content. Cholesterol and sphingomyelin are known to exhibit a particularly close
interaction. In addition, the reduction of sphingomyelin into its bioactive catabolites may also
play a role in cholesterol availability to the ABCAI pathway. To study this potential role of
sphingolipid metabolites, we treated human aortic smooth muscle (aSMCS) and Chinese
hamster ovary (CHO) cells with 0–20 M of C2-ceramide, a cell permeant ceramide, and
measured 3H-cholesterol efflux to 10 g/ml apoA-I over 24 hrs. These cell types were chosen
because a recent study has shown both to contain a significant basal level of ABCAI mRNA
without pharmacological treatment (i.e. cAMP, retinoic acid). Treatment of aSMCs with
C2-ceramide caused a dose dependent increase in cholesterol efflux to apoA-I with a maximum
increase of 2.5 fold. In CHO cells, the maximum increase in cholesterol efflux was 5 fold. To
test if the C2-ceramide induced cholesterol efflux was due to a membrane perturbation effect
and specific to the apolipoprotein-mediated pathway, apoA-I was replaced with 25 g/ml
phospholipid vesicles or 0.5 mM methyl-cyclodextran. No increase in cholesterol efflux to the
non-specific acceptors was noted in either cell type. Cell viability was evaluated by a metabolic
activity assay (MTT test) and was 90% in all experiments. Western blot analysis showed that
cholesterol efflux changes were not due to increased expression of ABCAI. We conclude that
metabolites of sphingomyelin may play a role in the regulation of the cholesterol pool(s)
available for ABCAI-mediated removal.
P420
Transcriptional Profiling and Quality Analysis of Microarrays Applied to the
Gene Expression of Vascular Graft Neointima under Normal and High Blood
Flow Conditions
Patrick C Hsieh, David Hasenstab, Richard D Kenagy, Eileen R Mulvihill, Alexander W
Clowes. University of Washington, Seattle; University of Washington, Seattle, WA
Neointimal hyperplasia is the major etiology of long-term failure of vascular stents and grafts.
In a baboon model of bilateral aortoiliac bypass with polytetrafluoroethylene grafts, a switch
from normal to high blood flow, created in one graft by a distal arteriovenous fistula, induces
neointimal atrophy. The objective of this study is to characterize the genes induced or
suppressed by increased blood flow. Grafts under normal and high flow conditions were
harvested at 1, 4 and 7 days after flow switch (5 baboons per time) and subdivided into a
portion for microarray and a portion for immunohistochemistry (ICC). Intimal RNA was extracted
and pooledby into guest 6 groups on (2 April flow conditions, 4, 2013 3 time points) to make probes for microarrays.

a-74 Arterioscler Thromb Vasc Biol. Vol 22, No 5
Each probe was hybridized onto 4 replicate sets of microarrays consisting of about 30,000
genes or ESTs per set (GF 200–205, Research Genetics). Hybridization images were collected
and analyzed by Pathways 3.0 software (Research Genetics). Regulated genes were selected
with a 95% confidence level and a minimal intensity of 0.25. Several genes were further
confirmed by RT-PCR and ICC. False positive frequency was 3.1% (14 of 467) with 2.4%
coming from hybridization variability (comparing the same probes with replicate filters), 0.6%
from image collection (same filters different exposure times) and 0.1% from image alignment
variability. Two percent (453 of 30,000) of genes were up- or down-regulated by high blood
flow and were classified into several groups, including proteases (e.g. chymotrypsin-like
protease, upregulated 2.4 fold), cell proliferation and death (e.g. PDGF-B, upregulated 2.1 fold),
signaling mediators (e.g. MAPK kinase, upregulated 1.9 fold), and extracellular matrix (e.g.
thrombospondin 4, upregulated 2 fold). We concluded that flow-induced neointimal regression
is a dynamic process involving multiple spatially and temporally coregulated pathways.
P421
Chlamydia pneumoniae Infection Induces a Shift in Atherosclerotic Lesion
Type and an Increase in T-Cell Influx in Atherosclerotic Lesions of APO
E*3-Leiden Mice
Rajaa Ezzahiri, Harrie Kurvers, Gert Grauls, Peter Kitslaar, Cathrien Bruggeman.
Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands
Atherosclerosis is an inflammatory process and is characterised by presence of influx of
T-lymphocytes in the lesions. To study the role of Chlamydia pneumoniae(CP) in this process
and the effect of infection on T-cell influx, we infected APO E*3-Leiden mice with CP and
investigated the effect on lesion development and T-cell influx in atherosclerotic lesions at
different time points post infection (pi). Methods: Nine week old APO E*3-Leiden mice were
either mock infected or infected twice with CP (strain TWAR 2043, 3.10 7 IFU i.p., n8/group)
and sacrificed at 1, 4, 6 and 9 months pi. Mice were on an atherogenic diet. Longitudinal
sections of 4m of the aortic arches of the mice were stained with hematoxylin-eosin to
analyse atherosclerotic lesion type and lesion area, or with rabbit-anti-CD3 to detect the
presence of CD3 T-cells in the atherosclerotic lesions. T- cell influx was expressed as
number of CD3 cells in lesion/lesion area. For statistical analysis Mann-Whitney test (T-cell
influx) and Chi-square test (aortic lesion type) were used. Results: All mice developed
atherosclerotic lesions. At 1 month pi type 1, 2 and 3 lesions were present in all mice. At 4,
6 and 9 months pi also type 4, 5a and 5b lesions were present. Infection with CP resulted in
a shift in the lesion formation respectively from type 3 to type 4 (p 0.022) at 6 months pi,
and from type 4 to type 5a (p0,002) at 9 months pi. Positive CD3 cells were observed at every
time point pi. At 1 month pi a significant increase in T-cell influx in atherosclerotic lesions was
observed (p 0.0005). In these lesions T-cells were concentrated in the subendothelial layer
of the plaque. Conclusion:This study shows that CP infection stimulates the formation of more
complex lesions and enhances the inflammatory process as shown by the increased influx of
T-lymphocytes in the atherosclerotic lesions.
Aggrenox Inhibits the Synthesis of Cytokines Generated by
Platelet-Monocyte Aggregates
Andrew S Weyrich, Jennifer R Eyre, Stephen M Prescott, Guy A Zimmerman, Wolfgang G
Eisert. University of Utah, Salt Lake City, UT; Boehringer Ingelheim GmbH, Ingelheim,
Germany
P422
Cigarette smoke, oxidized phospholipids, and thrombin induce inflammatory reactions that
increase the number of platelet-leukocyte aggregates found in circulating blood. The formation
of these aggregates results in the synthesis of inflammatory gene products that are associated
with increased cardiovascular disease. Here we test whether aggrenox, a fixed-dose
combination of dipyridamole (DIP) and aspirin (ASA), inhibits the synthesis of inflammatory gene
products that are generated by platelet-monocyte aggregates. Human platelets and monocytes
were pretreated with DIP (5 g/ml), ASA (625 ng/ml), or a DIP/ASA mixture (aggrenox; an 8:1
ratio of DIP/ASA). The cells were subsequently stimulated with thrombin and the synthesis of
cyclooxygenase-2 (COX-2), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1)
was measured in the cell suspensions. Thrombin did not induce COX-2, IL-8, or MCP-1 in
platelets or monocytes incubated separately from one another. Co-incubation of the platelets
with the monocytes increased the number of intercellular aggregates and the expression of
COX-2, IL-8 and MCP-1 in vehicle treated cells. Pretreatment with aggrenox or DIP, but not
ASA, blocked the synthesis of MCP-1 (p0.05) over an 18 hour time period (MCP-1 ng/ml;
untreated 47123503; aggrenox 734622; DIP 514317; ASA 50583876
[n6]). Synthesis of IL-8 was completely blocked at 2 hours and markedly reduced after 4
hours in the presence of aggrenox or DIP. ASA had no inhibitory effects on IL-8 synthesis at
any time point. None of the drugs inhibited IL-8 synthesis at later time points nor did they block
the formation of COX-2 demonstrating that some gene expression pathways remain intact
following drug treatment. These results demonstrate that aggrenox differentially inhibits the
expression of inflammatory gene products generated by platelet-monocyte aggregates. The
inhibitory actions of aggrenox on gene expression are attributable to the dipyridamole
component of this drug, indicating that extended release dipyridamole/aspirin mixtures may be
more beneficial in preventing secondary strokes and associated cardiovascular events than
aspirin alone.
not been fully defined. The present study was carried out to determine the effect of taxifolin
on key lipogenic enzymes involved in triglyceride synthesis and secretion, namely DGAT
(diacylglycerol acyltransferase) and MTP (microsomal triglyceride transfer protein), in the
human hepatoma cell-line, HepG2. Cells pretreated with taxifolin were initially shown to inhibit
triglyceride synthesis and secretion in a dose-dependent manner, with an optimal inhibition on
synthesis and secretion of 59Â 1% and 68Â 3%, respectively, at 200 M observed within
24h. At this concentration, cell viability was not compromised. As to the mechanism underlying
the inhibitory effect of taxifolin on triglyceride secretion, taxifolin was shown to inhibit the
activity of both DGAT (60Â 2%) and MTP (27Â 1%). The marked inhibition on DGAT activity,
as measured by esterification of 1,2 diacylglycerol using radiolabeled palmitoyl-CoA, appeared
to shift metabolic pathways from triglyceride to phospholipid synthesis. Phospholipid synthesis
was found to be only slightly inhibited (15Â 2%), despite a 57Â 1% inhibition on secretion.
The effect on DGAT activity by taxifolin was shown to be dose-dependent and to be specific as
other flavonoids, including quercetin and genistein, did not reduce its activity. Taken together,
the data suggests that taxifolin reduces triglyceride-rich lipoprotein secretion by inhibiting both
triglyceride synthesis via DGAT and lipidation of the lipoprotein particle via MTP. This study
suggests a potential role of plant flavonoids in the treatment of hypertriglyceridemia.
P424
Sexually Dimorphic and Time-Windows Response of the Balloon-Injured
Rat Carotid Artery to HMG-CoA Reductase Inhibitor Treatment
Tatsuhiko Mori Sr, Tetsuya Hayashi, Yasushi Kitaura. Osaka Medical College, Takatsuki,
Japan
Background: HMG-CoA reductase inhibitor (HMG) has shown to have vasoprotective effect
independent of cholesterol lowering. The current study was designed to test whether HMG
possesses sexually dimorphic pattern and short-term treatment effects for vasoprotection.
Methods: Ovariectomized female Sprague-Dawley rats (OVX) were randomly divided into four
with the following time windows of HMG (cerivastatin 1mg/kg/day (Ce)) treatment (n4–6
each). (1) Vehicle (V), (2) From 3 days before to 14 days after balloon injury (-3D to 14D), (3)
-3D to 3D and (4) 7D to 14D. Male rats were divided into two groups like OVX. (5)Intact male
(M)V and (6) MCe(-3Dto14D). Two weeks after balloon injury of the right common carotid
artery, neointimal thickening of the artery was evaluated using intima to media ratio (I/M).
Results and Conclusion: The summarized figure shows that the vasoprotective effects of HMG
depend on the timing of treatment and do not need prolonged treatment throughout the period
of neointimal formation. Furthermore, vasoprotective effects of HMG were more potent to
female than male. These findings have important clinical implications for the potential use of
HMG in the prevention and treatment of vascular disease.
P425
SB 242784, a Selective Inhibitor of the Osteoclastic V-H ATPase, Inhibits
Artery Calcification at Doses That Inhibit Bone Resorption
Paul A Price, Helen H June, Jessica R Buckley, Matthew K Williamson. University of
California, San Diego, La Jolla, CA
Secretion of Hepatic Triglyceride-Rich Lipoprotein Is Inhibited by the
Flavonoid, Taxifolin, via Reduced DGAT and MTP Activity
P423
The present experiments were carried out to test the hypothesis that artery calcification is
linked to bone resorption by determining whether the selective inhibition of bone resorption
with SB 242784, a specific inhibitor of the osteoclast V-H
Andre Theriault, Adele Casaschi, Dan Ota. University of Hawaii, Honolulu, HI
Taxifolin, a plant flavonoid, has recently been shown to reduce hepatic triglyceride synthesis
and secretion. However, the mechanisms responsible for this hypotriglyceridemic effect have
-ATPase, will inhibit artery
calcification. Artery calcification was induced by treating 49-day-old male rats with toxic doses
of vitamin D. Treatment for 96h with vitamin D caused widespread Alizarin red staining for
calcification in the aorta and the femoral, mesenteric, hepatic, renal, and carotid arteries, and
SB 242784 completely prevented calcification in each of these arteries at a dose of
40mg/kg/day, and significantly reduced calcification at a dose of 10mg/kg/day. Treatment with
vitamin D also caused extensive Alizarin red staining for calcification in the lungs, tracheal
cartilage, and kidneys, and treatment with SB 242784 prevented or reduced calcification at
each of these sites. Chemical analyses showed that treatment with vitamin D alone increased
the level of calcium in the abdominal aorta, lung, kidney, and trachea by 10- to 40-fold, and
that concurrent treatment with the 40mg/kg/day dose of SB 242784 reduced calcium to control
levels. Measurement of serum levels of cross-linked N-teleopeptides, a specific measure of
bone resorption activity, further showed that treatment with vitamin D alone increased bone
resorption activity by 2.5-fold, and that concurrent treatment with the 40mg dose of SB 242784
reduced bone resorption activity to below control levels. We conclude that doses of SB242784
that inhibit bone resorption in normal rats are able to potently counteract both effects of vitamin
D treatment, the dramatically increased rate of bone resorption and the extensive calcification
of arteries. These results are consistent with earlier work showing that the specific inhibition
of bone resorption with amino bisphosphonates or with osteoprotegerin completely inhibits
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817–824 and 1610–1616).

P426
Comparison of Endothelial Functions in Patients with Established CAD and
Individuals at Risk for CAD in Indian Population Using Brachial Artery
Flow-Mediated Vasodilatation
Sharad Tandon, Kartikeya Bhargava, Ravi R Kasliwal, Naresh Trehan. Escorts Heart Institute
and Research Centre, New Delhi, India
BACKGROUND: Endothelial dysfunction has been hypothesized as an initial step in the
atherogenesis.Endothelial assessed by brachial artery FMD has been shown to correlate with
coronary endothelial functions. Comparative studies of enothelial function assessment using
brachial artery FMD in Indian subjects with and without CAD are lacking. AIMS: To assess and
compair endothelial functions in patients with established CAD and those with risk factors for
CAD. METHODS: Endothelial functions were assessed in 170 individuals by flow mediated
vasodilation (FMD) in brachial artery. Individuals were divided into 3 groups- normal controls
grp1(n45), individuals with risk factors grp 2 (n58) and patients with CAD grp 3(n67).
Clinical evaluation, ECG, TMT and lipid profile were performed in all and coronary angiography
was done in indicated individuals. Endothelial functions were assessed using brachial artery
doppler evaluation at baseline and post-tourniquet release. RESULTS: As per table CONCLU-
SIONS: Endothelial functions are significantly depressed in patients with CAD and in individuals
at risk as compared to controls. Suppression of endothelial functions in individuals at risk for
CAD may be a marker of subclinical atherosclerosis.Role of FMD as a prognostic marker in
these individuals need to be evaluated in long term prospective studies.
PDGF and IGF-1 Differentially Regulate Phosphatidylinositol 3-Kinase
Activity in Human Aortic Smooth Muscle Cells
Lakshman Sandirasegarane, Mark Kester. Penn State College of Medicine, Hershey, PA
P427
Previous studies have shown that tyrosine kinase-linked receptor agonists, PDGF, IGF-1 and
insulin induce phosphatidylinositol 3-kinase (PI3K)-dependent activation of Akt / protein kinase
B in vascular smooth muscle cells (VSMC). However, the differences in the signaling
components that mediate PDGF and IGF-1 -induced activation of PI3K still remain to be verified.
The aim of the present study is to compare the relative increases in PDGF and IGF-1 -stimulated
PI3K activity in VSMC, using the immunocomplexes obtained with antibodies specific for
phosphotyrosine, p85alpha (SH2-domain containing regulatory subunit of class 1A PI3K), focal
adhesion kinase (FAK) and insulin receptor substrate-1 (IRS-1). Stimulation of human aortic
VSMC with PDGF (1 nM, 5 min) led to a pronounced increase in phosphotyrosine-associated
PI3K activity (23-fold, p 0.05, n 4), with modest increases in PI3K activity associated with
FAK (9-fold), p85alpha (3.2-fold) and IRS-1 (2.5-fold). In contrast, exposure of VSMC to a similar
concentration of IGF-1 (1 nM, 5 min) resulted in marked elevation of IRS-1-associated PI3K
activity (41-fold, p 0.05, n 5), with modest increases in PI3K activity associated with
phosphotyrosine (2.2-fold) and FAK (2.3-fold). Additionally, IGF-1 did not stimulate p85alphaassociated
PI3K activity. Exposure of VSMC to insulin (100 nM) produced a pattern of PI3K
signaling similar to that observed for IGF-1. Pretreatment of VSMC with PI3K inhibitor,
LY294002 (100 microM, 1 hr), led to complete inhibition of PDGF-stimulated, phosphotyrosineassociated
PI3K and IGF-1-stimulated, IRS-1-associated PI3K activity respectively. The present
data suggest that stimulation of human aortic VSMC with PDGF or IGF-1 may lead to the
differential recruitment of multiple SH2-domain containing proteins, other than p85alpha, to
tyrosine phosphorylated proteins to induce PI3K. Furthermore, the differences in growth
factor(s)-induced PI3K signaling may, in part, be attributable to differential regulation of VSMC
phenotypes by PDGF (competence growth factor) and IGF-1 (progression growth factor).
P428
Association of Hepatic Lipase Activity with the LIPC Gene Polymorphism
(C-514T) in Japanese, Black and Caucasian Americans
Molly C Carr, Samir S Deeb, John D Brunzell. University of Washington, Seattle, WA
A common genetic variant in the LIPC gene promoter (-514) has been reported to contribute
to variance in hepatic lipase activity (HLA). This study was designed to evaluate whether the
relationship of the LIPC polymorphism to HLA was similar in three different ethnic groups,
Poster Presentations a-75
Japanese (JA) (n79), Black (BA) (n70) and Caucasian (CA) (n106) Americans. Post
heparin hepatic lipase activity and LIPC genotype (C-514T) were obtained in all subjects. The
allele frequencies of the LIPC T allele in all three ethnic groups were consistent with those
published previously (Blacks 0.45, Japanese 0.50, Caucasians 0.22). We have shown that HLA
is partially determined by a common LIPC gene polymorphism in all three ethnic groups
studied. This is the first time this association has been reported in Japanese. The relationship
of the LIPC gene polymorphism with HLA is similar in Japanese to those reported in Blacks and
Caucasians. All three ethnic groups appear to have a dose effect of the T allele on HLA.
P429
Altered Matrix Metalloprotease-2 Regulation and Tissue Angiotensin and
Age-Associated Aortic Remodeling in Non-Human Primates
Mingyi Wang, Gen Takagi, Kuniya Asai, Dorothy E Vatner, Filipinas F Natividad, Edward G
Lakatta. National Institute On Aging, Baltimore, MD; Hackensack University Medical Center,
Hackensack, NJ
The diffuse arterial intimal thickening that occurs with aging, is considered to place these
vessels at high risk for the subsequent development of atherosclerosis. Recent studies indicate
that age-associated aortic intimal thickening occurs in a non- hypertensive, non-human
primates and is associated with endothelial dysfunction. The present study extends the
investigation of age associated intimal thickening in this monkey model. Specifically MMP-2
activity and its regulators, i.e., MT1MMP and TIMP-2, and angiotensin and ACE were measured
via immunohistochemical techniques in aortae of younger (aged 6.40.7 years) and older
(20.0 .9 years) Macaaq monkeys. With aging: the intimal thickness increases 3.2-fold and
contains numerous VSMC, but no inflammatory cells; Intimal MMP-2 antibody staining
increased by 80% (p 0.01)and, in situ zymography showed that MMP-2 activity increased
3-fold (p 0.01). Intimal MT1-MMP antibody staining increased by 150% (p 0.001), but the
TIMP-2 antibody staining did not change; Intimal steady levels of mRNA (via in situ
hybridization) for MMP-2 increased 7 fold, for MT1-MMP increased 9 fold and for TIMP-2 2 fold
(all p 0.001). Angiotensin II (Ang II) immunofluorescence was not detectable in the younger
intima, but was abundant in the older intima. AngII fluorescence co-localized not only with
angiotensin converting enzyme (ACE), but also with MMP-2 fluorescence. Thus,in the thickened
intima of the old primate aorta, MMP-2 transcription, protein levels and activity increase. The
later may be attributable to the observed changes in factors that regulate MMP-2 activity. The
increased MMP-2 activity within the aged intima may be involved in age-associated vascular
structural remodeling and altered vascular functions, e.g., fragmentation of the intimal elastic
membrane and facilitated migration of VSMC of the media into the intima. The co-localization
of Ang II, ACE and MMP-2 is intriguing, as Ang II signaling is known to directly and indirectly
modulate MMP-2 activity in addition to its multiple additional actions that affect vascular
structure.
P430
Smoking Is an Independent Predictor of Measurable Aortic Calcification
Ganesh Raveendran, Thomas Knickelbine. Minneapolis Heart Institute, Minneapolis, MN
Background: Electron beam computed tomography (EBCT) is a noninvasive tool for evaluating
coronary and aortic calcification (AC). While smoking is a strong risk factor for the development
of symptomatic peripheral vascular disease, there is no consensus on the traditional risk factor
predictors, prevalence, clinical significance, and long term outcome of AC detected by EBCT.
Methods: We evaluated AC in 6990 consecutive asymptomatic patients who underwent EBCT.
Baseline characteristics and risk factor profiles for coronary artery disease including smoking
history was obtained. Significant AC was defined as an AC score of 49 and was compared
for patients with smoking history and non-smokers. Multivariate analysis was performed using
a logistic regression model. Results: AC score was 49, in 2286 (32.7%) patients. Of the
smokers, 40.3% had significant AC, compared with only 27.8% of the patients with no smoking
history (Univariate chi square, p 0.001). After adjusting for age, gender, diabetes,
hypertension and hyperlipidemia, smoking was a significant multivariate predictor of AC (p
0.037). Conclusion: AC is relatively common (33%) in asymptomatic patients. Our data suggest
smoking is an independent risk factor for AC and that AC may be an early marker of
atherosclerosis and peripheral arterial disease. Further prospective studies are needed for
assessment of AC as an indicator of future symptomatic peripheral vascular disease and
precursor of atherosclerosis.
Gender and Apolipoprotein E Genotype Modulate the Impact of the
Apolipoprotein A-IV Q360H Polymorphism on Plasma High Density
Lipoprotein Cholesterol Levels
Brian J Mackenzie, Rachel A Anderson, Victoria R Cook, Richard B Weinberg. Wake Forest
University School of Medicine, Winston-Salem, NC
Background: The apo A-IV Q360H polymorphism has been reported to be associated with
increased plasma HDL concentration in some population studies, but not in others, suggesting
that other genetic factors could modulate its impact. Apo E genotype has multiple effects on
lipoprotein metabolism, yet to date, no study has examined its interaction with the apo A-IV
Q360H polymorphism on plasma lipids. Methods: We measured fasting plasma lipids, and
determined apo E genotype and the presence of the apo A-IV Q360H polymorphism by
PCR-RFLP analysis, in a cohort of 238 male and 162 female subjects. Single factor effects on
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gender-allele and allele-allele interactions
P431

a-76 Arterioscler Thromb Vasc Biol. Vol 22, No 5
were analyzed by two-way ANOVA. Results: Apo A-IV allele frequencies were f0.91 and
f0.09. Apo E allele frequencies were f®0.09, f0.77, and f0.14. In the whole cohort
of 400 subjects, females had higher HDL (p0.001) and lower triglyceride (p0.001) levels
than males, and LDL levels were related to apo E genotype in order 2/33/33/4 (p0.001),
but the apo A-IV 360-H allele had no impact on plasma lipids. Moreover, no gender x A-IV allele
or gender x E allele interactions were found. However, a significant A-IV x E allele interaction
was noted in males: in males with the apo E 2/3 genotype, those with the apo A-IV Q/H
genotype had lower HDL levels than those with the apo A-IV Q/Q genotype (31.5 4.2 vs
41.8 2.2, p0.03); a trend to higher HDL levels was noted in male Q/H subjects carrying
an apo E4 allele (41.7 3.8 vs 37.3 1.6). There was no A-IV x E allele interaction on plasma
lipids in females. Conclusions: The apo A-IV Q360H polymorphism is associated with lower HDL
levels in males carrying an apo E2 allele and higher HDL levels in males carrying an apo E4
allele, suggesting that a complex (Gender) x (A-IV allele) x (E allele) interaction may modulate
HDL metabolism. This also suggests that the apo E allele frequencies and gender ratios in
previously reported cohorts may have determined whether the apo A-IV Q360H polymorphism
was observed to affect plasma HDL concentration.
Hypertrophic Flow-Induced Arterial Remodeling Increases with Age in
Hyperlipidemic Mice
Henny Schulten, Rob Hilgers, Paul Schiffers, Gregorio Fazzi, Ebo De Muinck, Jo De Mey.
University Maastricht, Maastricht, Netherlands
P432
We used unilateral carotid artery ligation to study the influence of aging and hypercholesterolemia
on flow-induced arterial remodeling in normal mice and mice deficient in apolipoprotein
E (apoE-/-). We measured two-dimensional adaptations of the carotid artery due to chronic
changes in blood flow in young adult (approximately 6 months) and old (approximately 14
months) wild type mice (apoE/) on a normal diet, apoE/ mice on a high fat diet (D) and
apoE-/- mice on D. In all mice, left carotid artery (LCA) ligation eliminated BF in the occluded
vessel and nearly doubled BF in the contralateral right carotid artery. In young apoE/, LCA
outer diameter (OD) was significantly reduced (56412 mm versus 7135 mm), and RCA-OD
was significantly increased compared to sham arteries (7759 mm versus 7135 mm). The
same trend was observed for young apoE/ and apoE-/- on D. In old mice, LCA-OD was
significantly reduced in all experimental groups. However, RCA-OD was not modified in old
apoE/ on normal chow, but was significantly increased in the other mice. Four weeks of
left carotid artery ligation had no effect on media cross-sectional area (CSA) of both LCA and
RCA in young apoE/. However, in old mice hypertrophic inward remodeling was observed
in the LCA, and outward hypertrophic remodeling in the RCA. This same trend of inward
hypertrophic remodeling of the LCA was observed in young apoE/ and apoE-/- both on D.
Aging augmented this hypertrophy. An outward remodeling was observed for all apoE/ on
D and apoE-/- on D, aging and hyperlipidemia did not induce hypertrophy of the media due to
a 4-week exposure to a doubling of BF. These observations demonstrate that age and diet
determine the nature of flow-induced arterial remodeling in mice. In young adult mice
flow-induced arterial remodeling does not involve a change in arterial wall mass. Aging and
hyperlipidemia induce hypertrophic arterial remodeling, and these effects are more pronounced
during chronic reductions in BF.
P433
Symmetry of Atherosclerotic Plaque Volume and Calcification in Human
Cadaveric Carotid Arteries
Gareth J Adams, Giles W Vick III, Cassius B Bordelon Jr, Kay T Kimball, William Insull Jr,
Joel D Morrisett. Baylor College of Medicine, Houston, TX
Background: Atherosclerosis is the primary causes of stroke and myocardial infarction. The
carotid arteries provide a site at which progression of atherosclerosis can be monitored
noninvasively. This study was conducted to determine the degree of similarity of atherosclerotic
plaques in the left and right carotid arteries. This question was explored using perfusion-fixed
cadaveric carotid arteries and two noninvasive clinical imaging techniques, magnetic resonance
imaging (MRI) and electron-beam computed tomography (EBCT). Methods: Fifty pairs of
cadaveric carotid arteries were imaged using MRI and EBCT. Thirty-eight pairs contained an
entire plaque and were suitable for rigorous analysis. Carotid artery volumes were measured
from the MRI images using an active contour algorithm. Plaque volume was estimated using
an automated algorithm. Both Agatson and volumetric calcification scores were computed from
the EBCT images. Aggregate volumes for nine contiguous slices surrounding the bifurcation
were computed for each sample. Similarity of volume measurements and calcification scores
of the left and right carotids within the cadaveric pairs was quantified using Lin’s concordance
correlation (LCC) algorithm. Results: The total wall volumes of the left and right carotid arteries
were moderately correlated, with a LCC coefficient of 0.71. The plaque volumes were
moderately correlated, with a LCC coefficient of 0.58. Calcification scores were highly
correlated, with LCC coefficients of 0.95 for the Agatston scores and 0.94 for the volumetric
calcium scores. Conclusions: Noninvasive medical imaging techniques provided a nondestructive
method of measuring carotid artery volumes and quantitating calcification, a significant
component of atherosclerotic plaque. Analysis of the results suggests that carotid artery
atherosclerosis in elderly humans is moderately symmetric. These results suggest that
diagnostic information about atherosclerotic plaque in one carotid artery can be used to infer
information about the composition and volume of atherosclerotic plaque in the contralateral
artery.
P434
Role of Cysteine(Cys)31 and Cys184 in Optimizing the Anti-Inflammatory
Properties of Lecithin:Cholesterol Acyltransferase
Zhen Jia, Trudy M Forte, John K Bielicki. Lawrence Berkeley National Laboratory, Berkeley,
CA
Previous studies have shown that lecithin:cholesterol acyltrasferase (LCAT) can hydrolyze the
potent pro-inflammatory mediator, platelet-activating factor (PAF), in an apolipoprotein A-I
(apoA-I) independent reaction. The present study was undertaken to determine the structural
features of LCAT that influence PAF hydrolysis. A recombinant LCAT (rLCAT) enzyme and a
Cys31 3Gly, Cys184 3Gly double mutant were created, expressed in CHO-K1 cells, and
purified to homogeneity from conditioned medium. PAF hydrolysis was assessed using a
standard micelle substrate composed of labeled [3H]- and unlabeled-PAF. Both enzyme’s
hydrolyzed PAF in a dose-dependent manner; however, the double mutant consistently yielded
50 12% (n3, p0.01) less activity regardless of protein concentration. In contrast to
PAF-hydrolysis, the acytransferase activity, measured using an exogenous proteoliposome
substrate, was the same for both enzymes (8.5 1.6 versus 8.6 1.7 % cholesteryl ester
formed/h/20 mg for rLCAT and double mutant, respectively) indicating that the enzyme’s free
thiols played a specific role in maximizing the hydrolysis of PAF but not in cholesterol
esterification. ApoA-I stimulated PAF hydrolysis in a dose-dependent manner; enzymatic
activity of both enzymes was enhanced 2-fold using 40 mg/ml of apoA-I, but the double mutant
always had 50% lower activity compared to rLCAT. We conclude that LCAT possesses a
“PAF-acetylhydrolase like” activity in the absence of apoA-I, but the presence of apoA-I can
further stimulate this activity. The data suggest that Cys31 and Cys184, which are located at
the active site boundary of the enzyme, may play an important role in modulating the overall
PAF-acetylhydrolase activity of LCAT, perhaps by optimizing interaction of PAF with the catalytic
triad (Asp-Ser-His).
P435
Growth Enhancing Effects and Cytoskeletal Protein Interactions of AIF-1 in
Human Coronary Artery Smooth Muscle Cells
Michael V Autieri, Karl W Wendt. Temple University School of Medicine, Philadelphia, PA
Background: Characterization of proteins which regulate vascular smooth muscle cell (VSMC)
proliferation in response to injury is essential in understanding the pathogenesis of vascular
proliferative diseases. In particular, cytoskeletal elements are key regulators of the VSMC
response to injury. Allograft Inflammatory Factor-1 (AIF-1) is a cytoplasmic, calcium binding
protein which is expressed in aortic VSMC by allograft and balloon angioplasty injury. AIF-1 is
not present in cultured human VSMC but is induced by cytokines and is associated with
proliferative gene expression. The objectives of this study are to determine if over expression
of AIF-1 influences VSMC growth and to ascertain a potential mechanism of this growth
enhancement. Methods and results: We stably transduced primary human VSMC with retrovirus
containing the AIF-1 protein coding region (AIF-RV) and found that AIF-RV containing cells grew
an average of 47% faster than empty-RV control cells (p0.05 in three experiments). This
effect is more pronounced in serum reduced media, where AIF-RV cells grow an average of
90% more rapidly than empty-RV cells (p0.05 in three experiments). Up regulation of AIF-1
protein in transduced VSMC was verified by western blot. To elucidate a mechanism for this
growth enhancement, we identified AIF-1-interacting proteins by AIF-1/GST fusion protein
affinity. Several AIF-1-interacting proteins were identified which interact in an activationdependent
fashion, including those migrating at 115, 42, and several in the 25–10 kDa range.
Trypsin digestion and MALDI-TOF mass spectrometer amino acid analysis identified the most
abundant of these proteins as beta actin. This interaction was verified by coimmunoprecipitation
using specific antibody, and colocalization with F-actin in cultured human
VSMC. Conclusions: Many small cytoplasmic actin-binding proteins are involved in VSMC
signaling, proliferation, and pathophysiology. Considering the AIF-1 expression pattern in
injured VSMC, the data presented here suggest that AIF-1 may be involved in the cytoskeletal
signaling network leading to vascular proliferative diseases.
P436
Evidence That Catalytically Inactive Hepatic Lipase Can Be Used to Treat
Hypercholesterolemia
Kun Qian, Helén L Dichek. University of Washington, Seattle, WA
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Hepatic lipase (HL) lowers cholesterol via both catalytic and noncatalytic mechanisms. The
noncatalytic mechanism can lower cholesterol independently of the LDLreceptor. In humans,
deficiency of the LDLreceptor causes familial hypercholesterolemia(FH) and results in
premature coronary artery disease. Therefore FH is a prime target for cholesterol lowering
therapies, including genetherapy. To prepare for genetherapy studies we characterized the
cholesterol lowering potential of a catalytically inactive mutant of HL(HLS145G). Thus, we
determined whether HLS145G predominantly lowers cholesterol associated with ApoB48-or
apoB100-containing lipoproteins by transgenically expressing HLS145G in LDLreceptor deficient
mice homozygous for wildtype mouse apob (Ldlr-/-b), apob48 (Ldlr-/-b48/), or
apob100 (Ldlr-/-b100/). To exclude the possibility that lipoprotein remodelling by mouse
hepatic lipase was a prerequisite for HLS145G mediated cholesterol lowering we also examined
hepatic lipase deficient Ldlr-/- mice(Ldlr-/-hl-/-) expressing HLS145G. Fasting plasma from
nontransgenic and transgenic mice was collected for total cholesterol and triglyceride
measurements (see Table) and fractionation by FPLC. Our results demonstrate that in the
absence of the LDL receptor, HLS145G expression lowers cholesterol by 20%. Our results
also show that the cholesterol lowering occurs independent of whether apoB48 or apoB100 is
the predominant apolipoprotein. These results indicate the potential utility of HLS145G

genetherapy to lower cholesterol in FH as well as in other conditions associated with
hypercholesterolemia.
P437
High-Flow Augments Myo-Endothelial Contacts in Rabbit Common Carotid
Arteries before the Initiation of the Gaps of Internal Elastic Lamina
Yasushi Asari, Akihiro Sugita, Masayo Murakami, Hiroshi Nanjo, Eiketsu Sho, Mikio
Kobayashi, Koichi Kawamura, Tatsuro Sugiyama, Hirotake Masuda. Akita University School
of Medicine., Akita, Japan; Yuri-Kumiai General Hospital, Akita, Japan
The gaps of internal elastic lamina (IEL) are known to be a key event of high-flow induced
arterial remodeling. To understand their morphogenesis, ultrastructural study combined with
3-dimensional reconstruction of the rabbit common carotid arteries were performed at 3 days
(one day before the initiation of the IEL gaps) of high-flow created by arterio-venous fistula
(n3). By high-flow, fenestrae of IEL significantly enlarged( 66.344.1m2 compared to
16.112.3 m2 in controls, p0.05), occupying 10.54.3% of the whole arterial surface(2.91.9%
in controls, p0.05). Proliferation of smooth muscle cells were often observed
just beneath the fenestrae of IEL. Endothelial protrusions came close to smooth muscle cells
to form myo-endothelial contacts (M-E contact) in almost all the fenestrae of IEL (96.80.6%,
compared to 28.420.2% in controls, p0.05). By high-flow, M-E contacts were augmented
both in numbers and average area (Numbers: 4927 1740/mm2, compared to 607472/
mm2 in controls. Average area: 2.030.52m2, compared to 0.840.53 m2 in controls,
both p0.05) At high magnification TEM, there were occasional close appositions of the
plasma membranes, resembling gap-junction. We suggest that high-flow augments M-E
contacts and that they may lead to tear IEL by transmitting the structural distortions of the
smooth muscle cells into the fenastrae of IEL when they proliferate after 3 days of high-flow.
P438
Atheroslerotic Plaques of Carotid Arteries and Dyslipidemia in Essential
Hypertension and Type 2 Diabetes Mellitus
Zhiming Zhu, Zhigang Zhao. Department of Hypertension and Endocrinology, Daping
Hospital, Third Military Medical University, Chongqing, Chongqing, China
Background: Hypertension and diabetes mellitus are predisposing risk factors for coronary
heart disease (CHD) and atherosclerosis. Although both are related with atherogenic dyslipidemia,
differences in the development of atherosclerosis between hypertension and diabetes
mellitus remain to be determined. The present study aims to evaluate whether dyslipidemia is
associated with atherosclerotic plaques of carotid arteries in patients with hypertension and
type 2 diabetes mellitus without and with hypertension. Methods: We performed color-flow
doppler-assisted duplex imaging of carotid arteries in patients with essential hypertension (EH,
n42), patients with type 2 diabetes mellitus without (DM, n26) and with hypertension (HD,
n39). Ultrasound intima media thickness and plaques of carotid arteries were measured.
Results: Level of fasting glucose was 5.4/-0.7 mmol/l in EH, 11.9/-4.8 mmol/l in DM and
10.5/-3.7 mmol/l in HD. Intima media thickness was 0.84/-0.31 mm in EH, 0.81/-0.32
mm in DM and 0.93/-0.2 mm in HD. Plaques in carotid arteries were significantly more
frequent in DM (50%) and HD (54%) compared with EH (33%, p0.05). Fasting levels of total
cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol were not significantly different
between the three groups (p0.05). Profile of dyslipidemia (hypertriglyceridemia [Ht],
hypercholesterolemia [Hc], combined hyperlipidemia [Ch]) were similar in three groups (EH: Ht
12.9%, Hc 19.8%, Ch16.4%; DM: Ht 16.3%, Hc 22.7%, Ch 12.8%; HD: Ht 11%, Hc 26.5%, Ch
22.2%). Conclusions: Compared with high blood pressure, these findings indicate that
atherosclerosis of the carotid artery is more susceptible to hyperglycemia under the similar
dyslipidemic circumstance (Supported by NSFC grant 39725013).
Endothelial Dysfunction in Patients with End-Stage Renal Failure
P439
Ruben Dammers, Jeroen M Hameleers, Jan H Tordoir, Rob J Welten, Arnold P Hoeks, Peter
J Kitslaar. University Hospital Maastricht, Maastricht, Netherlands; University of Maastricht,
Maastricht, Netherlands; Atrium Medical Center Heerlen, Heerlen, Netherlands
Objectives. Carotid artery vessel wall mechanic and hemodynamic properties decline in
patients with end-stage renal disease (ESRD). Endothelial dysfunction is hypothesized to be an
underlying cause. Brachial artery (BA) data, however, are scarse. The purpose of this study is
to compare BA vessel wall mechanic and hemodynamic properties in healthy volunteers (group
I) and patients with ESRD (group II). Methods. Both groups consisted of 10 age-matched men
(group I: 55.7 8.5 yrs, group II: 63.5 11.5 yrs; p 0.103). Every five minutes, blood
pressure was obtained with an oscillometric bloodpressure device. A Wall track system was
used to measure BA diameter (D), distension (d), and wall thickness (IMT)in the non-dominant
arm, from which compliance (CC) and distensibility (DC) could be obtained. Shear stress (SS)
was determined using a shear rate estimating system. Downloaded For statistical from
analysis an analysis of
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covariance was used. Findings. IMT was significantly higher in group II (412 vs 351 m; p
0.05). D was not significantly different (4.7 vs 4.4 mm; p 0.077). The same holds for d (
80 m). CC was 0.07 mm 2 kPa -1 in both populations and DC was lower in group II (3.4 vs
5.1 MPa -1 ;p 0.01). Mean SS was 50% less in group II compared to group I (0.25 vs 0.52
Pa; p 0.001). Conclusion. Brachial artery shear stress is lower in patients with ESRD,
corroborating with findings in the carotid artery. Except for the distensibility, which is
significantly decreased, mechanical properties are not different. The brachial artery tends to
have a lower sensitivity for lower shear stresses in ESRD patients. Therefore, these results
suggest that endothelial dysfunction in ESRD is also apparent in the brachial artery.
Skin Cholesterol Predicts History of Myocardial Infarction
Poster Presentations a-77
P440
Dennis L Sprecher, Gregory L Pearce, Shaun G Goodman, Paul Kannampuzha, Anatoly
Langer. Cleveland Clinic Foundation, Cleveland, OH; St. Michaels Hospital, Toronto, Canada;
Trillium Health Centre, Toronto, Canada; Canadian Heart Research Centre, Toronto, Canada
Background: We previously reported that skin cholesterol levels predict the presence of
angiographic disease. It remains unknown whether a correlation with MI history can be
discerned; particularly after adjustment for extent of angiographic disease. Methods: Skin
cholesterol measurement, coronary angiography, and traditional risk factor evaluation was
performed in 649 individuals, at 3 sites (CCF n147, THC. n179, SMH, n323). Skin
cholesterol was determined non-invasively using the Cholesterol 1,2,3 system (IMI). Angiographic
outcome was determined qualitatively and categorized as 0%, 1–49% or 50% for
the LAD, LCX and RCA. History of myocardial infarction (MI) was also recorded. Results: Skin
cholesterol quintiles were significantly associated with MI history. Once adjustment for extent of
disease was performed the relationship remained significant. (Table) Conclusion: Individuals with
higher skin cholesterol levels were significantly more likely to have had a previous MI than patients
skin cholesterol values in the lowest quartile, even after adjusting for severity of angiographic
disease. This suggests skin cholesterol as a potential predictor of acute coronary events.
P441 WITHDRAWN
P442
Overexpression of the Tumor Autocrine Motility Factor Receptor-gp78, a
Ubiquitin Protein Ligase (E3), Results in Increased Ubiquitination and
Decreased Secretion of Apolipoprotein B in Hep G2 Cells
Junshan Liang, Shengyun Fang, Allan M Weissman, Henry N Ginsberg. College of
Physicians and Surgeons of Columbia University, New York, NY; Regulation of Protein
Function Laboratory, Center for Cancer Research, National Cancer Institute, Bethesda, MD
Recently, we identified the tumor autocrine motility factor receptor-gp78 as a RING
finger-dependent Ub protein ligase (E3), the first mammalian ER resident E3 (Fang, Setal:
PNAS 2001, 98:14422–14427). This protein localizes primarily to the ER and targets itself and
a well-characterized ER-associated degradation (ERAD) substrate, CD3-delta, for proteasomal
degradation. We, and others, have shown that the degradation of apolipoprotein B (apoB) is
mediated mainly by the cytosolic ubiquitin-proteasome pathway. However, the mechanism
whereby the degradation of newly synthesized apoB is regulated by Ub protein ligases remains
unclear. In this study, we investigate whether overexpression of gp78 affects the ubiquitination
and degradation apoB in Hep G2 cells. After being transiently transfected with gp78, Hep G2
cells were labeled for 2 hr with 35S-Met. After labeling, cellular and medium apoB were
analyzed by immunoprecipitation. Ubiquitinated apoB was also analyzed by two-step immunoprecipitation.
Specific expression of gp 78 in Hep G2 cells after transfection was confirmed
by Western blot analysis with an anti-gp78 antibody. Overexpression of gp78 in Hep G2 cells
resulted in significantly decreased secretion of apoB. However, overexpression of Itch, a
cytosolic E3, had no effect on secretion of apoB. The decreased secretion of apoB by
overexpression of gp78 was reversed by ALLN, a proteasome inhibitor. Furthermore, results
from the two-step immunoprecipitation showed that ubiquitinated apoB molecules were
consistently increased in cells transfected with gp78 in the presence of ALLN. Overexpression
of gp78 had no effect on the secretion of albumin or apoAI. Together, these results indicate that
overexpression of gp78 resulted in decreased secretion of apoB via increased ubiquitination
and targeting of apoB for proteasomal degradation.
P443
Immunoaffinity Isolation of Endoplasmic Reticulum from Total Microsomes
Junji Yamaguchi, Junshan Liang, Henry N Ginsberg. College of Physicians and Surgeons,
Columbia University, New York, NY
The isolation of pure, functionally intact subcellular organelles, including endoplasmic reticulum
(ER), is imperative for the study of processes regulating the secretion of such proteins. The
most commonly used methods of separating subcellular organelles are based on available
centrifugation techniques. These methods are time-consuming and are poorly reproducible. We
previously described a technique for the immunoaffinity isolation of apolipoprotein B
(apoB)-containing microsomes (Mcs) (ER and Golgi) using anti-apoB antibodies (Ab). We have
modified that approach to develop a novel method for the immunoaffinity isolation of ER from
total Mcs using by guest anti-calnexin on April (CN) C-terminal 4, 2013polyclonal
Ab. CN is an integral membrane protein

a-78 Arterioscler Thromb Vasc Biol. Vol 22, No 5
only found in the ER, with its N-terminal domain inside the ER lumen and its C-terminal domain
exposed to the cytosol. Total Mcs were isolated in the S100 fraction of homogenized cells by
high speed centrifugation. Mcs were further fractionated by incubation with either anti-CN
C-terminal polyclonal Ab or anti-human albumin Ab at 4 °C for 3 h. The Mcs-Ab complexes
were precipitated by an additional 2 h incubation with protein G-Sepharose CL-4B. The
immunoaffinity-isolated Mcs were lysed and analyzed by Western blotting using anti-CN Ab as
an ER marker, and anti-TGN38 monoclonal Ab as a Golgi marker. Lysates from Mcs isolated
by anti-CN Ab strongly reacted with anti-CN Ab. In contrast, no reaction with anti-CN Ab was
observed with lysates of Mcs isolated by anti-human albumin Ab. Neither Mcs lysates
immunoisolated by anti-CN Ab nor Mcs lysates immunoisolated by anti-albumin Ab reacted
with anti-TGN38 Ab. ApoB-containing lipoproteins were extracted from Mcs immunoisolated by
anti-CN Ab and separated by sucrose gradient ultracentrifugation. These extracted lipoproteins
were found mainly in the density range of HDL particles. Together, these results indicate that
Mcs which are immunoaffinity-isolated by anti-CN C-terminal polyclonal Ab are pure ER, not
contaminated with Golgi compartments. This is a simple, quick method for isolation of pure ER
that will facilitate the study of the assembly of lipoproteins in cells.
P444
Differences in Body Fat Distribution and Postprandial Lipemia in Men with
or without Hypertriglyceridemia
Yangsoo Jang, Jong Ho Lee, Ha Jung Ryu, Oh Yoen Kim, Jey Sook Chae, Seok Min Kang.
Cardiovascular Genome Center, School of Medicine, Yonsei University, Seoul, Korea; College
of Human Ecology, Yonsei University, Seoul, Korea
Background: We aimed to evaluate the differences in body fat distribution and the effects of
high-fat meal on postprandial lipemia in normortriglyceridemic and hypertriglyceridemic men
who usually consume typical high carbohydrate diet. Methods & Results: Seventy-one men
aged 25–49 yr were divided into two groups; normal fasting TG150mg/dl (n46) and high
TG150mg/dl (n25). The macronutrient composition of the subjects’ usual diet was 64% of
energy from carbohydrate, 19% from fat and 17% from protein. A high-fat meal (HFM) for the
test composed of 28g fat, 10g protein, and 432kcal. Blood samples were withdrawn at 0, 2,
3, 4 and 6 hours after a HFM. High postprandial lipemia or high response was defined as
postprandial TG maxima above the 70 th percentile (200mg/dl) of TG maxima distribution after
a HFM in normotriglyceridemic men. Normotriglyceridemic men were subdivided into 32
normal responders (control) and 14 high responders. There were no significant differences in
mean age and body mass index between groups. Postprandial lipemia including TG and
chylomicron-TG was about 182% higher in the hypertriglyceridemic men and about 69% higher
in the normotriglyceridemic men with high response than in the control, respectively.
Postprandial insulinemia was about 59% higher in hypertriglyceridemic men than in the control.
Compared with the control, both groups of hypertriglyceridemic men and normotriglyceridemic
men with high response had greater visceral fat area at the L1 and L4 vertebrae, higher fasting
insulin and the area under the curve for insulin during oral-glucose-tolerance test, higher levels
of LDL, total cholesterol and apoB, and lower concentrations of HDL cholesterol and apoA1.
Conclusion: These results indicate that hypertriglyceridemic men and normotriglyceridemic
men with high response are associated with delayed clearance of postprandial lepemia in
response to HFM and harmful effects on the caridiovascular risk factors related to visceral
obesity, hyperinsulinemia and dyslipidemia.
P445
Mutagenesis Studies on ACAT1 Reveal Several Residues, but Not S269,
that may be Essential for ACAT Catalysis
Xiaohui Lu, Song Lin, Catherine C Chang, Ta-Yuan Chang. Dartmouth Medical School,
Hanover, NH
Acyl-coenzyme A: cholesterol acyltransferase (ACAT) plays an important role in cellular
cholesterol homeostasis and in early stages of atherogenesis. ACAT has been investigated as
a pharmaceutical target, yet its active sites remain largely unknown. Earlier studies by other
investigators revealed that when a conserved serine residue (S269 in human ACAT1) was
mutated to leucine, and when the S269L mutant was expressed in mammalian cells, loss of
ACAT activity was observed. In the current study we replaced each of the sixteen conserved
polar amino acids in ACAT1, including S269, with alanine or with other selected residues. The
resultant mutants were expressed in insect cells using the baculovirus expression system. The
ACAT activities were measured in vitro. Our results showed that the S269A and S269T mutants
were fully active; the S269L and S269C mutants retained 30% to 50% of the wild-type ACAT1
activity. We next expressed these mutants in ACAT deficient CHO cells, and showed that the
S269A, S269T, S269C mutants were all expressed and active, while the S269L mutant was not
expressed. Thus, our results indicated that S269 does not play an essential role in ACAT
catalysis, and that the loss of ACAT activity in the S269L mutant is mainly due to its poor
expression in mammalian cells. Using the baculoviral expression system, additional mutagenesis
studies showed that point mutations in any of the six residues K266, D400, N421, S456,
H460, and E461 caused severe loss in ACAT1 activity (by 99% or more). Among these residues,
H460 is an invariant residue within a membrane-bound O-acyltransferases superfamily. It is
located within a long hydrophobic region. Separate study from this laboratory showed that
mutation in the equivalent histidine in ACAT2 also caused severe loss in ACAT activity. These
data suggest that H460, as well as K266, D400, N421, S456, E461, may constitute as part of
the ACAT active site. (Supported by NIH HL 60306) Downloaded from
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P446
Plasma Kallikrein Activity Increases and a Novel Presumptive Lipase
Activity Decreases in Response to Hyperlipidemia in Atherosclerosis-Prone
Mice
Snehal T Patel, David R Ruecker, Tony E Hugli, Carole L Banka. La Jolla Institute for
Molecular Medicine, San Diego, CA
Hyperlipidemia has been implicated in hypercoagulability and endothelial dysfunction in
humans. We profiled protease activity generated in mice during lipid-induced endothelial injury
and inflammation. Upon fractionation of EDTA plasma from male C57BL/6J (BL6) and low
density lipoprotein receptor-deficient (LDLRo) mice, we identified two peaks of enzymatic
activity using chromogenic protease substrates. Inhibitor studies, substrate profiles and
Western blot analyses revealed the first peak to be kallikrein. Pre-kallikrein activation increased
dramatically after 48 hrs of feeding a high-fat, high-cholesterol diet (HFD). Kallikrein activity
was highest in LDLRo HFD LDLRo chow BL6 HFD BL6 chow. Using the
protease substrate, AGLTR-pNA, we detected enzymatic activity in the second peak that,
surprisingly, was not affected by the protease inhibitors corn trypsin inhibitor (CTI), PPACKII,
lima bean trypsin inhibitor (LBTI), Hirudin, or soybean trypsin inhibitor (SBTI). The lipase
inhibitors, bromoenol lactone (BEL), dithio-nitrobenzoic acid (DTNB) and chlorpromazine
inhibited this enzyme, suggesting phospholipase activity. We examined this enzyme activity in
plasma from male BL6 and LDLRo mice fed HFD. Presumptive lipase activity in BL6 and LDLRo
mice dropped 48 hours after introducing a HFD and rose for 15 days thereafter. Preliminary
results suggest an inverse correlation between the putative lipase activity and plasma
triglyceride levels. Fasting cholesterol levels at baseline, 48 hours and 15 days of HFD were 88,
126 and 125 mg/dl in BL6 mice and 220, 638 and 734 mg/dl in LDLRo mice, respectively.
These findings suggest that pre-kallikrein activation and an unexpected lipase activity in
plasma, presumably a phospholipase A2, respond in opposite directions to the inflammation
associated with high plasma cholesterol levels. These markers may serve to monitor early
inflammatory events associated with hyperlipidemia and atherosclerosis. (Supported by
HL55517, CLB and AI41670, TEH).
P447
HDL Subfraction Metabolism in Human Apolipoprotein A-I Transgenic Mice
Lorraine M Lanningham-Foster, James W Furbee, Thomas Smith, Ellen R Burleson, John S
Parks. Wake Forest University School of Medicine, Winston-Salem, NC
Multicompartmental modeling of HDL subfraction turnover in monkeys has suggested a
unidirectional conversion of small apoA-I only HDL particles (LpA-I) to medium LpA-I or large
LpA-I, which are the end products of HDL metabolism and are degraded preferentially by the
liver. We examined small LpA-I and pre-beta LpA-I metabolism in human apo A-I transgenic
(Tg) mice, which have heterogeneous HDL subfractions similar in size to that of humans. LpA-I
particles were isolated from plasma of Tg mice, without centrifugation, by immunoaffinity
chromatography and radiolabeled with the residualizing compound 125I-tyramine cellobiose
(TC). The 125I-TC LpA-I was separated, using size-exclusion chromatography, into small and
pre-beta LpA-I and injected separately into recipient apoA-I Tg mice. Plasma die-away of
radiolabel was followed for 24 hours, at which point the animals were sacrificed and tissues
were harvested for quantification of the radiolabel uptake. The plasma die-away of the pre-beta
LpA-I was 14-fold faster than that of the small LpA-I (FCR3.71 Â 2.38 pools/hr and 0.27
 0.09 pools/hr, respectively). Small LpA-I increased to medium-sized HDL with time and
were degraded preferentially by the liver (58.7 Â 4.7% injected dose), whereas pre-beta
LpA-I were degraded almost exclusively by the kidney (79.5 Â 13.2% injected dose). Native
gel electrophoresis of plasma samples revealed two metabolic fates for pre-beta LpA-I, rapid
removal from plasma and movement of radiolabel into medium-sized HDL particles. We
conclude that small LpA-I are precursors of medium-sized HDL but not pre-beta HDL, and that
two pools of pre-beta HDL exist in plasma, one that is has a rapid turnover and is degraded
by the kidney and one that contributes to medium-sized HDL and, ultimately, is degraded by
the liver.
P448
Lysophosphatidic Acid Induces Tissue Factor Expression in Aortic Smooth
Muscle Cells
Mei-Zhen Cui, Guojun Zhao, Allison Winokur, Guy M Chisolm III, Xuemin Xu. University of
Tennessee, Knoxville, TN; Cleveland Clinic Foundation, Cleveland, OH
Lipid lysophosphatidic acid (LPA), one component of oxidized lipoprotein, markedly induces
tissue factor activity, tissue factor protein, and tissue factor messenger RNA (mRNA) in smooth
muscle cells, which are important in vascular remodeling and vascular diseases. LPA increased
tissue factor mRNA in a concentration- and time-dependent manner. Results from mRNA
stability and nuclear run-on experiments demonstrated that tissue factor gene induction by LPA
is primarily controlled at the transcriptional level. Our data also showed that phosphorylation
of MEK, MAPK, and ribosomal S6 kinase (RSK) was markedly induced by LPA at very early time
points. Gi protein inhibitor, pertussis toxin, and MEK inhibitors, U0126 and PD98059,
completely blocked LPA-induced phosphorylation of MAPK and RSK, as well as the induction
of tissue factor mRNA. Taken together, our data suggest that LPA is a thrombogenic risk factor
by regulating TF expression. Our data also demonstrated that the LPA-induced signaling
pathway, which leads to tissue factor gene regulation in smooth muscle cells, involves the
activation by of Gi guest proteinon andApril a series 4, of 2013 kinases including MEK, MAPK, and RSK.

P449
Human Acyl-Coenzyme A: Cholesterol Acyltransferase 2 Expressed in
Chinese Hamster Ovary Cells: Membrane Topography and Location of the
Active Histidine 434 Residue
Song Lin, Xiaohui Lu, Catherine C Chang, Ta-Yuan Chang. Dartmouth Medical School,
Hanover, NH
Acyl-coenzyme A: cholesterol acyltransferase (ACAT) is a membrane bound enzyme that
produces cholesteryl esters intracellularly. Two ACAT genes (ACAT1 and ACAT2) have been
identified. The expression of ACAT1 is ubiquitous, while the expression of ACAT2 is tissue
restricted. hACAT1 may contain seven transmembrane domains (TMDs) (Lin et al., J. Biol.
Chem., 1999, vol.274, 23276–23285). In the current study, we inserted two different antigenic
tags (HA, Mab1) at various hydrophilic regions flanking each putative TMD of the hACAT2
protein, and expressed the recombinant proteins in mutant Chinese hamster ovary cells lacking
endogenous ACAT. Each tagged ACAT2 was expressed as a single undegraded protein band
based on Western analysis, and remained at least partially active enzymatically. We then used
cytoimmunofluorescence and protease protection assays to monitor the sidedness of the HA
and Mab1 tags along the ER membranes. Certain regions were further examined by inserting
the factor Xa sites and probing with factor Xa digestions. The results indicated that ACAT2
contains only two detectable TMDs. A conserved serine (S245) as well as a conserved histidine
(H434) has been implicated as part of the ACAT active site(s). Our site-specific mutagenesis
results show that H434 is essential for ACAT activity, while S245 is not. H434 may be located
at or near the cytoplasmic side of the ER membrane.
P450
HDL2 Remodels to HDL3 in Humans and Estrogen Replacement Raises
HDL2 by Reducing This Remodeling in Postmenopausal Women
Lucian Saucan, Ruby J Sheffer, Zoltan Vajo, Matt Banaszak, Eliot A Brinton. Phoenix VAMC,
Phoenix, AZ
HDL is strongly antiatherosclerotic, especially HDL lacking apo A-II (Lp A-I), and especially large
HDL (HDL2). In monkeys, large and small HDL remodel unidirectionally into medium sized HDL,
but the size-specific turnover of HDL in humans is poorly understood. Estrogen replacement
(ERT) in postmenopausal women raises HDL levels, specifically large Lp A-I, which makes ERT
an interesting and clinically relevant model for study of human HDL metabolism. Six healthy
postmenopausal women underwent a paired crossover study of HDL turnover in three
conditions: basal state, unopposed estrogen replacement (ERT, conjugated equine estrogen,
0.625 mg/d), and combination estrogen plus progestin (HRT, adding medroxyprogesterone
acetate, 2.5 mg/d). Lp A-I HDL tracer was prepared from autologous plasma by immunoaffinity
and radioiodination then size-separation into HDL2 and HDL3. Tracers were given by
intravenous bolus injection and plasma die-away measured for one week. Size-specific
remodeling was measured by gel-filtration of plasma. Statistical analyses included ANOVA and
linear regression. As expected, ERT raised HDL-C levels vs. the basal state, while HRT produced
intermediate results. Although the FCR of the HDL2 and HDL3 tracers was not significantly
changed by ERT or HRT, FCR inversely predicted HDL-C levels across treatments (p.05).
HDL2 tracer moved rapidly and quantitatively into HDL3-sized particles, the HDL2 die-away
curve crossing the HDL3 curve at its peak. ERT substantially blunted the movement of HDL2
into HDL3, while HRT gave intermediate results. In contrast to the HDL2 tracer, little HDL3
tracer appeared in HDL2-sized particles, and ERT slightly enhanced this transfer. In conclusion,
for the first time we have shown that in humans HDL2 is a quantitative precursor to HDL3, and
that this precursor-product remodeling is reduced by ERT and HRT. This suggests a novel
mechanism for the selective increase in large Lp A-I by ERT. Surprisingly, the primary precursor
for HDL2 does not appear to be HDL3, and remains unknown.
Mice Heterozygous for Apolipoprotein B38.9-Related
Hypobetalipoproteinemia may have Reduced Susceptibility to Dietary
Ethanol-Induced Fatty Liver
P451
Nobuhiro Sakata, Xiaobo Lin, Pin Yue, Zhouji Chen, Gustav Schonfeld. Washington University
School of Medicine, St. Louis, MO
Humans and mice with familial hypobetalipoproteinemia (FHBL) due to truncated apolipoprotein
B (apoB) frequently accumulate triglycerides in their livers (fatty liver). Dietary ethanol is also
known to cause fatty liver. To assess whether dietary alcohol causes more severe fatty livers
in FHBL, wild type mice (n14) and apoB38.9 mice heterozygous for FHBL (n16) were fed
a liquid diet with or without ethanol (25% or 35% of total calories) for two weeks. Ethanol was
substituted for carbohydrate. Daily food intakes, body weights, serum aspartate aminotransferases
(AST), liver triglycerides and mRNAs of lipid-related enzyme were examined. Serum
AST level, a marker of hepatic toxicity was increased in both wild type and apoB38.9
heterozygous mice but only by the 35% ethanol diet. Hepatic triglycerides in wild type were
increased from 21.9 to 49.0 mg/g tissue (p0.05) by the 25% ethanol diet and from 23.1 to
38.9 mg/g tissue by the 35% ethanol diet. In apoB38.9 heterozygotes hepatic trigylceride
values were not significantly increased by either ethanol diet (from 77.3 to 64.8 mg/g tissue
on 25% ethanol diet, and from 39.1 to 44.3 mg/g tissue on 35% ethanol diet). Hepatic sterol
regulatory element binding protein 1c and carnitine palmitoyl transferase mRNAs were
relatively reduced by alcohol diet. Interestingly, the mRNA levels of the ATP binding cassette
half-transporter ABCG5 were also reduced by 50% but only in wild type suggesting that
ABCG5 may play a role in ethanol-induced lipid accumulation in wild types. I n conclusion,
although mechanisms are not understood apoB38.9 heterozygous mice may have less
susceptibility to dietary ethanol than wild type.
P452
A Novel Instrument for Measurement of Blood Viscosity and Shear Stress
Kenneth R Kensey, Bill Hogenauer. Rheologics, Inc, Exton, PA
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Poster Presentations a-79
Measuring the rheological properties of blood, including blood viscosity, may be important in
the study of shear stress and endothelial dysfunction. Until now, viscosity measurements were
performed on adulterated blood via in-vitro test methods. Measurements using unadulterated
blood are necessary to delineate the effect of anti-coagulants and deviations from body
temperature on blood viscosity. The non-Newtonian characteristics of whole blood also require
viscosity measurement over a wide range of shear rates in order to properly study the
relationship of viscosity, shear rate and shear stress. A new Scanning Capillary Tube
Viscometer (SCTV) intended for “point of care” applications has been tested. Preliminary results
indicate the device accurately measures blood viscosity over a range of shear rates including
very low shear. Following a venipuncture with a 21g needle, less than 3cc of blood flows into
a temperature controlled biocompatible-coated capillary tube/rising tube assembly. Highresolution
sensors measure the fluid levels in the rising tubes during a period of three minutes
and transmit the readings to a computer data file. The corresponding flow velocity in the rising
tubes is calculated using height vs. time curves. The velocity and shear rate at the capillary
tube are determined, rendering the shear-viscosity profile of the blood sample. The SCTV was
validated with known viscosity fluids such as water, diluted glycerin and bovine blood. Human
blood viscosity measurements from the SCTV were compared with measurements from a
rotating cone-and-plate viscometer (Brookfield Instruments.) The clinical utility of the SCTV is
clear. It can provide “point of care” measurement of blood viscosity at a full complement of
shear conditions, at body temperature and without the addition of anti-coagulants.