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

Abstracts of the 6th Annual Conference on Arteriosclerosis, Thrombosis, and Vascular 

Biology 

Arterioscler Thromb Vasc Biol. 2005;25:e43-e109 

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Genetic Determinants of Leukocyte Recruitment in Mice 

Jane Hoover-Plow, Aleksey Shchurin, Jengfeng Sha, Erika Hart, Yelena Shchurin, Cleveland 

Clinic Foundation, Cleveland, OH; Annie Hill, Case Western Reserve Univ, Cleveland, OH; 

Eric Lander, Jonathan Singer, MIT, Boston, MA; Joseph Nadeau; Case Western Reserve 

Univ, Cleveland, OH 

Multiple genetic factors contribute to the development of cardiovascular diseases (CVD). The 

objective of this study was to evaluate leukocyte recruitment in mouse models susceptible or 

resistant to CVD. Peritoneal leukocyte recruitment was evaluated in C57BL/6J (B6) mice, 

susceptible to atherosclerosis, and A/J mice, mice, resistant to atherosclerosis, using two 

inflammatory stimuli, a biopolymer implant and a thioglycollate injection. The number of 

responding neutrophil and monocyte/macrophage cells were quantified (number of cells x 10 6 , 

mean SEM, n number of mice). Marked differences were found between the two strains. 

Peritoneal macrophage recruitment induced by the implants, was slightly higher (p 0.05) in 

the A/J (9.8 0.4, n 8) mice compared to B6 (7.7 0.4, n 38) mice, but 6h after 

thioglycollate injection, recruitment of both neutrophils (A/J 0.6 0.2, n 8, B6 1.9 

0.3, n 16) and macrophages (A/J 1.9 0.3, n 8, B6 4.7 0.7, n 16) was 3-fold 

lower (P 0.02) in A/J mice than for B6 mice. At the peak macrophage recruitment time of 

72h, macrophage number was also decreased (P 0.001) in the A/J mice compared to B6 

mice (A/J 4.9 0.8, n 8, B6 12.8 1.2, n 22). To dissect the molecular basis 

for these differences, chromosome substitution strains (CSS), B6 strains of mice with individual 

A/J chromosomes, one strain for each chromosome, were evaluated for macrophage 

recruitment 72h after thioglycollate injection. One strain, B6.A10 (7.6 1.3, n 8), has been 

identified which carries the trait for decreased (P 0.05) macrophage recruitment, similar to 

the parent A/J strain. Five CSS strains, B6.A5, B6.A8. B6.A14, B6.A15, and B6.AX had 

significantly (P 0.05) increased macrophage recruitment. In the B6.AX strain, macrophage 

number was nearly 2-fold higher than for B6 mice. Thus, at least one gene or quantitative trait 

locus on each of the identified chromosomes contributes to macrophage recruitment in the 

peritoneal thioglycollate inflammatory model and will be evaluated for susceptibility to 

atherosclerosis. The CSS will ultimately allow us to identify genes that contribute to CVD. 

2 

The TSP-4 SNP Variants Trigger Distinct Effects in Human Neutrophils via 

Engagement of M 2 Integrin 

Elzbieta Pluskota, Eric J Topol, Olga I Stenina, Dorota Szpak, Edward F Plow; Cleveland 

Clinic Foundation, Cleveland, OH 

High-throughput genomic technology identified an unanticipated association between a 

particular single nucleotide polymorphism (SNP) in the thrombospondin-4 (TSP-4) gene and 

myocardial infarction. The disease-associated SNP, a proline at position 387 (P387) as 

compared to the predominant alanine (A387), is associated with an increased risk of MI 

(adjusted odds ratio: 1.89 P0.002) and occurs at high frequency within the Caucasian 

population. Little is known about the TSP-4 gene product and its functions in vivo. In view of 

the inflammatory hypothesis of atherosclerosis, which invokes prominent roles for leukocytes 

and cytokines in pathogenesis, the interactions of the TSP-4 variants with human neutrophils 

(PMN) were investigated. Adhesion of PMA-stimulated PMNs to the recombinant TSP-4 variants 

was equal and M 2-dependent as it was blocked by function blocking mAbs to this integrin 

and its high affinity ligand NIF (Neutrophil Inhibitory Factor). Involvement of M 2 in TSP-4 

recognition was corroborated using HEK 293 cells overexpressing this integrin. More 

importantly, despite equal adhesion of PMNs to both TSP-4 variants, PMNs adherent to the 

P387 TSP-4 were significantly more spread, showed higher expression and robustly more M 2 

clusters on their surface than the cells adherent to the A387 variant. In signaling experiments, 

the P387 variant induced a substantially more robust tyrosine phosphorylation of 48–55, 

36 – 42 and 28 –30 kDa proteins than the A387 variant. The P387 TSP-4 stimulated activation 

of stress-related MAPKs (Mitogen-activated Protein Kinases): p38 MAPK and SAPK/JNK 

(Stress-activated Protein Kinase/ c-Jun NH2-terminal Kinase) and also enhanced STAT1 and 

HSP27 (Heat Shock Protein 27) phosphorylation. In addition, PMNs adherent to TSP-4 (P387) 

released 4-fold more H 2O 2 and secreted 2-fold more IL-8 as compared to the A387. The p38 

MAPK activation was crucial for H 2O 2 release since this response was suppressed by a specific 

p38 inhibitor. PMN adhesion, H 2O 2 release and p38 MAPK activation were M 2 integrindependent 

as they were totally inhibited by M 2 blockade. Thus, M 2 plays a central role in 

proinflammatory activities of the TSP-4 (P387) variant more likely as a consequence of its 

distinct clustering and activation. 

3 

M-CSF Deficient Mice Develop Angiotensin II-Induced Aortic Intra-Laminar 

Hemorrhage in a Site and Gender Specific Manner 

Fjoralba Babamusta, Debra L Rateri, Jessica J Moorleghen, Lisa A Cassis, Alan Daugherty; 

Univ of Kentucky, Lexington, KY 

Objective: Angiotensin II (AngII) infusion into hyperlipidemic mice promotes macrophage 

infiltration. In our previous study, we demonstrated that AngII-infused osteopetrotic (Op) mice 

(which lack M-CSF and have reduced macrophages) have aortic intramural thrombus which 

extended from the proximal thoracic aorta to the arch. In the current study we defined the 

location and histological features of intra-medial hemorrhage as well as morphological 

characteristics of aortic segments under basal conditions. Methods and Results: Op-/- male 

mice were bred to apoE -/- female mice for four generations to generate wild type (Op/) 

and osteopetrotic male mice in apoE/ and -/- Downloaded backgrounds. AngII from 

and saline were infused 

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Oral Presentations E-43 

in all these groups of mice (n4–15 per group) and histological features of the vessel wall 

before and after AngII infusion were determined. Only AngII infused Op-/- male mice had upper 

thoracic and aortic arch hemorrhage. Tissue sections of the aortic arch and proximal thoracic 

aortas from AngII-infused mice revealed that intra-medial hematomas were localized to the 

external margins of the vessel wall. Small numbers of macrophages were present at the edge 

of the large hematoma. Interestingly, histological examination of thoracic aortas from Op-/male 

mice under basal conditions demonstrated regions of medial elastin detachment and 

increased space between elastin fibers in both upper and lower thoracic aorta. Maximal medial 

thickness in proximal and distal thoracic segments was increased by 28% in Op-/- mice 

compared to Op/ mice (P0.02), whereas minimal medial thickness was the same in both 

genotypes. Conclusion: M-CSF deficiency predisposes proximal thoracic aorta of male mice to 

intra-laminar hemorrhage localized to the external margins of the vessel. 

4 

C-Reactive Protein Decreases Tissue Plasminogen Activator Expression and 

Activity in Human Aortic Endothelial Cells: Further Evidence for a 

Procoagulant Phenotype 

Uma Singh, Sridevi Devaraj, Ishwarlal Jialal; UCDavis Med Cntr, Sacramento, CA 

C-reactive protein (CRP) is not only a risk marker but also a mediator of atherogenesis. CRP 

induces tissue factor in mononuclear cells and decreases nitric oxide synthase expression and 

prostacyclin in endothelial cells. Increased arterial thrombosis has been reported in transgenic 

mice overexpressing human CRP, following femoral artery injury. Recently, we showed that 

CRP induces the expression and activity of plasminogen activator inhibitor-1 in human aortic 

endothelial cells (HAECs). However there are no reports of the effect of CRP on tPA. Hence, the 

aim was to test the effect of CRP on tPA in HAECs. The cells were exposed to CRP (0 –50 ug/ml) 

for different time periods (3–24h). tPA activity and antigen levels were measured in culture 

supernatants. Cell lysates were probed for intracellular tPA expression by western blot. tPA 

mRNA levels were measured by Real-Time RT-PCR. tPA activity and antigen levels were 

significantly decreased by CRP treatment (12.5 ug/ml for 12 hours, p0.01). Similarly 

intracellular tPA was also decreased with CRP. However, tPA mRNA remained unchanged with 

CRP suggesting that the effect of CRP on tPA is a post-transcriptional event. In order to gain 

mechanistic insights into regulation of tPA by CRP, we tested the effect of following pathways: 

endothelin-1 (ET-1), reactive oxygen species (ROS), cyclic AMP and pro-inflammatory cytokines 

as all these pathways have been shown to alter tPA levels. Previously, we have shown that CRP 

promotes oxidative stress and CRP has also been shown to activate ET-1. However, the ET-1 

receptor blocker (Bosentan) or the cell permeable analog of superoxide dismutase (PEG-SOD) 

failed to reverse the CRP mediated downregulation of tPA. Similarly, adenyl cyclase inhibitors 

also had no effect on tPA. In the present study, CRP treatment resulted in a dose-dependent 

increase in IL-1 and TNF- secretion by HAECs. Furthermore, the use of neutralizing 

antibodies to IL-1 and TNF- reversed the CRP mediated downregulation of tPA (50% 

inhibition compared to CRP-treated cells, p0.05). To conclude, CRP decreases tPA antigen 

and activity from HAEC via generation of pro-inflammatory cytokines, IL-1 and TNF-. This 

study lends further support of CRP promoting a procoagulant phenotype in the vasculature. 

A Common Pathogenetic Link between Venous Thrombosis and 

Atherosclerosis: Increased Prevalence of Small, Dense LDL 

Alberto Zambon, Paolo Simioni, Sandra Bertocco, Valentina Polentarutti, D Tormene, Paolo 

Prandoni, Antonio Pagnan; Univ of Padova, Padova, Italy 

Background: Venous thromboembolism (VTE) is a serious, potentially fatal disease affecting 2 

persons per 1000/year. In a third of patients the cause of VTE is unexplained. An association 

between atherosclerosis and VTE has been demonstrated. Risk factors for atherosclerosis (i.e 

hypercholesterolemia) seem to play a marginal role in the pathogenesis of VTE. Small, dense 

low-density lipoproteins (sd-LDL) are highly atherogenic and susceptible to oxidation (ox-LDL), 

and associated with endothelial dysfunction. This study investigates the potential role of sd-LDL 

and ox-LDL as a risk factor for VTE. Patients and Methods: Fifty patients, aged 6015 yrs, 

with first diagnosis of VTE, confirmed by compression ultrasonography, and no history of VTE 

or symptomatic atherosclerosis were evaluated. Mutations and conditions associated with 

thrombophilia were excluded, and patients classified as having secondary (cancer, estrogen 

use, etc.) or spontaneous VTE. Diabetics and patients under lipid-lowering medications were 

excluded. Seventy healthy controls participated in the study. Lipoprotein subclasses were 

analyzed by density gradient ultracentrifugation, plasma ox-LDL concentration by ELISA and 

lipids by standardized enzymatic kits. Results: Twenty-one and 29 patients had secondary and 

spontaneous VTE respectively. As a whole, VTE patients had similar total, LDL, VLDL, IDL, HDL 

cholesterol and triglycerides than controls. No differences in these parameters were found 

between those with spontaneous and secondary VTE. Despite similar LDL-C, patients with VTE 

had higher sd-LDL cholesterol (p0.01) as well as ox-LDL (p0.05) than controls. Both the 

increased sd-LDL cholesterol as well as the increased ox-LDL levels in the VTE group were 

entirely due to the subgroup with spontaneous VTE (p0.01 vs. controls and secondary VTE). 

Cholesterol in the sd-LDL and levels of ox-LDL were significantly associated (r0.39;p0.05). 

Patients with secondary VTE and controls had similar ox-LDL levels and lipid phenotype. 

Conclusion: Spontaneous VTE is associated with significantly increased cholesterol in the 

sd-LDL particles and higher levels of ox-LDL, which may represent a common link in the 

pathogenesis by guest of both on atherosclerosis June 29, and 2013 VTE. 

5

E-44 Vol 25, No 5 May 2005 

The Role of Platelet Surface Ultralarge Complexes in Heparin Induced 

Thrombocytopenia 

Lubica Rauova, Children’s Hosp of Philadelphia, Philadelphia, PA; Bruce B Sachais, Douglas 

B Cines, Univ of Pennsylvania, Philadelphia, PA; Mortimer Poncz; Children’s Hosp of 

Philadelphia, Philadelphia, PA 

Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy 

caused by antibodies to complexes between high-molecular weight species of heparin and 

Platelet Factor 4 (PF4) leading to limb and/or life-threatening thrombosis in 50% of the 

diagnosed patients. We are interested in understanding the basis of an important clinical 

observation: HIT antibodies form in most heparinized patients, but only a small percentage of 

these patients develop thrombocytopenia and/or thrombosis We have shown recently by gel 

filtration that PF4 and heparin form antigenic ultralarge complexes (ULC) over a very narrow 

molar range (1:1) of these two molecules. These ULC are stable and visible by electron 

microscopy, but can be dissociated into smaller complexes (SC) with additional heparin. ULC 

form inefficiently with low molecular weight heparin, and none form with the pentasaccharide 

fondaparinux. Mutation studies show that formation of ULC depends on the capacity of PF4 to 

form tetramers. The ULC were more reactive as determined by their capacity to bind to a 

HITT-like monoclonal antibody KKO and showed greater capacity to promote platelet activation 

in an antibody- and FcRIIA-dependent manner than were the smaller complexes. We have 

also found that similar antigenic complexes form on the surface of platelets and other vascular 

cells presumably between PF4 and membrane glycosaminoglycans (GAG), again only over a 

narrow molar range. PF4 incubated with normal human or mouse platelets evokes binding of 

HIT-like monoclonal antibody in a dose-dependent, bell-shaped manner and induces FcRIIA 

dependent platelet activation. The capacity of PF4 to form ULC composed of multiple PF4 

tetramers arrayed in a lattice with several molecules of heparin and/or GAG may play a 

fundamental role in platelet activation and the propensity for thrombosis in patients with HIT. 

We propose a model involving these antigenic surface ULC that incorporates our observations 

and explains the infrequent nature of HIT in the heparinized population. This model assumes 

that the highest at-risk patients are those who have a high steady-state level of surface PF4 

and offers a testable model that may be useful in eliminating high-risk patients from heparin 

exposure. 

7 

Cardiovascular Consequences of Microsomal PGE Synthsase-1 Deletion in 

Mice 

Miao Wang, Yan Cheng, Emanuela Ricciotti, Wenliang Song, Julien Ferrari, John Lawson, 

Garret A FitzGerald; Univ of Pennsylvania, Philadelphia, PA 

Background: Accumulating evidences show increased cardiovascular hazards exist for COX-2 

selective NSAIDs. Interest in therapeutic target selection is shifting downstream in the 

prostanoid biosynthetic/response pathway, towards specific prostaglandin synthases and 

receptors. The microsomal (m) PGE synthsase (S)-1, which catalyzes the isomerization of PGH 2 

into PGE 2, has emerged as an alternative drug target. However, the rationale is based on the 

assumption that this isozyme is the dominant source of PGE 2 in vivo and that suppression of 

PGE 2 does not contribute to the cardiovascular hazard observed with the COX-2 selective 

NSAIDs. These assumptions have been tested in mPGES-1 knock out mice. Objectives: To 

examine role of mPGES-1 in production of PGE 2 in vivo and the thrombotic response to 

mPGES-1 deletion Methods and Results: Urine 7-hydroxy-5, 11-diketotetranorprostane-1, 

16-dioic acid (PGE-M), measured by mass spectrometry, was used as biomarker of PGE 2. The 

origin of the material in mouse urine was confirmed by systemic administration of authentic 

PGE 2. Urinary PGE-M was higher in wild type (WT) males (82.1 7.5 ng/mg creatinine ) than 

in females (28.0 2.3 ng/mg creatinine ) and was depressed in mPGES-1 knock out (KO) mice 

(males 14.74.0 ng/mg creatinine; females 15.8 2.2 ng/mg creatinine ). These differences 

were significant between mPGES-1 KO and WT in each gender (males: P 0.004; females: 

P0.0025), and between male and female mice (P0.0003). We have previously shown that 

inhibition of COX-2 and deletion of the receptor for prostacyclin (PGI 2) accelerated the 

thrombotic occlusive response to photochemical carotid vascular injury. Deletion of mPGES-1 

failed to alter the time to occlusion in male (WT 35.90 5.25 vs KO 40.438.69 min) or female 

(WT 34.507.32 vs KO 32.144.74 min) mice (One-way ANOVA, P0.85). Conclusion: 

mPGES-1 is the dominant source of PGE 2 in vivo under physiological conditions in mice and 

deletion of this enzyme, unlike inhibition or deletion of COX-2, fails to accelerate the occlusive 

response to thrombogenic stimulus in vivo. 

Vezf1 Regulates Early Vasculature Formation 

Zhongmin Zou, Huiqin Sun, Wendy LeVine, Frank Kuhnert, Heidi Stuhlmann; The Scripps 

Rsch Institute, La Jolla, CA 

Vascular endothelial zinc finger 1 (Vezf1) is expressed during early embryonal development. 

VEZF1 protein contains six zinc finger domains, a nuclear localization sequence (NLS) domain 

and a transcriptional activation domain at its C-terminus. Embryos carrying a Vezf1null allele 

die during midgestation due to angiogenic remodeling defects and loss of vascular integrity. 

Here we have further investigated the role of Vezf1 in the formation of vasculature by using the 

embryoid body (EB) in vitro differentiation system. Wild type ES cells, Vezf1/- and Vezf1-/- 

ES cell clones were used to generate EBs. In addition, these cells were used to study Vezf1 

function in the in vivo model of teratocarcinoma formation in nude mice. In parallel, cell 

biological studies were performed on mouse embryo fibroblasts (MEFs) isolated from E11.5 

wild type and mutant embryos. Vezf1-/- ES cell derived EBs failed to form a well-organized and 

differentiated vascular network in 2D attachment culture and in suspension culture. Abnormal 

collagen IV deposition and distribution were observed. In addition, using a 3D collagen gel 

angiogenesis assay, Vezf1-/- EBs showed dramatic retarded sprouting. Teratocarcinomas 

derived from Vezf1-/- ES cells were able to spontaneously Downloaded differentiate from 

into cells of different 

6 

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tissue types. However, histological and histochemical examination of these tumors showed 

decreased cell proliferation, delayed differentiation, and large foci of cells with extensive 

deposition of extracellular matrix. Vezf1-/- MEFs showed significantly reduced level of cell 

proliferation, adhesion and migration compared with wild type and Vezf1/- MEF controls. 

RT-PCR and immunostaining results indicated that integrin v/3, p57, cyclin B1, collagen IV, 

and FAK may be involved in regulating these cellular processes. Together, these results suggest 

that Vezf1 is involved early differentiation processes of the vascular system by regulating cell 

differentiation, proliferation, migration and ECM deposition. Our present studies focus on 

identifying downstream targets of Vezf1 as well as proteins that functionally interact with Vezf1. 

9 

Oxidized Phospholipids Stimulate Angiogenesis via Up-Regulation of VEGF, 

IL-8 and COX-2 

Valery N Bochkov, Institute of Vascular Biology and Thrombosis Rsch, Med Univ of Vienna, 

Austria; Maria P Filippova, Cardiovascular Laboratories, Univ Hosp Basel, Switzerland; 

Alexandra Kadl, Olga Oskolkova, Alexander Furnkranz, Erduan Karabeg, Johannes Breuss, 

Institute of Vascular Biology and Thrombosis Rsch, Med Univ of Vienna, Austria; Therese J 

Resink, Cardiovascular Laboratories, Univ Hosp Basel, Austria; Bernd R Binder, Norbert 

Leitinger; Institute of Vascular Biology and Thrombosis Rsch, Med Univ of Vienna, Austria 

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. Formation of 

vasa vasorum is thought to promote plaque growth, stimulate accumulation of inflammatory 

cells and decrease stability of lesions. Mechanisms regulating angiogenesis within the plaque 

are only partially understood. It is thought that in addition to hypoxia, some local factors within 

the plaque are responsible for enhanced growth of vessels. We hypothesized that oxidized 

phospholipids (OxPL) formed by non-enzymatic oxidation of phospholipids such as palmitoylarachidonoyl-phosphatidyl 

choline (PAPC), and known to accumulate in atherosclerotic vessels, 

can stimulate angiogenesis. To test this possibility, we analyzed effects of OxPAPC in 

endothelial cell sprouting assay. We found that OxPAPC in a time- and concentration-dependent 

manner stimulated formation of sprouts by human umbilical vein endothelial cells (HUVEC). 

Furthermore, OxPAPC promoted growth of new vessels in Matrigel plug assay in mice as 

demonstrated by morphologic analysis and quantitation of plug hemoglobin. Analysis of gene 

expression in HUVEC using microarrays and reverse transcription/real time PCR demonstrated 

that OxPAPC stimulated expression of VEGF, COX-2 and IL-8, but not of recognized angiogenic 

factors PlGF, PDGFB, FGF-2, angiopoietins 1, 2 and 4, or angiogenin. Furthermore, in vivo 

application of OxPAPC mixed with Pluronic F-127 gel to the adventitial surface of mouse carotid 

artery resulted in up-regulation of VEGF, COX-2 and KC/IL-8 messages. Low molecular weight 

inhibitors of VEGF receptors and COX-2, as well as blocking antibodies to IL-8 suppressed 

angiogenic effects of OxPAPC in sprouting assay. Thus, we conclude that OxPAPC stimulates 

angiogenesis via an autocrine mechanism mediated by VEGF, IL-8, and COX-2-derived 

prostaglandins. Our data show that OxPL demonstrate angiogenic properties in vitro and in vivo 

and thus accumulation of OxPL can partially explain increased growth of blood capillaries in 

advanced atherosclerotic lesions. 

10 

Atherosclerosis Regression is Characterized by Macrophages Altering their 

Phenotype into a Dendritic-Like State 

Jonathan E Feig, Eugene Trogan, Jeffrey Mayne, Yanqing Ma, Snjezana Dogan, James X 

Rong, New York Univ Sch of Medicine, New York, NY; Stephen G Young, UCLA Sch of 

Medicine, Los Angeles, CA; Gwendalyn J Randolph, Mount Sinai Sch of Medicine, New 

York, NY; Edward A Fisher; New York Univ Sch of Medicine, New York, NY 

There has been considerable interest to reduce the severity of atherosclerosis. We have 

developed two novel mouse models of atherosclerosis regression by changing the plasma 

environment from hyperlipidemia to normolipidemia. The first is accomplished by transplanting 

a lesioned aortic arch from a western diet (WD) fed apoE-/- mouse into the abdominal aorta 

of a wild type recipient (J Vasc Surg.2001;34:54). In this model, we reported rapid plaque 

regression associated with emigration of macrophages, which manifested some features of 

dendritic cells, monocyte-derived cells that travel from tissue sites to lymph nodes 

(PNAS.2004;101:11779). The second is accomplished by conditional inactivation of the MTP 

gene in WD-fed Reversa mice (LDLR-/- and express only apoB100; Circulation.2003;107:1315) 

after plaques form, thereby reducing hepatic lipoprotein secretion, normalizing lipid levels, and 

promoting regression. In the present study, we investigated whether a feature common to both 

regression models was the assumption by lesional macrophages of a dendritic cell phenotype. 

In both models, mice were fed WD for 16 weeks. In the transplant model, as before, grafts were 

isolated 3 days post-surgery. In the Reversa model, plaques were analyzed 1 week after MTP 

inactivation by pI-pC injection. In both, lesional macrophages expressed the dendritic marker 

CD11c in the regression, but not progression (apoE-/- recipients or Reversa mice saline 

injected) setting. Using laser capture microdissection, lesional macrophages were isolated and 

their RNA analyzed by real-time PCR. The mRNA for CCR7, a chemokine receptor required for 

homing to lymph nodes and expressed by mature dendritic cells, was essentially absent in the 

progression samples but was upregulated 5–6.4X in both of the regression models. This was 

confirmed at the protein level by immunostaining. In lesions under both progression and 

regression, lymphatic vessels in the vicinity of foam cells was evident by immunostaining with 

a lymphatic endothelial marker. In conclusion, independent of the mode of normalizing lipid 

levels, regression of plaques can be induced and that this process involves the assumption by 

lesional macrophages by guest on of key June features 29, 2013 of dendritic cells, which then emigrate to lymph nodes.

11 

Mice Lacking Catalytic Function of PI3kinase p110delta Develop Decreased 

Atherosclerotic Lesions, Concurrent with Decreased Lymphocyte Activation 

and Akt-Mediated Signal Transduction 

Laura J Pinderski, Karen M Lewis, Jason D Washington, Univ of Alabama, Birmingham, 

Birmingham, AL; Bart Vanhaesebroeck, Ludwig Institute for Cancer Rsch, London, United 

Kingdom; James F George; Univ of Alabama, Birmingham, Birmingham, AL 

Background: PI3kinases are a family of heterodimeric lipid kinases which can mediate 

proliferative and activation pathways downstream of cell-surface receptor signaling, through 

phosphorylation of phosphotidylinosital. PI3Kp110 is a catalytically active isoform subunit, 

found predominantly within leukocytes. As the adaptive immune response plays a key role in 

atherogenesis, we hypothesized catalytic inactivity of p110 (p110 m ) should significantly alter 

essential proliferative and activation responses of lymphocytes, decreasing atherosclerosis. 

Methods: Male LDL-R deficient mice received bone marrow transplantation with either WT 

(n10) or p110 m (n8) bone marrow and placed on a high fat, high cholesterol diet for 20 

weeks. At sacrifice, proximal aortas were obtained. Intracellular cytokines were measured in 

spleen and peri-aortic lymph nodes (LN). Downstream signaling from PI3K activity was 

measured by flow cytometric assessment of the activated form of AKT (p-Ser473 AKT). 

Results: WT BMT mice developed 52% increased atherosclerosis than p110 m BMT mice 

(43.8893.19 vs 22.3312.81mm 2 ;p0.0001), despite similar circulating lipids between the 

two groups. Intracellular TNF- expression in CD4 and CD8 lymphocytes within the spleen 

and LN was up to 24-fold higher in the WT BMT group (p 0.001). CD8 IFN- expression 

was markedly increased in WT compared to P110 m BMT group in LN (15.35% vs 0.91%; 

p0.03), and splenocytes (23.6% vs 3.3%; p0.0002). Intracellular IL-2 was increased nearly 

10-fold in CD4 and CD8 LN and spleen in WT vs P110 m . CD4 and CD8 splenocytes 

demonstrated a marked increase in pSer473AKT in WT BMT mice at 20 weeks, compared to 

WT baseline or P110 m BMT mice at 20 weeks, with a smaller but detectable difference noted 

in LN. Conclusions: Atherosclerosis progresses normally through inflammatory pathways 

incorporating PI3K mediated lymphocyte activation and Akt-mediated signal transduction. 

Interruption of this pathway, downstream of cell-surface mediated receptor signaltransduction, 

results in a significant diminution of atherosclerosis. 

Macrophage Expression of Active MMP-9 is Sufficient to Induce Plaque 

Rupture in ApoE-/- Mice 

Peter J Gough, GlaxoSmithKline, Stevenage, United Kingdom; Paul T Wille, Elaine W Raines; 

Univ of Washington, Seattle, WA 

It is becoming increasingly clear that the majority of the clinical consequences of atherosclerosis 

are primarily due to the physical rupture of advanced atherosclerotic plaques. It has been 

hypothesised that macrophages play a key role in inducing plaque rupture by secreting 

proteases that destroy the extracellular matrix components that provide physical strength to the 

fibrous cap. Despite many reports detailing the expression of multiple proteases by 

macrophages in rupture-prone shoulder regions, there is no direct proof that macrophagemediated 

matrix degradation can induce plaque rupture. We set out to test this hypothesis by 

using a novel macrophage-specific retroviral vector to overexpress the candidate enzyme 

MMP-9 in macrophages of advanced atherosclerotic lesions of ApoE-/- mice. Despite a greater 

than 10-fold increase in the expression of MMP-9 by macrophages, there was only a minor 

increase in the incidence of plaque rupture. Subsequent analysis revealed that macrophages 

secrete MMP-9 predominantly as a pro-form, and this form of the enzyme is unable to degrade 

the matrix component elastin. Expression of an auto-activating form of MMP-9 in macrophages 

in vitro greatly enhances elastin degradation, and induces significant plaque rupture when 

overexpressed by macrophages in advanced atherosclerotic lesions of ApoE-/- mice in vivo. 

These data show for the first time that enhanced macrophage proteolytic activity can induce 

plaque rupture, and highlight MMP-9, and factors that regulate its activation, as potential 

therapeutic targets for stabilising rupture-prone plaques. 

Diversified Regulation of IIb 3 Activation by two Cytoplasmic Tails 

Yan-Qing Ma, Jun Yang, Jun Qin, Edward F Plow; The Cleveland Clinic Foundation, 

Cleveland, OH 

Activation of integrin IIb 3 is pivotal for platelet aggregation, which mediates physiological 

hemostasis and also plays a key role in pathological processes, such as atherosclerosis and 

thrombosis. The process of activation is initiated at the two cytoplasmic tails (CT) of the integrin 

subunits, which then trigger a conformational change in the extracellular domain of the 

receptor for ligand binding. To further address the regulatory roles of membrane-proximal and 

membrane-distal regions of CT in integrin activation, a series of mutations in the CT were 

prepared and their effects on the conformational change of the integrin were evaluated using 

the monomeric ligand mimetic antibody PAC-1 binding by flow cytometry. We found that 

disrupting of membrane-proximal salt-bridge by point mutation IIb(R995D) or 3(D723R) only 

partially activated IIb 3 while truncation of either IIb (991) or 3 (717), or both CT resulted 

in much higher activation. These data indicate that full unclasping of membrane-proximal 

complex is required for maximal activation of integrin IIb 3. To investigate the regulatory 

capacity of membrane-distal regions of CT on activation, we employed double-mutations in 

both the membrane-proximal and membrane-distal regions of IIb and 3 CT. Deletion of the 

membrane-distal region of 3 CT, 754, or of IIb CT, 996, failed to activate the integrin. 

Deletion of IIb, 1001, also failed to alter activation induced by mutation of the membraneproximal 

region of IIb CT, R995D. However, truncation the 3 membrane-distal CT at 754 

blocked the activating effect of R995D. These data indicate that the membrane-distal region of 

IIb CT does not regulate integrin activation. In contrast, both the membrane-distal as well as 

the membrane-proximal region of the 3 CT play a regulatory role. Events in the membrane- 

12 

13 





distal region of 3 CT regulates events in the membrane-proximal complex of the IIb and 3 

CT. 

Microparticle Release from Coated-Platelets is Dependent on CD9 

George L Dale, Gyula Remenyi, Paul Friese; Univ. Oklahoma Health Sci. Cntr., Oklahoma 

City, OK 

Dual agonist stimulation of platelets with thrombin plus collagen results in generation of 

coated-platelets, a sub-population of cells known formerly as COAT-platelets (Curr. Opin. 

Hematol. 10:351, 2003). Coated-platelets retain several adhesive and procoagulant proteins on 

their surface, express phosphatidylserine and support formation of prothrombinase complexes. 

In this report, we utilize a new methodology to demonstrate that coated-platelets also release 

microparticles. Platelets labeled with 2 M Bodipy-maleimide were stimulated with convulxin 

plus thrombin, and fluorescent microparticles 0.3 to 0.5 m in diameter were observed by 

confocal microscopy. Microparticles were positive for glycoprotein IIb/IIIa, glycoprotein Ib, CD9 

and phosphatidylserine but negative for fibrinogen, thrombospondin and von Willebrand factor. 

This methodology utilizing Bodipy-labeled platelets was also applicable to flow cytometric 

quantitation of microparticles. Platelets stimulated with thrombin, convulxin, ADP or TRAP alone 

produced few microparticles; however, activation with convulxin plus thrombin or A23187 

ionophore plus thrombin produced 15 5 and 25 8 (mean 1SD; n7) microparticles per 

coated-platelet, respectively. Furthermore, the strong correlation (r0.87) between coatedplatelet 

levels and microparticle production indicated that microparticles originated from 

coated-platelets, not the non-coated-platelet population. With this insight, however, it is 

surprising that microparticles did not express the adhesive proteins (fibrinogen, vWF, etc.) that 

are characteristic of coated-platelets. Additionally, four separate monoclonal antibodies against 

the tetraspanin CD9 were found to prevent the release of microparticles during coated-platelet 

production. The anti-CD9 monoclonal antibodies did not impact synthesis of coated-platelets 

but did inhibit microparticle release by 60 to 104%, depending on the specific anti-CD9 mAb. 

Members of the tetraspanin superfamily are known to facilitate membrane fusion, but this is 

the first instance indicating a role for platelet CD9 in membrane vesiculation associated with 

microparticle release. 

Defining the Molecular Basis of Species Differences of IIb3 RGDS 

Sensitivity Provides Insights into Ligand Binding 

Mortimer Poncz, Univ of Pennsylvania; Sch of Medicine, Philadelphia, PA; Ramesh Basani, 

Children’s Hosp of Philadelphia, Philadelphia, PA; Michael Thornton, Florida A&M, 

Tallahassee, FL; Joel S Bennett; Univ of Pennsylvania, Philadelphia, PA 

Fibrinogen binding to IIb3 on rat (R) platelets is 100-fold less sensitive to inhibition by the 

tetrapeptide RGDS than IIb3 on human (H) platelets. Using R-H IIb3 chimeras expressed 

in Chinese Hamster Ovary cells, we localized this species difference in RGDS sensitivity to 2 

regions in the propeller domain of IIb: a region immediately upstream of Asp163 (mature 

H IIb numbering) and to a His substitution at Asp232. These residues are or are immediately 

adjacent to the amino acids (aa) involved in stabilizing the binding of the Arg of an RGD cyclic 

peptide based on the V3 crystal structure. However, a crystal structure of the activated 

IIb3 extracellular domain suggests that the basic moiety of the -carboxyl ligand of 

fibrinogen binds deeper within the IIb3 ligand pocket at Asp224. While R and H IIb differ 

in aa sequences upstream of the Asp224 site, these differences do not affect RGDS sensitivity. 

Tirofiban is a small molecule IIb3 antagonist that mimics the -chain ligand of fibrinogen. 

R IIb3 is resistant to tirofiban inhibition of fibrinogen binding, just as it is to RGDS. However, 

tirofiban resistance is not localized to the same regions of IIb as RGDS resistance. von 

Willebrand Factor (vWF) binds to IIb3 depends on the presence of an RGD motif located in 

its C1 domain. On the basis of the species differences in RGD sensitivity, we asked whether 

there also species-specific differences in the affinity of vWF binding to IIb3. Because R 

IIb3 does not bind H vWF, we measured vWF binding to IIb3 on mouse (M) platelets that 

is also insensitive to inhibition by RGDS. We found that H vWF binds to thrombin-stimulated H 

and M platelets and to M platelets solely expressing HIIbM3. As expected, both fibrinogen 

and vWF binding to M IIb3 was resistant to RGDS, but there was no difference in sensitivity 

to inhibition by tirofiban. These studies localize the species differences in RGD sensitivity to two 

spatially distinct regions of the IIb3 ligand-binding pocket and are consistent with the 

presence of two sites of ligand binding on IIb: (1) a more superficial RGD binding site that 

involves Arg163 and Arg232 involved in VWF binding, and (2) a deeper site at Arg224 involved 

in binding to the -carboxyl end of fibrinogen. 

16 

EPCs Derived from ex Vivo Expansion of Unmobilized Human Peripheral 

Blood Co-Express Hematopoietic and Endothelial-Specific Markers and can 

be Derived from Culture of a Highly Purified Population of CD14-Positive 

Monocytes 

Emerson E Sharpe, III, Amylynn A Teleron, Bin Li, Pampee P Young; Vanderbilt Univ Med 

Cntr, Nashville, TN 

Increasing data has suggested a more dynamic role of vasculogenesis, whereby bone marrow 

(BM)-derived endothelial progenitor cells (EPCs) home and contribute to new blood vessel 

formation during tumor growth, ischemic injury, and wound healing. EPCs can be obtained by 

isolating hematopoietic progenitor cells from BM or cord blood, or through ex vivo expansion 

of unmobilized human peripheral blood (PB). These PB-EPCs express endothelial markers and 

incorporate into developing neovasculature in vivo. The ease of obtaining unmobilized human 

PB has made PB-EPCs an attractive candidate with which to develop cell based therapy to treat 

ischemia. In parallel with clinical trials designed to understand their therapeutic potential, there 

is continued by effort guest to better on June characterize 29, 2013 the PB-EPC and understand its biology. It is currently 

14 

15

E-46 Vol 25, No 5 May 2005 

thought that EPCs are directly derived from a CD34/lin- faction of hematopoietic stem cells. 

However, we have confirmed prior reports that ex vivo expansion of human PB generates 

similar numbers of EPCs as compared to plating unfractionated human BM, which contains 

50-fold higher CD34/lin- content. This suggests that PB-EPCs may not be derived from the 

CD34/lin- population. Using immunofluorescence and FACS analysis we show that PB-EPCs 

not only express endothelial markers such as vWF, VEGF Receptor 1 and 2, VE-cadherin, UEA-1 

lectin, Tie-1, and Tie-2, but also hematopoietic markers such as CD45 and CD14, a marker 

enriched on monocytes. To test if PB-derived CD14-positive cells can give rise to PB-EPCs, we 

isolated them from human PB to 98% purity. Culture of CD14-positive cells generated 

adherent clusters of spindle shaped cells morphologically similar to EPCs, while the 

CD14-negative fraction failed to yield adherent cells. The CD14 derived cells stained positive 

for various endothelial and monocyte/hematopoietic cell markers, as well as demonstrated in 

vitro tube formation. Using a GUSB deficient mouse model we have shown that human CD14 

derived PB-EPCs will incorporate into sites of neovascularization in tumor and matrigel models 

in vivo. We have also shown, through the use of marked BM cells from transgenic mice, that 

culture expanded murine EPCs are derived from a monocyte intermediate. 

17 

Opposing Effects of Bone Morphogenetic Protein 2 (BMP-2) and Epidermal 

Growth Factor (EGF) on Nuclear Targeting of Extracellular-Regulated 

Kinase 1/2 (ERK1/2) and the Transcription Factor, Acute Myeloid Leukemia 

1 Protein (AML1), in Human Pulmonary Artery Smooth Muscle Cells 

(HPASMC) 

Georg Hansmann, Eliana C Martinez, Marlene Rabinovitch; Stanford Univ Sch of Medicine, 

Stanford, CA 

Mutations in the BMP receptor II and elevated elastase activity have been linked to progressive 

pulmonary arterial hypertension. Elastase activity was shown to depend on phosphorylation (p) 

and targeting of pERK to the nuclear extract and subsequent shuttling of AML1B from the 

nuclear matrix to a transcriptionally active state in the nuclear extract of PASMC. We therefore 

hypothesized that growth promoting (EGF) and inhibitory (BMP-2) stimuli have opposing effects 

on nuclear compartmentalization of pERK and AML1B in HPASMC. Methods: HPASMC were 

stimulated with BMP-2 (10ng/ml) or EGF (50ng/ml) for 5– 60min. The compartimental 

localization of phosphorylated ERK1/2 (pERK1/2) and of AML1B were determined by immunoblotting 

and confocal microscopy. Results: Under control condition (0.1% serum), western 

immnoblotting showed that BMP-2 transiently reduced the pERK1/ERK1 and pERK2/ERK2 ratios 

by 29% and 42% in the nuclear extract (p 0.01), and by 24% and 23% in the cytoplasmic 

extract (p 0.05; n 4). BMP-2 led to a 2.0 fold increase of AML1B in the nuclear matrix 

(p 0.01; n 3). Confocal microscopy was consistent with these observations, in verifying 

that BMP-2 augmented AML1B in the nuclear matrix as judged by its co-localization with the 

nuclear matrix protein NuMA. In contrast to BMP-2, EGF transiently increased pERK1/total ERK1 

and pERK2/total ERK2 ratios in the cytoplasmic extract by 2.3 fold and 3.0 fold and in the 

nuclear extract by 2.6 fold and 3.1 fold, respectively (p 0.01; n 4). EGF had no significant 

effect on the amount of AML1B in the 3 cellular compartments (n 3). Confocal microscopy 

showed that nuclear pERK1/2 was not distributed to the nuclear matrix and did not co-localize 

with NuMA. Conclusions: In HPASMC, BMP-2 increases AML1B in the nuclear matrix in a 

pERK-independent manner, where it has previously been shown to be non-transcriptionally 

active. EGF induces pERK in the nuclear extract where it can contribute to regulation of 

transcriptionally active genes. An additional factor independent of EGF mediated signaling is 

required for AML1B release from the nuclear matrix to the nuclear extract. 

Overexpression of PTEN Inhibits Beta3 Integrin-Mediated Signal 

Transduction in Vascular Smooth Muscle Cells 

Jianhua Huang, UNC at Chapel Hill, Chapel Hill, NC; Christopher D Kontos, Duke Univ Med 

Ctr, Durham, NC; Alok Pathak, George A Stouffer; UNC at Chapel Hill, Chapel Hill, NC 

Vascular smooth muscle cell proliferation, migration, adhesion and survival play an important 

role in neointimal hyperplasia resulting from vascular injury. PTEN, a tumor suppressor protein, 

which functions as a phosphatidylinositol 3’-phosphatase and protein phosphatase, has been 

identified to regulate cellular survival, proliferation and migration. Our previous studies 

demonstrated that adenovirus-mediated intraarterial delivery of PTEN inhibits neointimal 

hyperplasia following rat carotid artery injury. Since 3 integrin expression is upregulated by 

vascular injury and 3 integrin antagonists inhibit neointima formation, we investigated the 

effects of PTEN overexpression on 3 integrins levels and integrin-mediated signaling. Human 

aortic smooth muscle cells (HASMC) were infected with a recombinant replication-deficient 

adenovirus encoding either wild type PTEN, dominant negative PTEN or an empty vector used 

as control. Overexpression of PTEN downregulated expression of 3 integrins by Western 

Blotting, and inhibited fibronectin-mediated phosphorylation of FAK, Akt and p44/42 MAPK and 

fibronectin-mediated proliferation. In contrast, inhibition of endogenous PTEN by adenovirusmediated 

overexpression of dominant negative PTEN enhanced fibronectin-mediated phosphorylation 

of FAK, Akt and p44/42 MAPK, and also enhanced proliferation in HASMCs. 

Immunofluorescence assay shown PTEN overexpression inhibited focal adhesion formation, 

while dominant negative PTEN enhanced focal adhesion formation. Taken together, these 

findings demonstrate that PTEN downregulates 13 integrins and modulates integrin/ECMmediated 

signaling and biological functions in HASMC. Downloaded from 

18 



Activation of PPARgamma Inhibits Telomerase Activation in Vascular 

Smooth Muscle Cells by Negative Cross-Talk with ETS-1-Dependent 

Activation of the hTERT Promoter 

Daisuke Ogawa, Takafumi Nakamachi, Jeff Stone, Dennis Bruemmer; Univ of Kentucky, 

Lexington, KY 

Proliferation of vascular smooth muscle cells (VSMC) plays a decisive role for cardiovascular 

diseases. Activation of the peroxisome proliferator-activated receptor (PPAR) by thiazolidinediones 

(TZD) prevents neointima formation and inhibits VSMC proliferation. In the present 

study we demonstrate that ligand-induced and constitutive activation of peroxisome 

proliferator-activated receptor (PPAR) inhibits mitogen-induced telomerase activation. PPAR 

activation by rosiglitazone (RSG), pioglitazone (PIO) and a novel non-TZD partial PPAR agonist 

(nTZDpa) dose-dependently inhibited platelet-derived growth factor and insulin induced 

telomerase activity in rat aortic VSMC (RSG 62.4 8.41 %, PIO 82.9 7.75 %, nTZDpa 

98.3 5.53 % inhibition at 10 M vs. PDGF/insulin, n3, p0.05). Using adenoviralmediated 

overexpression of a constitutively-active and dominant-negative PPAR mutant, we 

provide evidence that PPAR ligands inhibit telomerase activity through a receptor-dependent 

pathway. All PPAR ligands markedly inhibited Telomerase reverse transcriptase (hTERT) 

protein and mRNA expression (RSG 65.2 8.1%, PIO 71.9 9.1, nTZDpa 92.3 6.3 % 

inhibition of mRNA expression at 10 M vs. PDGF/insulin, n3, p0.05), the subunit 

mediating the catalytic activity of telomerase. Transient transfection experiments revealed a 

potent PPAR-dependent inhibition of mitogen-induced hTERT promoter activity by these 

ligands. Inhibition of hTERT promoter activity was mediated through negative cross-talk of 

ets-1 binding to the hTERT promoter as evidenced by 5’-deletion analysis of the hTERT 

promoter and overexpression of ets-1. Thus, inhibition of telomerase activation by PPAR 

ligands in VSMC may occur through an inhibition of hTERT transcription involving a 

PPAR-dependent mechanism. These findings provide a previously unrecognized novel 

mechanism for the antiproliferative effects of PPAR ligands and support the concept that 

PPAR ligands may constitute a novel therapeutic approach for the treatment of proliferative 

cardiovascular diseases. 

20 

Point Mutations in ApoA-I Mimic the Phenotype Observed in Patients with 

Classical LCAT Deficiency 

Angeliki Chroni, Adelina Shkodrani, Horng-Yuan Kan, Tong Liu, Vassilis I Zannis; Boston 

Univ Med Sch, Whitaker Cardiovascular Institute, Boston, MA 

We have analyzed the effect of charged to neutral amino acid substitutions around the kinks 

flanking helices 4 and 6 of apoA-I and the deletion of helix 6 on the in vivo activity of 

lecithin:cholesterol acyl transferase (LCAT) and the biogenesis of HDL. The LCAT activation 

capacity of apoA-I in vitro was nearly abolished by the helix 6 point (apoA-I[R160V/H162A]) and 

deletion (apoA-I[(144–165)]) mutant, but was reduced to 50% in helix 4 point mutant 

(apoA-I[D102A/D103A]). Following adenovirus-mediated gene transfer in apoA-I deficient 

(apoA-I -/- ) mice, plasma HDL cholesterol was greatly reduced in helix 6 point and deletion 

mutants. Electron microscopy and two-dimensional gel electrophoresis showed that the helix 

6 point mutant formed predominantly high levels of discoidal particles and had increased 

pre1-HDL/-HDL ratio. The helix 6 deletion mutant formed few spherical particles and had 

increased pre1-HDL/-HDL ratio. Mice infected with adenovirus expressing the helix 4 

mutant or WT apoA-I had normal HDL cholesterol and formed spherical -HDL particles. 

Co-infection of mice with adenoviruses expressing human LCAT and the helix 6 point mutant 

increased dramatically plasma HDL and apoA-I levels, indicating that the LCAT activity was rate 

limiting for the biogenesis of HDL. The LCAT treatment caused only a small increase in HDL 

cholesterol and apoA-I and -HDL particle numbers in the helix 6 deletion mutant. The findings 

indicate a critical contribution of residues 160 and 162 of apoA-I in the in vivo activity of LCAT 

and the subsequent maturation of HDL and explain the low HDL levels in heterozygous subjects 

carrying these mutations. 

21 

Oxidized Phospholipids Mediate the Induction of Unfolded Protein 

Response in Human Aortic Endothelial Cells: Athero-Protective Role of HDL 

Peter S Gargalovic, Nima M Gharavi, Michael J Clark, UCLA, Los Angeles, CA; Wen-Pin 

Yang, Aiqing He, Amy Truong, Bristol-Myers Squibb, Pharmaceutical Rsch Institute, 

Princeton, NJ; Tamar Baruch-Oren, Judith A Berliner, UCLA, Los Angeles, CA; Todd G 

Kirchgessner, Bristol-Myers Squibb, Pharmaceutical Rsch Institute, Princeton, NJ; Aldons J 

Lusis; UCLA, Los Angeles, CA 

The interaction of oxidized phospholipids with endothelial cells results in an inflammatory 

response, likely representing a crucial step in the initiation of atherosclerosis. In this study we 

used gene expression array analysis to comprehensively examine the effects of oxidized 

palmitoyl-arachidonyl-phosphatidyl choline (oxPAPC) on cultured primary human aortic endothelial 

cells (HAEC). HAEC were treated with increasing doses of oxPAPC (10, 30 and 50 g/ml) 

for 4 hours and changes in gene expression were compared to cells treated with bacterial 

lipopolysacharide (LPS). Our data show that in addition to inflammatory genes such as 

interleukin-8 (IL8), monocyte-chemotactic protein-1 (MCP1) and GRO chemokines, oxPAPC but 

not LPS treatment resulted in a dose- and time-dependent induction of a large number of genes 

involved in the endoplasmic reticulum stress-mediated Unfolded Protein Response (UPR) 

pathway, including ATF4, ATF3 and XBP1. The UPR activation was associated with changes in 

gene expression characteristic of the cell cycle arrest, without induction of apoptosis. These 

effects were endothelial cell-specific, as oxPAPC failed to induce the UPR in human 

monocyte-derived macrophages. Treatment of HAEC with UPR inducers, tunicamycin and 

dithiothreitol (DTT), resulted in significant induction of MCP1 and IL8. Furthermore, treatment 

of HAEC with HDL inhibited oxPAPC-mediated activation of the UPR, and the induction of IL8 

and MCP1by but guest not heme on oxygenase June 29, 12013 and heat shock 70kDa protein 1A. These data are the 

19

first to show activation of the UPR pathway by oxidized phospholipids, and provide an additional 

mechanism for the anti-atherogenic effects of HDL. 

22 

Placental Growth Factor (PlGF) Promotes Atherosclerotic Intimal Thickening 

and Macrophage Accumulation 

Rohit Khurana, Univ College London, London, United Kingdom; Lieve Moons, Cntr for 

Transgene Technology & Gene Therapy, Univ of Leuven, Belgium; Shahida Shafi, Univ 

College London, London, United Kingdom; Aernout Luttun, Desire Collen, Cntr for Transgene 

Technology & Gene Therapy, Univ of Leuven, Belgium; John F Martin, Univ College London, 

London, United Kingdom; Peter Carmeliet, Cntr for Transgene Technology & Gene Therapy, 

Univ of Leuven, Belgium; Ian C Zachary; Univ College London, London, United Kingdom 

Background: Placental Growth Factor (PlGF) is implicated in pathophysiological angiogenesis 

and monocyte recruitment underlying chronic inflammatory disease, but its role in atherosclerosis 

has not been examined. We investigated the effects of adenoviral PlGF delivery on 

atherogenic intimal thickening and macrophage accumulation induced by collar placement 

around the rabbit carotid artery, and examined the effects of PlGF deficiency on atherosclerosis 

in ApoE -/- mice. Methods and Results: Periadventitial transfer of PlGF2-encoding adenoviruses 

significantly increased intimal thickening, macrophage accumulation, endothelial VCAM1 

expression and adventitial neovascularization in the collared arteries of hypercholesterolaemic 

rabbits, and also increased the intima to media ratio in rabbits fed a normal diet. Neointimal 

macrophages were associated with increased expression of the PlGF receptor, Flt-1. The size 

and macrophage content of early (10 weeks) atherosclerotic aortic root lesions were reduced 

in mice deficient in both ApoE and PlGF compared to ApoE-deficient mice. This effect was not 

sustained for more mature lesions (25 weeks). PlGF deficient mice also developed fewer lesions 

in the descending thoracic and abdominal aorta at the early timepoint, but plaque number and 

size were similar in both genotypes at the later timepoint. The total leukoyte count in peripheral 

blood was also reduced in PlGF deficient mice with early but not late lesions. Conclusions: 

Local adenoviral PlGF2 delivery promotes atherogenic neointima formation in hypercholesterolaemic 

rabbits and PlGF is required for macrophage infiltration in early atherosclerotic lesions 

in ApoE -/- mice. These findings support a novel role for PlGF in the pathogenesis of 

atherosclerotic disease. 

Effects of Ad.PlGF2 Gene Delivery on Lesion Formation in Collared Rabbit Carotid Arteries 

1.5% CHOL DIET NORMAL DIET 

Ad.LacZ Ad.PlGF2 Ad.LacZ Ad.PlGF2 

Intima/Media 0.15.0.02 0.28.0.01* 0.097.0.006 0.16.0.02* 

Macrophages/ mm2 

neointima 

24630 47066* ND ND 

Macrophages/ mm2 

adventitia 

13.51.5 22.53.6* 24.43.3 47.14.1* 

CD31/mm2 adventitia 6.72.3 28.72.3* 9.32.7 22.44.4* 

% Endothelial VCAM1 24.01.6 47.84.4* ND ND 

AXL is a Novel Mediator of Flow-Induced Vascular Remodeling 

Vyacheslav A Korshunov, Amy M Mohan, Bradford C Berk; Univ of Rochester, Rochester, NY 

Vascular remodeling in response to flow is a physiologic response that requires coordinated 

signaling between endothelial cells and vascular smooth muscle cells (VSMC). Previously we 

identified Axl, a receptor tyrosine kinase, to be highly induced in VSMC after carotid injury. 

Because Axl function is essential for VSMC survival, proliferation and migration, we 

hypothesized that Axl would play a role in vascular remodeling. Vascular remodeling in mice 

deficient in Axl expression (Axl-/-) and wild-type littermates (Axl/) was induced by ligation 

of the left external and internal carotid arteries maintaining flow via the left occipital artery. 

Axl-/- and Axl/ mice had the same body weights (18 –22g, dependent on gender), systolic 

blood pressure (130mmHg), heart rate (620 b/min), and carotid artery structure. Partial 

ligation altered blood flow equally in both genotypes: increased by 60% in the right carotid 

(RCA) and decreased by 80% in the left carotid (LCA). There were no significant differences in 

RCA remodeling between Axl-/- and Axl/. However, the increases in intima and media at 

2 weeks were significantly less in LCA from Axl-/- compared to Axl/ mice (32 and 282 

vs. 63 vs. 364 x10(-6)m(3), respectively). Quantitative immunohistochemistry analyses 

suggested that increased apoptosis (10-fold) in the intimal layer accounted for altered vascular 

remodeling in Axl-/- compared to Axl/ mice. These data demonstrate an important role for 

Axl in flow-dependent remodeling and demonstrate that a single gene contributes significantly 

to vascular remodeling. 

24 

Forkhead Signaling Inhibits Vascular Smooth Muscle Cell Proliferation and 

Neointimal Hyperplasia 

Ruhul Abid, Kiichiro Yano, Shaodong Guo, Katherine C Spokes, Virendra I Patel, Gautam 

Shrikhande, Christiane Ferran, William C Aird; Harvard Med Sch, BIDMC, Boston, MA 

Background: Vascular smooth muscle cell (VSMC) proliferation and migration contribute 

significantly to atherosclerosis, post-angioplasty restenosis, and transplant vasculopathy. 

Forkhead transcription factors belonging to the FoxO subfamily have been shown to inhibit 

growth and cell cycle progression in a variety of cell types. We hypothesized that forkhead 

proteins may play a role in VSMC biology. Methods and Results: Under in vitro conditions, 

platelet derived growth factor (PDGF)-BB, tumor necrosis factor (TNF)- and insulin-like growth 

factor 1 (IGF-1) stimulated phosphorylation of FoxO in human coronary artery smooth muscle 

cells (CASMC) via MEK1/2 and/or PI3K-dependent signaling pathways. PDGF-BB, TNF- and 

IGF-1 treatment resulted in the nuclear exclusion Downloaded of FoxO, from 

whereas PDGF-BB alone 

23 

downregulated the FoxO target genes, p27 kip1 and B-cell translocation gene (BTG) 1, and 

enhanced cell survival and progression through the cell cycle. These effects were abrogated by 

over-expression of a constitutively active, phosphorylation-resistant mutant of the FoxO family 

member, FKHRL1. In a rat balloon carotid arterial injury model, adenovirus-mediated gene 

transfer of FKHRL1 caused an increase in the expression of p27 kip1 in the VSMC and inhibition 

of neointimal hyperplasia in vivo. Conclusions: These data suggest that FoxO activity inhibits 

VSMC proliferation and activation, and that this signaling axis may represent a therapeutic 

target in vasculopathic disease states. 

25 

Unsaturated Fatty Acids Phosphorylate and Destabilize ABCA1 through a 

Phospholipase D2 Pathway 

Yutong Wang, John F Oram; Univ of Washington, Seattle, WA 




Abnormal high-density lipoprotein (HDL) metabolism among patients with diabetes and insulin 

resistance may contribute to their increased risk of atherosclerosis. ATP-binding cassette 

transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to 

HDL apolipoproteins and thus modulates HDL levels and atherogenesis. We previously reported 

that unsaturated fatty acids (FAs), which are elevated in diabetes, destabilize ABCA1 in murine 

macrophages and ABCA1-transfected baby hamster kidney (BHK) cells by a novel mechanism. 

Here we examined the pathway mediating the fatty acid inhibition of ABCA1 in both cell types. 

The long-chain acyl-CoA synthetase inhibitor Triacsin C completely reversed FA-induced 

ABCA1 destabilization, indicating that FAs need to be activated to their CoA derivatives to 

enhance ABCA1 degradation. Phospholipase D (PLD) activity was stimulated by unsaturated but 

not saturated FAs, and the inhibitory effect of unsaturated FAs on ABCA1 was reduced by PLD 

inhibitor 1-butanol, but not by 2-butanol, indicating a role for PLD in FA-induced ABCA1 

degradation. Serine phosphorylation of ABCA1 was enhanced by unsaturated FAs and the PLD 

activator mastoparan. PLD2 siRNA abolished the FA-induced degradation and serine phosphorylation 

of ABCA1, indicating that unsaturated FAs destabilize ABCA1 by increasing 

phosphorylation of its serine residues through a PLD2 pathway. The diacylglycerol analog 

oleoylacetylglycerol also reduced ABCA1 protein levels and activity. These data suggest that 

unsaturated acyl-CoA derivatives activate an ABCA1-destabilizing signaling pathway involving 

generation of diacylglycerol by PLD2, activation of a serine protein kinase, and phosphorylation 

of ABCA1. 

Transfection of Apolipoprotein A-IV Induces Secretion of Larger 

Triglyceride-Rich Lipoproteins in McA-RH7777 Rat Hepatoma Cells 

James W Gallagher, IV, Richard Weinberg, Gregory S Shelness; Wake Forest Univ Baptist 

Med Cntr, Winston Salem, NC 

Background: Recent evidence suggests that apolipoprotein A-IV (apoAIV) may increase 

intestinal lipid transport by facilitating chylomicron assembly. Moreover, we have shown that 

expression of apoAIV modified with the ER retention signal KDEL (apoAIV-KDEL) in COS cells 

decreases secretion of cotransfected apoB41, suggesting that apoAIV and apoB interact with 

each other within the secretory pathway. Here we have explored the functional consequences 

of this interaction. Methods: We generated stable human apoAIV and apoAIV-KDEL-expressing 

McA-RH7777 rat hepatoma cells, radiolabeled cellular lipids with [ 3H]oleate, and measured 

secretion of endogenous apoB and triglyceride (TG). Results: As expected, in oleate-stimulated 

cells, tranfection of apoAIV-KDEL reduced apoB secretion by 64% with a parallel 60% 

reduction of TG secretion. However, tranfection with native apoAIV resulted in a 34% 

reduction in apoB secretion with no change in TG secretion and a 2.5-fold increase in the 

TG:phospholipid ratio in the media (p0.001). Conclusions: These data suggest that apoAIV 

interacts with apoB within the secretory pathway, thus resulting in secretion of larger 

apoB-containing nascent TG-rich particles. Given the slow kinetics of native apoA-IV secretion, 

we hypothesize that an interaction between apo AIV and apoB retards the intracellular transport 

of apoB, thereby increasing its residence time in a compartment responsible for nascent 

TR-rich particle expansion, which enhances addition of core TG, and ultimately facilitates 

intestinalby lipidguest transport. on June 29, 2013 

26

E-48 Vol 25, No 5 May 2005 

Mechanism of HDL Endocytosis by Scavenger Receptor SR-BII 

Erik Eckhardt, Lei Cai, Shoba Shetty, Nancy R Webb, Deneys R van der Westhuyzen; Univ 

of Kentucky, Lexington, KY 

Scavenger Receptor BI (SR-BI) mediates selective uptake of lipids from HDL, but the exact 

mechanism of this process remains unknown. It is still not clear, for example, whether 

lipoprotein endocytosis contributes to selective uptake. Our group previously identified an 

alternative mRNA splicing variant of SR-BI, named SR-BII, with an entirely different, yet highly 

conserved cytoplasmic carboxy-terminus. We recently reported that, in transfected CHO 

fibroblasts, the SR-BII isoform mediates endocytosis of HDL particles at a markedly faster rate 

than SR-BI. In the present study we have investigated the pathways responsible for SR-BI and 

SR-BII internalization and recycling. SR-BI and SR-BII exhibited different subcellular distributions 

and the majority of HDL endocytosed by SR-BII appeared to be localized in the 

endosomal-recycling compartment (ERC), where most of GFP-tagged SR-BII resided. A specific 

dileucine- motif responsible for rapid receptor and ligand internalization and recycling was 

identified in the C-terminal tail of SR-BII through mutational analysis. A less well conserved 

tyrosine-based YXXL motif, which partially overlaps with the dileucine motif, may modulate 

internalization. Endocytosis of SR-BII occurred via a clathrin-dependent pathway that was 

distinct from the pathway responsible for SR-BI internalization. Due to the differences in 

receptor- and ligand- internalization and recycling pathways, SR-BI and SR-BII might have 

different effects on cellular cholesterol flux. 

The Molecular Clock and Circadian Variability in Vascular Biology 

Elizabeth J Westgate, Anne M Curtis, Yan Cheng, Ekaterina Kostetskaia, Garret A FitzGerald; 

Univ. of Pennsylvania, Philadelphia, PA 

Cardiovascular physiology, as well as pathophysiology, undergoes marked diurnal variation, 

which may be influenced by the master circadian clock located in the suprachiasmatic nucleus. 

Peripheral clocks have been characterized in cardiovascular tissue and cells which may 

influence the known circadian rhythms in blood pressure (BP). Using telemetry to record BP in 

a free running environment, we observe a robust daily rhythm in BP in wild-type (WT) C57Bl6 

mice that attains peaks and troughs at approximately 1am and 1pm, respectively. We used 

mice with disrupted (BMAL KO) or mutated (NPAS2 mutant) core components of the molecular 

clock to determine their role in regulating BP rhythms. NPAS2 mutants display a modest 

hypotensive phenotype and the time at which peak BP occurs is phase delayed. The 

behaviorally arrhythmic BMAL1 KO mice also show no apparent BP rhythm in comparison to 

WT controls. Genes relevant to vascular injury and integrity have been shown to oscillate in 

mouse aorta. Therefore, we explored the influence of circadian clocks in conditioning the 

response to a thrombogenic injury in the mouse femoral artery. Laser-induced photochemical 

injury at 9AM and 9PM led to significantly faster occlusion times (16.9 /- 1.1 and 13.4 /- 

2.8min, respectively) compared to injury performed at 3PM (26.6 /- 2.0min, p0.05 and 

p0.01, respectively). We next addressed the role of platelet aggregation, which is known to 

oscillate in humans, by whole blood (WB) aggregation assays in WT mice. A shift in the dose 

response curve to the potent thromboxane receptor agonist I-BOP was evident, with a 6-fold 

increase in the response to 250nM I-BOP at 3PM versus 9AM. These studies suggest that the 

molecular clock profoundly influences circadian variation in BP. The detection of circadian 

variation in platelet function and the response to a thrombogenic stimulus in vivo suggests that 

mice will afford a convenient model system in which to investigate the role of the molecular 

clock in cardiovascular biology. 

Genetic Reduction of Tissue Factor Does not Affect the Progression of 

Atherosclerosis in Mice 

Rachel Tilley, Brian Pederson, Yuichiro Sato, The Scripps Rsch Institute, La Jolla, CA; Y. C 

Shen, Sharlene Day, David Eitzman, Univ of Michigan Med Cntr, Ann Arbor, MI; Linda 

Curtiss, The Scripps Rsch Institute, La Jolla, CA; William Fay, Univ of Michigan Med Cntr, 

Ann Arbor, MI; Nigel Mackman; The Scripps Rsch Institute, La Jolla, CA 

The clinical manifestations of atherosclerosis, including acute coronary syndrome, myocardial 

infarction and stroke, are often consequences of acute plaque rupture that induces the 

formation of a platelet-rich thrombus. Tissue factor (TF), the primary cellular activator of the 

coagulation cascade, is abundantly expressed in atherosclerotic plaques and likely contributes 

to thrombosis after plaque rupture. In addition, TFDownloaded has been shown tofrom enhance macrophage and 

27 

28 

29 

vascular smooth muscle cell migration and restenosis. In this study, we determined if TF plays 

a role in the progression of atherosclerotic lesions using two mouse models. Transgenic mice 

that express either 50% (TF /- ) or 1% (low TF) levels of TF were crossed into an apolipoprotein 

E deficient (ApoE -/- ) background. In the first experiment, we compared the extent of 

atherosclerosis in the aorta and major arteries between ApoE -/- /TF /- and ApoE -/- /TF / in mice 

fed a high fat diet for 34 weeks. No differences in atherosclerosis were observed between the 

two groups. Unfortunately, the ApoE -/- /low TF mice died prematurely on a high fat diet, which 

precluded the analysis of the extent of atherosclerosis. Macrophages are a major source of TF 

in atherosclerotic plaques, and are critical for lesion formation in mice. Therefore, we 

investigated the effect of specifically reducing hematopoietic cell (macrophage) -derived TF 

using bone marrow transplantation of low TF or TF /o ( o indicates /- or / ) mice into LDLR 

deficient (LDLR -/- ) recipients. Atherosclerotic lesions were measured after 4 or 16 weeks on a 

high fat diet. In the LDLR -/- /low TF and the LDLR -/- /TF /o controls, plasma cholesterol levels 

were increased due to the high fat diet; however there were no differences between the two 

groups. Atherosclerotic lesions within the aorta and heart valves were similar in LDLR -/- /low TF 

and LDLR -/- /TF /o controls after both 4 and 16 weeks on a high fat diet. These data suggest 

that either a 50% reduction of TF in all cells or a reduction in hematopoietic cell 

(macrophage)-derived TF does not affect the development of atherosclerotic lesions in two 

mouse models. 

GPG-290: A vonWillebrand Factor Antagonist That Prevents Occlusive 

Thrombus Formation in Models of Arterial Thrombosis and can be 

Reversed with DDAVP 

Gray D Shaw; Wyeth Rsch, Cambridge, MA 



Background: Under the high shear conditions of arterial blood flow, the initial capture and 

accumulation of platelets on the vessel wall is highly dependent on subendothelial collagenbound 

von Willebrand factor (vWF) binding to GPIb on the platelet surface. GPG-290 is a 

soluble recombinant Ig chimera form of GPIb that can act as a competitive antagonist to this 

high shear interaction with less effect on low shear platelet adhesion events. Structure/function 

analysis of GPG-290 indicates that, in the dose ranges studied, its activity is primarily due to 

blockade of the vWF A1 domain and not antagonism of -thrombin exosite II. The efficacy of 

GPG-290 was evaluated in a canine Folt’s model of coronary artery injury as well as an 

electrolytic injury model. Results: In the Folt’s model, a single intravenous bolus injection of 

GPG-290 at doses of 50 or 100mcg/kg immediately abolished cyclic flow reductions (CFRs) in 

100% of treated dogs (n 14). After high dose GPG-290, (500mcg/kg) subsequent treatment 

of animals with DDAVP (0.3mcg/kg) could rapidly reversed prolonged bleeding times by 

releasing stored endothelial vWF, without causing CFR recurrence. In the electrolytic injury 

model, time to thrombotic occlusion (TTO) in control arteries was 326 min compared to 

625, 9421, and 15623 min in the 50, 100 and 500mcg/kg dose groups respectively 

(p0.05 vs. control). Treatment with clopidogrel (4.3mg/kg loading dose and 2 days of 

1.1mg/kg) as a monotherapy resulted in a prolonged TTO (88.2 20.7 min) compared to 

control (p0.05). Combination therapy, clopidogrel GPG290 (100mcg/kg), resulted in an 

additional prolongation of time to occlusion (127.8 32.3 min) together with a complete 

prevention of occlusion in 3/5 dogs. In combination studies with aspirinLMWH, GPG-290 

(50mcg/kg) prolonged bleeding significantly less than eptifibatide. GPG-290 activity in human 

whole blood could be readily monitored using either a PFA-100 assay or ristocetin induced 

platelet aggregation (RIPA) assay. Conclusions: GPG-290 represents a potentially advantageous 

new therapy for the management of patients with unstable angina or other acute 

thrombotic disorders. 

31 

Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1): A Modulator of 

Thrombosis 

Jenny J Zhang, Purba Biswas, Puyao Li, Joseph A Madri; Yale Univ, New Haven, CT 

Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoglobulin family vascular 

adhesion molecule, is expressed on hematopoietic and endothelial cells. PECAM-1 can function 

in modulating apoptosis, cell proliferation, migration and maintaining endothelial integrity as 

well as neutrophil transmigration. PECAM-1 is also known to inhibit platelet function and 

thrombus formation. The present study demonstrated that PECAM-1 knock out mice (KO) 

exhibited increased thrombi in peritubular microvessels and increased TUNEL positive staining 

in renal tubules as compared to wild type animals after 30 minutes renal ischemia followed by 

24 hours reperfusion, suggesting that PECAM-1 has a role in modulating thrombosis, which 

may, in turn affect parenchymal cell survival. We demonstrated an increase in tissue factor (TF) 

expression (a known inducer of coagulation) in the KO kidneys. In in vitro studies using human 

umbilical vein endothelial cells (HUVEC) transfected with scrambled (Scr) and antisense (AS) 

PECAM-1 oligonucleotides to selectively down-regulate PECAM expression we documented a 

robust 1.7-fold induction of TF in the AS-treated HUVEC compared to Scr-treated HUVEC 

following thrombin stimulation. This TF induction was mediated through thrombin receptor 

PAR-1 and was dependent on Rho activation, phosphorylation of p38 MAPK. P85 and Akt were 

dephosphorylated after thrombin treatment in both cell types, but the PECAM-1 knock down 

cells showed 2.5-fold less Akt phosphorylation compared to wild type cells. This may be 

attributed to an increase of PTEN expression in the PECAM-1 knock down cells in response to 

thrombin stimulation. Interestingly, we found an inverse correlation of PI3K-Akt phosphorylation 

with p38 phosphorylation. Selective pharmacological inhibition of PI3K and/or p38 confirmed 

this correlation. The studies outlined here suggest a signaling pathway involving PECAM-1 

modulation of PI3K, which, in turn regulates p38 phsophorylation state by Akt activation. This 

study illustrates that PECAM-1 can modulate TF induction in endothelial cells by regulating 

Rho/Rho-kinase, p38 MAPK, PI3K-Akt and PTEN. These findings provide new insights into the 

action of by PECAM-1 guest ason a modulator June 29, of2013 thrombosis. 

30

32 

RNA Interference Targeting 8 Integrin Sheds Light on the Dichotomy of 

Vascular Smooth Muscle Cell Migration and Proliferation 

Ramin Zargham, Gaétan Tibault; McGill Univ and IRCM, Montreal, Canada 

The central dogma in the development of many vascular diseases, including atherosclerosis, 

hypertension and restenosis after angioplasty, is phenotype modulation of vascular smooth 

muscle cell (VSMC) from differentiated to de-differentiated mainly mediated by the effect of 

growth factors. We have shown that 8 integrin is a differentiation marker of VSMCs, and its 

downregulation is correlated with heightened VSMC migratory activity, but it coudn‘t affect their 

proliferation rate in response to growth factors’ stimulation. Therefore, it raised the question 

that whether 8 integrin could affect the phenotype machinery of VSMCs and, if it is so, why 

the proliferation rate was not changed. In the present study, first we elucidated the possible 

influence of 8 integrin on VSMC phenotype. Then, we aimed to shed light on the dichotomy 

between migration and proliferation regarding the VSMC phenotype. To downregulate 8 

integrin expression, we used short interference RNA (siRNA) method of gene silencing. To 

induce de-differentiated phenotype in vivo, a rat model of carotid angioplasty was performed. 

Immunoblotting and Immunohistochemical analysis were used to detect the expression of 

phenotype dependent markers. Our finding presented evidence that 8 knock down induces a 

de-differentiated phenotype by inhibiting the expression of differentiation markers through 

RhoA signaling pathway. To address whether there is a correlation between mitogenic effect 

of growth factors and a specific, de-differentiated phenotype, VSMCs were stimulated by 

different growth factors, including PDGF-BB, b-FGF, EGF and Thrombin. Our results demonstrated 

that a growth factor with low mitogenic effect (PDGF-BB) induced the highest migratory 

effect with the de-differentiated phenotype. Interestingly, another growth factor with highly 

mitogenic effect (thrombin) could induce differentiated (contractile) phenotype and at the same 

time increased the expression of cyclin D1, as an indicator of cell cycle progress, as well as 

increased thymidine incorporation. Taken together our results suggest that phenotype 

modulation of VSMC elicited by growth factors is more related to their chemotactic effect rather 

than their mitogenic effect. 

Valsartan Inhibition of Intima Formation Requires Angiotensin II Type 2 

Receptor Function 

Thomas A Barker, Michael P Massett, Vyacheslav A Korshunov, Amy M Mohan, Bradford C 

Berk; Univ of Rochester, Rochester, NY 

The effects of angiotensin II type 1 receptor (AT1R) blockers are due in part to angiotensin II 

type 2 receptor (AT2R) signaling. Interactions between the AT2R and kinins modulate 

cardiovascular function. Because AT2R expression increases following vascular injury we 

hypothesized that the effects of valsartan on vascular remodeling require AT2R signaling 

through the bradykinin 1 and 2 receptors (B1R and B2R). To test this hypothesis Brown Norway 

rats had telemetry probes implanted. They were then assigned to one of six treatments 

(n16/group): valsartan, valsartanPD123319 (AT2R inhibitor), valsartandes-arg9-[Leu8]bradykinin 

(B1R inhibitor), valsartanHOE140 (B2R inhibitor), amlodipine and vehicle. After 1 

week of treatment carotid balloon injury was performed. Two weeks later carotids were 

harvested for morphometry and analysis of receptor expression by immunohistochemistry and 

western blotting. All treatments significantly reduced mean arterial pressure compared to 

vehicle (p0.01). Valsartan significantly reduced intima:media ratio compared to both the 

blood pressure control amlodipine (1.8 vs. 0.68, a 62% reduction, p0.001) and vehicle (1.78 

vs. 0.68, a 61% reduction, p0.005). Blockade of AT2R, B1R or B2R in the presence of 

valsartan prevented the reduction seen with valsartan alone. Injury increased AT1R, AT2R, B1R 

and B2R expression as shown by western blotting. Using immunohistochemistry (IHC) receptor 

expression was mainly seen in the intima. AT1R, B1R and B2R were also present in the media. 

Quantitation of IHC showed that valsartan treatment significantly increased intima AT2R 

expression 2-fold compared to vehicle (p0.004). This was not reversed by inhibition of AT2R, 

B1R and B2R. Conclusions: These results show the beneficial effects of valsartan on arterial 

remodeling require increased AT2R expression, and AT2R signaling via B1R and B2R. 

34 

Tissue Specific Deletion of Enterocyte ABCA1 Identifies the Intestine as a 

Significant Source of Plasma HDL Cholesterol in Vivo 

Liam R Brunham, Terry D Pape, Univ of British Columbia, Vancouver, Canada; Catherine 

Fievet, Institut Pasteur, Lille, France; Jenelle M Timmins, Wake Forest Univ, Winston-Salem, 

NC; Nagat Bissada, Bryan A Coburn, Univ of British Columbia, Vancouver, Canada; Bart 

Staels, Institut Pateur, Lille, France; John S Parks, Wake Forest Univ, Winston-Salem, NC; 

Michael R Hayden; Univ of British Columbia, Vancouver, Canada 

The ATP-binding cassette transporter A1 (ABCA1) controls the rate-limiting step in HDL particle 

formation by mediating the efflux of cellular cholesterol and phospholipids to an apolipoprotein 

acceptor. ABCA1 is widely expressed throughout the body; however, the quantitatively 

important sites of HDL formation are unknown. We have recently used tissue specific gene 

targeting to selectively delete ABCA1 in the liver, which we have shown to result in a substantial 

(80%) decrease in plasma HDL cholesterol. These results indicated that the liver is the major, 

but not the sole contributor to plasma HDL cholesterol levels, and raised the question of what 

are the extrahepatic sources of HDL production in vivo? The intestine is a candidate tissue for 

HDL production because it, along with the liver, is an important source of ApoA-I, the principal 

apolipoprotein component of HDL, and because studies in rats have demonstrated the presence 

of nascent HDL in intestinal lymph. To specifically assess the contribution of the intestine to 

plasma HDL levels we generated mice with enterocyte-specific deletion of ABCA1 by crossing 

Abca1 floxed mice to mice transgenic for Cre recombinase under the control of the 

enterocyte-specific Villin promoter. Tissue specific recombination of the Abca1 locus was 

confirmed by Southern blot. ABCA1 intestinal specific knock-out (Abca1-I/-I) mice had 

undetectable ABCA1 protein expression in theDownloaded intestine, whereas from 

hepatic ABCA1 protein 

33 



expression was comparable to wildtype animals. HDL cholesterol concentrations in Abca1/, 

Abca1/-I and Abca1-I/-I mice were 74.913.3, 66.821.4 and 47.422.4 mg/dL, 

respectively, indicating a 35% decrease in plasma HDL cholesterol between / and -I/-I 

mice (p0.05). Plasma ApoA-I and ApoB levels were similarly decreased by approximately 

25% and 30%, respectively. These results provide physiological evidence for the role of the 

intestine in HDL production, and establish intestinal ABCA1 as a significant contributor to 

plasma HDL cholesterol levels in vivo. 

35 

Identification of the N-Terminal Domain of ApoB-100 Critical for Initiation 

of Lipoprotein Assembly 

Nassrin Dashti, Medha Manchekar, Univ of Alabama, Birmingham, AL; Paul E Richardson, 

Coastal Carolina Univ, Conway, SC; Zhihuan Sun, Jere P Segrest; Univ of Alabama, 

Birmingham, AL 

We have proposed that the N-terminal 1 domain (residues 1–1000) of apoB-100 forms a 

“lipid pocket” that initiates the assembly of apoB-containing lipoproteins. We demonstrated 

that apoB:1000 (residues 1–1000) is secreted by stable transformants of McA-RH7777 cells as 

a monodisperse particle with HDL 3 density. In contrast, apoB:931 (residues 1–931) was 

secreted as a lipid-poor particle considerably more dense than HDL 3. We showed that the “lipid 

pocket” is formed without a structural requirement for MTP, that it has a maximum capacity 

of 50 molecules of phospholipids, and that it has a surface to core lipid ratio of 4:1, 

supporting a small bilayer-type organization. These results are supported by our proposed 

all-atom model of the “lipid pocket” in which the charged residues 717–720 (localized in the 

amphipathic helical hairpin formed by residues 667–746), form salt bridges with the 

complementary charged residues 997-1000 at the C-terminus of the model completing the 

“lipid pocket” without the structural requirement for MTP. This “lipid pocket” model can hold 

approximately 48 POPC molecules. These results indicated that a portion, or perhaps all, of the 

amino acid residues between 931 and 1000 are critical for the formation of the lipoprotein 

particle. To map the structural domain required for the initiation of particle assembly, we 

generated two C-terminally truncated apoB cDNA constructs between residues 931 and 1000, 

i.e., apoB:956 and apoB:986. To test the hairpin bridge mechanism for the formation of the 

“lipid pocket”, we generated apoB:996 which lacks the C-terminus residues required to lock 

the bridge. Characterization of the secreted particles showed that (i) ApoB:956 is secreted as 

a lipid-poor unstable particle with a high tendency to form aggregates; (ii) ApoB:986 and 

apoB:996 form two types of particles, a proportion is secreted as large aggregates, and a 

smaller proportion as lipid-containing particles that appear to be monomeric; (iii) In 

contradistinction, apoB:1000 is predominantly secreted as monodisperse, stable relatively 

lipid-rich particle. Our results indicate that the entire 1 domain is essential for the initiation 

of stable particle assembly and suggest that residues 997-1000 may play a key role in this 

process. 

36 

Inflammation and the Reverse Cholesterol Transport Pathway in Humans 

Margarita de la Llera- Moya, Childrens Hosp of Philadelphia, Philadelphia, PA; Michael 

Byrne, Megan L Wolfe, Christine C Hinkle, Jennifer Tabita-Martinez, Kimberly F Sellers, Univ 

of Pennsylvania Med Cntr, Philadelphia, PA; George H Rothblat, Daniel J Rader, Childrens 

Hosp of Philadelphia, Philadelphia, PA; Muredach P Reilly; Univ of Pennsylvania Med Cntr, 

Philadelphia, PA 

A chronic inflammatory state coincides with reduced HDL cholesterol (HDL-C) and apoA-I in 

insulin resistance and is believed to contribute to reduced reverse cholesterol transport (RCT) 

and atherosclerosis. However, the mechanisms of low HDL-C/apoA-I and altered RCT in human 

inflammatory states remain poorly characterized. We employed low level human endotoxemia 

(LPS 3ng/kg bolus intravenous dose; n20 healthy volunteers; 3 day inpatient GCRC protocol) 

to assess the acute effects of inflammation on plasma lipids, apolipoproteins, serum and HDL 

efflux capacity as well as whole blood mRNA expression of cholesterol efflux genes. Plasma, 

serum and whole blood RNA were collected serially at 8 time points before and 8 time points 

after LPS. Compared to the 24 hours before LPS (repeated measures ANOVA), plasma levels of 

HDL-C (5% reduction, F3.4, p0.03) fell slightly whereas there were greater reductions in 

levels of plasma apoA-I (13% reduction, F 13.0, p0.01) and HDL associated phospholipids 

(22% reduction; F5.0, P0.01) were markedly reduced, with the nadir at 12-hours following 

LPS. The ability of serum (16% reduction; F 22.2, p0.001), and the HDL fraction (18% 

reduction; F 15.8, p0.001), to efflux 3H-cholesterol from FU5AH cells was reduced after 

LPS, also reaching a nadir at 12 hours. Compared to pre-LPS, whole blood cell mRNA 

expression of SR-BI fell by 76% (p0.001) and ABCG1 fell by 62% (p0.01), whereas there 

was no significant reduction in ABCA1. These studies suggest that acute inflammation induces 

HDL remodeling, with reduced HDL capacity to efflux cellular cholesterol as well as 

down-regulation of efflux protein expression, consistent with a marked attenuation of RCT in 

humans. 

Skin Cholesterol Adds to Framingham Risk Assessment 


Dennis L Sprecher, Gregory L Pearce; Univ of Pennsylvania, Philadelphia, PA 

Introduction: It has been demonstrated that skin cholesterol (SC) is associated with 

angiographic disease. Now, we further delineate the relative risk of multivessel disease (50% 

stenosis in at least two vessels) in the conjoint presence of high SC and high traditional risk 

burden. Methods: Patients scheduled for angiography (N649) had SC measured immediately 

prior to the procedure. Patients were classified according to the presence of high (110) SC 

and high (10) Framingham global risk scores. Multivariable logistic regression models were 

used to estimate relative risk of multivessel disease for patients with isolated high skin 

cholesterol, isolated high Framingham risk or conjoint high skin cholesterol and high 

Framingham by risk guest (eachon compared June 29, to neither 2013factor 

elevated). Results: The mean age was 63 

37

E-50 Vol 25, No 5 May 2005 

12 years and 33% (n214) were women. Thirty seven percent (n237) had angiographically 

determined multivessel disease. Table 1 shows that both isolated high SC and isolated high 

Framingham risk tended to infer increased risk of multivessel disease. However, when both 

scores were elevated, risk of multivessel disease was increased over 4-fold compared to 

neither elevated. Table 1. Presence and relative risk of multivessel disease by high 

Framingham, high Skin Cholesterol. Conclusion: We see an independent, additive risk of 

concurrent multivessel disease when Framingham risk and skin cholesterol are both elevated. 

Skin cholesterol may have value in further stratifying subjects with Framingham scores 10. 

Framingham, 

SC MVDx Rate 

Odds Ratio, 95% CI, 

p-value 

10, 110 41/163 (25%) - 

10, 110 107/313 (34%) 1.55 (1.01–2.36) 0.04 

10, 110 23/61 (38%) 1.80 (0.96–3.37) 0.07 

10, 110 66/112 (59%) 4.27 (2.55–7.16) 0.001 

38 

Effects of Estrogen Receptor-Alpha Gene on HDL Cholesterol Response to 

Atorvastatin 

Kouji Kajinami, Ryoko Sato, Hironobu Akao, Kanazawa Med Univ, Uchinada, Japan; Ernst J 

Schaefer; Tufts Univ, Boston, MA 

Objectives: To test the hypothesis that common polymorphisms in the estrogen receptor alpha 

(ESR1) and apolipoprotein A-I (APOA1) genes are associated with the plasma lipid response to 

atorvastatin therapy. Background: In addition to lowering LDL cholesterol, statins can modestly 

raise the levels of both HDL cholesterol and apolipoprotein A-I, a major apolipoprotein of HDL 

particle. Recently, associations between common polymorphisms in ESR1 and the HDL 

cholesterol response to hormone replacement therapy were reported. Methods: ESR1 haplotype 

with two polymorphisms (PvuII and XbaI) and two APOA1 polymorphisms (G-75A and 83) 

were examined in 338 hypercholesterolemic patients treated with atorvastatin 10mg. Results: 

The ESR1 PvuII(-)XbaI() haplotype was significantly, and independently, associated with a 

greater response to atorvastatin in terms of HDL raising in women (13% vs. 7%, p0.010) 

but not in men (9% vs. 7%, p0.248). Effects of the APOA183 variant allele on HDL 

cholesterol response also differed significantly by gender (p0.012); 8% vs. 1% in men, 

8% vs. 14% in women. The APOA183 variant allele was associated with higher 

pre-treatment LDL cholesterol levels in men as well (p0.013) but not in women (p0.256). 

Finally, significant interactions were observed between the ESR1 PvuII(-)XbaI() haplotype and 

the APOA183 variant allele with respect to the HDL (p0.042) and LDL (p0.031) 

cholesterol responses. Conclusions: In hypercholesterolemic patients, the ESR1 PvuII(-)XbaI() 

haplotype was associated with a greater response to atorvastatin in terms of HDL-raising in a 

gender-specific manner. Interactions between the ESR1 and APOA1 genotypes in terms of HDL 

and LDL cholesterol response were also gender-specific. 

39 

Apoliprotein B/apolipoprotein A-I Ratio is Closely Related to the Metabolic 

Syndrome in 64-Year old Women 

Bjorn Fagerberg, Gerhard Brohall, Carl Johan Behre, Johannes Hulthe; Wallenberg 

Laboratory, Goteborg, Sweden 

The apoliprotein B/apolipoprotein A-I ratio (apoB/A-I) has been reported to be a more powerful 

predictor of coronary disease than LDL-cholesterol. Hypothetically, apoB/A-I might be closely 

related to the metabolic syndrome. The aim was to test this hypothesis in a population-based 

sample of 64-year old women consisting of 230 women with diabetes mellitus (DM), 211 with 

impaired glucose tolerance (IGT), and either randomly selected women with normal glucose 

tolerance (NGTr, n91) or body mass index (BMI) matched to the IGT group (NGTm, n103). 

The results showed that apoB/A-I was linearly associated with glucose tolerance status (DM 

0.760.23, IGT 0.760.73, NGTm 0.740.19, IGTs 0.710.23, respectively, p0.05 for 

trend). ApoB/A-I correlated to BMI (r0.34, p0.001), waist-hip-ratio (r0.31, p0.001), HDL 

(r-0.71,p0.001), triglycerides (r0.59, p0.001), insulin (r0.37 p0.001), HbA1c 

(r0.15), systolic blood pressure (r0.10, p0.022). Subjects fulfilling the ATP III/NCEP 

criteria of the metabolic syndrome (MetS) had higher apo B/ApoA-I ratio than those not fulfilling 

the criteria (0.850.24, n242, 0.680.17 respectively, p0.001). There was a correlation 

between the number of fulfilled MetS criteria in the individual subjects and the apoB/A-I ratio 

(r0.31, p0.001). The ratio also correlated to C-reactive protein (r0.22, p0.001). In 

conclusion, the apoB/apoA-I ratio was associated with all established components in the MetS 

and was higher among subjects with the MetS compared to those without the syndrome. The 

apoB/apoA-I ratio was also associated with low-grade inflammation that seems to be a risk 

marker for coronary disease. 

40 

Postprandial Lipoprotein Metabolism in Familial Hypobetalipoproteinemia 

due to Truncated Apolipoprotein B Variants 

Amanda J Hooper, Univ of Western Australia, Perth, Australia; Ken Robertson, Royal Perth 

Hosp, Perth, Australia; Klaus G Parhofer, Univ of Munich, Munich, Germany; P Hugh R 

Barrett, Frank M van Bockxmeer, Univ of Western Australia, Perth, Australia; John R 

Burnett; Royal Perth Hosp, Perth, Australia 

Familial hypobetalipoproteinemia (FHBL; OMIM 107730) is a rare autosomal co-dominant 

disorder of lipoprotein metabolism characterized by decreased plasma concentrations of 

LDL-cholesterol and apolipoprotein (apo) B. Over 50 mutations in the APOB gene causing FHBL 

have been reported, most of which result in a truncated apoB molecule. We examined the effect 

of truncated apoB variants on postprandial triglyceride-rich lipoprotein (TRL) metabolism. A 

standardized oral fat load containing retinol was given after a 12 h fast to seven heterozygous 

[apoB-6.9 (n3), apoB-25.8 (n1), apoB-40.3 (n2), Downloaded apoB-80.5 (n1)] from 

FHBL subjects (age, 



3713 yr; BMI, 262 kg/m 2 ) and ten normolipidemic controls (age, 302 yr; BMI, 223 

kg/m 2 ). Plasma was obtained every 2 h for 10 h. Large TRLs [containing chylomicrons (CM)] 

and small TRLs (containing CM-remnants) were isolated by ultracentrifugation and total 

cholesterol, triglyceride (TG), and retinyl palmitate (RP) measured. As expected, when 

compared to controls, FHBL subjects had significantly decreased fasting plasma total 

cholesterol (2.20.6 vs. 4.80.6 mmol/L), TG (0.40.2 vs. 1.50.5 mmol/L), LDLcholesterol 

(0.70.3 vs. 3.00.5 mmol/L), and apoB (0.210.06 vs. 0.950.14 g/L) 

concentrations (all P0.001). The incremental area under the curve (iAUC) in FHBL subjects 

was decreased for both large TRL-cholesterol (-35%; P0.20) and TRL-TG (-61%; P0.001) 

and small TRL-cholesterol (-84%; P0.001) and TRL-TG (-83%; P0.001) compared to 

controls. Moreover, the peak postprandial TRL-TG was earlier (2 vs. 4 h) in FHBL subjects. 

However, neither large nor small TRL-RP parameters were affected. We conclude that 

compared to normolipidemic controls, heterozygous FHBL subjects have increased in vivo 

lipolysis, but normal postprandial TRL particle clearance. 

Effects of Obesity on Monocyte Chemoattractant Protein-1 Expression in 

Mouse and Man 

Huaizhu Wu, Abu R Vasudevan, Peter H Jones, Antonios M Xydakis, C W Smith, John 

Sweeney, William Fisher, Christie M Ballantyne; Baylor College of Medicine, Houston, TX 

Obesity is associated with insulin resistance and cardiovascular disease. Adipokines such as 

TNF- and IL-6 may be an important link between obesity and obesity-related disease. 

Monocyte chemoattractant protein-1 (MCP-1), which plays an important role in cardiovascular 

disease, has been recently recognized as an adipokine. In these experiments, we first used a 

high-fat diet (HF)-induced mouse obesity model and examined MCP-1 mRNA levels in 

perigonadal adipose tissue, liver, heart, and spleen with RNAse protection assay (RPA), and 

measured serum MCP-1 level with ELISA. After 24 weeks, HF mice were significantly heavier 

than mice fed normal chow diet (body weight 47.72.3 g vs. 30.80.6 g, n6 in each group, 

P0.001). RPA showed that MCP-1 mRNA levels were significantly increased in obese mice 

in adipose tissue (relative intensity to GAPDH [RI] 23033 vs. 7018 in lean mice, P0.01) 

and livers (RI 9410 vs.164, P0.001) with no difference in hearts and spleens. Serum 

MCP-1 level was significantly higher in obese mice (17636 vs. 303 pg/ml in lean, 

P0.001). We also found that obese mice had increased expression of CD11b and CD11c on 

peripheral leukocytes which are markers of leukocyte activation (percentage of CD11b / 

CD11c cells 8.80.4% vs. 3.00.3% in lean, n4 in each group, P0.001) by flow 

cytometry. We also measured serum MCP-1 levels in obese humans with metabolic syndrome 

(BMI 38.40.7) and lean controls (BMI 22.70.6). Serum MCP-1 level in obese subjects 

(536.5316.1 pg/ml, n20) was significantly higher than that in lean controls (263.396.5 

pg/ml, n14; P0.001). To identify the potential source of MCP-1 in humans, we examined 

MCP-1 mRNA levels in both subcutaneous and omental (equivalents to mouse perigonadal) 

adipose tissue from obese humans, and found that MCP-1 mRNA level was significantly higher 

in omental than subcutaneous adipose tissue (RI 610113 vs. 37562, n9, 

P0.05).summary, MCP-1 mRNA expression in visceral adipose tissue was increased in 

diet-induced obese mice and obese humans, and this may contribute to the increased serum 

levels of MCP-1 observed in obesity. 

Beneficial Effects of Exercise on Age-Related Reductions in Endothelial 

Progenitor Cell Number and Migratory Activity 

Greta L Hoetzer, Heather M Irmiger, Gary P Van Guilder, Rebecca S Keith, Jared J Greiner, 

Univ of Colorado, Boulder, CO; Brian L Stauffer, Univ of Colorado, Health Sciences Cntr, 

Denver, CO; Christopher A DeSouza; Univ of Colorado, Boulder, CO 

Bone marrow-derived circulating endothelial progenitor cells (EPCs) are critical to vascular 

health as they contribute to both reendothelialization and neovascularization. Numerical and 

functional impairment of these cells has recently been linked to endothelial dysfunction and 

atherogenesis. Moreover, EPC dysregulation is associated with a number of pathologies 

associated with human aging including hypertension, type 2 diabetes, myocardial ischemia, 

coronary artery disease and stroke. However, it is currently unclear whether aging per se is 

related to diminished EPC number and function. This represents a critical void in our 

understanding of vascular aging. Indeed, reduced EPC bioavailability and reparative capacity 

may contribute to the increased risk of atherosclerosis and the prolonged and often 

complicated recovery from ischemic events in older adults. We tested the hypotheses that: 1) 

circulating EPC number and migratory activity decrease progressively with age in healthy, 

sedentary adult humans; and 2) regular aerobic exercise (EX) will restore some, if not all, of the 

loss in EPC number and migration in previously sedentary middle-aged and older adults. 

Peripheral blood samples were collected from 42 healthy sedentary men: 8 young (261 yr: 

Y), 13 middle-aged (481 yr: MA) and 21 older (631 yr; O). The number of circulating EPCs 

was determined ex vivo using a colony-forming assay. EPC migratory activity was assessed by 

the fluorescence of migrated cells through a modified Boyden chamber. Number of EPC colony 

forming units was 75% lower (P0.01) in MA (114) and O (82) compared with Y (407). 

There was no difference in colony count between MA and O. EPC migration (fluorescent units) 

was significantly blunted in O (452 72) compared with Y (813114) and MA (760134). To 

date, 8 sedentary O men have completed a 3-month EX intervention (walking 5.3 d/wk, 45 

min/d @ 67% of maximal heart rate). EX increased (P0.05) both EPC colony forming units 

(113to245) and migratory activity (62587 to 975130). These results demonstrate that 

circulating EPC number and migratory activity declines with age in healthy men. Importantly, 

regular aerobic exercise is an effective strategy for increasing EPC number and migratory 

function in by older guest adults. on June 29, 2013 

41 

42

43 

Impact of Differently Processed Forms of Soybean Based Foods on Plasma 

Lipid and Lipoprotein Profiles in Hypercholesterolemic Women 

Nirupa R Matthan, Susan M Jalbert, Lynne M Ausman, Ernst J Schaefer, Alice H Lichtenstein; 

Jean Mayer USDA Human Nutrition Rsch Cntr on Aging at Tufts Univ, Boston, MA 

Data on the effect of soy relative to animal protein on plasma lipid and lipoprotein levels, 

independent of the fatty acid profile of the diet, are inconsistent. It has been hypothesized that 

a putative component of the soybean, as yet to be identified, maybe lost during processing. One 

candidate, soybean isoflavones, has been ruled out. In order to address this issue further, diets 

(55% energy as carbohydrate, 30% energy as fat, 15% energy as protein with 7.5% energy as 

experimental protein) were designed to contain products made from either whole soybeans 

(SB), soyflour (SF) or soymilk (SM), and compared to an equivalent amount of protein derived 

from common animal sources (AP). Cholesterol, fiber and fatty acid profile of the diets were 

held constant (80mg cholesterol/1000kcal, 15g fiber/1000kcal, 7% energy as SFA, 10–15% 

energy as MUFA and 10% energy as PUFA). Twenty women (50 years with LDL-C 130 

mg/dL) were provided with each diet in a random cross over design for 6 week periods. Body 

weight was maintained at a constant level throughout the study. Total cholesterol levels were 

223, 216, 222 and 219 mg/dL (AP, SB, SF and SM, respectively); LDL-C levels were 150, 145, 

149 and 145 mg/dL (AP, SB, SF and SM, respectively); HDL-C levels were 57, 56, 57 and 58 

mg/dL (AP, SB, SF and SM, respectively) and triglyceride levels were 138, 134, 128 and 133 

mg/dL (AP, SB, SF and SM, respectively). In no case did the differences reach statistical 

significance. These data suggest that type of protein (animal versus soy) and method of 

soybean processing has little impact on plasma lipid and lipoprotein levels, when other major 

dietary variables are held constant. Hence, relatively high intakes of soy protein, per se, appear 

to have little effect on CVD risk. 

44 

Lower Levels of Adiponectin among Japanese than American Men Despite 

Less Obese Composition 

Takashi KADOWAKI, Akira SEKIKAWA, Univ of Pittsburgh, Pittsburgh, PA; Tomonori 

OKAMURA, Shiga Univ of Med Science, Otsu, Shiga, Japan; Tomoko TAKAMIYA, Univ of 

Pittsburgh, Pittsburgh, PA; Atsunori KASHIWAGI, Shiga Univ of Med Science, Otsu, Shiga, 

Japan; Wahid R ZAKY, Univ of Pittsburgh, Pittsburgh, PA; Hiroshi MAEGAWA, Shiga Univ of 

Med Science, Otsu, Shiga, Japan; Aiman EL-SAED, Univ of Pittsburgh, Pittsburgh, PA; 

Yasuyuki NAKAMURA, Kyoto Women’s Univ, Kyoto, Japan; Rhobert W EVANS, Univ of 

Pittsburgh, Pittsburgh, PA; Naomi MIYAMATSU, Shiga Univ of Med Science, Otsu, Shiga, 

Japan; Daniel EDMUNDOWICZ, Univ of Pittsburgh Med Cntr, Pittsburgh, PA; Yoshikuni KITA, 

Shiga Univ of Med Science, Otsu, Shiga, Japan; Lewis H KULLER, Univ of Pittsburgh, 

Pittsburgh, PA; Hirotsugu UESHIMA; Shiga Univ of Med Science, Otsu, Shiga, Japan 


http://atvb.ahajournals.org/ by guest on June 29, 2013 



[Background] Adiponectin, one of the adipocytekines, has potential anti-atherogenic, antidiabetic, 

and anti-inflammatory properties. Despite being solely expressed and secreted from 

adipose tissue in humans, the levels of adiponectin are paradoxically reduced in obesity. 

[Objective] We hypothesized that Japanese men, who are much leaner, have higher levels of 

adiponectin that may in part be protective against coronary heart diseases. The purpose of this 

study was to examine this hypothesis using stored blood samples. [Subjects and Methods] 

This study was done as a part of a collaborative study to compare the levels of atherosclerosis 

between American and Japanese men aged 40–49. We examined the levels of adiponectin in 

American (n100) and Japanese (n93) men. Venous blood was drawn after participants had 

fasted for at least 12 hours. The centrifugation procedure was done on site immediately after 

the venipuncture procedures, and samples were stored at -80 °C. The samples were examined 

in the same laboratory after shipped on dry ice. [Results and Conclusion] Adiponectin levels 

were significantly lower in Japanese men (7.3 4.2 vs. 13.2 5.8 (g/ml)), despite a smaller 

waist circumference and a lower body-mass index. They were lower at any measured levels 

of waist circumference (Figure 1). The reasons remain unknown, but this may be due to 

differences in polymorphisms of genes affecting adiponectin levels, visceral and subcutaneous 

fat distribution, or environmental factors such as diet and physical activity.

E-52 Vol 25, No 5 May 

Apolipoprotein A-I-Mediated Cholesterol Efflux is Impaired in 

Intimal-Phenotype Arterial Smooth Muscle Cells 

Hong Y Choi, Teddy Chan, Gordon A Francis; Univ of Alberta, Edmonton, Canada 

Arterial smooth muscle cells (SMC) contain a large percentage of the excess cholesterol 

accumulated in atherosclerotic lesions. The presence of less differentiated SMC in the intimal 

compared to medial layer of atherosclerotic arteries suggests intimal SMC may have an 

impaired ability to release lipids to apolipoproteins for HDL particle formation. To test this 

hypothesis, we examined the ability of clones of arterial SMC from adult (3 month old) or pup 

(12 day old) Wistar Kyoto rats to bind and release lipids to apolipoprotein A-I (apoA-I). The 

pup-derived SMC clone maintains a less differentiated (intimal) phenotype in continuous 

culture. Intimal-phenotype SMC showed low levels of apoA-I binding compared to medial 

(adult) SMC, which bound apoA-I with high affinity. Incubation of medial SMC with 10 g/mL 

apoA-I for 16 h removed 70 5% (mean SD) of cholesterol available for esterification by 

acyl-CoA:cholesterol acyltransferase, and 10 –12% of total cell plus medium [ 3 H]cholesterol 

and [ 3 H]phosphatidylcholine to the medium of these cells over 24 h. The same incubations of 

intimal-phenotype SMC resulted in little or no depletion of cholesterol available for esterification 

or efflux of these lipids to the medium. Impaired cholesterol mobilization to apoA-I from 

intimal-phenotype SMC was confirmed by measuring cholesterol mass in medium and cells. 

The level of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein, the key protein 

required for apoA-I-mediated lipid efflux, was much lower in basal and cholesterol-loaded 

intimal-phenotype SMC compared with medial SMC. Increased ABCA1 protein levels in the 

plasma membrane (as assessed by biotinylation) following transfection with full-length murine 

ABCA1 cDNA, or treatment of intimal-phenotype SMC with the liver X receptor agonist 

T0901317, however, failed to correct the impaired binding and cholesterol efflux to apoA-I. 

These findings suggest that, in addition to ABCA1, other factor(s) involved in apoA-I-mediated 

lipid efflux are lacking in intimal phenotype SMC, but present in medial phenotype arterial SMC. 

The marked difference in response to apoA-I between intimal- and medial-phenotype rat SMC 

provides an excellent model to study additional cellular requirements for this interaction. 

Lysosomal Cholesterol Accumulation in Macrophage Foam Cells 

Brian E Cox, Jody C Ullery, Evelyn E Griffin, W G Jerome; Vanderbilt Univ Med Cntr, 

Nashville, TN 

Macrophage foam cells in atherosclerosis develop via massive accumulations of free (FC) and 

esterified (CE) cholesterol, much of which is within lysosomes. In cell culture, macrophages 

incubated with mildly oxidized LDL (ox-LDL), small aggregates of LDL (agg-LDL) or CE rich lipid 

dispersions (LD) develop into foam cells with significant lysosomal accumulation and inhibition 

of lipoprotein CE hydrolysis. The inhibition of CE hydrolysis was not immediate and did not 

require oxidized lipids. Lysosomal accumulation of FC preceded inhibition of CE hydrolysis, 

suggesting a linkage of inhibition to FC accumulation. In contrast, treatment with acetylated 

LDL (ac-LDL) did not produce lysosomal FC accumulation nor inhibition of CE hydrolysis. 

Accompanying the inhibition of CE hydrolysis, we found a neutralization of the lysosomal pH, 

suggesting that a factor shared between ox-LDL, agg-LDL, and LD inhibits lysosome 

acidification and thus interrupts hydrolysis. In the current studies we used THP-1 macrophages 

to further define the mechanism of lysosomal neutralization. To explore the role of FC, we 

inhibited FC exit from ac-LDL treated lysosomes by incubating with progesterone (10 g/ mL). 

A rapid neutralization of the lysosomes occurred. This effect was reversible with short term (1 

day) progesterone treatment but not with longer term (3 day). The cells remained viable even 

though lysosomal hydrolysis was disrupted. Pretreatment of cells with ox-LDL for 3 days (but 

not 6 hours) resulted in inhibition of ac-LDL-derived CE hydrolysis. This suggests a change in 

lysosomes via ox-LDL treatment rather than a direct inhibition of enzyme. Lysosomal 

acidification is produced by lysosomal membrane v-ATPases. Immunoblots suggest that 

cholesterol-engorged lysosomes produced by incubation with 100 g/mL agg-LDL have 

control levels of v-ATPase. The simplest explanation of our present data is that the initial FC 

accumulation in lysosomes inhibits v-ATPase activity leading to an inability of lysosomes to 

maintain an acidic pH. This neutralization of the lysosome appears to inhibit acid hydrolase 

activity. The sequestration of FC and CE in lysosomes away from normal cholesterol 

homeostatic mechanisms would be expected to profoundly affect foam cell biology. 

The Contribution of Various Pathways of Cellular Cholesterol Efflux to 

Human Serum 

MyNgan Duong, The Children’s Hosp of Philadelphia, Philadelphia, PA; Weijun Jin, Univ of 

Pennsylvania Sch of Medicine, Philadelphia, PA; George H Rothblat; The Children’s Hosp of 

Philadelphia, Philadelphia, PA 

Reverse cholesterol transport is accepted as an important pathway in which excess cellular 

cholesterol is removed from periphery tissue. An essential step in this pathway is the 

movement of cholesterol out of cells and this can be achieved by several mechanisms, 

including those mediated by ATP-binding cassette AI (ABCAI) and scavenger receptor BI (SR-BI). 

This study examines the relative contributions of these mechanisms in cholesterol efflux to 

human serum. WI38VA13, embryonic human fibroblast were used. Cells were stably 

transfected with SR-BI and ABCA1 was upregulated with cis retinoic acid (cRA) and 22 

hydroxycholesterol (22-OH). Quantitation of free cholesterol efflux to a pool of human serum 

added at 2.5% demonstrated that efflux from cells without either receptor was 2.3%/2h, 

whereas expression of ABCA1 produced a small Downloaded increase in efflux from 

to 3.7%. SR-BI had a 

Poster Presentations 

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dramatic impact on efflux with a value of 8.8%. To further quantitate the contribution of 

different efflux pathways we used pretreatment with Probucol to block ABCA1-mediated efflux 

and BLT (block lipid transporter)-1 to inhibit SRBI activity. Treatment of SR-BI expressing cells 

with BLT-1 inhibited efflux by 71%. Probucol pretreatment of cells expressing ABCA1 reduced 

efflux by 28%. In cells expressing both receptors treatment with the two inhibitors resulted in 

60% inhibition. Under all conditions a background efflux was present in cells without receptors, 

or in cells that had both receptors inhibited, of 2.7% 0.8% / 2h. When fibroblasts are 

exposed to human serum, SRBI is responsible for the majority of the cholesterol efflux. The 

ABCAI-mediated component makes a much smaller contribution to efflux. SR-BI and the 

background efflux increase with increasing serum concentrations, whereas the ABCA1mediated 

efflux does not change over the serum concentration range of 2.5 % to 7.5%. 

Background efflux is unlikely to be mediated by ABCG1 since the level of this receptor appears 

to be low, and the background efflux does not change upon treatment with cRA/22-OH. We 

propose that background efflux, that is not inhibited by either Probucol or BLT-1, is mediated 

by aqueous diffusion or by a yet unknown transporter, and makes a significant contribution to 

the overall efflux of cholesterol from cells to serum. 

Increased ATP-Binding Cassette Transporter A1-Mediated Cholesterol 

Efflux Potential of Sera from ApoA-I Milano Carriers 

Elda Favari, Ilaria Zanotti, Francesca Zimetti, Franco Bernini, Univ of Parma, Parma, Italy; 

Miriam Lee, Petri T Kovanen, Wihuri Rsch Institute, Helsinki, Finland; Ceare R Sirtori, Guido 

Franceschini, Laura Calabresi; Univ of Milan, Milan, Italy 

The first step in RCT is the efflux of unesterified cholesterol from peripheral cells to either free 

apolipoproteins or lipoprotein acceptors present in the extracellular space. ATP-binding 

cassette transporter A1 (ABCA1) mediates cellular cholesterol and phospholipid efflux to 

lipid-poor apolipoproteins and its activity is believed to be antiatherogenic. ApoA-I Milano (A-I M)is 

the first described mutant of human apolipoproteins. Thirty-eight heterozygous carriers have 

been identified, who represent the largest group of individuals with low plasma HDL levels 

because of a single defect in the ApoA-I gene. The severe hypoalphalipoproteinemia and the 

partial LCAT and CETP deficiencies suggest a defective RCT, but the carriers do not suffer from 

premature coronary heart disease. To investigate the mechanism(s) behind this apparent 

paradox, we compared in the present study the ABCA1-madiated cholesterol efflux potential of 

sera from A-I M carriers and control subjects. To evaluate the participation of ABCA1-dependent 

pathways we used J774 murine macrophages expressing high levels of ABCA1 upon treatment 

overnight with 0.3mM cpt-cAMP. We measured either cholesterol than phospholipid efflux and 

tested the effect of sera in human fibroblasts from normal and Tangier patients. The results are 

showing that ABCA1-mediated cholesterol efflux to sera from A-I M carriers was significantly 

increased (from 2- to 4-fold) compared with sera from control subjects. Chymase treatment of 

both set of sera showed a marked reduction on the ABCA1-mediated efflux and eliminated the 

difference on efflux potential between sera from A-I M carriers and control subjects. Chymase 

treatment cleaved the pre-beta particles of both set of sera and small particles (7–8nm) that were 

specifically present only into sera from carriers. These particles contains one molecule of A-I M 

dimers and agarose gel analysis showed a migration in a area between the - and - positions. 

We conclude that despite the dramatic hypoalphalipoproteinemia, sera from A-I M carriers have an 

efficient efflux potential. The enhanced ability of sera from A-I M carriers to promote the 

ABCA1-mediated efflux is specifically related to the presence of specific HDL particles. 

The Role of Ceramide in Modulating ABCA1-Mediated Cellular Cholesterol 

Efflux 

Amy B Ghering, James L Dressman, Scott R Witting, W. S Davidson; Univ of Cincinnati 

Genome Rsch Institute, Cincinnati, OH 

Elevated high density lipoprotein (HDL) levels correlate with decreased risk of atherosclerosis 

due in part to its role in reverse cholesterol transport. Most human HDL is generated by 

ATP-Binding Cassette Transporter AI (ABCA1) which effluxes cellular lipids to apoA-I. 

Unfortunately, gene induction strategies for ABCA1 to increase HDL have proven problematic. 

While investigating other modes of ABCA1 regulation, we found that treating cultured cells with 

ceramide, a lipid signaling molecule, causes increases in cellular and cell surface ABCA1 with 

a concurrent increase in cholesterol efflux to apoA-I. Ceramide is believed to act via a 

posttranscriptional mechanism as the effect requires prior expression of ABCA1 and does not 

depend on native regulatory gene elements. Ceramide increases cellular ABCA1 without 

increases in other proteins such as the transferrin receptor and -actin and does not induce 

general toxicity. To determine if ceramide acts through a physical lipid ordering effect or by a 

protein-mediated pathway, we measured cholesterol efflux from monolayers treated with chiral 

ceramide analogs that exhibit highly similar physical properties to the naturally occurring 

ceramide but vary in their interactions with biological molecules. Changes in the native spatial 

orientation at either of two chiral carbon centers resulted in 50% decrease in cholesterol 

efflux, and changing the arrangement at both carbons decreased efflux a further 15% (65% 

total decrease). This chirality dependence supports the possibility that ceramide mediates its 

effects via a protein signaling pathway to affect ABCA1. We also studied the cellular fate of 

ABCA1 by tracking its cellular degradation rate and its duration at the cell surface. Western 

blots monitoring cellular protein degradation after cycloheximide treatment showed that 

ceramide prolongs ABCA1 cellular lifetime. Similarly, in Western blots of surface ABCA1, 

ceramide prolongs ABCA1 surface residence. The chiral analog and series of Western blot 

experiments taken together indicate that ceramide acts through a protein-based cell signaling 

pathway to slow ABCA1 degradation and increase cell surface ABCA1 with a concomitant 

increase by in cholesterol guest on efflux June to apoA-I. 29, 2013 

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Non-Class Effects of Calcium Channel Blockers on Expression of 

ATP-Binding Cassette Protein 

Kazuhiro Hasegawa, Shu Wakino, Kouichi Hayashi, Takeshi Kanda, Kyouko Yoshioka, Satoru 

Tatematsu, Kouichirou Honma, Ichiro Takamatsu, Naoki Sugano, Takao Saruta; Keio Univ 

Sch of Medicine, Tokyo, Japan 

Calcium channel blockers (CCBs) have been reported to be effective in preventing the 

progression of atherosclerosis by multiple mechanisms. Verapamil, a phenylalkylamine-class 

CCB, is reported to increase mRNA and protein expressions of cellular cholesterol transporter, 

ATP binding cassette transporter A1 (ABCA1) via a liver X receptor (LXR)-independent pathway, 

and is considered to potential mechanism for anti-atherosclerotic effects of CCBs. We 

examined whether dihydropyridine-class CCBs also exerted this effect. Undifferentiated 

monocytic cell lines, THP-1 cells were maintained in RPMI-1640 medium and were treated with 

different kinds of dihydropyridine CCBs. Cell lysates were obtained 24 hours after the treatment 

and ABCA1 expression was measured by real-time PCR and immunoblotting. Among the CCBs 

tested, aranidipine and efonidipine increased ABCA1 protein expression, without the increase 

in ABCA1 mRNA expression. To delineate the mechanisms by which aranidipine and efonidipine 

induced ABCA1 protein expression, THP-1 cells were pretreated with a protein kinase A (PKA) 

inhibitor (H89), a tyrosine kinase (TK) inhibitor (genistein), and a JAK2 inhibitor (AG490). 

Intracellular cAMP concentration was measured by an enzyme immunoassay. H89 inhibited 

aranidipine-induced ABCA1 protein expression, whereas other inhibitors had no effects. 

Furthermore, efonidipine-induced ABCA1 protein expression was not affected by either 

inhibitor. Consistent with these results, intracellular c-AMP levels were enhanced by only 

aranidipine. In conclusion, among dihydropyridine CCBs, aranidipine and efonidipine have the 

potential to induce ABCA-1 protein by a distinct mechanism. These non-class effects of CCBs 

may provide novel strategies for the development of anti-atherosclerotic drugs. 

Inhibition of AIF-1 Expression Suppresses Inflammation-Initiated Signal 

Transduction and Macrophage Migration and Proliferation 

Ying Tien, Sheri Kelemen, Michael Autieri; Temple Univ Sch of Medicine, Philadelphia, PA 

Introduction. Activated macrophages are an important component in the genesis and 

progression of atherosclerosis. Characterization of proteins which regulate macrophage 

pathophysiology is essential to better understand the etiology of this disease. Allograft 

Inflammatory Factor-1 (AIF-1) is a cytoplasmic, calcium-binding inflammation-responsive 

signaling protein involved in activation of vascular smooth muscle cells. The function of AIF-1 

in inflammatory cells, and specifically, in the development of atherosclerosis, is unknown at 

this time. The objective of this work is to determine a role for AIF-1 in macrophage activation. 

Methods and Results. AIF-1 expression co-localized with CD68-positive macrophages in 

atherosclerotic human coronary arteries. Stable expression of AIF-1 siRNA in a macrophage cell 

line, RAW264.7, reduced AIF-1 protein expression by 79%, and effects on migration, 

proliferation, and activation of intracellular signaling proteins were measured. AIF-1 siRNA 

expression reduced macrophage proliferation by 49% (mean 422.8 Vs 215.7 for control and 

siRNA, respectively, P0.05 for 3 experiments). AIF-1 siRNA expression reduced macrophage 

migration by 60% (mean 87.6 Vs 35.1 for control and siRNA, respectively, P0.05 for 3 

experiments). Both proliferation and migration of siRNA expressing macrophages could be 

rescued by adenoviral infection of AIF-1 (P0.05 for all experiments). Phosphorylation of p90, 

Akt, p44/42, and S6 kinase were reduced 29, 23, 26, and 49% in siRNA macrophages 

challenged with inflammatory stimuli (P0.05 for all experiments). Interestingly, basal levels 

of Akt phosphorylation were reduced 37% in unstimulated cells. This suggests that AIF-1 

participates in inflammation-initiated signaling, and that inhibition of migration and proliferation 

may be due to suppression of signaling pathways. Conclusions. Together, these data indicate 

that AIF-1 expression is key component in the activation of macrophages, and may have 

implications in the etiology of atherosclerosis. 

Protection of Small Heterodimer Partner (SHP) Knockout Mice from Diet 

Induced Hypercholesterolemia and Liver Inflammation 

KehDih Lai, Wyeth, Collegeville, PA; Kim Kanki, Wyeth, Andover, MA; Brenda Lager, Wyeth, 

Princeton, NJ; Mark J Evans; Wyeth, Collegeville, PA 

Mice consuming a diet containing cholesterol and cholic acid show both diet-induced 

hypercholesterolemia and hepatic inflammation. The nuclear hormone receptor SHP can 

promote inflammation by functioning as a coactivator for the transcription factor NFB. To 

determine whether SHP plays a role in hepatic inflammation, SHP knockout mice were 

generated. When fed a diet containing 2% cholesterol plus 0.5% cholic acid for 5 weeks, serum 

cholesterol levels increased from 130 mg/dl to 250 mg/dl in WT mice. In female SHPKO, this 

increase was attenuated (210 mg/dl) while in male SHPKO the increase was completely 

eliminated. The diet treatment also repressed triglyceride levels by 50% in WT mice. This 

repression was completely abolished in both female and male SHPKO, consistent with FXR 

induction of SHP as being critical for regulation of triglyceride levels in the mouse. Analysis of 

gene expression in the liver indicated that diet-mediated induction of inflammatory genes 

(RANTES, VCAM, ICAM-1 and invariant chain) was completed abolished in both female and 

male SHPKO. Similarly, SHPKO mice fed a diet containing 1% cholic acid for 5 days were 

protected from induction of inflammatory gene expression in the liver. We conclude that the 

loss of SHP provides significant beneficial effects on plasmid cholesterol levels, potentially by 

inhibiting bile acid-induced hepatic inflammation. Downloaded from 

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Poster Presentations E-53 

Non-Anticoagulant Fondaparinux Retains Anti-Inflammatory Activity in a 

Mouse Model of Kidney Ischemia-Reperfusion Injury 

Todd Holscher, The Scripps Rsch Institute, La Jolla, CA; Rolf D Frank, Univ Hosp Aachen, 

Aachen, Germany; Gernot Schabbauer, Yuichiro Sato, Michael Tencati, Rafal Pawlinski, The 

Scripps Rsch Institute, La Jolla, CA; Jeffrey I Weitz, McMaster Univ and Henderson Rsch 

Cntr, Hamilton, Canada; Nigel Mackman; The Scripps Rsch Institute, La Jolla, CA 

In a previous study, we investigated the effect of the synthetic pentasaccharide fondaparinux, 

a selective antithrombin (AT)-dependent inhibitor of coagulation factor Xa (FXa), in a lethal 

murine model of kidney ischemia-reperfusion (I/R) injury that is associated with coagulation 

and inflammation. Fondaparinux treatment of I/R-injured mice significantly reduced serum 

creatinine levels, lowered fibrin deposition, reduced IL-6 and MIP-2 expression and decreased 

neutrophil accumulation in the injured kidneys. In addition, we showed that fondaparinux 

reduced recruitment of neutrophils into the peritoneum in a model of acute peritonitis and 

inhibited the binding of U937 cells to P-selectin in vitro. These results indicate that 

fondaparinux has anti-inflammatory activity. It is unclear however, whether the anticoagulant 

properties of fondaparinux also contribute to its protective effect in this model. To address this 

issue, fondaparinux was oxidized with periodate and reduced with sodium borohydride to 

abolish its affinity for antithrombin. The activity of this modified fondaparinux was then tested 

in the mouse kidney I/R model. Unlike fondaparinux, modified fondaparinux did not prolong the 

activated partial thromboplastin time (APTT) or produce detectable anti-FXa activity in the 

mouse plasma consistent with its lack of anticoagulant activity. However, modified fondaparinux 

significantly lowered interleukin-6 (IL-6) levels in the plasma at 24hrs vs. saline controls 

(1108 /- 634 pg/ml vs. 3938 /- 1618 pg/ml, P0.008) and inhibited the binding of U937 

cells to P-selectin coated plates (P0.003) in a similar fashion to fondaparinux. These data 

indicate that the protective effect of fondaparinux in the mouse kidney I/R model reflects its 

anti-inflammatory properties and is not dependent on its anticoagulant activity. 

Apolipoprotein C-III in ApoB Lipoproteins Enhances the Adhesion of 

Monocytes to Endothelial Cells by Increasing the Active Form of -1 

Integrin 

Akio Kawakami, Harvard Sch of Public Health, Boston, MA; Masanori Aikawa, Peter Libby, 

Harvard Med Sch, Boston, MA; Frank M Sacks; Harvard Sch of Public Health, Boston, MA 

(Background) Recently, apoCIII has been implicated as an independent risk factor for coronary 

heart disease (CHD). We reported that LDL with apoCIII strongly predicts recurrent CHD risk. 

However, the direct effects of VLDL with apoCIII (VLDL CIII) and LDL with apoCIII (LDL CIII) 

on vascular cells have not been studied. We investigated the effect of VLDL CIII and LDL CIII 

on monocyte-endothelial interaction. (Methods and Results) VLDL CIII and LDL CIII were 

isolated from the fasting plasma of normolipidemic volunteers by immunoaffinity chromatography 

and ultracentrifugation. THP-1 cells, a human monocytic cell line, were incubated with 

VLDL CIII or LDL CIII, and their adhesion to human saphenous vein endothelial cells 

(HSVECs) was examined. Preincubation with of VLDL CIII and LDL CIII (10 mg apoB/dL, 24 

hours) increased THP-1 cell adhesion to HSVECs by 2.40.3 times (p0.01) and 1.80.7 

times (p0.05), respectively. In contrast, VLDL without apoCIII and LDL without apoCIII had no 

effects. Interestingly, incubation with apoCIII (10mg /dL, 24 hours) also increased THP-1 cell 

adhesion by 2.10.6 times (p0.01). To elucidate the mechanism of VLDL CIII and LDL 

CIII-induced THP-1 cell adhesion, we examined the expression and affinity of integrins on 

THP-1 cells. VLDL CIII and LDL CIII each increased the active form of 1-integrin on THP-1 

cells. Western blotting analysis revealed that VLDL CIII and LDL CIII-induced 1-integrin 

activation was dependent on PKC and RhoA. PKC inhibitor and Rho-kinase inhibitor inhibited 

VLDL CIII and LDL CIII-induced THP-1 cell adhesion. ApoCIII also activated PKC and RhoA 

in THP-1 cells, which resulted in 1-integrin activation. When VLDL CIII or LDL CIII were 

pretreated with anti-apoCIII antibody, their effects on THP-1 cell adhesion were abolished 

whereas antibodies to other apolipoproteins had no effect. (Conclusion) VLDL CIII and LDL 

CIII increased THP-1 cell adhesion to HSVECs via PKC and RhoA-mediated 1-integrin 

activation. ApoCIII itself played a direct dominant role in these processes. These results indicate 

that apoCIII not only modulates lipoprotein metabolism, but also may contribute to the 

development of atherosclerosis through monocyte recruitment on vascular endothelium. 

Naringenin Mimics the Kinetics of Insulin-Induced Inhibition of 

ApoB100-Containing Lipoprotein Assembly in HepG2 Cells through a 

Mechanism Independent of the Insulin Receptor 

Emma M Allister, Jane Y Edwards, Robarts Rsch Institute, London, Canada; P Hugh R 

Barrett, Univ of Western Australia, Perth, Australia; Erin E Mulvihill, Murray W Huff; Robarts 

Rsch Institute, London, Canada 

Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins is characteristic of 

the dyslipidemia associated with insulin resistance and reflects the inability of insulin to 

attenuate hepatocyte apoB assembly and secretion. Recently, we demonstrated that the 

grapefruit flavonoid, naringenin, like insulin, decreased apoB secretion from HepG2 hepatocytes 

by activation of both the phosphoinositide 3-kinase (PI3-K) and the mitogen activated 

protein/extracellular regulated kinase (MAPKerk ) signaling pathways. The objective of the 

present study was to determine whether naringenin-induced insulin signaling required 

activation of the insulin receptor and if the kinetics of apoB assembly and secretion in cells 

exposed to naringenin were similar to those observed for insulin. Immunoblot analysis revealed 

that insulin (100nM) stimulated maximal tyrosine phosphorylation of the insulin receptor 

-subunit and insulin receptor substrate 1 or 2 after 10 min whereas naringenin (100M) did 

not affect either at any time point up to 60 min. Multicompartmental modeling of apoB 

pulse-chase studies, using SAAMII, demonstrated that a model which included an intracellular 

compartment by guest from which on June apoB can 29, be2013 secreted or degraded by a rapid (proteosomal) or slow 

P9 

P10 

P11

E-54 Vol 25, No 5 May 2005 

(non-proteosomal) pathway best fit the data. Curves for total and secreted radiolabelled apoB 

in naringenin- or insulin-treated cells were similar. Naringenin and insulin decreased apoB 

secretion by 57% and 43% respectively and both stimulated intracellular degradation via the 

kinetically defined rapid pathway. The slow degradation pathway, which accounted for less 

than 15% of total degradation, was unaffected by naringenin but was stimulated by insulin. We 

conclude that naringenin mimics the insulin-induced inhibition of hepatocyte apoB secretion 

through activation of both PI3-K and MAPK erk signaling, resulting in similar kinetics of apoB 

secretion. However, the mechanism for naringenin is independent of the insulin receptor and 

therefore, naringenin represents a possible strategy for reduction of hepatic apoB secretion, 

particularly in the setting of insulin resistance. 

P12 

Role of High Density Lipoprotein Particles in Cellular Cholesterol Efflux 

Bela F Asztalos, Ernst J Schaefer, Tufts Univ, Boston, MA; George H Rothblat; Children’s 

Hosp of Philadelphia, Philadelphia, PA 

Introduction: Low HDL-cholesterol (40 mg/dl) is an important CHD risk factor. HDL promotes 

cholesterol efflux from various cells via ABCA1 and SRB1 (a bidirectional process). ApoA-Icontaining 

HDL can be seperated by charge into pre-beta, alpha, and pre-alpha particles and 

by size into 5.4 –12.7 nm particles. Patients with apoA-I deficiency lack these particles while 

Tangier patients with ABCA1 defects have only pre-beta1 HDL. In contrast, homozygous CETP 

deficient subjects have marked increases in the largest HDL particles and have abnormal 

LpAI:A-II:E particles. CHD patients have significantly (p0.001) increased pre-beta1 and 

decreased alpha1 and pre-alpha1 HDL particles than controls (ATVB 2004;24: 2181–87). 

ApoA-I content in alpha1 HDL are far superior to HDL-cholesterol in CHD risk prediction. Statins, 

niacin, and torcetrapib (CETP inhibitor) specificaly increase alpha1 and decrease pre-beta1 

HDLs. Moreover, increases in alpha1 HDL predict less progression of coronary stenosis on 

angiography. Objectives: Our purpose was to examine the association of individual HDL 

particles with cellular cholesterol efflux. We carried out incubation studies with HDL from 93 

subjects with varying HDL particle levels and J774 macrophages to assess ABCA1 function and 

with FU5AH hepatoma cells to assess SRB1 function. Results: On multivariate analysis only 

pre-beta1a (p0.036) and alpha2 HDL (p0.002) were significantly associated with ABCA1mediated 

efflux, with pre-beta1 being more than twice as effective as alpha2. SRB1 mediated 

liver cell efflux was significantly related to alpha1 (p0.001), alpha2 (p0.006), and alpha3 

(p0.010) HDL particle levels, with alpha1 having twice the efficacy of either alpha2 or alpha3 

HDL. The most efficient ABCA1- and SRB1-mediated efflux was observed using HDL from 

subjects with both high pre-beta1 and high-alpha1 levels, while the least efficient samples 

were from subjects with the lowest levels of pre-beta1 or low levels of both pre-beta1 and 

alpha1. Conclusions: These data indicate that pre beta1a (small) are the most effective particles 

in promoting ABCA1 mediated macrophage cholesterol efflux, while alpha1 HDL (large) are the 

most effective in promoting liver cell SRB1 cholesterol efflux and possibly uptake. 

Distinct Residues in the Carboxyterminus of Endothelial Lipase are 

Important for Heparin-Binding and Enzymatic Activity 

Karen O Badellino, Daniel J Rader; Univ of Pennsylvania, Philadelphia, PA 

Endothelial lipase is a lipase expressed on endothelial cells that has been shown to modulate 

HDL cholesterol levels in humans and in mouse models of atherosclerosis. In a manner similar 

to lipoprotein lipase and hepatic lipase, it has been shown to bind to heparin and to be released 

in vivo from the surface of the vasculature by the administration of intravenous heparin. We 

previously reported that an area , R 427 ,R 428 ,R 430 ,K 432 in the carboxyterminus of the protein was 

important for heparin-binding and that mutation of these basic residues to asparagines resulted 

in loss of enzymatic activity. We have now further examined this area through alanine-scanning 

mutagenesis of individual basic residues. Wild-type endothelial lipase binds to heparin with a 

K DApp of 5.9 0.9 nM, while changing the R 428 to alanine decreased the affinity of the protein 

to 13.4 2.4 nM. The mutation of R 427 to alanine had no effect on heparin binding, and 

mutation of R 430 and K 432 had little effect. In contrast, the R 427 to alanine (R 427 A ) mutant retained 

approximately 30% of the enzymatic activity compared to wild type endothelial lipase, the R 428 

A mutant retained approximately 60% and the R 430 A and K 432 A mutants had almost complete 

loss of activity in both triglyceride lipase and phospholipase assays. We conclude that R 428 is 

important for heparin-binding and that R 430 and K 432 may be involved in intramolecular 

interactions important for the conformation of the protein. 

P14 

Selective Up-Regulation of LXR Sensitive Genes, ABCA1, ABCG1 and APOE, 

in THP-1 Macrophages through Partial Inhibition of Oxidosqualene: 

Lanosterol Cyclase: Implications for Attenuation of Foam Cell Formation 

Michael M Beyea, Claire L Heslop, Cynthia G Sawyez, Jane Y Edwards, Janet G Markle, 

Robert A Hegele, Murray W Huff; Robarts Rsch Institute, London, Canada 

Activation of the liver X receptor (LXR) by oxysterols or synthetic ligands represents an 

attractive mechanism to prevent macrophage foam cell formation. Recently, we demonstrated 

that partial inhibition of oxidosqualene:lanosterol cyclase (OSC), a cholesterol biosynthesis 

enzyme, stimulated synthesis of the LXR-ligand 24(S), 25-epoxycholesterol (24(S), 25-epoxy) 

leading to enhanced ABCA1-mediated cholesterol efflux. In contrast to a synthetic, nonsteroidal 

LXR activator, TO901317, triglyceride accumulation was not observed. In the present 

study, we hypothesized that endogenous 24(S), 25-epoxy synthesis would selectively activate 

macrophage LXR-sensitive genes involved in cholesterol efflux, but not those regulating fatty 

acid metabolism. Incubation of THP-1 human macrophages with the OSC inhibitor (OSCi) 

RO71– 4565 (15nM) reduced cholesterol synthesis by 50% and maximized synthesis of 24(S), 

25-epoxy. This resulted in a 3-fold and 2-fold increase in ABCA1 and ABCG1, respectively, and 

a 2-fold increase in APOE mRNA, coinciding with a significant 1.5 fold increase in cholesterol 

efflux to apoA1. The expression profile of these Downloaded genes was similar from 

in cells exposed to 

P13 

exogenous 24(S), 25-epoxy (1M) and TO901317 (2nM). In contrast, expression of the 

LXR-sensitive genes lipoprotein lipase and fatty acid synthase was unchanged by OSCi, 

exogenous 24(S), 25-epoxy (1M) or TO901317 (2nM). High doses of TO901317 (10nM) 

enhanced expression of all examined LXR-regulated genes 2.5- to 7-fold. Sterol regulatory 

element binding protein 1c (SREBP1c), is activated by LXR and regulates genes involved in fatty 

acid and triglyceride metabolism. Immunoblotting revealed that all 3 treatments increased 

SREBP1 precursor. However, its conversion to the active nuclear form was blocked by OSCi and 

exogenous 24(S), 25-epoxy but not TO901317 (10nM), which instead induced a 2.3-fold 

increase. Our results demonstrate that partial OSC inhibition selectively upregulates expression 

of LXR-sensitive genes involved in cholesterol efflux without stimulating genes linked to fatty 

acid metabolism. Thus, prevention of macrophage cholesterol deposition by endogenous 

synthesis of the oxysterol 24(S), 25-epoxy represents a novel therapeutic approach. 

P15 

Phosphatidylinositol and High Density Lipoprotein Composition Regulate 

Hepatic Lipase Activity 

Jonathan Boucher, Tanya A Ramsamy, Daniel L Sparks; Univ of Ottawa Heart Institute, 

Ottawa, Canada 

Phosphatidylinositol (PI) is a negatively charged phospholipid that is currently being evaluated 

as a novel lipid therapeutic in clinical trials. Oral administration of PI to healthy volunteers for 

two weeks lowered plasma triglyceride (TG) levels by 40% and increased HDL cholesterol by 

almost 20%. PI appears to lower TG levels by directly stimulating lipolysis. Increasing the 

negative charge of plasma lipoproteins by inclusion of PI stimulates lipid hydrolysis by hepatic 

lipase (HL). PI enrichment of human serum significantly stimulated HL activity compared to 

control sera. PI increased lipolytic rates by almost 2-fold in postprandial serum samples. We 

have previously shown that HDL composition and charge uniquely regulate HL activity. HDL 

phospholipid content and electrostatic properties appear to affect HL-mediated lipolysis by 

controlling the binding and association of HL. Enrichment of native or synthetic HDL particles 

with negatively charged phospholipids (PI or PG) significantly stimulated VLDL hydrolysis 

relative to enrichment with uncharged phospholipid (PC). Increased VLDL hydrolysis was 

associated with a reduced high affinity association of HL with the HDL particles. Under normal 

conditions, HL preferentially associates with HDL particles in the plasma and only small 

amounts of HL bind to VLDL. However, PI enrichment of HDL almost completely blocked the 

binding and association of HL with HDL particles. In contrast, enrichment of HDL with apoA-II 

increases the association of HL with HDL and inhibits lipolysis. An increase in the high affinity 

association of HL with HDL may therefore inhibit HL by restricting the interlipoprotein 

movement of the enzyme between substrates. We have shown that small dense HDL 3 are 

inhibitory to HL activity whereas the larger and less dense HDL 2 are stimulatory to VLDL 

hydrolysis. Higher levels of HDL 2 in the bloodstream are associated with a more efficient TG 

clearance. These data suggest that enrichment of HDL with PI may also stimulate TG clearance. 

Increased clearance of TG would be expected to positively affect HDL levels and may reduce 

HDL clearance from the bloodstream. HL is therefore sensitive to the electrostatic properties of 

the plasma lipoproteins and lipolysis can be stimulated by anionic lipids. 

P16 

Cellular Proteoglycans Mediate Uptake of Group V Secretory Phospholipase 

A 2 Modified Low Density Liporoteins by Macrophages 

Boris Boyanovsky, Deneys van der Weshuyzen, Nancy R Webb; Univ of Kentucky, Lexington, 

KY 

Background: Secretory phospholipase A 2 (sPLA 2) enzymes hydrolyze the sn-2 fatty acyl ester 

bond of glycero-phospholipids to produce a free fatty acid and a lysophospholipid. Group V 

sPLA 2 (GV sPLA 2) is expressed in mouse peritoneal macrophages and has high affinity for 

phosphatidylcholine-containing substrates. Our studies have shown the presence of GV sPLA 2 

in human and mouse atherosclerotic lesions, its activity towards LDL phospholipids, and the 

ability of such modified LDL (GV-LDL) to induce foam cell formation. The goal of this study is 

to investigate the mechanisms involved in the uptake of GV-LDL by macrophages. Methods 

and results: Peritoneal macrophages isolated from C57BL/6 (WT) and SR-A/CD36 -/- (DKO) mice 

were treated with control LDL, GV-LDL, oxidized LDL (ox-LDL) or LDL aggregated by vortexing 

(vx-LDL). Ox-LDL promoted more cholesterol ester accumulation in WT compared to DKO 

macrophages. In contrast, there was no difference in the uptake of GV-LDL or vx-LDL between 

the two cell types. 125 I labeled ox-LDL exhibited high affinity, saturable binding to WT cells, that 

was significantly reduced in DKO cells. In contrast, vx-LDL and GV-LDL showed low-affinity, 

non-saturable binding that was similar for both cell types and significantly higher compared to 

control LDL. Degradation assays revealed that GV-LDL are internalized and degraded by 

macrophages in a manner independent of SR-A and CD36. Analyses by confocal microscopy 

indicated distinct intracellular distributions for Alexa-568 labeled GV-LDL and Alexa-488 

labeled ox-LDL. Uptake of GV-LDL, but not ox-LDL or vx-LDL, was significantly reduced in cells 

pre-incubated with heparin or NaClO 3, suggesting a role for proteoglycans in GV-LDL uptake by 

macrophages. Conclusions: Our data indicate that macrophage uptake and degradation of 

GV-LDL involves cell-surface proteoglycans, and not SR-A or CD36. These data point to a 

physiological modification of LDL that has the potential to promote macrophage foam cell 

formation by mechanisms independent of scavenger receptors. 

P17 

Effects of Insulin Resistance on Apolipoprotein A-I Kinetic and Selective 

Uptake in Dog 

François Briand, Edwige Bailhache, Patrick Nguyen, Michel Krempf, Thierry Magot, Khadija 

Ouguerram; Cntr de Recherche en Nutrition Humaine, Nantes, France 

HDL play an important role in the reverse cholesterol transport. This pathway includes a hepatic 

selective uptake of HDL cholesteryl ester (HDL-CE). In humans, insulin resistant states are 

http://atvb.ahajournals.org/ characterized by guest by a low on HDL June cholesterol 29, 2013 concentration and disturbed HDL-apolipoprotein A-I 

Abstracts are embargoed until time of presentation.

(HDL-apo A-I) metabolism. Beagle dog model of obesity-associated insulin resistance has 

previously demonstrated similar lipoprotein abnormalities. The aim was to study the reverse 

cholesterol transport in the insulin resistant dog, a species lacking cholesteryl ester transfer 

protein activity. We used a combined tracers infusion allowing the calculation of selective 

uptake of HDL-CE. Five male Beagle dogs were overfed with a high-fat diet for 28 2.5 

weeks. Obesity was associated with insulin resistance, assessed by euglycemic hyperinsulinemic 

clamp technique. Kinetic studies were conducted in dogs, in healthy and insulin 

resistant states, using a primed constant infusion of [1,2 13 C 2]acetate and [5,5,5-D 3]leucine, as 

labeled precursors of CE and apo A-I, respectively. Isotopic enrichment was measured by mass 

spectrometry. A compartmental model (SAAMII) was used for the analysis of tracer kinetics 

data and calculated selective uptake. HDL-apo A-I did not change with insulin resistance 

whereas production and catabolic rates of apo A-I were both higher (0.612 0.260 vs 

1.334 0.512 mg/kg/h; 0.005 0.002 vs 0.012 0.005 h -1 , respectively, p0.05). HDL-CE 

were lower with insulin resistance (3.61 0.20 vs 3.12 0.21 mmol/l, p0.05) whereas 

HDL-triglycerides were higher (0.027 0.016 vs 0.249 0.189 mmol/l, p0.05). Production 

and catabolic rates of HDL-CE were both lower (15.18 2.18 vs 6.60 1.40 mol/kg/h; 

0.094 0.014 vs 0.046 0.008 h -1 ,p0.05). The selective uptake of HDL-CE was also lower 

(0.089 0.015 vs 0.034 0.010 h -1 ,p0.05). We concluded that insulin resistant dog exibits 

different disturbances of apo A-I kinetics than those described in humans. This study also 

demonstrates that HDL-CE metabolism and its selective uptake is impaired with insulin 

resistance. This animal model could be useful to test treatments that improve dyslipidemia in 

the insulin resistant states. 

SAA Promotes Cellular Cholesterol Efflux through SR-BI 

Lei Cai, Maria C de Beer, Wei Shi, Frederick C de Beer, Deneys R van der Westhuyzen; 


Serum Amyloid A (SAA) is an acute phase protein whose expression is greatly up-regulated 

during inflammation and infection. However, the physiological function of SAA remains unclear. 

We have shown recently that SAA is a ligand for SR-BI and inhibits the selective CE uptake from 

HDL. Class B scavenger receptor type I (SR-BI) mediates selective CE uptake from HDL and also 

facilitates cellular free cholesterol efflux to HDL. In this study, we report that SAA promotes 

cellular cholesterol efflux mediated by SR-BI. In CHO cells expressing SR-BI, SAA promoted 

cellular cholesterol efflux in an SR-BI dependent manner whereas apoA-I did not. Similarly, SAA 

but not apoA-I, promoted cholesterol efflux from HepG2 cells in a SR-BI dependent manner as 

shown using the SR-BI inhibitor, BLT-1. In order to test whether endogenously produced SAA 

can influence efflux, SAA was over-expressed in HepG2 cells using adenovirus mediated gene 

transfer. Endogenously expressed SAA promoted efflux. This efflux was inhibited by BLT-1 

indicating it was SR-BI dependent. In plasma, the majority of SAA is associated with acute 

phase HDL (ApHDL). To assess the effect of SAA mediated efflux, we compared the ability of 

normal HDL, ApHDL (prepared from mice injected with Lipopolysaccharide) and SAA-HDL (HDL 

prepared from mice over-expressing SAA) to function as acceptor for cholesterol efflux from 

CHO-SRBI and HepG2 cells. Both ApHDL and SAA-HDL promoted 2-fold greater FC efflux than 

normal HDL. In summary, lipid-free SAA and HDL-associated SAA promoted SR-BI dependent 

cholesterol efflux suggesting that SAA may play an important role in influencing cholesterol 

metabolism during inflammation through its interaction with SR-BI. 

Glycosylation Regulates the Endothelial Lipase-Mediated Hydrolysis of 

Phospholipids in Reconstituted High Density Lipoproteins 

Daniela Caiazza, Kate Shearston, Chatri Settasatian, Kerry-Anne Rye; The Heart Rsch 

Institute, Sydney, Australia 

Human endothelial lipase (EL), a recently identified member of the triglyceride lipase gene 

family, is a glycosylated enzyme that contains four N-linked glycosylation sites (Asn62, Asn375, 

Asn118 and Asn473). In this study, we examined the hypothesis that glycosylation at one or 

more of the four N-linked glycosylation sites regulates the EL-mediated hydrolysis of 

phospholipids in reconstituted high density lipoproteins (rHDL). To test this hypothesis we 

determined the contribution of each glycosylation site to the activity of the enzyme by using 

site-directed mutagenesis. The rHDL employed in this study were well characterised, 

homogenous preparations of apolipoprotein-specific, spherical rHDL. The rHDL contained either 

apoA-I as the only apolipoprotein, (A-I)rHDL, apoA-II as the only apolipoprotein, (A-II)rHDL, or 

apoA-I as well as apoA-II, (A-I/A-II)rHDL. The rHDL were comparable in terms of size and lipid 

composition, and contained cholesteryl esters (CE) as their sole core lipid. The EL-mediated 

phospholipid hydrolysis was quantified as the mass of non-esterified fatty acids (NEFA) 

released from the rHDL during incubation with EL. Kinetic analysis showed that wild-type EL 

had a greater affinity for the phospholipids in (A-I)rHDL than in (A-I/A-II)rHDL. In contrast, the 

rate of phospholipid hydrolysis was much greater for (A-I/A-II)rHDL than for (A-I)rHDL. Very little 

hydrolysis of phospholipids was observed for (A-II)rHDL with the wild-type EL. Mutation at site 

Asn473 reduced the overall activity of the enzyme, however, the trend for the rate of hydrolysis 

and binding affinity of the Asn473 mutant with the rHDL substrates remained the same. 

Interestingly, the Asn118 mutant afforded a much greater rate of hydrolysis in (A-II)rHDL than 

that observed with the wild-type enzyme. Mutation of either Asn62 or Asn375 resulted in a 

significant decrease in the hydrolysis of the rHDL substrates compared to wild-type, and the 

kinetic parameters could not be determined for these systems. These results establish that 

glycosylation of EL at specific sites is a major determinant of the kinetics of EL-mediated 

phospholipid hydrolysis in rHDL. Downloaded from 

P18 

P19 



Hyperlipidemia in Diet Versus Genetically-Induced Obese Mice 

Kimberly R Causey, Marnie L Gruen, Alyssa H Hasty; Vanderbilt Univ, Nashville, TN 

Obesity is a growing epidemic in the United States and is a major health care concern because 

of its role in other diseases related to the metabolic syndrome, such as atherosclerosis and 

diabetes mellitus. One of the main pathological components of the metabolic syndrome is 

hyperlipidemia, including elevated levels of free fatty acids (FFA), LDL, and triglycerides (TG), 

along with decreased levels of HDL. In order to investigate the relative impact of diet versus 

genetically-induced obesity on lipoprotein metabolism, we placed the obese yellow agouti (A y /a) 

mice on an LDLR-/- background. Non-obese (a/a) LDLR-/- littermates were used as controls. 

At four months of age, A y /a;LDLR -/- and a/a;LDLR -/- mice were either maintained on a chow diet 

(CD) or placed on a Western diet (WD) for 12 wks. Thus, we obtained 4 groups of mice: 1) 

a/a;LDLR-/- on CD, 2) Ay/a;LDLR-/- on CD, 3) a/a;LDLR-/- on WD, and 4) Ay/a;LDLR-/- on WD. 

After 12 weeks on their respective diets, body weights increased in the following order: 

a/a;LDLR-/- (CD) A y /a;LDLR-/- (CD) a/a;LDLR-/- (WD) A y /a;LDLR-/- (WD). Total body fat 

and abdominal adipose tissue weight mirrored these results. Plasma total cholesterol (TC) 

levels were 30% higher and TG levels were 2-fold elevated in CD-fed Ay/a;LDLR-/- compared 

to a/a;LDLR-/- mice suggesting that obesity alone can induce hyperlipidemia. WD-fed 

a/a;LDLR-/- mice displayed greater than 4-fold increase in TC and TG levels and WD fed 

Ay;LDLR-/- mice displayed 7-fold and 12-fold increases in TC and TG levels compared to 

CD-fed a/a;LDLR-/- mice. FFA and insulin levels were also dramatically elevated (3-fold and 

20-fold, respectively) in WD-fed Ay/a;LDLR-/- mice compared to CD-fed a/a;LDLR-/- mice. 

These data indicate that increasing body weight via genetic manipulation and high fat diet 

feeding has a synergistic effect on hyperlipidemia. This model will provide a useful tool in which 

to study mechanisms by which obesity induces hyperlipidemia, diabetes and atherosclerosis. 

n 

Body 

Wt (g) 

Cholesterol 

(mg/dl) 

Triglycerides 

(mg/dl) 

FFA 

(mEq/ml) 

P20 

Insulin 

(ng/ml) 

a/a;LDLR-/- (CD) 9 27 (.3) 203 (12) 88 (19) .56 (.07) .42 (.04) 

Ay/a;LDLR-/- (CD) 7 36 (1.2) 263 (12) 167 (15) .59 (.06) 2.6 (.5) 

a/a;LDLR-/- (WD) 11 37 (1) 990 (162) 345 (60) 1.3 (.31) 2.3 (.4) 

Ay/a;LDLR-/- (WD) 11 49 (.7) 1435 (83) 1075 (64) 1.8 (.18) 8.5 (1.6) 

ApoB100-Only Ob/Ob Mice: A Novel Model for Studying 

Diabetes/Obesity-Induced Dyslipidemia 

Zhouji Chen, Xiucui Ma, Robin L Fitzgerald, Donglin Cheng; Washington Univ Sch Med, Saint 

Louis, MO 

Overproduction of triglyceride (TG)-rich VLDL by the liver is a key physiologic base of 

dyslipidemia associated with metabolic syndrome in humans. To develop an animal model to 

understand the underlying mechanism, we have produced an apoB100-only leptin-deficient 

ob/ob mice (ApoB100/Ob-Ob) by breeding a targeted apoB100-specifying allele into the ob/ob 

mice. Like the human liver, the liver of this mouse produces only apoB100. In vivo TG secretion 

in these mice was determined at age of 10-week old using Triton-WR1339 to block VLDL 

clearance. Compared to apoB100-only lean mice (either Leptin-wild or heterozygous for the 

ob-allele), in vivo rate of hepatic triglyceride export by ApoB100/Ob-Ob was increased by 

2.5-fold. Density-gradient ultracentrifugation analysis of lipoprotein showed that this increase 

was mainly due to an increased amount of TG in the VLDL1 fraction of the ApoB100/Ob-Ob 

mouse plasma. Metabolic labeling experiments on primary hepatocytes showed that secretion 

rates of 35 S-labled apoB100 by ApoB100/Ob-Ob hepatocytes were increased by 1.4-fold, 

compared to that of the hepatocytes isolated from the apoB100 lean littermates. Concomitantly, 

there was a 4-fold increase in the rates of secretion of 3 H-glycerol-labled TG by ApoB100/ 

Ob-Ob hepatocytes. Pulse-chase studies demonstrated that pre-secretory degradation of 

apoB100 was retarded in ApoB100/Ob-Ob hepatocytes. Supplementation of oleic acid (0.6 mM) 

to culture media stimulated secretion of both apoB100 and TG by the hepatocytes of 

apoB100-only lean mice but it enhanced only TG secretion by the ApoB100/Ob-Ob hepatocytes, 

indicating that even in the absence of exogenous fatty acid supply, the newly synthesized 

apoB100 in ApoB100/Ob-Ob hepatocytes may be already fully recruited for VLDL production, 

probably due to the enhanced lipogenesis in the ob/ob mouse liver. Together, these results 

suggest that assembly and secretion of enlarged TG-rich VLDL may be a key mechanism for 

the overproduction of VLDL-TG by the liver of ApoB100/Ob-Ob mice. Further studies into the 

pathways and cellular events accompanied with the biogenesis of these enlarged VLDL 

particles in this mouse model may provide new insights into the mechanism of diabetes/ 

obesity-induced dyslipidemia. 

P22 

Mechanism of ApoE-Induced VLDL Secretion: Evidence for a Critical Role 

of ApoE in Bulk Triglyceride Addition During VLDL Assembly in the ER 

Zhouji Chen, Xiucui Ma; Washington Univ Sch Med, Saint Louis, MO 


ApoE participates in VLDL synthesis and stimulates VLDL-triglyceride (TG) secretion but the 

underlying mechanism is poorly understood. In this study, we determined first the effect of 

apoE deficiency on apoB100-VLDL secretion. Deletion of apoE gene in apoB100-only mice 

reduced hepatic TG secretion by 40%. Rates of apoB-100 synthesis and secretion were not 

affected by the loss of apoE function but secretion of TG-rich VLDL-1 was diminished, with 

increased amounts of apoB100 being secreted as TG-poor IDL. Thus, apoE may have a critical 

role in the second step of VLDL synthesis. Subsequently, apoE3-overexpressing HepG2 cells 

were used to determine the underlying mechanism. Synthetic rate of endogenous apoE in 

HepG2 cells was very low, corresponding to only 15% of that of mouse hepatocytes. A 

recombinant adenovirus (Ad-E3) was used to overexpress human apoE3 in HepG2 cells to 

achieve an apoE expression level similar to that found in mouse hepatocytes. GFP-expressing 

adenovirus (Ad-GFP)-infected HepG2 cells were used as controls. ApoE3 overexpression did not 

affect synthetic by guest rates of on apoB100 June 29, and TG. 2013 However, it increased TG-secretion 3-fold, with only 

P21

E-56 Vol 25, No 5 May 2005 

a modest (35%) increase in apoB100 secretion. In the presence of oleic acid (0.6 mM), 

Ad-E3-infected HepG2 cells secreted a majority (65%) of apoB100 as VLDL whereas only 18% 

of apoB100 secreted by Ad-GFP-infected HepG2 floated as VLDL. Density-distribution analyses 

of lumenal apoB-lipoproteins within the secretory compartments indicated that an enhanced 

conversion of LDL/HDL- to VLDL-sized particles within the ER could account for the increased 

VLDL secretion by Ad-E3-infected HepG2 cells. Overexpression of an ER-retained form of apoE3 

(apoE3-KDEL) decreased secretion of both apoB100 and TG by 3– 4 fold but it coordinately 

increased the contents of newly synthesized TG and apoB100 in microsomal membranes, 

providing direct evidence for an interaction of apoE with apoB100-containing particles in the 

ER. In sum, these results suggest that apoE may stimulate VLDL-TG secretion by promoting 

bulk TG addition to VLDL precursors in the ER during the second step of VLDL assembly. 

Furthermore, an inherently low rate of apoE synthesis may be responsible for the defective 

VLDL-secreting capability in HepG2 cells. 

P23 

Apolipoprotein A-I Kinetics within Pre1 HDL, HDL and TRL in Fed State 

maud chetiveaux, Khadija Ouguerram, Yassine Zair, Michel Krempf; inserm U539, Nantes, 

France 

Stable isotope method was used to determine the kinetic behavior of Apo A-I within HDL 

subclasses, pre 1 and HDL, in fasted and fed states. Six normolipidemic male subjects were 

given a 14h constant infusion of d 3-leucine. Blood sample were collected during infusion and 

at time 24, 48, 72, and 96. Subjects were studied in fasted state and in fed state providing 

100% of usual total calorie intake with small hourly meals. Pre 1 and HDL were isolated by 

FPLC and VLDL and TRL by ultracentrifugation. Apo A-I, B100 and B48 were separated by 

SDS-PAGE. Apo tracer/tracee enrichment curves were obtained by GC-MS. Data were fitted to 

a compartmental model (SAAM II) using the plateau enrichment of VLDL-Apo B100 and 

TRL-Apo B48 for an estimation of the hepatic and intestinal pool precursor, respectively. In 

fasted or fed state, kinetic parameters of Apo A-I - HDL subclasses were not statistically 

different, excepted for a lower clearance rate of Apo A-I - pre 1 HDL in fed state (0.447 

0.165 vs. 0.235 0.093 d -1 ,p0.05). Mean Apo A-I TRL and HDL clearance rate were 4.29 

2.66 and 0.235 0.093 d -1 , respectively. The mean Apo A-I - TRL retention time was 5.59 

hours compared to 4.25 days for Apo A-I - HDL and 3.58 hours for Apo B48 - TRL. Hepatic Apo 

A-I synthesis was estimated 17.17 6.74 mg.kg -1 .d -1 in fed state and 18.67 4.14 

mg.kg -1 .d -1 in fasted state. Intestinal Apo A-I secretion, characterised by the secretion of Apo 

A-I in TRL, was estimated 2.20 1.50 mg.kg -1 .d -1 (13 9% of the total Apo A-I secretion rate 

(2.20 of 19.38 mg.kg -1 .d -1 )). Our result indicate that Apo A-I - HDL kinetic parameters are 

similar in fasted and fed state, and that intestine contribute to about 13% of the total Apo A-I 

secretion rate in fed state. 

Pcsk9 Expression is Altered in Diabetic Rats and its Expression is 

Regulated by Glucose and Insulin 

Philippe Costet, INSERM, Nantes, France; Bertrand Cariou, Institut Pasteur-Université Lille 2, 

Lille, France; Florent Lalanne, Gilles Lambert, Bernard Lardeux, Anne Laure Jarnoux, 

INSERM, Nantes, France; Bart Staels, Institut Pasteur-Université Lille 2, Nantes, France; 

Michel Krempf; INSERM-CRNH, Nantes, France 

Proprotein Convertase Subtilisin/kexin type 9 (PCSK9, Narc-1) is a new gene involved in familial 

hypercholesterolemia. Our group showed that patients heterozygote for the S127R mutation 

develop hypercholesterolemia and an increased production of apoB containing lipoparticles. 

Others showed that hepatic PCSK9 affects the amount of Low Density Lipoprotein receptors at 

the cell surface (LDLr). Wild-type PCSK9 therefore appears to regulate plasma clearance of 

atherogenic particles. In an effort to assess a potential role of PCSK9 in the metabolic 

syndrome, we studied the regulation of PCSK9 by insulin and glucose. In vivo studies suggested 

a combined influence of glucose and insulin on PCSK9 expression. Real time Q-PCR analysis 

revealed that PCSK9 expression increases with age in ZDF fatty diabetic rats (2-fold increase 

in 10 and 20 weeks old compared to lean ZDF rats or ZDF fatty rats of 6 weeks old, p0,05, 

student test). In rats treated with streptozotocin, PCSK9 mRNA levels dropped by two fold ( 

p0,05), and insulin injections partially restored it. In mice exposed to a 24h starving period 

a dramatic decrease in PCSK9 mRNA and protein was observed which returned to normal 

within 24h after re-feeding. In vitro, glucose dose dependently increased PCSK9 mRNA levels 

in HepG2 cells, in the absence of insulin (2.5 fold increase between 5 and 10mM after 24h 

treatment, p 0,05,). In immortalized human hepatocytes (IHH), a 15h exposure to 25nM 

insulin increased PCSK9 mRNA levels by 4 fold compared to cells cultured without insulin 

(p0,05). In primary cultures of mouse hepatocytes, an 18h period exposure to 100nM insulin 

increased 2 fold PCSK9 mRNA levels, at low (5mM) or high doses of glucose (25mM) (p0,05) 

. Together these results show that PCSK9 is activated by glucose and insulin. Although the 

molecular mechanisms are still under investigation, this suggests that PCSK9 could play a role 

in the development of dyslipidemia associated with alterations of glucose homeostasis, as 

found in patients with the metabolic syndrome and Downloaded diabetes. from 

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Lysophosphatidic Acid Induces Early Growth Response Gene Egr-1 

Expression in Vascular Smooth Muscle Cells 

Mei-Zhen Cui, Essam Laag, Guojun Zhao, Longsheng Sun, Mingqi Tan, Xuemin Xu; Univ of 

Tennessee, Knoxville, TN 

Evidence is accumulating from in vitro and in vivo studies that suggest that the early growth 

response gene-1 (Egr-1) is a key transcription factor in regulating a cluster of genes, which are 

implicated in the pathogenesis of vascular diseases. However, the regulation of the key 

transcription factor Egr-1 gene, by atherogenic factors in atherosclerotic lesions is little 

understood. Specifically, how the Egr-1 gene transcription is regulated in vascular smooth 

muscle cells (SMC) is currently unknown. Lysophosphatidic acid (LPA) is a component of 

oxidized lipoprotein and an agent released by activated platelets. Our data show that LPA 

markedly induces Egr-1 messenger RNA (mRNA) and Egr-1 protein in SMC. Results of the 

nuclear run-on experiments demonstrate that LPA-induced Egr-1 gene expression is principally 

controlled at the transcriptional level. Transfection studies using a series of deleted Egr-1 

promoters revealed that a -140- to 21- base pair region of the Egr-1 promoter contains 

regulatory elements required for LPA-mediated induction. Electrophoretic mobility shift assays 

revealed that the DNA-binding activities of both cAMP-response element binding protein (CREB) 

and serum response factor (SRF) to the CRE and SRE motifs are markedly elevated in response 

to LPA, suggesting a role of both CREB and SRF in regulating LPA-induced Egr-1 expression. 

Transfection of site-directed mutants of the Egr-1 promoter demonstrated that CRE and SRE 

cis-acting elements in the Egr-1 promoter were required for LPA-induced Egr-1 gene induction. 

Our data show for the first time that lysophosphatidic acid induces Egr-1 gene expression in 

smooth muscle cells and that this Egr-1 gene expression is mediated by CREB and SRF 

transcription factors. Understanding the mechanisms of induction of the key transcription factor 

Egr-1 expression in response to atherogenic factors in vascular smooth muscle cells is 

important toward understanding the formation and development of atherosclerotic lesions. 

P26 

SR-BI Selective Lipid Uptake: Subsequent Metabolism of Acute Phase and 

Normal HDL Remnants 

Maria C De Beer, Nancy R Webb, Nathan L Whitaker, Deneys R Van Der Westhuyzen, 

Frederick C De Beer; Univ.of Kentucky, Lexington, KY 

In vivo processing of polydisperse human HDL2 in apoA-I-deficient mice over expressing SR-BI 

gives rise to distinct smaller remnant particles that contain both apoA-I and apoA-II, or apoA-I 

only. Remnant particles containing only apoA-I are not able to re-associate with HDL. Data 

presented here focus on the sequelae of SR-BI interaction with monodisperse acute phase 

mouse HDL (AP-HDL) and normal mouse HDL (N-HDL). This approach allows study of the 

metabolic fate of the respective HDL apoproteins derived from a single monodisperse HDL. 

Inflammation alters HDL and serum amyloid A protein (SAA) can become the major apoprotein. 

Here we show that SR-BI processing of N-HDL and AP-HDL generates similar discrete small 

remnants. A stable remnant of 7.7 nm diameter, containing only apoA-I, is rapidly generated 

and it becomes the only detectable entity at 6 h of SR-BI processing. SAA and apoA-II 

segregate with larger remnants. SDS-PAGE analysis of plasma collected from C57BL/6 mice at 

timed intervals after injection of labeled AP HDL showed preferential loss of SAA and apoA-II 

with SAA clearing most rapidly of all HDL apoproteins. Isoelectric focusing showed that the two 

major acute phase SAA isoforms cleared at similar rates. When AP HDL remnant particles were 

incubated with normal mouse HDL, only SAA-and apoA-II-containing remnants converted to 

larger particles by associating with HDL, whereas the 7.7 nm apoA-I only remnant did not. 

Remodeling potential is thus associated with SAA and apoA-II. Analysis of remodeled remnants 

obtained from incubating AP HDL remnant particles with human HDL3 indicated further 

segregation of remnant particles into those containing apoA-I and SAA and remnants containing 

all three apoproteins but remarkably enriched in apoA-II. Our data indicate that SR-BI 

processing of monodisperse mouse AP-HDL generates distinct HDL remnants that segregate 

apoA-I from SAA and apoA-II. These distinct remnant particles differ in their subsequent 

interactions with normal mouse HDL and human HDL3. SAA acts in this system analogous to 

apoA-II but distinctly different from apoA-I. These studies identify a novel pathway whereby 

SR-BI processing of a monodisperse HDL gives rise to polydisperse particles that have distinct 

metabolic properties. 

P27 

Characterization of High Density Lipoprotein Particles Formed by 

ATP-Binding Cassette Transporter A1-Mediated Efflux of Cellular Lipids to 

Apolipoprotein A-I 

Phu T Duong, Heidi L Collins, Margaret Nickel, Sissel Lund-Katz, George H Rothblat, 

Michael C Phillips; The Children’s Hosp of Philadelphia, Univ of Pennsylvania Sch of 

Medicine, Philadelphia, PA 

The purpose of the study is to characterize the nascent high density lipoprotein (HDL) particles 

formed by interaction of human plasma apolipoprotein (apo) A-I with J774 mouse macrophages 

in which the ATP-binding cassette transporter A1 (ABCA1) is up-regulated. Non-denaturing 

two-dimensional gradient polyacrylamide gel electrophoresis shows that 3 sizes of apo 

A-I-containing HDL particles are formed; these particles exhibit alpha-electrophoretic mobility. 

The results of chemical cross-linking using bis (sulfosuccinimidyl) suberate and SDS-PAGE 

show that apo A-I is the sole protein and that the 9 nm particles contain 2 apo A-I molecules 

whereas the 12 nm particles contain 3–4 apo A-I molecules. Far-ultraviolet circular dichroism 

data indicate that the apo A-I alpha-helix contents are about 50% in both cases. The ratio of 

phospholipid (PL)/apo A-I is 96/1 and 196/1 (mol/mol) for the 9 and 12 nm particles, 

respectively. The PL compositions of both particles are similar: 20–24% phosphatidylcholine 

(PC), 47–40% phosphatidylethanolamine (PE) and phosphatidylserine (PS), 9–8% phosphatidylinositol 

(PI), 9–13% sphingomyelin (SM), 8–10% lyso PC, 7–5% free fatty acid (FA). The 

alpha-mobility by guest and negative on June charge 29, on2013 these HDL particles can be explained by the presence 

P25

of PS, PI and FA molecules. When sufficient apo A-I is available, the 9 nm particles do not seem 

to enlarge to form the 12 nm species. From the mass analysis of PL and unesterified (free) 

cholesterol (FC), the ratio of PL/FC is 8:1 and 5:1 (mol/mol) for the 9 and 12 nm HDL particles, 

respectively. The higher FC content of the latter perhaps arises because they are created by 

ABCA1 molecules in a relatively FC-rich domain of the plasma membrane. 

P28 

Pitavastatin Induces Arrest of the Cell Cycle in S Phase Associated with 

the Downregulation of CCR2 and CCR5 Expression 

Masahiro Fujino, Shin-ichirou Miura, Hiroyuki Tanigawa, Yoshino Matsuo, Akira Kawamura, 

Keisuke Okamura, Keijirou Saku; Fukuoka Univ Hosp, Dept of Cardiology, Fukuoka, Japan 

The pleiotropic effects of statin, including its anti-inflammatory effects, via chemokines may be 

independent of statin-induced cholesterol reduction. Therefore, we examined the effect of 

pitavastatin on cell proliferation and the association between chemokine receptors (CCR2 and 

CCR5) and their ligands, RANTES (regulated upon activation, normal T cell-expressed and 

secreted) and MCP-1 (monocyte chemotactic protein-1), in monocytes. Pitavastatin but not 

pravastatin inhibited cell proliferation in a dose-dependent manner and showed S phase arrest 

associated with the downregulation of CCR2 and CCR5 expression in human monocytic tumor 

cells (U937 cells). The anti-proliferative effects of pitavastatin were not inhibited by lower 

concentrations of RANTES and MCP-1, which were the physiological concentrations in human 

plasma. Pitavastatin induced upregulation of p21waf1 but not p27kip1, and the upregulation 

was blocked by RANTES and MCP-1. In addition, RANTES and MCP-1 upregulated cyclin D1 in 

the presence of pitavastatin. In conclusion, the anti-proliferative effect of pitavastatin, but not 

pravastatin, through cell cycle regulation associated with the downregulation of CCR2/CCR5 

may be a pleiotropic effect. This effect may be anti-atherogenic in monocytes. 

P29 

Hepatocyte CETP Mediates HDL-CE Selective Uptake by a Process Which is 

Only Partially Susceptible to Inhibition by Torcetrapib 

Andre Gauthier, Ruth McPherson; Univ Ottawa Heart Inst., Ottawa, Canada 

Cholesteryl ester transfer protein (CETP) has an established function as a neutral lipid transfer 

protein in the plasma compartment. We have demonstrated that CETP also plays a direct role 

in HDL-derived cholesteryl ester (CE) selective uptake. Inhibition of plasma CETP activity by 

torcetrapib effectively increases HDL-C but the effects of this compound on reverse cholesterol 

transport and atherosclerosis in humans are not yet clear. In these studies, we have established 

that CETP mediates selective uptake by mouse hepatocytes studied both ex vivo and in vivo. 

Mice do not express CETP. Using adenovirus-mediated CETP (adCETP) expression in primary 

mouse hepatocytes from either wildtype, LDL receptor -/- or SR-BI-/- mice, we demonstrate 

accumulation of HDL-derived CE without degradation of the HDL particle, labeled in its 

apolipoprotein moiety, thus demonstrating bona fide selective uptake not dependent on either 

SR-BI or reuptake of lipoproteins by the LDLr. Hepatocyte CETP-mediated selective uptake was 

also not impaired by treatment with RAP or heparin. Addition of recombinant CETP to media 

containing labeled HDL also resulted in accumulation of CE by wildtype or LDLr -/- hepatocytes. 

Notably, hepatocyte CE uptake, mediated by endogenous adCETP expression, only partially 

inhibited by torcetrapib and preincubation of torcetrapib with exogenous CETP and HDL did not 

further attenuated CETP-induced selective uptake. In further studies, following tail vein injection 

of adCETP, C57Bl/6J mice received daily IV injections of torcetrapib (0.125 mM) or vehicle 

(DMSO) in Intralipid. Discontinuous density gradient ultracentrifugation of plasma 7d post 

adCETP injection demonstrated that hepatic CETP expression resulted in a 55% decrease in 

cholesterol in the HDL density range when mice were treated with vehicle alone as compared 

to a 35% decrease with IV torcetrapib treatment. Thus, hepatic CETP expression in vivo acutely 

lowers HDL-C and this effect is only moderately attenuated by torcetrapib treatment. These 

data are consistent with a direct role for hepatocyte CETP in mediating selective uptake. This 

may involve both transfer-mediated and partial-fusion-mediated processes; the latter mechanism 

appears insensitive to torcetrapib. (CIHR #44360) 

Regulation of Human Macrophage Cholesteryl Ester Hydrolase by High 

Density Lipoprotein (HDL) 

Jennifer S Hill, Bin Zhao, Shobha Ghosh; Virginia Commonwealth Univ, Richmond, VA 

Cholesteryl ester hydrolase (CEH) catalyses the rate limiting step in free cholesterol efflux from 

macrophage foam cells and difference in the levels of CEH correlate with lipid accumulation in 

the foam cells and susceptibility to atherosclerosis. Free cholesterol released as a result of 

CEH-mediated hydrolysis of intracellular cholesterol esters is effluxed to extra-cellular 

cholesterol acceptors such as HDL. In addition to its role as a cholesterol acceptor, HDL is 

increasing being recognized as a signaling molecule shown to initiate a multitude of 

intracellular signaling events including activation of MAP kinases. In the present study, we 

examined the regulation of human macrophage CEH by HDL. Human THP-1 cells were 

differentiated for 4 days with 100 nM PMA and subsequently PMA was withdrawn for two days 

to restore the sensitivity to extra-cellular stimuli. These cells regained their ability to activate 

MAP kinases (e.g., Erk1/2 phosphorylation) in response to GM-CSF. THP-1 macrophages 

cultured as described above were exposed to human HDL. Total cell extracts were prepared 

and analyzed by western blot analyses for CEH. CEH protein levels were determined by 

densitometry and normalized to -actin. As shown in the Figure, exposure to HDL for as low 

as 15 minutes resulted in greater than 4-fold increase in CEH protein. These data are consistent 

with the reported stabilization of cholesterol transporter ABCA1 protein by ApoA1 and suggest 

that HDL not only accepts the free cholesterol released Downloaded by CEH mediated from 

hydrolysis but also 

P30 




enhances this process by increasing intra-cellular levels of CEH protein. Effect of HDL on CEH 

protein stability and/or de novo synthesis will be discussed. 

Lipidation and Dimerization of Apo A-II and Apo E by HepG2 Cells 

Baiba K Gillard, Hu-Yu A Lin, Yuen-Shing A Chen, John W Gaubatz, Henry J Pownall; Baylor 

College of Medicine, Houston, TX 

Human apo A-II and the E2 and E3 but not E4 isoforms of apo E contain cysteine, and can form 

disulfide-linked homo- and heterodimers. Human plasma apo A-II is 96% homodimer, and apo 

E in E3/E3 homozygous individuals occurs as 45% monomer, 26% homodimer and 29% apo 

A-II heterodimer. Lipidation and dimerization of these apos affects biological function. Dimeric 

but not monomeric human apo A-II expression reduces mouse plasma HDL size; lipid-bound 

but not lipid-free apo E binds to the LDL receptor. The roles of apo A-II and apo E dimers in 

HDL formation and remodeling have not been defined. We identified the mechanism for apo A-II 

dimerization, which is second order with respect to apo A-II and “catalyzed” by lipid, protein, 

and lipoprotein surfaces, with reconstituted HDL (rHDL) increasing the dimerization rate by a 

factor of 3000. These observations led to hypotheses that lipidation may catalyze disulfide 

bond formation intrahepatically and in plasma HDL, and that apo homo- and heterodimers 

affect HDL formation, stability and remodeling. We have analyzed lipidation and dimerization of 

apo A-II and apo E in the human hepatoma cell line HepG2. Media was collected after 

incubation for 2 and 24 hr and free sulfhydryls were alkylated with iodoacetamide. Lipid-bound 

and free apos were separated by density centrifugation (d1.25), and samples were analyzed 

under reducing and non-reducing conditions by SDS-PAGE/ Western blotting to determine the 

content of monomers and dimers. Apo A-II at both 2 and 24 hr was essentially all 

lipid-associated and present as homodimer, with a trace of apo E heterodimer at 2 hr and more 

at 24 hr. Apo E was also all lipid-associated at 2 hr, but 5% was in the lipid-free fraction at 

24 hr. Unlike apo A-II, at 2 hr apo E was mostly monomeric, with a trace (1%) of heterodimer 

and no homodimer. By 24 hr, half of the apo E had dimerized, with 20% homodimer and 

30% heterodimer. This data suggests that apo A-II and apo E are either lipidated 

intracellularly, prior to secretion, or acquire lipid rapidly upon secretion. Apo A-II is fully 

dimerized either intracellularly or shortly upon secretion, while apo E is secreted as a monomer, 

but forms dimers after secretion. Our results suggest that apo E dimerization and HDL 

remodeling may be concerted processes. 

Pioglitazone Therapy Reduces Plasma Triglycerides by Inhibiting 

Production of Apolipoprotein C-III and Increasing Plasma Lipoprotein 

Lipase Concentration 

Henry N Ginsberg, Kazunori Nagashima, Colleen Ngai, Nelson Fontenez, Columbia Univ 

College of Physicians and Surgeons, New York, NY; Andre Bensadoun, Cornell Univ, Ithaca, 

NY; Steve Holleran, Rajasekhar Ramakrishnan, Columbia Univ College of Physicians and 

Surgeons, New York, NY; Jeffrey S Cohen; Clinical Rsch Institute of Montreal, Montreal, 

Canada 

Insulin resistance is thought to be linked to elevated plasma very low density lipoprotein (VLDL) 

triglycerides (TG) in patients with type 2 diabetes mellitus (T2DM) via several possible 

mechanisms. Increased flux of fatty acids (FA) from adipose tissue to the liver, increased de 

novo hepatic lipogenesis, and decreased intracellular degradation of newly synthesized 

apolipoprotein B (apoB) can each lead to increased secretion of VLDL TG and apoB. Additionally, 

lipoprotein lipase (LpL)- mediated lipolysis of VLDL TG can be reduced in T2DM because LpL 

is decreased and/or apoC-III plasma levels are increased. ApoC-III inhibits LpL-mediated 

lipolysis of TG. We studied the effects of Pioglitazone (Pio), a peroxisome proliferator-activated 

receptor (PPAR)gamma agonist that improves insulin sensitivity, on VLDL metabolism in 8 

patients with T2DM. Hepatic secretion rates (SR) of VLDL apoB and TG, and of plasma apoC-III 

were determined using stable and radioactive isotope methods. Although Pio treatment 

improved insulin sensitivity and reduced plasma FA levels, the observed reduction of 25% in 

VLDL TG concentration was not associated with reductions in SR of either VLDL apoB or TG. 

Instead, the reduction in TG levels during Pio treatment was accounted for by increased 

fractional clearance (FCR) of VLDL TG from the circulation (increased 57%, p0.015), without 

any change in direct removal of VLDL particles. This indicated increased lipolysis of VLDL TG 

during Pio treatment, a mechanism supported by our finding of increased LpL mass in plasma 

(increased 24%, p0.013) and decreased levels of plasma apoC-III (decreased 20%, p0.05) 

due to reduced apoC-III SR (decreased 34%, p0.02). Why VLDL TG and apoB SR were not 

reduced by Pio treatment remains to be determined. Thus, Pio, a PPAR gamma agonist, 

reduced VLDL TG levels by increasing LpL mass and inhibiting apoC-III production. These two 

changes were associated with an increased FCR of VLDL TG, almost certainly due to increased 

LpL-mediated by guest lipolysis. on June 29, 2013 

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P32

E-58 Vol 25, No 5 May 2005 

Cell-Free Assay to Determine Inflammatory Properties of High Density 

Lipoproteins (HDL) 

Timothy A Gong, Greg Hough, Naeimeh Kamranpour, Susan Hama, Mohamad Navab, 

Srinivasa T Reddy, Alan M Fogelman; Atherosclerosis Rsch Unit, Div of Cardiology, Dept of 

Medicine, Univ of California, Los Angeles, Los Angeles, CA 

P33 

Introduction: The inflammatory properties of HDL play a significant role in the development of 

atherosclerosis. We developed a new cell free assay (CFA) to distinguish the inflammatory 

properties of HDL. This new assay is a modified version of a CFA we previously reported (Navab 

et al. 2001; J Lipid Res. 42:1308 –1317) based on the ability of HDL to inactivate oxidized 

phospholipids in LDL. Methods: HDL was isolated from plasma or serum by Magnetic Bead 

Reagent (Polymedco). A standard mixture of LDL (90.0g/mL) and oxidized 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphorylcholine 

(62.5g/mL) were incubated (30min) and the 

oxidation of the mixture was measured in the presence of DCFH (dichlorofluorescein) that 

generates a fluorescent signal (485/530 nm) when oxidized. HDL was tested for its ability to 

inhibit the fluorescent signal, which in turn is a measure of HDL’s ability to inactivate oxidized 

phospholipids in LDL. Results: Using this new method, we were able to accurately distinguish 

the inflammatory properties of HDL, i) from mouse models of atherosclerosis, ii) from animal 

models of atherosclerosis in which an anti-atherogenic peptide, D-4F (an ApoAI mimetic 

peptide) was administered, iii) from monkeys that were administered D-4F, and iv) from known 

proinflammatory and anti-inflammatory human samples. Moreover, the new CFA was equally 

effective on HDL prepared from both serum and plasma samples. Conclusion: HDL function 

has become the target for therapeutic interventions of cardiovascular diseases. We have 

developed a simple, rapid, versatile, and effective CFA to determine the inflammatory 

properties of HDL in serum samples, which can be used as a biomarker for drug efficacy. 

Assembly of Apo100-Containing Very low Density Lipoproteins Occurs 

Post- Endoplasmic Reticulum (ER) and may Require ApoB-100 

Conformational Changes and ApoE 

Viktoria Gusarova, New York Univ Sch of Medicine, New York, NY; Jeffrey L Brodsky, Univ 

of Pittsburgh, Pittsburgh, PA; Edward A Fisher; New York Univ Sch of Medicine, New York, 

NY 

We have previously reported that the final step of VLDL assembly occurs post-ER using a 

cell-free ER-budding assay. Some investigators taking different approaches also suggested that 

VLDL maturation occurs post-ER while others concluded that the ER is the site of final 

assembly. To address this controversy and to explore the role of apoE in the VLDL assembly 

process, we repeated ER budding assays and performed similar Golgi assays using microsomal 

membranes purified from rat hepatoma McA-RH7777 cells that had been incubated with oleic 

acid to stimulate triglyceride synthesis. Again, in the ER-derived vesicles, apoB100 was only 

partially lipidated. In addition, apoB100 and apoE were found in different subfractions of 

ER-derived vesicles, implying that they travel to the Golgi in distinct types of vesicles, and that 

apoE was not involved in the initial phase of apoB100 lipidation. In contrast, in Golgi-derived 

vesicles, apoB100 was fully lipidated to VLDL buoyancy, and 75% of apoB100 was found in the 

same subfraction of vesicles as apoE. By protease susceptibility, apoB100 had domain 

exposure to the cytosol in ER-derived vesicles, but was found 3 times more protected from 

trypsin in Golgi vesicles, suggesting a conformational change associated with VLDL maturation. 

In conclusion, direct examination of Golgi-derived vesicles support: 1) that this organelle is the 

site of VLDL maturation, 2) that this maturation involves apoB100 conformational changes, and 

3) that the reported ability of apoE to enhance VLDL secretion may reflect an interaction 

between apoE and apoB100-lipoproteins in the Golgi that results in the stabilization of a larger 

particle or the provision of bulk lipids. 

P35 

Cholesteryl Ester Trafficking from the Plasma Membrane to Lipid Droplets 

Does not Require Scavenger Receptor BI 

Christopher J Harder, Heidi McBride, Ruth McPherson; Univ Ottawa Heart Inst., Ottawa, 

Canada 

Scavenger receptor BI (SR-BI) plays a critical role in the delivery of HDL cholesterol and 

cholesteryl esters (CE) to liver and steroidogenic tissues by a selective process that does not 

result in significant degradation of HDL protein. Although SR-BI mediated recycling of HDL has 

been proposed, it is not clear how HDL recycles or whether efficient SR-BI mediated selective 

uptake occurs strictly at the plasma membrane or at additional sites along its endocytic 

itinerary. To examine live-cell intracellular trafficking of SR-BI, we created a SR-BI cyan 

fluorescent fusion protein (SR-BI-CFP). We outlined the intracellular trafficking of SR-BI-CFP 

and fluorescently labeled HDL and determined that SR-BI-CFP exists in both intracellular and 

cell surface microdomains. Importantly, we demonstrate that the process of HDL endocytosis 

and recycling is not required for efficient SR-BI mediated selective uptake and that the majority 

of HDL remains bound to SR-BI at the cell surface. Using energy depletion experiments, we 

show that BODIPY labeled HDL CE puncta align along actin filaments on the cell surface of 

SR-BI expressing cells in a pattern reminiscent of surface caveolae. However, the majority of 

these puncta do not colocalize with SR-BI-CFP cell surface microdomains. Monensin treatment 

revealed that increased internalization of SR-BI leads to decreased CE selective uptake. These 

data supports a model where SR-BI-mediated HDL CE selective uptake occurs exclusively at 

the plasma membrane and CE is separated from HDL by SR-BI and subsequently transferred 

into an SR-BI independent microdomain. Downloaded from 

P34 



P36 

Positive Association between Plaque Regression Determined by 

Trans-Esophageal Magnetic Resonance Imaging and Low-Density 

Lipoprotein Reduction after 12 months of Simvastatin Therapy in Patients 

with Documented Atherosclerosis 

Sachin Agarwal, Sandeep Gautam, Milind Desai, Henning Steen, William P Warren, Joao A 

Lima; Johns Hopkins Hosp, Baltimore, MD 

Background: Clinical trials have shown the efficacy of statin therapy in promoting plaque 

regression. However, the exact mechanism of this effect remains incompletely understood. 

Low-density lipoprotein (LDL) lowering, high-density lipoprotein (HDL) increase and antiinflammatory 

mechanisms have been postulated. Our hypothesis is that the relationship 

between LDL reduction and plaque regression is strong and directly proportional in nature. 

Methods: Twenty- seven patients with at least moderate atherosclerosis in / 1 vascular 

bed were examined by MRI at baseline and then after 12 months of simvastatin therapy (20 

mg-80 mg) to monitor aortic plaque. Changes in LDL (baseline to 12 months) were categorized 

into no reduction or an increase i.e. 0(n6), reduction 0 and 31 mg/dl (n8) and 

reduction 31 mg/dl (n13), given 31mg/dl is 50th percentile value of LDL change. Paired 

t-test and multiple linear regression with generalized estimating equation was used to test the 

hypothesis. Results: There was a 23 % or 28.4 mg/dl (p0.0001) reduction in LDL, 17 % or 

559 mm 3 reduction in plaque volume (p0.008) and 18 % or 29.4 mm 2 reduction in plaque 

area (p0.005) from baseline to 12 months. Changes were found to be non-significant for HDL 

and triglycerides levels. Compared to LDL reduction 0 group, plaque volume reduction was 

1005.73 mm 3 (p0.049) greater in the 31 mg/dl LDL reduction group, and 660.73 mm 3 (p 

NS) greater in the reduction 0 and 31 mg/dl group. Similarly, compared to LDL 

reduction 0 group, plaque area reduction was 46.9 mm 2 (p0.05) greater in the 31 mg/dl 

group and 35.5 mm 2 (p NS) greater in the reduction 0 and 31 mg/dl group. Univariate 

analysis and then after adjusting for confounders including age, baseline statin, body mass 

index, HDL levels and triglyceride levels , there was a reduction of 45.2 mm 3 (p0.013) in 

plaque volume and 2.2 mm 2 (p0.010) in plaque area with each mg/dl reduction in LDL. 

Conclusion: LDL, plaque volume and area reduced significantly after 12 months of simvastatin 

therapy. The amount of regression in plaque was found to be directly proportional to the 

amount of LDL reduction.There is a strong relationship between plaque volume and area 

reduction with LDL lowering. 

Polymorphisms in ABCA1 Gene Affect Plasma HDL Cholesterol 

Concentration 

Usman Ahmad, Danish Saleheen, Philippe M Frossard; The Aga Khan Univ, Karachi, 

Pakistan 

Introduction: Plasma levels of HDL cholesterol are an independent predictor of coronary artery 

disease with low HDL being a risk factor for early and more severe CAD. The relationship holds 

true even for HDL levels within the desirable range. The South Asian population has been 

shown to have a higher risk of CAD. It has also been seen that south Asians have inherently 

low HDL levels even in the absence of any overt disease. Family and twin studies have shown 

that about 50% of the variation in plasma HDL level is genetically determined. Genetic 

mutations in ATP-Binding Cassette Transporter 1 gene (ABCA1) were found to cause severe 

HDL deficiency in Tangier disease. We checked the association of four SNPs in ABCA1 with HDL 

levels in normal Pakistani subjects. Methods: This study was conducted on normal healthy 

adults of Pakistani origin. The selected subjects were free of all cardiovascular diseases and 

comorbids. Informed consent was obtained and 10ml venous blood was collected from all 

subjects after 8 hours of fasting. Total cholesterol (TC), serum triglyceride (TG) and HDL-C 

concentrations were measured in all plasma samples using enzyme assays The Friedewald’s 

method was used to estimate LDL-C levels. DNA was extracted from whole blood and 

genotyping was performed by PCR-RFLP. Results: Two hundred and fifty subjects (160 males, 

90 females), average age 53.7 years were included. Mean TC level was 188.2 mg/dl, mean 

HDL-C level was 41.2 mg/dl and mean TG level was 171.5 mg/dl. The four SNPs screened in 

these subjects were G1051A, T1591C, A2715C and G2868A. The A2715C genotypes were 

found to confer significantly different levels of HDL-C (p 0.007). Subsequent analysis, with 

age, gender, TC and TG level adjustment did not affect the significance of difference in the 

HDL-C levels in A2715C genotypes. The other polymorphisms did affect plasma HDL-C levels 

but the difference was not found to be significant. Conclusion:Single nucleotide polymorphisms 

in ABCA1 gene can affect the plasma cholesterol level. The A2715C genotypes are associated 

with varying plasma HDL-C levels, with the A allele being associated with higher HDL-C levels, 

in the Pakistani population. 

Plasma Osteopontin Levels are Associated with Vascular Inflammation 

Hironobu Akao, Michihiko Kitayama, Akihiro Fukuda, Hiroaki Uenishi, Masamitsu Asano, 

Ryoko Sato, Atsushi Motoyama, Shinji Okubo, Hiroichi Tsugawa, Kouji Kajinami; Kanazawa 

Med Univ, Uchinada, Japan 

Osteopontin (OPN) contributes to smooth muscle cell proliferation as well as vascular 

calcification in atherosclerotic lesions. Coronary rotational atherectomy (RA) is the procedure 

ablating advanced atherosclerotic lesions. To investigate the hypothesis that RA may release 

OPN from vessel wall into blood, we measured peripheral levels of OPN before and after RA, 

and investigate the determinant of their levels with special reference with inflammatory 

markers. We enrolled consecutive 35 patients (mean age: 70 years, M/F23/12) treated with 

RA. Plasma OPN levels (meanSD, ng/ml) significantly (p0.001) increased immediately after 

RA (from 623244 to 777302), further increased to 1089466 three hours later, and 

returned to 714329 at the time of 24 hours after procedure. None of the extent of coronary 

calcification, lesion length, total ablation time, and maximum burr size was significantly 

associated by with guest the magnitude on June of 29, OPN2013 increase. In pre-procedural state, OPN levels showed 

P37 

P38

highly-significant and positive associations with the values of IL-6 (r0.715, p0.0001) and 

of log-transformed hs-CRP (r0.544, p0.0009). Increase of OPN after RA showed 

highly-significant and positive associations with pre-procedural IL-6 levels (r0.591, 

p0.0002) and log-transformed hs-CRP levels (r0.565, p0.0005). In conclusion, plasma 

OPN levels, before and after RA, are associated with pre-procedural inflammatory marker 

levels, which suggests the causal relationship between vascular inflammation and OPN 

expression in atherosclerotic lesions. 

Interleukin-1 Regulation of CCAAT Enhancer Binding Protein Gene 

Transcription 

Saira Ali, BSc; Cardiff Univ, Cardiff, United Kingdom 

Atherosclerosis can be considered to be a chronic form of inflammation not only associated 

with dramatic changes in the function of vascular cells but also macrophages and hepatocytes. 

Inflammation in the liver leads to increased expression and secretion of acute phase proteins 

(e.g. C-reactive protein) which are considered to be risk factors for cardiovascular disease. The 

expression of acute phase protein genes is triggered in response to inflammatory mediators, 

particularly cytokines such as interleukin (IL)-1. The transcription factor CCAAT Enhancer 

Binding Protein (C/EBP) is critical in regulating the expression of key genes in both 

macrophages and hepatocytes (including the acute phase response genes) implicated in 

normal cellular function and the inflammatory response. We have previously demonstrated that 

the action of several cytokines, including IL-1 activates the expression/activity of C/EBP. 

However, the molecular mechanisms underlying the activation of C/EBP gene transcription as 

mediated by IL-1 remain poorly understood. Therefore for this study, we employed semiquantitative 

RT-PCR and western blot analysis to determine the effects of pharmacological 

inhibitors, designed against specific components of known signalling pathways on IL-1 

mediated C/EBP expression. The experimental evidence generated by these investigations 

suggests a potentially novel role of the SAPK/JNK MAPK pathway in the regulation of IL-1 

mediated C/EBP expression. Pre-treatment of human hepatoma, Hep3B cells with curcumin 

and SP600125 (pharmacological inhibitors of SAPK/JNK pathway) attenuated IL-1 mediated 

C/EBP mRNA and protein expression. We used a phospho-SAPK/JNK specific antibody to show 

that the dual phosphorylation (threonine 183/tyrosine 185) and therefore activation of p54 

SAPK/JNK occurs in Hep3B cells in response to IL-1, maximally between 15–30 minutes 

following treatment. Furthermore, the IL-1 mediated p54 SAPK/JNK dual phosphorylation was 

impaired when cells were pre-treated with curcumin and SP600125. The role of this pathway 

is currently being investigated in detail using dominant negative constructs and RNAi. These 

studies will provide a novel insight into IL-1 mediated regulation of C/EBP gene expression. 

COX-1 Deficiency in Bone Marrow-Derived Cells Increases Early 

Atherosclerosis in ApoE and LDL-Receptor Null Mice 

Vladimir R Babaev, Lei Ding, Youmin Zhang, Jason D Morrow, Matthew D Breyer, Sergio 

Fazio, MacRae F Linton; Vanderbilt Univ, Nashville, TN 

Cyclooxygenase-1 (COX-1) has been implicated in promoting atherothrombosis. The beneficial 

effects of aspirin in reducing cardoivascular events is largely attributed to inhibition of 

COX-1-mediated platelet thromboxane (Tx). Inhibition of COX-1 has been reported to decrease 

atherosclerosis in apoE -/- mice, and platelet Tx has been proposed to promote atherogenesis. 

To study the role of COX-1 expression from bone marrow-derived cells in atherosclerotic leson 

formation, 7-week-old male LDLR -/- mice were lethaly irradiated, transplanted with male 

COX-1 / (n14) or COX-1 -/- (n17) bone marrow and, six weeks later, fed a Western diet for 

8 weeks. There were no significant differences between the groups of recipient mice in body 

weight, lipoprotein distributions, or serum lipid levels. Mice transplanted with COX-1 -/- had 

completely suppressed levels of platelet (0.30.1 vs. 139.415.8 ng/ml; p0.001) and 

urinary TxB 2. Compared to COX-1 / 3LDLR -/- mice, COX-1 -/- 3LDLR -/- recipients had 

increased atherosclerotic lesions in the proximal aorta (12419627631 vs. 615661610 

m 2 ; p0.03). In a separate set of experiments, 8-week-old female apoE -/- mice were lethaly 

irradiated, transplanted with COX-1 / /apoE -/- (n13) or COX-1 -/- /apoE -/- (n13) marrow and 

fed a normal chow diet for 9 weeks. Again, there were no significant differences between the 

recipient mice in lipoprotein distributions, or serum lipid levels. Hovewer, the extent of 

atherosclerotic lesions in COX-1 -/- /apoE -/- 3apoE -/- mice was significantly increased in the 

proximal aorta (394633090 vs. 283522791 m 2 ; p0.02) and distal aorta (0.200.01 vs. 

0.180.01%; p0.05) compared to apoE -/- 3apoE -/- mice. In addition, macrophages isolated 

from COX-1 -/- /apoE -/- 3apoE -/- mice have decreased levels of TxB 2 released by calcium 

ionophore, but increased basal and LPS-mediated levels of COX-2 expression. These data 

demonstrate that the elimination of COX-1 bone marrow-derived cells (e.g. platelets and 

macrophages) worsens early athersclerosis in apoE -/- and LDLR -/- mice. The data also suggest 

that platelet Tx does not appear to contribute to early lesion formation and up-regulation of 

COX-2 in the COX-1-/- macrophages may play a significant role in promoting early 

atherogenesis. 

P41 

Effectiveness of Multiple Interventions on Carotid Artery Atherosclerosis in 

a Clinical Setting 

Bradley F Bale, Amy L Doneen, Heart Attack Prevention Clinic, Spokane, WA; Thomas F 

Smith; The Austin Diagnostic Clinic, Austin, TX 

Interventions that lower LDL and raise HDL have been shown to slow the rate of progression 

of atherosclerosis. Regression of atherosclerosis has been demonstrated with statins. However, 

this did not occur in many patients in the treatment groups. We determined the effect of 

aggressive use of multiple evidence-based interventions in a clinical setting over one year in 

54 consecutive patients with known hyperlipidemia and at least moderate Framingham Risk 

Score. Mean age was 548 years. All had beenDownloaded prescribed medicine from 

for their hyperlipidemia. 

P39 

P40 




Carotid arterial intima-media thickness (CIMT) and atheromas were assessed using highresolution 

B-mode ultrasound. Lifestyle modifications based on ApoE genotype, including diet 

and exercise, were recommended to all patients. Forty-eight patients were treated with a statin 

and six also were treated with ezetimide. Patients with low HDL, sub-particle problems, or high 

triglycerides were prescribed a fibrate (18 patients) or niacin (39 patients). Forty-three patients 

were treated with an ACE inhibitor or an ARB. Six patients were treated with a beta-blocker and 

six were treated with a calcium channel blocker. Twenty-two patients with diabetes or insulin 

resistance were prescribed a thiazolidinedione. Adherence to individualized treatment was 

encouraged by regular follow-up every 3 to 6 months. After one year, LDL decreased from a 

mean of 11237 mg/dL to 8827 mg/dL. HDL was 5919 mg/dL initially and did not change 

significantly. TC/HDL ratio decreased from 3.34 to 2.41. Triglycerides decreased from 13311 

mg/dL to 10058 mg/dL. CIMT decreased from a mean of 0.7700.104 mm to 0.6990.096 

mm (p0.0001). All but four had a CIMT decrease of 0.010 mm. Initially, 47 patients had 

one or more plaques. Of these, 39 had soft plaque. One year later, only 3 patients had soft 

plaque. Fourteen of fifteen patients with a single soft plaque moved to no soft plaque, and 

22/24 patients with multiple soft plaques moved to no soft plaque. We conclude that aggressive 

use of multiple interventions in an office setting reverses early, preintrusive atherosclerosis of 

the carotid artery. This demonstrates the need for formal studies of the effect of multiple 

interventions on consequences of atherosclerotic artery disease. 

P42 

Inhibition of Hepatic ACAT2 by Antisense Oligonucleotides Reduces 

Atherogenic Lipoprotein Concentrations in ApoB100 Only/LDL Receptor Null 

Mice 

Thomas A Bell, III, Wake Forest Univ Sch of Medicine, Winston-Salem, NC; Mark Graham, 

Kristina Lemonidis, ISIS Pharmaceuticals, Carlsbad, CA; Matthew Davis, Janet Sawyer, 

Ramesh Shah, Wake Forest Univ Sch of Medicine, Winston-Salem, NC; Rosanne Crooke, 

ISIS Pharmaceuticals, Carlsbad, CA; Lawrence Rudel; Wake Forest Univ Sch of Medicine, 

Winston-Salem, NC 

ACAT2 or acylCoA: cholesterol 0-acyltransferase 2 is the enzyme responsible for the formation 

of cholesteryl esters that are available for incorporation into nascent lipoproteins secreted by 

the small intestine and liver. Recent studies examining the role of ACAT2 in atherosclerosis 

have found that mice lacking a functional ACAT2 were protected from significant lesion 

formation. To elucidate the relationship between ACAT2 and atherosclerosis, antisense 

oligonucleotides (ASOs) have been developed by ISIS Pharmaceuticals that specifically reduce 

murine hepatic ACAT2 through an RNaseH-mediated degradation process. To test the 

effectiveness of the ACAT2 ASOs, apoB100 only/LDL receptor null mice were injected biweekly 

with three ASOs (two different ACAT2-specific and the third an ACAT2-mismatch control ASO) 

at a dosage of 25 mg/kg; a saline control group was also studied. Throughout the treatment 

period mice were fed a diet consisting of 20% of calories from saturated fat and 0.2% 

cholesterol by weight. After 8 weeks of biweekly injections, the mice treated with the 

ACAT2-specific ASOs showed at least a 40% reduction in plasma cholesterol when compared 

to mice injected with saline or the control ASO. Plasma cholesterol values for mice given the 

ACAT2-specific ASO were not significantly different from those in ACAT2 knockout mice of the 

same genotype. A 2-week pilot study demonstrated that differences in plasma cholesterol in 

the mice given the ACAT2-specific ASOs were due to a reduction in LDL cholesterol associated 

with significant reductions in hepatic ACAT2 message and protein. These studies provide early 

evidence that antisense oligonucleotides that target hepatic ACAT2 can effectively suppress the 

ability of the enzyme to generate apoB-lipoprotein cholesteryl ester and ultimately lower 

plasma cholesterol levels. The contribution of ACAT2 to the progression of atherosclerotic 

disease can now be directly assessed. 

Selective Inihibition of MMP-3 and MMP-9 by an Amino-Sulphone- 

Hydroxamate Derivative 

Rachele Parente, Univ of Milan, Milan, Italy; Gabriele Candiani, INSERM Unite 99, Creteil, 

France; Monica Canavesi, Univ of Milan, Milan, Italy; Francoise Pecker, INSERM Unite 99, 

Creteil, France; Monica Sani, Matteo Zanda, C.N.R., Milan, Italy; Stefano Bellosta; Univ of 

Milan, Milan, Italy 

Excessive breakdown of extracellular matrix (ECM) by metalloproteinases (MMPs) occurs in 

many pathological conditions, such as cancer and atherosclerosis, and thus inhibition of MMPs 

activity might have therapeutic potential. Numerous broad-spectrum MMPs inhibitors caused 

drug-related adverse events due to their lack of selectivity. In the search for a more selective 

inhibitor we have synthesized a new compound named MS560 (alpha-trifluoromethyl-alphaamino-beta-sulphone 

hydroxamate). The introduction of the trifluoromethyl group was expected 

to be an effective strategy for changing and tuning the binding properties of the 

hydroxamate to MMPs’ catalytic site. We then tested MS560 capacity to interfere with MMPs’ 

expression and activity. The effect on MMP-2 and MMP-9 secretion and gelatinolytic activity 

was evaluated by gelatin gel zymography using cell-conditioned media by smooth muscle cells 

(SMCs) and macrophages, respectively. The activity on MMP-1 and MMP-3 was measured 

using the purified enzyme and a collagen zymography or a fluorescent assay, respectively. The 

incubation of macrophages or SMCs with increasing concentrations of the compound (from 

0.05 up to 25 M) affected the secretion of MMP-9 (up to 45% inhibition) in macrophages 

while it did not have any effect on MMP-2 in SMCs, in the absence of any effect on cellular 

viability. MS560 was able to inhibit directly the gelatinolytic activity of pro-MMP-2 and 

pro-MMP-9 (up to 90% and 92%, respectively, p0.01) released into the conditioned medium 

(IC50: MMP-2 970 nM; and MMP-9 87 nM). Interestingly, MS560 was more effective in 

inhibiting MMP-3 activity (IC50 MMP-3 14 nM), while it inhibited MMP-1 activity only at 

concentrations higher than 5 M. In conclusion, our results show that MS560 inhibits 

specifically MMP-3 and -9 activity at nanomolar concentration highlighting the potential 

beneficial effect of this type of compounds in the therapeutic control of excessive ECM 

breakdown. by guest on June 29, 2013 

P43

E-60 Vol 25, No 5 May 2005 

Early Chronic Renal Disease and Subclinical Atherosclerosis in 

Asymptomatic Subjects 

Oscar Beloqui, Dept of Internal Medicine, Univ Clinic, Pamplona, Spain; Inmaculada Colina, 

Félix Alegre, Dept of Internal Medicine, Univ Clinic, Pamplona, Spain; José APáramo, 

Josune Orbe, Arantxa González, Javier Díez; Cntr for Applied Med Rsch, Univ of Navarra, 

Pamplona, Spain 

P44 

Advanced renal disease is an independent risk factor for cardiovascular disease. In apparently 

healthy subjects we have investigated if early stages of chronic renal disease (E-CKD) are 

associated with markers of subclinical atherosclerosis. In the course of a general health 

check-up and vascular risk assesment we studied 894 consecutive subjects (710 men, mean 

age 55 years, range 20 to 81 years) with negative history of cardiovascular disease. E-CKD was 

considered as the stages 1 and 2 of the K/DOQI CKD classification. Carotid intima-media 

thickness (C-IMT) and the presence of atheromas were determined by ultrasonography and the 

Coronary Artery Calcium Content (CAC) by CT. Results: The prevalence of E-CKD was 16.2% 

(stage 1 in 6,7%, 60 of 894, and stage 2 in 9,5%, 85 of 894). E-CKD was more prevalent in 

males (17,7%, 126 of 710) than in females (10,3%, 19 of 184), (p0.015). The prevalence and 

severity (stages 1 or 2) of E-CKD increased with age (p for trend 0.000 in males and 0.025 in 

females) and in subjects with hypertension (p for trend 0.000), diabetes (p for trend 0.000), 

obesity (p for trend 0.003) and metabolic syndrome (p for trend 0.000), but not in smokers or 

in subjects with hypercholesterolemia. After adjustment for all factors, hypertension and 

diabetes remained associated with E-CKD, with ORs of 3,02 (95% CI, 1.92 to 4.74) and 2.69 

(95% CI, 1.65 to 4.38), respectively. Both C-IMT and the prevalence of carotid atheromatosis 

progressively increased as renal function deteriorates (p for trend 0.000 in both cases). After 

full adjustment, E-CKD remained significantly associated with a pathological C-IMT (0.75 

mm), with an OR of 1,78 (95% CI, 1.16 to 2.74) and with carotid atheromatosis, with an OR 

of 1.62 (95% CI, 1.00 to 2.60). Finally, CAC in 33 patients with E-CKD (217 79 Agatston 

Units) was significantly higher than in 156 subjects with normal kidney function (120 31 

Agatston Units, p0.000); after full adjustment, E-CKD remained significantly associated with 

high CAC ( 100 Agatston units), with an OR of 5,93 (95% CI 1,97–17,83). Conclusions: 

E-CKD is remarkably prevalent in asymptomatic subjects and behaves as an independent risk 

factor for the development of subclinical atherosclerosis. 

Enhanced Cardioprotective Efficacy of Combining Sildenafil with Oral 

Low-Dose Atorvastatin against Ischemia-Reperfusion Injury 

Yochai Birnbaum, Salvatore Rosanio, Yumei Ye, Atiar M Rahman, Sheldon Y Freeberg, 

Ming-He Huang, Barry F Uretsky; Univ of Texas Med Branch, Galveston, TX 

Background: Atorvastatin (ATV) and sildenafil (SL) each reduce myocardial infarct size (IS) 

through augmenting nitric oxide synthase (NOS) expression. The oral doses of ATV needed to 

exert maximal cardiac protection are high and may enhance SL-induced hypotension. It is 

unknown whether ATV and SL have synergistic effects in reducing IS and enhancing NOS 

expression without causing hypotension. Methods and Results: Rats were randomized before 

30 min ischemia-4 hr reperfusion to pre-treatment with high dose ATV (10 mg/kg (ATV-10)); 

low-dose ATV (1 mg/kg (ATV-1)); SL 1 mg/kg (SL-1); SL 0.7 mg/kg (SL-0.7); ATV-1SL-0.7; 

and controls. ATV was given orally once daily for 3 days and SL IP 18hr before coronary 

occlusion. IS (triphenyltetrazolium chloride staining, % risk area, meanSEM) was smaller in 

the ATV-10 (131%), SL-1 (112%), SL-0.7 (182%) and ATV-1SL-0.7 (91%) groups as 

compared with controls (343%; P0.001 for each comparison), whereas ATV-1 had no 

cardioprotective effects (292%). ATV-1SL-0.7 reduced IS more than SL-0.7 alone 

(p0.012). SL-1 significantly lowered mean blood pressure (MBP) as compared to controls 

whereas MBP in the ATV-10, SL-0.7 and ATV-1SL-0.7 groups were comparable to controls. 

SL-0.7 increased phosphorylated endothelial and inducible NOS expression (2132% and 

1511%, respectively), whereas ATV-1 did not. These changes were significantly enhanced by 

ATV-1SL-0.7 (P-eNOS, 2562%, iNOS 1951%). SL-1 resulted in marked augmentation of 

P-eNOS (3452%), and a similar increase in iNOS (1861%). Conclusions: Adding low-dose 

ATV to SL potentates the IS limiting effects through enhanced P-eNOS and iNOS expression. 

Although IS reduction is similar to SL at 1 mg/kg, combining therapy with low-dose ATV and 

SL 0.7mg/kg causes no profound effects on MBP. 

P46 

Sphingosine-1-Phosphate Prevents Monocyte: Endothelial Interactions in 

Type 1 Diabetic NOD/Bdc Mice 

David T Bolick, Suseela Srinivasan, Angela Whetzel, Marcia McDuffie, Catherine C Hedrick; 

Univ of Virginia, Charlottesville, VA 

Atherosclerosis and vascular disease are serious complications of Type 1 diabetes. Monocyte 

recruitment and adhesion to activated vascular endothelium in the vessel wall are key early 

events in atherosclerosis and vascular inflammation. In the present study, we examined the 

effect of sphingosine-1-phosphate (S1P) on modulating monocyte:endothelial interactions in 

the NOD/Bdc (NOD) mouse model of Type 1 diabetes. We isolated whole aortas from 

nondiabetic and diabetic NOD mice. Aortas were immediately placed into media and incubated 

for 4h at 37C in the absence or presence of 100nM S1P. Fluorescently-labeled monocytes were 

added to aortas for a monocyte adhesion assay. Unbound monocytes were rinsed, and bound 

monocytes were counted using a fluorescent microscope. We found that whole aortas from 

NOD diabetic mice bound 8-fold more monocytes than non-diabetic littermate mice (61 

monocytes/bound/field for non-diabetic mice versus 648 monocytes bound/field for diabetic 

mice, p0.0001). Incubation of diabetic aortas with 100nM S1P completely blocked monocyte 

adhesion. We confirmed these findings using in vitro cultures of freshly isolated aortic 

endothelial cells (EC) from non-diabetic and diabetic NOD mice. In an in vitro static monocyte 

adhesion assay, we found that diabetic NOD EC bound 3-fold more monocytes than 

non-diabetic EC (9 monos/field for non-diabetic Downloaded EC vs. 273 monos/field from 

for diabetic EC, 

P45 



p0.001). S1P (100nM for 4h) significantly reduced monocyte adhesion to diabetic NOD EC by 

90%. We found expression of S1P1, S1P2, and S1P3 receptors on NOD aortic EC, although the 

levels of expression were modulated in the setting of Type 1 diabetes. We also found that 

expression of VCAM-1, ICAM-1, and E-selectin was significantly increased in diabetic NOD EC 

compared to non-diabetic EC. Incubation of EC with 100nM S1P for 4h significantly reduced 

expression of all 3 adhesion molecules to levels observed for non-diabetic control EC. 

P-selectin was not changed by S1P. Thus, S1P functions in an anti-inflammatory manner in 

Type 1 diabetic vascular endothelium to prevent monocyte:EC interactions. Further understanding 

of the mechanism of action of S1P in diabetic endothelium could be important for 

prevention of vascular complications of Type 1 diabetes. 

P47 

12/15 Lipoxygenase Regulates ICAM-1 Expression and Monocyte Adhesion 

to Endothelium through Activation of RhoA and PKC 

David T Bolick, A. W Orr, Suseela Srinivasan, Angela Whetzel, Martin A Schwartz, Catherine 

C Hedrick; Univ of Virginia, Charlottesville, VA 

12/15-lipoxygenase (12/15 LO) activity leads to production of the pro-inflammatory eicosanoids 

12-S-hydroxyeicosatetraenoic acid (12SHETE) and 13-S-hydroxyoctadecadienoic acid 

(13SHODE). We have previously shown a 4-fold increase in endothelial ICAM-1 expression in 

mice overexpressing the 12/15LO gene. In the current study, we found that regulation of 

ICAM-1 expression on endothelium by 12/15LO is mediated by co-activation of PKC and the 

small GTPase rhoA. RhoA activation in EC by 100nM 12SHETE results in increased stress fiber 

formation with alignment of ICAM-1 along the actin fibrils. Addition of 100nM 12SHETE to EC 

also directly activated PKC and RhoA. RhoA activation by 12SHETE in EC causes translocation 

of the p65 subunit of NFkB to the nucleus, which induces ICAM-1 gene transcription. Inhibition 

of rhoA in EC using either C3 toxin or the rho kinase inhibitor Y27632 blocked both 

12SHETE-mediated ICAM-1 induction and monocyte adhesion to endothelium. Inhibition of 

PKC in EC using either Go6976 or an siRNA against PKC blocked 12SHETE-mediated rhoA 

activation, and prevented the increase in both ICAM-1 expression and monocyte adhesion to 

endothelium. Thus, the 12/15-LO pathway product 12SHETE stimulates multiple signaling 

events in EC to mediate vascular inflammation and monocyte:endothelial interactions. 

P48 

Retroviral Vector-Mediated Overexpression of Group V Secretory 

Phospholipase A2 in Hematopoietic Cells Promotes Atherosclerosis in Low 

Density Lipoprotein Receptor Deficient Mice 

Meredith Bostrom, Kathy Forrest, Boris Boyanovsky, Alan Daugherty, Nancy R Webb; Univ of 

Kentucky, Lexington, KY 

Previous studies in our laboratory have shown that Group V secretory phospholipase A 2 (GV 

sPLA 2) hydrolyzes LDL in vitro, resulting in smaller particles that are susceptible to aggregation. 

These hydrolyzed LDL particles are readily taken up by macrophages to form foam cells. We 

have shown by immunostaining that GV sPLA 2 is present in both human and mouse 

atherosclerotic lesions. Based on these findings, we have hypothesized that LDL trapped in the 

vascular subendothelium is hydrolyzed by locally secreted GV sPLA 2 to form particles that are 

taken up by macrophages, and promoting atherosclerotic lesion formation. To test this 

hypothesis in vivo, GV sPLA 2 was overexpressed in hematopoietic cells of LDL receptordeficient 

mice using retrovirus-mediated gene transfer. Green fluorescent protein (GFP) was 

co-expressed by the retroviral vector to provide a marker for retrovirus transduction. Five 

weeks after repopulation with bone marrow cells transduced with GV sPLA 2 and GFP or GFP 

only, mice were placed on a high fat diet for 16 weeks to induce atherosclerosis. Animals 

overexpressing GV sPLA 2 in hematopoietic cells had significantly increased lesion area in the 

aortic sinus compared to control animals (1.5 0.2 mm 2 and 1.0 0.1 mm 2 respectively, 

p0.05). Using immunohistochemistry, GV sPLA 2 was detected in lesional macrophages 

co-localized with GFP, indicating that the retroviral-encoded genes were expressed. Unexpectedly, 

plasma total cholesterol concentrations after high fat diet feeding were significantly higher 

in mice over-expressing GV sPLA 2 compared to control mice (1100 40 mg/dL and 960 

45 mg/dL respectively, p0.05). These data indicate that GV sPLA 2 overexpression in 

hematopoietic cells promotes atherogenesis, either through a localized mechanism in the 

vascular subendothelium, or by altering systemic lipoprotein metabolism. 

Myeloperoxidase is Associated with Macrovascular Disease in Type 2 

Diabetes 

Marie-Luise Brennan, Cleveland Clinic, Cleveland, OH; Aramesh Saremi, Carl T. Hayden 

VAMC, Phoenix, AZ; Jerome Sacks, Hines VAMC, Hines, IL; Dawn Schwenke, Carl T. Hayden 

VAMC, Phoenix, AZ; Stanley L Hazen, Cleveland Clinic, Cleveland, OH; Peter D Reaven; Carl 

T. Hayden VAMC, Phoenix, AZ 

There is increasing evidence that inflammatory markers such as myeloperoxidase (MPO) may 

be related to the development of atherosclerosis and onset of clinical events. We therefore 

evaluated the relationship of MPO measured on a sub-sample of Type 2 diabetes subjects as 

they enrolled into the VA Diabetes Trial (VADT) to assess their association with baseline 

atherosclerosis (as measured by coronary artery calcium (CAC)) and prevalence of cardiovascular 

disease (CVD)events, i.e,myocardial infarction,stroke and revascularization. A total of 286 

diabetic individuals (269 male/ 17 female) aged 40 years or older were included in this analysis. 

After adjustment for age, MPO was negatively correlated with total cholesterol (r -0.11, P 

0.05), and positively with CRP (r0.14, p0.02), but not associated with other cardiovascular 

risk factors, including CAC. In contrast, MPO levels were significantly (p 0.01) higher among 

subjects with CVD (482 pmol/l vs. 332 pmol/l) and the prevalence of CVD increased across 

tertiles of MPO levels (23%, 36%, and 42%), and after controlling for age the trend remained 

statistically by significant guest on (p-trend June 29, 0.01). 2013 Furthermore, in a logistic regression model adjusted 

P49

for age, MPO was significantly associated with CVD (OR: 1.35, CI: 1.04 –1.76), however, this 

did not remain significant after adjusting for CAC. To determine whether the association of MPO 

with CVD may depend on the extent of underlying vascular disease as has been suggested, we 

estimated the odd ratios for CVD stratified by low and high levels of CAC (dichotomized around 

the median). MPO was not associated with CVD among those with low CAC, but was 

significantly (p0.03) associated with CVD (OR: 1.47, 1.03–2.11) in those with high CAC levels. 

Moreover, after additional adjustment for other relevant CVD risk factors, the odds ratio for MPO 

did not change 1.46 (0.99 –2.14), and MPO remained significantly (P0.05) associated with 

CVD in this relatively high risk group. These data indicate that MPO levels in type 2 diabetes 

are associated with prevalence of CVD, and that this risk factor may be particularly relevant in 

those with greater amounts of atherosclerosis. 

Expression and Role of Interleukin-15 in Intimal Thickening 

Miha Cercek, Michiaki Matsumoto, Kuang-Yuh Chyu, Prediman K Shah, Bojan Cercek, Paul 

C Dimayuga; Cedars-Sinai Med Cntr, Los Angeles, CA 

Background: While interleukin-15 (IL-15) is expressed in atherosclerotic plaques, its effect on 

atherogenesis is not known. We investigated the role of IL-15 in intimal thickening after arterial 

injury. Methods: IL-15 and IL-15 receptor chain (IL-15R) mRNA expression were 

determined by RT-PCR of RNA from pooled injured carotid arteries of C57BL/6 mice before and 

21 days after carotid arterial cuff injury. IL-15 and IL-15R protein were detected by 

immunostaining on sections from injured arteries. The effect of IL-15 on intimal thickening in 

vivo was tested with anti-IL-15 specific antibody treatment (IL-15 Ab), 100g i.v./wk, for three 

weeks (n6). Injured carotid arteries from non-treated mice (n6) served as controls. In vitro, 

the effect of IL-15 on SMC proliferation was measured by cell cycle analysis of serumstimulated 

cells treated with recombinant mouse IL-15 (rmIL-15). IL-15 and IL-15R 

expression relative to -actin were determined by RT-PCR. Fractalkine receptor CX3CR1 

expression was studied as a likely pathway of IL-15 effect on SMC. Results: Injury did not 

affect IL-15 expression but resulted in 2-fold increase in IL-15R expression in the carotid 

arteries (Table). IL-15 protein expression was increased at the site of arterial injury, paralleled 

by increased IL-15R protein expression. Specific inhibition of IL-15 function by IL-15 Ab 

increased intimal thickening in vivo (Table). Serum stimulation of SMC increased IL-15R 

mRNA expression in vitro. Treatment with rmIL-15 attenuated serum-stimulated SMC 

proliferation and down regulated CX3CR1 expression. Conclusion: We provide evidence that 

IL-15 attenuates intimal thickening after arterial injury by inhibiting SMC proliferation. The 

potential mechanism of action is suppression of growth promoting CX3CR1 in SMC. Table: 

Results. 

Arterial mRNA † Uninjured 21 days after injury 

IL-15 0.28 0.28 

IL-15R 0.56 0.98 

Intimal area [mm2 ] Control IL-15 Ab 

0.010 0.004 0.021 0.006 * 

SMC mRNA in vitro Control Serum 

IL-15 0.05 0.04 0.16 0.13 

IL-15R 0.25 0.17 0.61 0.13 * 

In vitro cells in S phase 

[%] 

Control Serum SerumrmIL-15 

4.0 2.4 24.0 1.9 19.2 0.6 # 

† pooled samples (n4); * p0.05; # p0.01 vs. serum; mRNA values expressed as densitometric units relative to -actin 

P51 

Impact of Ace Polymorphism on Structural and Functional Arterial Changes 

in Patients with First Clinical Manifestation of Coronary Artery Disease 

Andreja Cerne, Igor Kranjec, Borut Peterlin; Med Cntr Ljubljana, Ljubljana, Slovenia 

Objective. Angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism was 

recently associated with increased risk for coronary artery disease (CAD), however the 

mechanism of this association remains unknown. Higher level of circulating ACE activity, 

described in DD genotype carriers, may well predispose to vascular wall thickening and 

increased vascular tone. Aims. 1) to access ACE I/D polymorphism in patients (pts) with new 

onset CAD, and 2) to investigate whether ACE I/D polymorphism modulates structural and 

functional arterial changes in these pts. Methods. One hundred consecutive pts (unstable AP 

in 47 pts, acute MI in 53 pts) with angiographically-proven obstructive CAD (50% stenosis) 

and 80 age- and gender-matched healthy subjects underwent ACE I/D genotyping. The carotid 

intima-media thickness (IMT) and brachial endothelium-dependent (flow-mediated) as well as 

-independent (nitroglycerin-induced) dilation were measured by echo Doppler technique. 

Results. The ACE DD occurred more frequently in CAD pts than in controls (37% vs. 21%, 

p0.05). By multivariate regression analysis accounting for traditional CAD predictors, the ACE 

DD genotype conferred a 2.4-fold increased risk for CAD (95% CI 1.1–6.6, p0.05). IN CAD 

pts, the ACE DD genotype was associated with significantly increased carotid IMT (DD vs. II; 

0.940.20mm vs. 0.790.20 mm; p0.05) and impaired endothelial-dependent response 

(DD vs. II; 5.94.2% vs. 8.72.9%, p0.05). Endothelium-independent dilation was 

unaffected by the ACE I/D genotypes (pns). Conclusion. ACE genotype polymorphism 

emerged as an independent risk factor for CAD in our population. A positive association 

between ACE DD genotype and early structural and functional arterial changes was detected, 

suggesting a possible pathogenetic link between ACE I/D polymorphism and development of 

CAD. 


P50 

Antioxidants Inhibit the Ability of Lysophosphatidylcholine to Regulate 

Synthesis of the Proteoglycan Form of Macrophage Colony Stimulating 

Factor (PG-MCSF) 

Mary Y Chang, Chang-Yeop Han, Univ of Washington, Seattle, WA; Thomas Wight, Hope 

Heart / Benaroya Rsch Institute, Seattle, WA; Alan Chait; Univ of Washington, Seattle, WA 

We have shown that lysophosphatidylcholine (lysoPC) mimics the ability of oxidized LDL to 

regulate multiple aspects of proteoglycan synthesis by smooth muscle cells (SMC) that impact 

on lipoprotein retention within the arterial wall. Among these effects, lysoPC (i) regulates 

glycosaminoglycan chain elongation on all secreted proteoglycans, while (ii) selectively 

up-regulating expression of the core protein for PG-MCSF, a survival, growth, and differentiation 

factor for mononuclear phagocytic cells that also can interact with lipoprotein particles. 

Both of these effects of lysoPC on proteoglycan synthesis are likely to contribute to increased 

lipoprotein retention within the arterial wall. Given the accumulating evidence for reactive 

oxygen species (ROS) as mediators of a variety of effects of both oxidized LDL and lysoPC, the 

present study evaluates the role of reactive oxygen species as intermediate molecules in the 

regulation of proteoglycan synthesis by lysoPC. The major findings are four-fold. First, lysoPC 

(10 microM)was found to stimulate rapid (5 mins) and sustained (up to 36 hrs) generation of 

ROS in SMC, as indicated using a fluorescent probe for measuring intracellular oxidants, 

CM-H 2DCFDA, and analysis by fluorescence-activated cell sorting (FACS). This was not 

associated with cytoxicity, as evaluated by fluorescence microscopy using MitoTracker Red or 

propidium iodide, cell number, cell protein, or lactate dehydrogenase release. Second, this ROS 

production could be blocked by pretreatment with the enzymatic antioxidants catalase or 

superoxide dismutase. Third, these antioxidants also blocked the ability of lysoPC to regulate 

proteoglycan synthesis (both glycosaminoglycan chain elongation and core protein regulation), 

as indicated by SDS-PAGE. Fourth, and most importantly, these antioxidants prevent the ability 

of lysoPC to stimulate synthesis of proteoglycans with enhanced lipoprotein-binding properties, 

as quantified by a gel shift binding assay. Results of this study strongly suggest that ROS are 

key mediators in the ability of lysoPC to regulate proteoglycan synthesis and to influence 

lipoprotein retention in the arterial wall. 

P53 

Soluble Cd40 Ligand and Two-Year Prognosis in Ischaemic Heart Disease 

Patients 

Alex O Chevtchenko, MD, Russian Med State Univ, Moscow, Russian Federation; Nadezhda 

Tchervjakova, Rsch Cntr of Transplantology and Artifical Organs, Moscow, Russian 

Federation; Olga F Prirodova, Oleg P Shevchenko, MD; Russian Med State Univ, Moscow, 

Russian Federation 

Background. The CD40 ligand is produced by activated inflammatory cels and platelets, and 

may participate in the pathogenesis of plaque progression and acute coronary syndromes 

develpement, being involved in activation of both coagulant and inflammation systems 

activation. Aim: the study was objected to evaluate prognostic significance of circulating 

sCD40L levels in ischaemic heart disease (IHD) patients. Methods: 24 patients with stable 

angina (62.310.2 y/o, 14 females) and 25 unstable angina (UA) (64.19.7 y/o, 13 females) 

with preserved myocardial function were included. Patients with inflammatory conditions and 

elevated trponin T and creatinphosphokinase were excluded. Plasma levels of sCD40L, hsCRP, 

IL-6, fibrinogen, neopterin, and sVCAM-1 were measured at admission using commercially 

available immunoassays. Patients were followed-up for 48 months, and death, acute MI, any 

revascularisation, and hospitalisation for UA or progressive effort angina were considered as 

end points. Results. There were no deaths, AMI or revascularizations at the end of follow-up 

period. Thirty (61.2%) patients were hospitalised with UA or angina progression. The prognosis 

did not correlated with the diagnosis of UA, lipid levels, smoking or hsCRP, IL-6, fibrinogen, 

neopterin, and sVCAM-1 levels, but sCD40L. We observed 6 events in 18 patients with low 

sCD40L (1.5 ng/ml) and 24 events in 31 patients with high sCD40L (1.5 ng/ml) 

(chi-square7.6, p0.006). The comparison of event-free survival curves showed significantly 

better outcome (logrank p0.007) in patients with lower sCD40L levels (1.5 ng/ml). 

Circulating sCD40L levels were not correlated with age, UA diagnosis, conventional risk factors 

and other inflammatory markers levels. Conclusion: the results show independent relation of 

sCD40L levels to the short-term prognosis of ischaemic heart disease patients. 

A Murine Model of Deep Vein Thrombosis 




Brian C Cooley, Linda Szema, Chao-Ying Chen, Jeffrey P Schwab, Gregory Schmeling; Med 

College of Wisconsin, Milwaukee, WI 

Deep vein thrombosis (DVT) occurs with high prevalence in association with a number of risk 

factors, including major surgery, trauma, obesity, bed rest ( 5 days), cancer, a previous 

history of DVT, and several predisposing prothrombotic mutations. A novel murine model of DVT 

was developed for applications to preclinical studies of transgenically constructed prothrombotic 

lines and evaluation of new antithrombotic therapies. A transient direct-current electrical 

injury was induced in the common femoral vein of adult C57Bl/6 mice. A non-occlusive 

thrombus grew and stabilized at the site. Histomorphometric volume reconstruction of the clot 

within harvested veins revealed a highly consistent size at 10 and 60 minutes (0.0198 

0.0045 mm3 and 0.0175 0.0050 mm3 , respectively; mean SEM), which was significantly 

reduced with pre-heparinization (0.0020 0.0009 mm3 and 0.0035 0.0015 mm3 at 10 and 

60 minutes respectively; p 0.05 versus nonheparinized). Homozygous Factor V Leiden mice 

(analogous to the clinical Factor V Leiden prothrombotic mutation) on a C57Bl/6 background 

had clot volumes more than twice those of wild-types (0.0412 0.0060 mm3 ;p 0.01), 

whereas Factor VIII-null mice had significantly smaller clots (0.0024 0.0010 mm3 ;p 0.05). 

Scanning electron microscopy revealed a clot surface dominated by fibrin strands, in contrast 

to arterial by thrombi guest which on June showed 29, a 2013 platelet-dominated structure. This new model of DVT 

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E-62 Vol 25, No 5 May 2005 

presents a quantifiable approach for evaluating thrombosis-related murine transgenic lines and 

for comparatively evaluating new pharmacologic approaches for prevention of DVT. 

P55 

C-Reactive Protein-Mediated Inhibition of Endothelial Cell PAI-1 Production 

is Prevented by Ethanol and Resveratrol 

John P Cullen, Nicholas von Offenberg Sweeney, Univ of Rochester Med Cntr, Rochester, 

NY; Paul A Cahill, Dublin City Univ, Dublin, Ireland; Eileen M Redmond; Univ of Rochester 

Med Cntr, Rochester, NY 

C-reactive protein (CRP), a prototypic marker of inflammation, is an important predictor of 

future cardiovascular events. Also, recent evidence suggests that CRP directly participates in 

the development of atherosclerosis. Endothelial cell-derived plasminogen activator inhibitior-1 

(PAI-1) is known to play an important role in regulating smooth muscle migration. We 

determined the effect of CRP on endothelial PAI-1 expression and the involvement of protein 

kinase C (PKC) and nitric oxide (NO) in mediating these effects. As moderate alcohol 

consumption, especially red wine, is associated with a reduced incidence of cardiovascular 

disease, we also determined whether ethanol and the red wine polyphenol, resveratrol, could 

protect against any CRP-induced effect on PAI-1. Human umbilical vein endothelial cells 

(HUVEC), passages 3–5, were used in all experiments. HUVEC were treated with CRP (0.1–100 

g/ml) in the absence or presence of either (i) bisindolylmaleimide (BIM) (5 M), (ii) L-NAME 

(100 M), (iii) ethanol (10 –100 mM) or (iv) resveratrol (10 –100 M). HUVEC were harvested 

and PAI-1 protein expression determined by Western blot analysis. CRP dose-dependently 

inhibited HUVEC PAI-1 protein expression; 37%, 51% and 81% inhibition for 1, 10 and 100 

g/ml CRP, respectively, at 24 hours (n4, p0.05 vs untreated). The effects of CRP (10 

g/ml) on PAI-1 protein expression were totally reversed when cells were incubated either in 

the presence of the PKC inhibitor, BIM (5 M), or the NO inhibitor, L-NAME (100 M). While 

low dose ethanol (10 mM) had no effect, co-treatment with 50 and 100 mM ethanol completely 

prevented the CRP-induced inhibition of PAI-1. Likewise, co-treatment with resveratrol (50 and 

100 M) ablated the CRP-induced inhibition of PAI-1. In conclusion, CRP potently inhibited 

PAI-1 protein expression in HUVEC via a PKC and NO dependent pathway. This inhibitory effect 

was reversed by both ethanol and resveratrol. Given that PAI-1 has been implicated in the 

setting of restenosis and atherosclerosis, these effects of CRP could be of clinical importance 

and warrant further investigation. In addition, whether the cardioprotective effects of 

ethanol/resveratrol are mediated in vivo by a counteraction of CRP actions merits further 

investigation. 

In Vivo and ex Vivo Quantification of Angiotensin II-Induced Abdominal 

Aortic Aneurysms by High Frequency Ultrasound 

Alan Daugherty, Deborah A Howatt, Debra L Rateri; Univ of Kentucky, Lexington, KY 

Objectives: Infusion of angiotensin II (Ang II) into hyperlipidemic mice promotes the formation 

of abdominal aortic aneurysms (AAAs). Studies of experimental AAAs have been hampered by 

the lack of techniques to quantitate the development of this disease both in vivo and ex vivo. 

The recent availability of a commercial high frequency ultrasound machine (40 MHz, 

Visualsonics) with a 30 micron resolution has the potential to facilitate AAA measurements in 

vivo and ex vivo. Methods and Results: To determine the utility of ultrasound imaging, AngII was 

infused into apoE deficient mice for selected intervals. AAAs, defined as an expansion of 

luminal diameters, were detected noninvasively within the first week of AngII infusion. To 

determine the accuracy of noninvasive ultrasound measurements, aortas were perfusion-fixed 

with paraformaldehyde at a pressure of 100 mmHg and excised. Adventitial tissue was 

removed and aortas were immersed in a saline bath. The ultrasonic probe acquired images at 

0.05 mm intervals along the length of the aortas. The measurements by this approach were 

within 5% of the dimensions achieved in vivo. Luminal diameters were verified by 

cross-sectioning of the aortas. While the conventional approach of histological analysis can 

provide luminal and external measurements, the use of high frequency ultrasound provides 

noninvasive and nondestructive quantitative information on AAA formation. Conclusions: This 

study demonstrates that high frequency ultrasound provides an approach to quantify AAAs both 

in vivo and ex vivo. 

AT1a Receptor Deficiency Reduces Hypercholesterolemia-Induced 

Atherosclerosis via an Effect on Resident Cells of the Arterial Wall 

Qingwei Zhao, Debra L Rateri, Lisa A Cassis, Alan Daugherty; Univ of Kentucky, Lexington, 

KY 

Objectives: We have demonstrated that deficiency of AT1a receptors profoundly reduced the 

development of atherosclerosis in LDL receptor -/- mice fed a diet enriched in saturated fat and 

cholesterol. AT1a receptors are expressed on all cell types present in atherosclerotic lesions. 

The purpose of this study was to determine the role of AT1a receptors on bone marrow-derived 

cells versus resident vascular wall cells on hypercholesterolemia-induced atherosclerosis. 

Methods and Results: Two month old male LDL receptor-/- mice that were either AT1a receptor 

/ or -/- were irradiated and repopulated with donor cells of these genotypes. Therefore, 4 

groups of chimeric mice were created to define the effects of recipient versus donor phenotype. 

Six weeks after irradiation, mice were placed on a diet enriched in saturated fat (21% wt/wt) 

and cholesterol (0.15% wt/wt) for 12 weeks. The success of the engraftment was defined by 

the genotype of bone marrow-derived DNA. AT1a receptor genotype of either recipient or donor 

did not significantly influence blood cell numbers, systolic blood pressure, plasma cholesterol 

concentrations, or lipoprotein cholesterol distributions. The size of atherosclerotic lesions was 

determined both on the aortic intimal surface by en face analysis and in sections of the aortic 

root. In both regions, the size of atherosclerotic lesions was decreased in the AT1a receptor 

deficient recipient groups, while the genotype ofDownloaded the bone marrow-derived from 

cells had no effect 

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P57 



on lesion size. Conclusions: AT1a receptor deficiency dramatically reduced hyperlipidemiainduced 

atherosclerosis due to resident cells of the arterial wall. 

Exogenous Interferon- Increases the Severity and Incidence of 

Angiotensin II-Induced Abdominal Aortic Aneurysms in Hyperlipidemic 

Mice 

Vishwesh Mokashi, Alan Daugherty; Univ of Kentucky, Lexington, KY 

Objective: Infusion of angiotensin II (AngII) into hyperlipidemic mice causes rapid formation of 

atherosclerosis and abdominal aortic aneurysm (AAAs). Interferon-gamma (IFN-gamma) is an 

inflammatory cytokine that has been invoked in both forms of vascular pathology. Previous 

studies from our laboratory have demonstrated that exogenous IFN-gamma is atherogenic in 

apoE-/- mice, but the genetic deficiency of IFN-gamma augments the incidence and severity 

of AngII-induced AAA formation in apoE-/- mice. Based on the above observations, we 

investigated the possibility that administration of exogenous IFN-gamma would ameliorate 

AngII-induced AAAs. Methods and Results: IFN-gamma / and -/- mice in an apoE-/background 

fed a regular diet were infused with 500 ng/kg/min AngII for 4 weeks. Groups of 

these mice were subjected to daily injections of either recombinant mouse IFN-gamma (100 

U/gm body weight) or saline (n5 to 9/group). Systolic blood pressure was monitored 

throughout the study, with no significant differences among groups. In addition plasma 

cholesterol concentrations were also not significantly altered. Unexpectedly, administration of 

rmIFN-gamma increased the incidence of AAAs in mice with both IFN-gamma genotypes. Mice 

which received rmIFN-gamma and AngII had increased incidence of AAAs (7/9 and 6/9 for 

rmIFN-gamma / and -/- genotypes, respectively) compared to their saline injected controls 

(P 0.01). Also, greater number of AAAs ruptured in mice injected with IFN-gamma (4/9 and 

3/9 for IFN-gamma / and -/- genotypes, respectively). Conclusion: Unexpectedly, these 

studies demonstrated that exogenous IFN-gamma augmented the incidence and severity of 

AngII-induced AAAs both in IFN-gamma / and -/- hyperlipidemic mice. 

P59 

Angiotensin II Infusion Promotes Medial Hypertrophy and Matrix Changes 

throughout the Aorta 

A. Phillip Owens, III, Jessica Moorleghen, Lisa A Cassis, Alan Daugherty; Univ of Kentucky, 

Lexington, KY 

Objective: Angiotensin II (Ang II) infusion into mice promotes the development of abdominal 

aortic aneurysms (AAAs). Medial dissection and subsequent remodeling of abdominal aortic 

tissues initiates this pathology. However, the basis for this highly localized aneurysmal 

response is unknown. The objective of this study was to determine whether any region-specific 

response could be discerned in the aorta during AngII infusion. Methods and Results: Control 

male C57BL/6 mice were compared to a group infused with AngII (1,000 ng/kg/min) for 28 days 

(n5 per group). Mice were perfusion-fixed and the aortas were dissected free. Specific 

regions were sectioned including the infra-renal portion of the abdominal aorta, the upper 

thoracic aorta, and the aortic root. Sections of aortic tissue (at least 5 per region) were 

subjected to computer-assisted morphological analysis to determine the medial area and 

thickness in these selected regions. Infusion of AngII led to increases in medial thickness in the 

abdomen (65%, P 0.001), thorax (45%; P 0.001) and the aortic root (24%; P 0.01). 

Furthermore, AngII infusion did not lead to overt differences in the integrity of elastin fibers in 

the different aortic regions. Conclusion: Although AngII infusion leads to the formation of 

aneurysms in a region- specific manner, the effects were uniform throughout the aorta in 

regard to medial hypertrophy. Thus, the basis for the regional selectivity of AAA formation 

remains unknown. 

Acetylated LDL-Induced Lipid-Loading of Macrophages Upregulates 

Angiotensinogen via the AT1a Receptor 

Hong Lu, Debra L Rateri, Katsuya Tashiro, Lisa A Cassis, Alan Daugherty; Univ of Kentucky, 

Lexington, KY 

Objective: Recently we have described that cultured macrophages express all the components 

of the classic renin-angiotensin system and secrete angiotensin peptides via an ACE dependent 

pathway. Since macrophages become engorged with lipid in atherosclerotic lesions, the aim of 

this study was to determine whether lipid loading increased the secretion of angiotensinogen, 

the only known precursor of angiotensin peptides. Furthermore, we sought to determine the 

mechanism of this effect. Methods and Results: Peritoneal macrophages were harvested from 

6–8 week old male C57BL/6 mice. Acetylated LDL(AcLDL)-induced lipid loading of macrophages 

increased the abundance of angiotensinogen mRNA relative to beta-actin (206%, 

P0.001). Cell associated angiotensinogen was increased following incubation with AcLDL as 

shown by both immunocytochemistry and Western blotting (90%, P0.035). Quantitative 

analysis by Western blotting also demonstrated lipid loading led to an increased secretion of 

angiotensinogen (51%, P0.036). AT1 receptors can regulate angiotensinogen synthesis. In 

accordance with the results above, lipid loading increased the relative abundance of AT1a 

receptor mRNA (410%, P0.045). We were unable to detect AT1b receptor mRNA in these 

cells under any condition. A functional role for the AT1a receptor was demonstrated by the lack 

of effect of AcLDL on angiotensinogen secretion in cultured macrophages lacking this receptor. 

Further evidence for a functional role of AT1a receptors was demonstrated by the incubation 

with losartan that ablated the secretion of angiotensin peptide production. Conclusions: 

AcLDL-induced lipid loading of macrophages increases the expression and secretion of 

angiotensinogen. This augmentation was ablated by the genetic or pharmacological attenuation 

of AT1a receptors. by guest on June 29, 2013 

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P61 

The Glucocorticoid Receptor (GR) Mediates Monocyte Chemoattractant 

Protein-1 (MCP-1) mRNA Stability: Evidence for Direct Binding of GR to 

MCP-1 Message 

Latika Dhawan, Bin Liu, Burns B Blaxall, Mark B Taubman; Univ of Rochester, Rochester, 

NY 

Monocyte chemoattractant protein-1, a potent chemoattractant for lymphocytes, plays an 

important role in the earliest events of atherogenesis. Our previous studies demonstrate that 

dexamethasone (Dex), a synthetic glucocorticoid, specifically destabilizes MCP-1 mRNA in 

vascular smooth muscle cells (SMC), and a 224 nt fragment from the 5’ end of the mRNA is 

necessary for Dex-mediated decay. Antibodies to the glucocorticoid receptor blocked the ability 

of extracts from Dex-treated SMC to degrade in vitro transcribed MCP-1 mRNA, whereas 

antibodies to PDGF or to Dex failed to do so. Additionally, extracts from Dex-treated COS cells, 

which do not constitutively express GR or MCP-1, failed to enhance MCP-1 RNA degradation. 

Based on these experiments, we hypothesized that GR is directly involved in the Dex-mediated 

MCP-1 mRNA destabilization. On a non-denaturing gel, a pronounced shift in the mobility of in 

vitro transcribed 32P-labeled full length human MCP-1 mRNA (probe) was observed after 

incubation with recombinant human GR for 5, 30, and 60 minutes, but not with other members 

of the steroid receptor family. Incubating the probe with human GR resulted in a concentrationdependent 

shift; at higher concentrations two bands were observed suggesting GR homodimerization. 

The addition of GR to Dex-treated SMC extracts inhibited the ability of extracts to decay 

MCP-1 mRNA in a dose-dependent manner, suggesting that the recombinant GR is acting as 

a competitive inhibitor of a degradative complex comprised of Dex, a potentially altered GR and 

a ribonuclease that binds specifically to MCP-1 mRNA. Interestingly, the destabilization of 

MCP-1 mRNA was not prevented by the addition of estrogen receptor to the Dex-treated SMC 

extracts. The involvement of the GR in a complex that degrades MCP-1 mRNA is being used 

to purify this complex. Control and Dex-treated SMC extracts were immunoprecipitated with 

antibody to GR and the precipitates were analyzed by PAGE under denaturing conditions. Mass 

spectrometry and microsequencing is ongoing to identify the proteins co-precipitated with the 

GR. This report suggests a novel role for the GR as a component of a degradative complex that 

regulates mRNA stability and thus may provide new strategies for regulating MCP-1 expression. 

P62 

Carotid Artery Intima-Media Thickness in Patients with Type 2 Diabetes 

Mellitus and Impaired Glucose Tolerance: A Systematic Review 

Bjorn Fagerberg, Gerhard Brohall, Anders Odén; Wallenberg Laboratory, Goteborg, Sweden 

The aims were to review the difference in carotid artery intima media thickness (IMT) between 

patients with type 2 diabetes (DM), or impaired glucose tolerance (IGT), and control subjects, 

and also to relate these differences in IMT to age, and risk for myocardial infarction and stroke. 

Systematic reviews were made in order to identify studies using the ultrasound method in a 

cross-sectional design. The difference between IMT in DM or IGT and control subjects were 

calculated. Meta-analysis using random effects modeling was used to calculate summary 

measures. The results showed that 23 included studies comprised 24111 subjects; 4019 with 

DM and 1110 with IGT. In 20 of 21 studies, the diabetic patients had larger carotid artery IMT 

than the subjects in the control groups. The estimated mean difference in IMT was 0.13 (95% 

CI:0.12– 0.14) mm, with similar results in both sexes. Heterogeneity was observed and likely 

sources were study size, diabetes-duration, and ultrasound method. In 3 out of 9 studies, the 

IGT patients had significantly larger carotid artery IMT than the subjects in the control groups. 

The estimated mean difference in IMT between IGT patients and controls was 0.04 (95% 

CI:0.014 – 0.071) mm. The conclusion is that type 2 diabetes was associated with a 0.13 mm 

increase in IMT compared to normal subjects. In patients with IGT the increase in IMT was 

about one third of that observed in diabetes. The observed difference in IMT can be interpreted 

as if the diabetes patients were more than 10 years older than the control groups, and that the 

relative risks of myocardial infarction and stroke were increased by almost 40 %, respectively. 

C-Reactive Protein in Atherosclerotic Lesions: Its Origin and 

Pathophysiological Significance 

Tomonari Koike, Huijun Sun, Tomonaga Ichikawa, Univ of Tsukuba, Tsukuba, Japan; Shuji 

Kitajima, Saga Univ, Saga, Japan; Kinta Hatakeyama, Yujiro Asada, Univ of Miyazaki, 

Miyazaki, Japan; Masatoshi Morimoto, Saga Univ, Saga, Japan; Bo Zhang, Fukuoka Univ, 

Fukuoka, Japan; Masashi Shiomi, Kobe Univ, Kobe, Japan; Jianglin Fan; Univ of Tsukuba, 

Tsukuba, Japan 

Background - C-reactive protein (CRP) is not only a predictor of cardiovascular events but also 

may be a potential risk factor for the development of atherosclerosis. It has been reported that 

CRP is frequently deposited in the lesions of the arterial intima; however, the origin and 

pathologic significance of CRP in these lesions are not completely understood. Methods and 

Results -Atherosclerotic lesions obtained from cholesterol-fed rabbits, Watanabe heritable 

hyperlipidemic rabbits, and human autopsy were used for the determination of CRP expression 

at both the mRNA and protein levels. CRP levels were significantly elevated in both 

cholesterol-fed and Watanabe heritable hyperlipidemic rabbits compared to normal rabbits, and 

correlated with aortic atherosclerotic lesion size. Immunohistochemical staining revealed that 

CRP-immunoreactive proteins were invariably found at all stages of atherosclerosis in the early 

to advanced lesions. CRP tended to be present extracellularly and colocalized with apolipoprotein 

B but was rarely associated with macrophages and foam cells. Real-time RT-PCR analysis 

revealed that CRP mRNA in atherosclerotic lesions was barely detectable and isolated 

macrophages did not express CRP mRNA. Administration of simvastatin to cholesterol-fed 

rabbits significantly reduced the plasma cholesterol levels as well as plasma and lesion CRP 

contents compared to those in the control group. Conclusions - Our results provide compelling 

evidence that there is a link between hypercholesterolemia, Downloaded plasma from 

CRP and the degree of 

P63 

atherosclerosis, and that the inhibition of hepatic CRP production (rather than arterial wall 

production) may represent a therapeutic modality for the treatment of cardiovascular disease. 

P64 

A Novel 3D Imaging Technique of En Face Preparations of the Arterial Wall 

Using Quantum Dots Nanocrystals and Two-Photon Excitation Laser 

Scanning Microscopy 

Dardo E Ferrara, Daiana Weiss, Emory Univ, Atlanta, GA; Xiaohu Gao, Shuming Nie, Emory 

Univ/Georgia Institute of Technology, Atlanta, GA; W. R Taylor; Emory Univ, Atlanta, GA 

Background: Traditional fluorescence imaging with single-photon confocal microscopy and 

organic fluorophores poses several challenges for imaging the endothelium, including tissue 

autofluorescence, fluorophore crosstalk, photobleaching, limited penetration and loss of 

resolution with depth. Methods: We studied human coronary arteries (HCAs) and mouse aortas 

with a modified immunohistochemical en face method using quantum dot (Qdot) bioconjugates 

as secondary antibodies. Two-photon excitation laser scanning microscopy (TPELSM) was 

performed with a Zeiss LSM 510 META equipped with a titanium-sapphire femtosecond laser. 

Results: We demonstrated the feasibility of multicolor labeling of endothelial cell (EC) markers 

by exciting Qdots of different emission spectra with only one wavelength (750nm). Detailed cell 

structures, such as the granular appearance of vWF, were visualized using green dots (525 

nm), even when the emission maximum of the Qdots overlapped that of tissue autofluorescence 

(510–520 nm). In addition, the localization of markers at areas of laminar or turbulent flow 

showed a crescentic or triangular EC arrangement upstream of intercostal ostia along with a 

higher frequency of VCAM-1 expression around these branches. We also noticed EC 

disorganization and preferential VCAM-1 expression over plaques in apo E knock-out (KO) mice. 

Interestingly, 3D orthogonal views uncovered a uniform distribution of VCAM-1 over plaques as 

opposed to prominence at the shoulder region, as previously reported with epifluorescence 

microscopy. We also identified early VCAM-1 expression at lesion-prone areas of 4 week-old 

apo E KO mice. Finally, elastin autofluorescence and Hoechst-stained nuclei at depths of 100 

m below the EC surface of HCAs were imaged without significant loss of definition. Large 

z-stack series were also obtained without photobleaching. Conclusion: We report the 

application of TPELSM with Qdot bioconjugates for the en face study of arterial walls. This 

innovative method allows highly-sensitive 3D visualization of the vascular endothelium with 

excellent spatial resolution. These results demonstrate the potential of this technique to 

become a useful tool to image early atherosclerotic changes ex vivo. 

Atheromas Drain Their Contents via “Exit” Tracts 




Richard J Frink; Heart Rsch Foundatilon of Sacramento, Sacramento, CA 

Objective: To present histologic evidence showing many atheromas are associated with tracts 

that connect to the lumen and serve to drain plaque contents. Secondly, to show evidence of 

endothelial injury associated with these tracts. Method: The hearts of 83 patients who died of 

acute coronary disease were studied by injecting a colored barium gelatin mass into the 

coronary arteries, followed by dissection of the major branches of the coronary tree, 

decalcification, cutting segments at 2–3 mm intervals and mounting all segments for histologic 

study. On average 86 segments were examined for each heart. Three to five subserial sections 

were mounted for each coronary segment. Atheromas from 17 patients, 13 males, age range 

33–81, and 5 females, age range 50–76, were selected to illustrate the features of exit tracts. 

Observations: Photographs showing different types of exit tracts and their communication with 

the lumen will be presented. The presence of red blood cells, colored injection mass or fibrin 

within the tract were used as evidence that these tracts existed in vivo. Plaque contents within 

the tract or near the mouth of the tract indicated the tracts served to drain the necrotic core. 

The tracts were often serpiginous and frequently entered the lumen near the plaque shoulder 

and were present in association with both large and small atheromas, but were unrelated to 

the severity of luminal stenosis. Adventitial inflammatory infiltrates associated with these tracts, 

suggested active atherosclerotic disease. These tracts were frequently associated with 

evidence of endothelial injury, suggesting injury by toxic chemical agents, contained in the 

necrotic core. Conclusions: Atheromas of all sizes are frequently associated with tracts that 

connect to the lumen and appear to serve as a route for plaque contents to exit the atheroma 

and decompress the necrotic core, a form of reverse transport. The contents of the necrotic 

core appear to injure the endothelium near the mouth of the exit tract, suggesting the necrotic 

core contains toxic chemical agents. 

P66 

Decreased Annexin V-Binding to Endothelium Caused by Antibodies - A 

Novel Mechanism in Atherothrombosis 

Anna Cederholm, Elisabet Svenungsson, Ann-Christin Ulfgren, Tina Trollmo, Jesper 

Swedenborg, Guo-zhong Fei, Johan Frostegard; Karolinska Institutet, Stockholm, Sweden 

OBJECTIVE: The cause of the high risk of atherothrombosis in systemic lupus erythematosus 

(SLE) is an important clinical problem, and may also shed light on the relation between immune 

mechanisms and atherothrombosis in general, not least in women. Annexin V has been 

described as a putative antithrombotic plasma protein, and antiphospholipid antibodies (aPL) 

are known since long time to cause thrombosis, though the mechanisms are only partly known. 

METHODS AND RESULTS: Twenty-six women (52/-8.2 years) with SLE and a history of 

cardiovascular disease (CVD) (SLE cases) were compared with 26 women with SLE but no CVD 

(SLE controls) and 26 healthy women (population controls). Common carotid intima-media 

thickness (IMT) was determined by B-mode ultrasound as a surrogate measure of atherosclerosis. 

Risk factors for CVD included increased: dyslipidemia, inflammation (CRP and TNFactivity), 

LDL-oxidation, aPL. We then determined Annexin V binding to human umbilical vein 

endothelial cells (HUVECs) by flow cytometry. After 24-hour culture Annexin V binding was 

decreased when plasma from SLE cases was used (SLE cases versus population controls: 

P0.002; by SLE guest caseson versus June SLE29, controls 2013P0.02). 

Antibodies against cardiolipin but also 

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E-64 Vol 25, No 5 May 2005 

Annexin V, S Pneumonae and oxidized LDL were among IgG antibodies causing decreased 

binding. There was a surprising positive association between Annexin V binding and IMT 

(R0.73; P0.001) among SLE cases, indicating that the same factor, at least at late stage 

of disease, can promote atherosclerosis and still cause thrombosis. Immunohistochemical 

analysis revealed presence of annexin V in all human atherosclerotic plaques tested, especially 

at sites prone to rupture. Preliminary experiments indicate that Ig from a large pool of donors 

(IVIG) neutralized the Annexin-inhibiting antibodies. CONCLUSIONS: Decreased annexin V 

binding to endothelium caused by antibodies against phospholipids but also infectious agents 

may represent a novel mechanism of atherothrombosis. We hypothesize that even though 

annexin V may promote plaque growth at some disease stages, it may also stabilize plaque. 

Increasing Annexin V binding could represent a new therapeutic possibility, egbyuseof 

neutralizing antibodies. 

P67 

Activation of Endothelial Nitric Oxide Synthase by Oxidized Phospholipids 

Regulates Interleukin 8 Expression in Endothelial Cells 

Nima M Gharavi, Nancy Baker, Henry Honda, UCLA, Los Angeles, CA; Eric J Smart, Univ of 

Kentucky, Lexington, KY; Judith A Berliner; UCLA, Los Angeles, CA 

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), which accumulates 

in atherosclerotic lesions and other sites of chronic inflammation, induces endothelial 

cells (EC) to synthesize atherogenic chemotactic factors, such as interleukin 8 (IL-8). In this 

study, we found that treatment of human EC with Ox-PAPC and its component phospholipid, 

1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), activated endothelial 

nitric oxide synthase (eNOS), as measured by phosphorylation of serine residue 1177. eNOS 

activation was observed as early as ten minutes following treatment, sustained for up to two 

hours, and was independent of other Ox-PAPC-induced pathways, such as cAMP and c-Src 

kinase. Ox-PAPC and POVPC treatment also induced the dose-dependent synthesis of nitric 

oxide (NO). Activation of eNOS was necessary for Ox-PAPC-induced IL-8 synthesis as 

pretreatment of human EC with the eNOS inhibitor, N w-nitro-L-arginine methyl ester (L-NAME), 

inhibited Ox-PAPC-induced IL-8 expression dose-dependently. Our data also demonstrated that 

treatment of human EC with the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), induced 

IL-8 expression. Furthermore, Ox-PAPC was found to induce oxidative stress in human EC, at 

similar time points as NO synthesis; NO, in the presence of oxidative stress, can react to 

generate peroxynitrite. Interestingly, pretreatment of human EC with HDL, which has potent 

anti-inflammatory and anti-atherogenic effects, inhibited the activation of eNOS and the 

induction of IL-8 by Ox-PAPC. These findings demonstrate that Ox-PAPC-induced eNOS 

activation regulates IL-8 transcription, through the production of either NO or peroxynitrite. In 

addition, these findings suggest a novel mechanism for the atheroprotective effect of HDL. 

P68 

High Field Magnetic Resonance Imaging of Human Peripheral Arteries with 

Low Grade Atherosclerotic Lesions - Comparison to Histology 

Kristof Graf, Thore Dietrich, Uwe Koehler, Philipp Stawowy, Burkhard Zipfel, Holger 

Haensch, Eike Nagel, Eckart Fleck; DHZB, Berlin, Germany 

The non-invasive characterisation of the vessel wall requires imaging techniques capable of 

near microscopic resolution. High field MRI provides sufficient signal for the acquisition of 

high-resolution datasets. The aim of this study was the detailed analysis of the internal 

structure of the human vascular wall ex vivo with MRI and a comparison to histology. Tissue 

samples with low-grade atherosclerotic lesions (Stary 0 –2; n12) from iliac and femoral 

arteries were obtained from patients undergoing vascular surgery, fixed in formalin and 

enclosed in agarose. These samples were scanned at 7 Tesla (Pharmascan Bruker) using 3D 

slabs of 256x256x256 pixels resulting in a resolution of 79x79x109 m: spin echo sequences 

were obtained with TE/TR of 13/500 ms (T1 weighted), 12/2500 ms (proton density weighted 

(PD)) and 63.5/2500 (T2 weighted). Combined evaluation using T1, PD and T2 enabled a clear 

differentiation of the intimal, neointimal, medial and adventitial layers in each sample using 

high field MRI. For histological comparison arterial crossections were stained with Elastica-von 

Giesson and trichrome stain. Corresponding crossections of each sample obtained by MRI and 

histology were analyzed for intima, media and adventitia thickness (n16/sample). Average 

width for intima-media was 92946 m using histometry and 101149 m using MRI 

analysis. Correlations of data sets from histology and MRI using linear regression analysis and 

Pearrson correlation revealed a highly significant correlation (p0.001) between data obtained 

by MRI and histology for intima, media thickness. A borderline significance was observed for 

adventitia size. The MRI and histological images show a close correlation for the spatial 

structure of the vessel wall. Furthermore, image data revealed a differentiated image of early 

lesion composition, which corssponded to the histology. A clear differentiation of the vessel 

wall is possible with MRI. These initial results prove 7 Tesla MRI to be a valuable tool in 

accessing the complex composition of human vessel wall in-vitro. However, concessions for 

spatial resolution and spatial coverage need to be made when transferring this knowledge to 

in-vivo imaging. 

P69 

Up-Regulation of Monocyte Chemoattractant Protein-1 and the Monocyte 

Chemoattractant Protein-1 Receptor (CCR2) in Macrophages by the 

Pro-Inflammatory Cytokine Interferon- 

Elizabeth J Harvey, BSc, Dipak P Ramji; Cardiff Univ, Cardiff, United Kingdom 

The chemokine monocyte chemoattractant protein (MCP)-1 has been shown in several 

independent studies to be a proatherogenic factor, contributing to the development of 

atherosclerosis primarily through the recruitment of monocytes/macrophages to the lesion. The 

pro-inflammatory cytokine interferon (IFN)- has also been implicated in the pathogenesis of 

atherosclerosis. Induction of MCP-1 expression by Downloaded IFN- has been demonstrated from 

in several cell 



types including macrophages. The signal transduction pathways required for the IFN-mediated 

regulation of MCP-1 expression have not yet been fully elucidated and may provide 

additional targets for therapeutic intervention in atherosclerosis. MCP-1 mRNA expression was 

increased in the mouse macrophage J774.2 cell line following treatment with IFN-, reaching 

maximal levels at 3h. Cellular protein levels of MCP-1 followed a similar pattern of induction 

but declined after 12h treatment, correlating with increased levels of secreted MCP-1. The 

induction of MCP-1 expression was attenuated by incubation with the Jak2 inhibitor AG490, the 

phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and wortmannin, and the casein kinase 

2 (CK2) inhibitor apigenin. IFN- produced a five-fold increase in reporter gene activity, 

regulated by a fragment of the MCP-1 promoter, in transfected human hepatoma Hep3B cells 

and differentiated cells of the human monocytic U937 cell line. Co-transfection assays showed 

that this IFN--mediated induction of MCP-1 promoter activity was inhibited by constructs 

specifying for dominant negative forms of JAK1, JAK2, STAT1, CK2 and PI3K, confirming a role 

for these signalling mediators in the regulation of this gene. The expression of the cell surface 

receptor for MCP-1 (CCR2) was also investigated in order to determine whether similar 

regulatory pathways govern the expression of both the chemokine and its receptor by IFN- in 

macrophages. RT-PCR analysis showed that mRNA expression for CCR2 was up-regulated by 

IFN- and that the same range of pharmacological inhibitors prevented the response, indicating 

that a similar regulatory mechanism may be involved. These studies provide novel insights into 

the IFN--mediated regulation of MCP-1 and its receptor in atherosclerosis. 

Androgen Administration Increases Angiotensin II-Induced Abdominal 

Aortic Aneurysm Formation in Apolipoprotein E Deficient Mice 

Tracy A Henriques, Alan Daugherty, Lisa A Cassis; Univ of Kentucky, Lexington, KY 

Objective: Chronic infusion of angiotensin II (AngII) into apolipoprotein E deficient (apoE-/-) mice 

accelerates atherosclerosis and causes abdominal aortic aneurysm (AAA) formation. Male mice 

exhibit a 3-fold greater incidence of AngII-induced AAA compared to females. Our recent 

studies demonstrate that removal of male sex hormones reduces AAA incidence to a level 

observed in females. The purpose of this study was to determine whether administration of 

androgens to male and female apoE-/- mice would increase AngII-induced AAAs. Methods: 

Male and female apoE-/- mice were castrated and 2 weeks later implanted with pellets to 

deliver equivalent doses (10 mg pellets/60 day release, or an average dose of 8mg/kg/day) of 

testosterone (T) or dihydrotestosterone (DHT). One week after implantation, mice were infused 

with either AngII (1,000 ng/kg/min) or saline for 28 days. Androgen administration significantly 

increased body weight in both male and female apoE-/- mice. Blood pressure responses to 

AngII were not altered in mice administered T, but were decreased in mice given DHT. 

Cholesterol concentrations and lipoprotein-cholesterol distributions were not altered by 

androgen in either male or female mice. Administration DHT significantly increased the 

incidence of AAAs in female mice compared to vehicle (22% placebo vs. 57% T vs. 64% DHT; 

P0.03). Similar results were obtained in male mice treated with androgens (27% placebo vs. 

67% T vs. 75% DHT: P0.05). T and DHT increased the severity of AAAs in both male and 

female mice compared to vehicle (P 0.05). Conclusion: Administration of androgen to 

castrated male and female mice increases the incidence and severity of AngII-induced AAA, 

demonstrating a primary role for male sex hormones as mediators of gender differences in AAA 

formation. 

P71 

Interactions among Matrix Metalloproteinases (MMPs), Tissue Inhibitors of 

Metalloproteinases (TIMPs), and Calcification Proteins in Carotid 

Endarterectomy (CEA) Lesions 

Catherine L Higgins, Salman Choudhary, Salim Isbilir, Baylor College of Medicine, Houston, 

TX; Michael J Reardon, Gerald M Lawrie, The Methodist Hosp, Houston, TX; Joel D 

Morrisett; Baylor College of Medicine, Houston, TX 

Background: Vascular calcification is frequently involved in atherosclerosis. Osteoporosis and 

arterial calcification often coexist. MMPs are critical for vascular remodeling by regulating 

degradation of extracellular matrix while TIMPs inhibit degradation. Osteoprotegerin (OPG) 

inhibits differentiation of osteoclasts but also protects against mineralization. Osteopontin (OPN) 

is associated with mineralization. Osteocalcin (OC) is involved in bone formation. Matrix GLA 

protein (MGP) is an inhibitor of soft tissue calcification. Objective: The goal of this study was 

to determine potential interactions among MMPs, TIMPs, and calcification proteins in vascular 

calcification. Methods: CEA specimens were obtained from surgery and cut into 5mm 

segments starting at the common, extending up to the bifurcation, and into the internal and 

external branches. Each segment was digitally photographed and homogenized. The homogenate 

was centrifuged, and supernatant and precipitate fractions were assayed for protein 

concentration levels by ELISA. Results: MMP2 and MMP9 levels were significantly higher than 

MMP1 levels in all CEA specimens. The MMPs and TIMPs had strong correlations, suggesting 

that they balance one another: MMP1 with TIMP2 (r 0.60, p0.0001); MMP2 with TIMP2 (r 

0.84, p0.0001); and MMP9 with TIMP1 (r 0.42, p 0.01). OPN levels were higher and OPG 

levels were lower in highly calcified lesions, whereas OPG levels were higher and OPN levels 

were lower in lowly calcified lesions; therefore, a negative correlation existed between OPN and 

OPG (r -0.41, p 0.04). OPN had positive correlations with MMP1 (r 0.52, p 0.002), 

MMP2 (r 0.72, p0.0001), and TIMP2 (r 0.58, p 0.0004). TIMP1 was negatively 

correlated with OC (r -0.36, p 0.05). MGP had positive correlations with MMP1 (r 0.38, 

p 0.05), OC (r 0.79, p0.0001), and OPG (r 0.43, p 0.05) but a negative correlation 

with MMP9 (r -0.39, p 0.04). Conclusion: Correlations among MMPs, TIMPs, and 

calcification proteins suggest that these proteins undergo significant interactions in the 

vascular calcification by guest on process. June 29, 2013 

P70

WITHDRAWN 

P72 

P73 

Development of Multiplexed Immunoassays for Simultaneous Quantification 

of Soluble Cardiovascular and Metabolic Biomarkers in Serum or Plasma 

Shaoquan Ji, Qiang Xiao, Terry Whitehead, Hank Hwang, Rick Ryan, Jehangir Mistry; LINCO 

Rsch, St Charles, MO 63304, MO 

Quantification of multiple circulating biomarkers (e.g. apolipoproteins, adhesion molecules, 

acute phase proteins, and proinflammatory cytokines, etc) is imporant for understanding the 

physiological and pathological processes associated with cardiovascular disorders such as 

vascular changes, atherosclerosis, and thrombosis. According to the range of serum 

concentrations of analytes, four separate multiplexed immunoassay panels were developed, 

using Luminex technology, to simultaneously quantify apolipoprotein A1, A2, B, C2, C3 and E 

(Panel 1); adiponectin, sE-Selectin, sICAM-1, sVCAM-1, MMP-9, MPO, and PAI-1 (Panel 2); 

C-reactive protein (CRP), fibrinogen, haptoglobin, serum amyloid A (SAA), and serum amyloid 

P (SAP) (Panel 3); IL-1, IL-6, IL-8, IL-10, IFN, MCP-1, NT-proBNP, TNF, and VEGF (Panel 

4). Briefly, samples were incubated 1hatRTorovernight at 4°C in a 96-well microtiter filter 

plate with a mixed population of polystyrene beads, which were covalently immobilized with 

specific capture antibodies and contained two internal fluorophores for bead identification. After 

washing, captured analytes on beads were incubated for 30 min or 1hatRTwith a cocktail 

of biotinylated detection antibodies. Following subsequent incubation with streptavidinphycoerythrin, 

fluorescent signals on beads were quantified using a Luminex 100 Reader. The 

total assay time is 2 hours (Panels 2, 4) or overnight (Panels 1,3). Each antibody pair used for 

individual analyte is highly specific, with no or negligible cross reactivity to other analytes 

within the panel. The assay robustness is demonstrated by acceptable precisions (CV 15% 

for inter-assay variations; CV 10% for intra-assay variations), linearity of dilution (100 

20%), and accuracy (95 20%) for serum or plasma samples. Serum or plasma samples 

require dilutions of approximately 1:10,000, 1:50, 1:5,000 (or higher), and no dilution, for 

Panels 1, 2, 3, and 4, respectively. Sample volume is 25 l per well. The assays may be used 

for other sample types, e.g. cell culture supernatant, tissue/cell lysate, etc. These novel, rapid, 

robust assays provide simple and economic tools for simultaneously quantifying multiple 

cardiovascular and metabolic biomarkers in biological samples. 

P74 

Hyperhomocysteinemia Impairs Endothelial Function and Enos Activity via 

PKC Activation 

Xiaohua Jiang, Fan Yang, Hongmei Tan, Dan Liao, Chuantao Jiang, Robert M Bryan Jr, 

Jaspreet K Randhawa, Rolando E Rumbaut, Xiaofeng Yang, William Durante, Baylor College 

of Medicine, Houston, TX; Andrew I Schafer, Univ of Pennsylvania Sch of Medicine, 

Philadelphia, PA; Hong Wang; Baylor College of Medicine, Houston, TX 

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease (CVD), 

and is associated with endothelial dysfunction. However, the mechanism for homocysteine 

(Hcy)-induced endothelial dysfunction is largely unknown. In this study, we examined the role 

of HHcy in endothelial dysfunction using two functional models, aortic rings and intravital 

videomicroscopy of the cremaster, in cystathionine -synthase (CBS) null mice. We found that 

arterial relaxation in response to acetylcholine, an endothelium-dependent vessel relaxant, was 

significantly impaired in severe HHcy. Impaired endothelium-dependent relaxation was restored 

by a nitric oxide donor, but not by superoxide dismutase and catalase. Plasma levels of 

asymmetric dimethylarginine were not increased in CBS -/- mice. Endothelial nitric oxide 

synthase (eNOS) activity was significantly reduced in aortic endothelial cells from CBS -/- mice, 

as well as in Hcy-treated mouse and human aortic endothelial cells. This was correlated with 

decreased protein expression and increased serine 495 phosphorylation of eNOS. Hcymediated 

eNOS inhibition was not rescued by adenoviral-transduction of superoxide dismutase 

and glutathione peroxidase, or by tetrahydrobiopterin and arginine supplementations. The 

protein kinase C (PKC) inhibitor GF109203X reversed Hcy-mediated eNOS inactivation and 

serine 495 phosphorylation. These data indicate that HHcy impairs endothelial function and 

eNOS activity through PKC activation. 

P75 

StatinsIinhibited the ADP-Stimulated Activation of Integrins Alpha-v Beta-5 

and Alpha-v Beta-3 of Vascular Smooth Muscle Cells 

Seung-Jae Joo, Ki-Seok Kim; Cheju National Univ College of Medicine, Jeju, Republic of 

Korea 

Background: Integrins mediate the migration, adhesion and proliferation of vascular smooth 

muscle cells, and ADP can activate vascular integrins. We assessed the hypothesis that statins 

inhibit the ADP-stimulated activation of integrins alpha-v beta-5 and alpha-v beta-3 in human 

aortic smooth muscle cells (HASMC). Methods: The expressions of integrins alpha-v beta-3 and 

alpha-v beta-5 on HASMC were analyzed by flow cytometry. Activations of integrins alpha-v 

beta-3 and alpha-v beta-5 were evaluated by the adhesion assay using prothrombin as an 

activation-dependent ligand. The MTT assay was used to evaluate the proliferation of HASMC. 

Results: Simvastatin and fluvastatin (5 M) did not suppress the expressions of integrins 

alpha-v beta-3 and alpha-v beta-5 of HASMC after 1 day- co-culture. HASMC had more integrin 

alpha-v beta-5 than alpha-v beta-3. ADP increased the adhesion of HASMC to prothrombin and 

the proliferation in a dose-dependent manner, resulting in the maximal adhesion and 

proliferation at 100 M. The adhesion was prevented partially by LM609, which blocks integrin 

alpha-v beta-3 (13% inhibition) and markedly by P1F5, which blocks integrin alpha-v beta-5 

(76% inhibition; n5, p0.05). However, the proliferation was inhibited by c7E3 and LM609 

(88%; n4, p0.05), but not P1F5. SimvastatinDownloaded and fluvastatin inhibited from 

the ADP-stimulated 

adhesions of HASMC in a dose-dependent manner (at 10 M, 15% and 27% inhibition; at 100 

M, 45% and 72% inhibition, respectively; n5, p0.05) after 15 min pretreatment. After 

incubating HASMC with statins at the concentration of 5 M for 1 day, simvastatin and 

fluvastatin inhibited the adhesion by 70% and 66%, respectively (n5, p0.05). Simvastatin 

and fluvastatin inhibited the ADP-stimulated proliferation of HASMC in a dose-dependent 

manner (at 10 M, 18% and 10% inhibition; at 100 M, 53% and 23% inhibition, respectively; 

n6, p0.05). Conclusions: ADP activated integrins alpha-v beta-5 and alpha-v beta-3 of 

HASMC. Simvastatin and fluvastatin did not suppress the expression of integrins alpha-v beta-5 

and alpha-v beta-3, but inhibited the activation of these integrins. The adhesion of HASMC to 

prothrombin appeared to be mediated mainly by integrin alpha-v beta-5, and the proliferation 

by alpha-v beta-3. 

Risk Factors Reduction and Thromobotic Risk Relapse in Vulnerable 

Patients 

Giorgio Corinaldesi, Medicina Generale ASL 7, 60100, Italy; Christian Corinaldesi; Università 

Politecnica delle Marche, Ancona, Italy 

The aim of this study was to determine the impact of the reduction of risk factors in the 

development and progression of atherothrombosis. We evaluated BMI and the abdominal 

circumference, blood pressure, number of involved vessels and entity of the atherosclerotic 

plaque, fasting glucose (110 mg/dl), total cholesterol (200mg/dl), HDL-cholesterol (40 

mg/dl in men, 50 mg/dl in women), triglyceride’s (TG)level (150 mg/dl), presence of 

pro-thrombotic targets (C reactive protein (CPR), fibrinogen, CD40L, a biomarker of platelet 

activation, and biochemical link between the inflammatory state, coagulative activation, and 

endothelial damage, PAI-I, D-dimer, FVIII:C, omocysteine, Lpa), and of flogistic markers (IL-6, 

IL-10, TNF-alfa, ICAM-I, VCAM-I). We have studied for 5 years 206 patients (148 men, 58 

women) aged among 56 and 68 years, and we have found that 108 of them (thus named group 

A) were affected by a central and/or peripheral symptomatic atherosclerotic vasculopathy, 

while 98 patients (group B) were oligo- or asymptomatics and they did not show any sign of 

atherothrombotic disorder. 97 patients (86 from the group A, and 11 from the group B) 

developed an ischemic CVD with a variable clinical expression of the symptoms. These data 

suggest that there is an important association between visceral and/or central adiposity and the 

development of insulin-resistance, and also they show how levels of LDL-cholesterol, 

HDL-cholesterol, TG, Lpa, the presence and the entity of plaques and hypercoagulability are 

important promoters of cardiovascular events. The overall risk is also highly amplified by the 

CPR over-expression, which in our study has a fundamental role in the progression of the 

disease (84 out of 97 patients which developed an ischemic CVD had altered CPR, and 30% 

of them had levels higher than 3.0 mg/l, 77 from group A, 7 from group B), and also by CD40L 

level measured by ELISA. In conclusion, the evaluation of CRP, and CD40L are fundamental 

prognostic markers, they are also very useful for risk stratification; the overall prognosis is not 

clearly influenced by weight reduction 10%, by the reduction of lipids levels 30%, and 

glucidic levels 20%, and from blood pressure normalisation. 

P77 

Prevalence of Peripheral Arterial Disease is a Predictor of Stroke in the 

Elderly 

Giorgio Corinaldesi, Medicina Generale ASL 7, 60100, Italy; Christian Corinaldesi; Università 

Politecnica delle marche, 60100, Italy 

We evaluated the prothrombotic risk profile of stroke in the elderly with peripheral arterial 

disease which is considered a risk factor with an important predictor of stroke. We evaluated 

vascular alterations in the epaortic vessels (through color doppler US, angio-MNR, angio-CT); 

ultrasound criteria included the presence of echogenic clot, or exacerbate neointimal 

hyperplasia, augmented fibrin deposition, absence or marked reduction of flow, presence and 

grade of vascular meandering, and we evaluated laboratory tests, in particular we studied 

markers of platelet and leukocytic activation. Data obtained demonstrate a close relationship 

between the neointimal hyperplasia (NH, greater than 1.5mm), the ankle brachial index (ABI 

0.9), the presence of atherosclerotic plaques and the index of plaque vulnerability (flapping, 

platelet activation, elevated CD40L, elevated PCR-hs). We studied 78 patients (56 male, 22 

female) affected by peripheral arterial disease aged between 64–76 years for five years, 

evaluating the thrombophilic risk profile and excluding patients with genetic prothrombotic 

factors; we evaluated their clinical evolution every six-months and on demand. Our results 

demonstrate a close correlation between stroke the progression of NH, which is considered as 

the main promoter of endothelial disfunction, and also between increased level of CD40L, 

and/or PCR-hs which are considered progression factors, and the between the presence of 

glyco-lipidic alterations (GLA) which are considered amplifying factors. 34 patients with 

progression of lesion developed a stroke, 16 of them had also increased level of CD40L, 8 

increase of PCR-hs level, 8 had associated GLA; the remaining 44 patients developed TIA in 15 

cases, of which 8 with increased level of CD40L, 6 with increase of PCR-hs, 8 with associated 

GLA; 29 patients did not show any clinical outcome, however, a MNR demonstrated the 

presence of silent infarct in 12 patients, an increase of CD40L in 4 patients, and an increase 

of PCR-hs in 3 patients, and associated GLA in 16 patients. 

Age-Associated Decrease in Vein Graft Neointimal Thickening 

Brian C Cooley; Med College of Wisconsin, Milwaukee, WI 




Most experimental studies of vein graft neointimal formation are done in young adult animals, 

whereas the clinical population for interpositional vein grafting is predominantly older/elderly. 

Arterial injury models (e.g., balloon angioplasty) have shown increased neointimal thickening in 

aged versus young animals. This study used a murine model of interpositional vein grafting in 

an old:young (20–22 months:2–3 months) cross-transplantation design: old-to-old, young-toold, 

old-to-young, by guest andon young-to-young June 29, 2013 grafting. The Rosa26 transgenic marker-gene mouse 

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E-66 Vol 25, No 5 May 2005 

(constitutive LacZ gene expression in all cells) was used for young animals in several of the 

cross-transplants. One-month neointimal thickness at the proximal end of the graft was 

significantly reduced with age when statistically evaluated for both graft age and recipient age 

(p0.02). Older grafts also showed reduced mid-graft neointimal thickening (p0.02). Table: 

Neointimal thickness (in microns; mean SEM) The neointima arose predominantly from the 

donor graft regardless of donor/recipient age, as determined by histochemical evaluation for 

Rosa26 marker-gene cells within young-to-young and old-to-young cross-transplanted grafts. 

These findings are in contrast to those of arterial balloon injury (increased neointima with age), 

suggesting that different mechanisms are involved between arterial and vein graft neointimal 

formation with respect to age-associated influences. 

Graft/Recipient Age Number 

Proximal 

graft Mid-graft 

Distal 

Graft 

Young/Young 11 11018 368 517 

Young/Old 9 8020 214 4311 

Old/Young 7 5810 284 559 

Old/Old 8 394 182 408 

Discovery and Pre-Clinical Evaluation of an Anti-vWF Aptamer for 

Treatment of Acute Coronary Syndrome (ACS) 

HA D Lagasse, Patricia G Merlino, Madaline C Gilbert, Jason R Killough, Kathleen Mills, 

Ryan M Boomer, Scott D Lewis, III, Ankit S Makim, Claude R Benedict, Thomas G 

McCauley, James B Rottman, H N Marsh, John L Diener; Archemix Corp., Cambridge, MA 

One of the primary responses to vascular damage is the binding of vWF multimers to exposed 

collagen via the vWF-A3 domain. Under the high shear force conditions observed in stenosed 

arteries, platelets bind to immobilized vWF multimers through the vWF-A1 domain and platelet 

gpIb receptor. The vWF-A1/gpIb binding interaction is the first event in the platelet activation 

pathway at sites of arterial damage. Using the technique of in vitro selection (SELEX), we have 

identified a DNA aptamer that binds to the A1 domain of vWF and blocks the interaction of vWF 

with platelets. The DNA aptamer has been rendered highly resistant to plasma nucleases in 

vitro through an extensive structure activity relationship (SAR) study involving the systematic 

replacement of standard deoxy-nucleotides with stabilizing nucleotides. In binding assays, the 

aptamer has high affinity for vWF (K D 1 nM) and flow cytometric analysis reveals that the 

aptamer blocks the interaction of vWF with human platelets. Functionally, the anti-vWF aptamer 

inhibits botrocetin-induced platelet aggregation (BIPA) in human platelet rich plasma (IC 90 

100 –300 nM) and inhibits shear stress induced platelet aggregation in citrated whole blood 

(IC 90 100 nM) as measured using the PFA-100 platelet function analyzer. Finally, stabilized 

aptamer has been dosed in cynomolgus macaques and inhibits platelet function as assessed 

by BIPA. Thus the anti-vWF aptamer may be useful as a novel anti-platelet agent for the 

treatment of ACS patients. 

P80 

Dose Dependent Effects of PAR1 Signaling by the Protein C Pathway and 

by Thrombin on Endothelial Permeability 

Clemens Feistritzer, Ross Lenta, Matthias Riewald; The Scripps Rsch Institute, La Jolla, CA 

The endothelial cell monolayer at the blood-tissue interface controls the exchange of proteins 

and cells, and breakdown of this barrier plays a major role in inflammation. Proinflammatory 

signaling by the blood coagulation protease thrombin through protease activated receptor-1 

(PAR1) is known to disrupt endothelial barrier integrity and thrombin concentrations of 80 pM 

or higher rapidly increased endothelial permeability in a dual chamber system using 

HUVEC-derived EA.hy926 cells. Activated protein C (APC) can also activate PAR1 but only very 

high APC concentrations (200 nM) increased basal permeability in our system, consistent with 

the concept that APC is a poor PAR1 agonist in comparison to thrombin. In contrast, a 3 hour 

preincubation with lower APC concentrations enhanced endothelial barrier integrity in 

subconfluent cells and diminished thrombin induced hyperpermeability in confluent cells 

dependent on binding to endothelial protein C receptor, activation of PAR1 and crossactivation 

of sphingosine 1-phosphate (S1P) pathway signaling. Preincubation of endothelial cells with a 

PAR1 specific agonist peptide or low concentrations of thrombin (20 – 40 pM) had a similar S1P 

pathway-dependent barrier enhancing effect. Preincubation with higher concentrations of 

thrombin did not lead to barrier protection and did not desensitize the subsequent PAR1dependent 

hyperpermeability response to 5 nM thrombin. A thrombin variant (W215A/E217A) 

with increased relative specificity for protein C activation than PAR1 activation was used to 

establish that the endogenous protein C activation pathway on the cell surface is linked to 

barrier protective PAR1 signaling. These results indicate that the same receptor PAR1 can 

mediate opposite effects on endothelial barrier integrity dependent on the kinetics of receptor 

activation by the interdependent thrombin and protein C pathways. 

Adjunctive Therapy During Percutaneous Coronary Intervention with 

Bivalirudin Reduces Myeloperoxidase (MPO) Levels as Compared with 

Eptifibatide Plus Unfractionated Heparin 

Hilary A Berlin, Valerie L Fierschnaller, Margaret J Hall, Susan S Smyth, Univ of North 

Carolina at Chapel Hill, Chapel Hill, NC; Steven R Steinhubl, Univ of Kentucky, Lexington, 

KY; Allison C Keenan, Justin C Young; Univ of North Carolina at Chapel Hill, Chapel Hill, NC 

Previous studies using only unfractionated heparin (UFH) and aspirin for adjunctive therapy in 

the setting of percutaneous coronary interventrions (PCI) have found increased levels of platelet 

activation and inflammation post-procedure. The addition of platelet GPIIb/IIIa receptor 

antagonists to this regimen has been shown to have beneficial effects in preventing platelet 

aggregation and acute ischemic events. The REPLACE-2 trial demonstrated that the directthrombin 

inhibitor bivalirudin during PCI providedDownloaded equivalent protection from 

periprocedural and 

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chronic ischemic complications compared with UFH plus planned use of GPIIb/IIIa antagonists. 

Therefore, we sought to determine the effect of bivalirudin on systemic markers of platelet 

activation and inflammation in the first week after PCI. 24 patients undergoing a non-urgent PCI 

of a coronary artery were randomized to eptifibatide (180 g/kg double bolus and a 2 

g/kg/min infusion for 18 to 24 hours) plus unfractionated heparin (50–70 U/kg) or bivalirudin 

(0.75 mg/kg bolus then 1.75 mg/kg/hr infusion until end of PCI). Clopidogrel 300 mg was given 

at the end of the procedure in all patients. Platelet activation and inflammatory markers were 

determined immediately prior to and after the PCI, the next morning, at 3–4 days, and at 7 

days. No significant differences between groups were observed in platelet activation as 

determined by P-selectin expression, PAC1 binding, sCD40L, or plasma RANTES levels. In both 

treatment groups, the inflammatory markers IL-6 and CRP levels transiently increased on the 

day after the procedure. Levels of MPO, an enzyme released by neutrophils and associated with 

increased cardiac risk in acute coronary syndromes, were significantly higher in the UFH 

eptifibatide treatment group immediately after the procedure. In an in vitro assay, platelet 

P-selectin induced neutrophil MPO release, suggesting that platelet-neutrophil interactions may 

contribute to MPO levels in vivo. Given that MPO levels may be both a marker and mediator of 

vascular inflammation, the reduction in MPO levels associated with bivalirudin use during PCI 

may contribute to the drug’s short-term and long-term efficiacy. 

P82 

Up-Regulation of the Leptin Receptor on Activated Platelets may Increase 

the Proaggregatory Effect of Circulating Leptin 

Claudia Dellas, David J Loskutoff; The Scripps Rsch Institute, La Jolla, CA 

Background: Administration of the satiety factor leptin to mice promotes arterial thrombosis, 

while mice that lack leptin develop unstable thrombi. These results suggest that endogenous 

leptin contributes to the thrombotic process in mice. Despite the potential clinical importance 

of these observations for obese hyperleptinemic humans, little is known about the underlying 

mechanisms. In this report, we test the hypothesis that platelet activation results in the release 

of stored leptin and in the up-regulation of the leptin receptor. Methods and Results: ELISA 

assays demonstrate that leptin levels are similar in human plasma compared to lysates from 

platelet rich plasma (9812% of plasma), releasates from concentrated platelets in plasma 

(9419%) and serum (9718%) from 12 healthy donors. Moreover, only traces of leptin 

(0.140.15 ng/10 9 platelets or less than 5.35.8 molecules/platelet; n6) were detected in 

lysates prepared from washed platelets whether tested in ELISA, immunoprecipitation or 

receptor pull down experiments. Thus, human platelets do not contain significant amounts of 

leptin as postulated. Platelets do express the leptin receptor as confirmed in flow cytometry and 

western blotting experiments. Furthermore, only the long isoform of the receptor is expressed 

and this isoform is highly glycosylated with a MW of 200 kDa (132 kDa after deglycosylation). 

Activation of platelets with thrombin, ADP, TRAP-6, or epinephrine induced a rapid upregulation 

of the leptin receptor as determined in leptin binding experiments (e.g., 93%2% 

positive platelets after activation with thrombin vs. 41%6% without stimulation; n3 

donors). The binding of labelled leptin to platelets was specific since it could be competed by 

soluble leptin receptor. Conclusion: Platelets do not contain significant amounts of leptin, and 

are probably not the source of endogenous leptin in thrombi. It seems more likely that activation 

of platelets leads to the up-regulation of the leptin receptor on platelets, increased binding of 

circulating leptin to platelets, and enhanced platelet activation. 

Matrix GLA Protein Affects Receptor Preference of the Transforming 

Growth Factor- Family 

Yucheng Yao, Alejandra Torres, Amina F Zebboudj, Kristina I Boström; UCLA, Los Angeles, 

CA 

Matrix GLA protein (MGP), an alleged calcification inhibitor, enhances transforming growth 

factor-1 (TGF-1) signaling and VEGF expression in bovine aortic endothelial cells (BAEC). It 

is also known to bind and inhibit bone morphogenetic protein-2 (BMP-2). We hypothesized that 

MGP exerts its effect on signaling by changing the preference of the TGF-/BMP ligands for 

particular receptors. Using HA-tagged receptors and luciferase reporter genes specific for 

TGF- signaling through the activin-like kinase receptor 1 (ALK1) or ALK5, we studied the 

relationship between MGP and TGF- receptors in BAEC. Our results showed that MGP bound 

to ALK1 and ALK5 only in the presence of the TGF-1 ligand, and that increased expression 

of TGF- type II receptor (TRII) increased the binding to ALK5. Correspondingly, increased 

expression of MGP enhanced TGF-1 signaling through ALK1, but increased expression of 

TRII superseded this enhancement and promoted ALK5 signaling. We then hypothesized that 

MGP may counteract or enhance the effect of other type II receptors. Using luciferase reporter 

gene assays, we studied the effect of increased expression of TRII, the BMP type II receptor 

(BMPRII), and the activin type II receptors (ActRII and ActRIIB) on signaling in absence or 

presence of MGP expression. BAEC are known to endogenously express TGF-, BMP and 

activin. Our results showed that MGP expression had minimal effect on the TRII induced 

decrease in ALK1 signaling consistent with above results. BMPRII induced signaling was 

inhibited by MGP expression in a way resembling the effect of exogenous Noggin, a BMP 

inhibitor. ActRII and IIB induced signaling was in both cases enhanced by MGP expression. 

ActRII induced signaling was inhibited by Noggin suggesting that BMP was the responsible 

ligand. ActRIIB induced signaling, on the other hand, was unaffected by both anti-TGF- 

antibodies and Noggin suggesting that activin was the responsible ligand. Together our results 

suggest that MGP affects the receptor preference of TGF- superfamily growth factors, most 

likely by binding to the growth factors. The findings may explain the defects in calcification and 

morphogenesis by guest in MGP on deficiency. June 29, 2013 

P83

P84 

Low-Dose Spironolactone Promotes Adaptive Arterialization of Vein Grafts 

but Does not Prevent Stenosis of Angioplastied Coronary Arteries 

Federico Milla, Matthew D Bacchetta, Mun K Hong, Ying Zhou, Rong Chen, Edward 

Southard, Leonard Y Lee, Charles A Mack, Karl H Krieger, O W Isom, Wilson Ko, Daniel F 

Catanzaro; Weill Med College, Cornell Univ, New York, NY 

The efficacy of vein grafts used in coronary artery bypass and peripheral artery repair is limited 

by excessive hyperplasia and fibrosis that may occur during adaptive remodeling. Aldosterone 

blockade has antifibrotic effects on the heart and has been shown to prevent restenosis in 

coronary artery angioplasty. In the present study, we sought to determine whether low-dose 

spironolactone might alleviate maladaptive vein graft arterialization. Twenty-four Yorkshire pigs 

weighing 40 –50kg received a daily dose of aspirin 325mg and Plavix 75mg throughout the 

study. Twelve pigs were randomized to treatment with a daily oral dose of spironolactone 25 

mg. All 24 animals underwent a right carotid artery interposition graft using a segment of the 

right external jugular vein, and 5 days after surgery, underwent angiography of carotid and 

coronary arteries. At that time, a bare metal stent was placed in the left anterior descending 

artery (LAD), and balloon angioplasty was performed on the circumflex coronary artery (LCX). 

Repeat carotid and coronary angiograms were performed just prior to euthanasia and graft 

excision at 30 days. Angiography revealed that venous grafts of animals treated with 

spironolactone had 2-fold greater lumen diameters than controls at 5 days and that this 

difference persisted at 30 days. Lumen areas determined histologically at mid-graft were 

10-fold greater in spironolactone-treated animals than in controls. Neointima and total vessel 

wall area were also 3–4-fold greater in spironolactone-treated animals compared to controls. 

However, vessels from spironolactone-treated animals showed lower collagen accumulation in 

the remodeled vessel wall. In the coronary circulation there were no differences between 

treatment groups in intimal hyperplasia of either the stented LAD, or the unstented LCX. Taken 

together, these observations suggest that spironolactone facilitates constructive remodeling 

that does not depend on the reduction of hyperplastic changes, but may involve structural 

changes of the remodeled vein wall. At low-dose, spironolactone does not prevent intimal 

hyperplasia in angioplastied arteries. 

Impaired Endothelium-Dependent Vasorelaxation was Predictive for 

Intima-Media Thickening in Mice 

Chun Chen, Vyacheslav A Korshunov, Michael P Masset, Bradford C Berk; Univ of 

Rochester, Rochester, NY 

We recently showed that there was greater intima-media thickening (IMT) induced by low flow 

in FVB and SJL mouse strains compared to C3H. The purpose of this study was to investigate 

whether differences in vascular reactivity exist among these three mouse strains. We tested the 

hypothesis that mice with increased IMT would have greater vasoconstrictor responses in 

response to phenylephrine (PE) and impaired relaxation in response to acetylcholine (Ach) and 

calcium ionophore (A23187). Thoracic aortic rings from FVB, SJL and C3H mice were subjected 

to increasing doses of PE, Ach and A23187. The responses of aortic rings to Ach and A23187 

were assessed after 70% submaximal precontraction with PE. The maximal responses for Ach 

were markedly less in aortas from FVB and SJL compared to C3H mice (Max vasodilation to 

Ach, mN: FVB-2.5/-0.4; SJL-1.24/-0.2 and C3H-4.4/-0.7). EC50 for Ach also were 

significantly less in aortas from FVB and SJL compared to C3H (Log [Ach] EC50, mM: 

FVB-4.3/-1.5; SJL -4.1/-0.6 and C3H-6.9/-0.2). Similar to responses to Ach, the 

endothelium-dependent vasodilator A23187 exhibited less relaxation in aortas from FVB and 

SJL mice compared to C3H mice. There was no significant difference in ED50 to PE among 

FVB, SJL and C3H mice. Significant strain-dependent variation in aortic vascular responsiveness 

in mice correlated with vascular remodeling: decreased vasodilation was associated with 

increased IMT. These data suggest that the genes involved in endothelium-dependent 

vasodilation contribute to vascular remodeling induced by low flow. 

P86 

Cyclic Strain Induces Changes in EP24.15 Subcellular Localization, and 

Peptide Cleavage; Possible Implications for Endothelial Cell Fate Decisions 

Eoin J Cotter, Paul A Fitzpatrick, Yvonne Birney, Vascular Health Rsch Cntr, Dublin 9, 

Ireland; T J Wu, Uniformed Services Univ of the Health Sciences, Bethesda, MD; Mark J 

Glucksman, Rosalind Franklin Univ of Medicine and Science, Chicago, IL; Paul A Cahill, 

Philip M Cummins; Vascular Health Rsch Cntr, Dublin 9, Ireland 

Introduction: Endopeptidase EC3.4.24.15 (EP24.15, thimet oligopeptidase) is a cyclic straininducible 

zinc metalloendopeptidase expressed within the vascular endothelium where it 

participates in regulation of vascular tone and remodeling via the hydrolysis of endotheliumderived 

peptides. In this study, we investigate if strain modulates EP24.15 subcellular 

localization and EP24.15-specific cleavage of the vasopeptide substrates bradykinin (BK) and 

angiotensin-I (Ang-I) in the extracellular compartment. A putative role for EP24.15 in 

strain-induced endothelial cell fate decisions was also investigated. Methods: A Flexercell® 

Tension Plus FX-4000T System (Flexcell International Corp., NC) was employed to culture 

bovine aortic endothelial cells (BAECs) under conditions of defined equibiaxial cyclic strain (5% 

strain, 1 Hz, 24 h). Post strain, cells were monitored for EP24.15 protein expression by 

immunocytochemistry in conjunction with confocal microscopy (total cell and extracelullar 

plasma membrane-associated expression). HPLC was employed to monitor cleavage of BK and 

Ang-I incubated with control and strained BAECs for 3 h following selective attenuation of 

EP24.15 function using pharmacological and molecular inhibition strategies. The effect of 

EP24.15 inhibition on strain-induced BAEC tube formation and migration was also monitored. 

Results: Cyclic strain increased both total and extracellular (i.e. plasma membrane-associated 

and secreted) levels of EP24.15. Strain also increased cleavage of exogenous BK and Ang-I by 

whole BAECs [2.20.3 and 1.50.1 fold, n3, P0.05], respectively, increases which were 

almost completely attenuated (90%) by EP24.15 Downloaded inhibition. Finally, from 

strain-induced increases 

P85 




in BAEC tube formation and migration (1.90.2 and 1.60.1 fold, respectively, n3, P0.05), 

were significantly attenuated following EP24.15 inhibition. Discussion: Our results suggest that 

cyclic strain significantly impacts on EP24.15 localization and function in vascular ECs. 

Moreover, the apparent sensitivity of strain-induced BAEC tube formation and migration to 

EP24.15 blockade suggests that this enzyme may play an important role in hemodynamic 

regulation of endothelial cell fate decisions. 

P87 

Egln3 Regulates Gene Expression and Differentiation of Skeletal Muscle 

Jian Fu, Keon Menzies, Mark B Taubman; Univ of Rochester, Rochester, NY 

The members of the EGLN family including EGLN 1, 2, and 3 have been shown to endow the 

a subunit of hypoxia-inducible factor with prolyl hydroxylase activity. However, the biological 

function of the EGLN family of hydroxylases remains largely unknown. We have previously 

reported that EGLN3 mRNA and protein are expressed in the arterial wall only by smooth 

muscle cells and are upregulated in the intima of injured arteries and atherosclerotic plaques. 

EGLN3 mRNA and protein are also up-regulated when C2C12 skeletal muscle cells are induced 

to differentiate, whereas they are barely detectable in undifferentiated, proliferating myoblasts. 

This expression profile raised the possibility that EGLN3 might be involved in the regulation of 

muscle gene expression and skeletal myogenesis. We employed several approaches to test this 

hypothesis. Antisense oligonucleotides to EGLN3 markedly inhibited myotube formation when 

C2C12 cells were allowed to differentiate in low serum (3% horse serum), whereas control 

oligonucleotides had no effect. Treatment of C2C12 cells with the prolyl hydroxylase inhibitors 

(defreoxamine and DMOG, 100 uM and 400 uM, respectively, used in the current study) also 

resulted in impaired formation of myotubes and decreased expression of muscle differentiation 

markers such as myogenin and troponin T. Treatment of C2C12 cells with EGLN3 small 

interference RNAs also decreased myogenin expression, whereas overexpression of exogenous 

EGLN3 increased levels of myogenin protein. These findings suggest that EGLN3 may be an 

important regulator of gene expression and differentiation in skeletal muscle. Studies are 

underway to further elucidate the molecular mechanism(s) underlying the effects of EGLN3 on 

myogenin and skeletal muscle differentiation. 

Genetic and Pharmaceutical Reduction of Epidermal Growth Factor 

Receptor (egfr/erbb1) Signaling Results in Valvular and Arterial 

Calcification 

Delia J Barrick, Hilary Berlin, Elizabeth L Clore, Susan Smyth, David W Threadgill; Univ of 

North Carolina at Chapel Hill, Chapel Hill, NC 

Vascular calcification is a prominent feature of atherosclerosis, and in this context refers to the 

deposition of calcium phosphate mineral in the intima of the vessel wall. Calcification was 

previously thought to be a degenerative, end-stage process of vascular disease, as calcification 

of the blood vessels and heart valves occurs with advanced age. However, the presence of 

proteins that are associated with bone formation, such as bone morphogenic proteins (BMPs), 

osteonectin and osteocalcin in calcified tissues suggests that calcification is an organized 

regulated process similar to mineralization in bone tissue. Accumulating evidence from in vivo 

and in vitro studies substantiates a role for the epidermal growth factor receptor (EGFR) in 

chondrocyte and osteoblast differentiation and bone formation. Recently, EGFR signaling was 

also shown to be critical for normal cardiac valvulogenesis, as mice null for HB-EGF, an EGFR 

ligand, or homozygous for the Egfr wa2 hypomorphic mutation have hyperplastic cardiac 

valves. This phenotype is thought to result from lack of EGFR regulation of the BMP signaling 

pathway. Additionally, we have detected numerous osteoblasts and chondrocytes within the 

cardiac valves of young C57Bl/6J (B6) Egfr wa2/wa2 mice and cartilagenous metaplasia in the 

aortic outflow tract. Von Kossa staining reveals increased calcification of cardiac valves, 

coronary arteries and aortic outflow tract compared to B6 Egfr wa2/ littermates. Wild-type B6 

mice chronically exposed to the specific EGFR inhibitor, EKB-569, have similar morphologic and 

histologic changes. Together, these observations suggest that EGFR may have an active role 

in curtailing valvular and arterial calcification. Future studies will investigate gene expression 

changes in markers for active mineralization as well as atherosclerotic plaque formation in the 

aortas of EGFRwa2 mice and EGFR inhibitor-exposed mice. 

P89 

CREB and TYPE 1 Collagen Impact on Vascular Smooth Muscle Cell Gene 

Regulation and Phenotype 

Yolanda E Bogaert, Jody Grippa, Raphael Nemenoff, Jane Reusch; UCHSC, Denver, CO 

Atherosclerosis is a major cause of morbidity and mortality in the U.S. In response to vascular 

injury, vascular smooth muscle cells (VSMC) undergo phenotypic modulation, a switch in 

phenotype from quiescent (contractile) to active (proliferative, migratory) that plays an 

important role in atherosclerotic plaque instability. Structural matrix proteins affect VSMC 

phenotype, but little is known about the mechanism of this effect. To better understand the 

impact of matrix protein on VSMC phenotype we cultivated VSMC on native Type 1 collagen 

fibrils (NC) or denatured collagen (DNC). Adult VSMC showed dramatic decreases in VSMC 

markers of differentiation, smooth muscle -actin (SMA) and SM22 in cells plated on NC for 

24 hours. These decreases involved transcriptional control, as there was a significant reduction 

in SMA promoter activity. We hypothesized that NC was leading to de-differentiation of VSMC 

by decreasing expression of key transcriptional regulators of differentiation SRF (serum 

response factor) and CREB (cAMP response element binding protein). VSMC plated on NC for 

24 or 48 hours had significant decreases in CREB and SRF expression. We investigated the 

mechanisms involved in decreased SMA and CREB expression on NC. Growth of VSMC on NC 

resulted in increased activation Akt, part of the PI3kinase signaling cascade, as compared to 

cells on DNC. Pharmacological disruption of Akt signaling blocked NC downregulation of SMA 

and CREB. We next assessed the in vivo relationship between Type 1 collagen, CREB, SRF and 

SMA expression. by guest Aortic on lysates June from 29, fatty 2013 Zucker rats were examined for expression of Type 1 

P88

E-68 Vol 25, No 5 May 2005 

collagen, phospho-Akt, CREB and SRF by Western blot. Type 1 collagen expression increased 

in fatty rats by 200% between 4 –14 weeks, phospho-Akt increased by 80%, CREB content 

decreased to 60% of baseline and SRF protein decreased to 20% of baseline. These alterations 

were prior to changes in SMA and were associated with decreases in the active form of focal 

adhesion kinase. Summary: Exposure of VSMCs to Type 1 collagen correlates with VSMC 

de-differentiation both in vivo and in vitro. Accumulation of Type 1 collagen following vascular 

injury may be an important mechanism for VSMC de-differentiation associated with atherosclerotic 

plaque instability. 

A New Antagonist Screening Model of Interleukin 6-Receptor 

Bingsheng Chang; UTSouthwestern Med Cntr, Dallas, TX 

Interleukin 6 (IL-6), which displays a broad range biological activities on many kinds of cells, 

such as lymphocytes, hematopoietic stem cells, liver cells and neuronal cells, is a kind of 

important cytokine. Additionally, IL-6 is involved in many pathology processes of disease such 

as inflammation, rheumatoid arthritis, tumor, cardiovascular diseases and autoimmune 

diseases. IL-6 exhibits its role on target cells through a double-stranded receptor complex 

consisting of IL-6R and a signal transduction subunit (gp130).IL-6R is one of transmembrane 

glycoprotein which has two types of natural receptors: gp130 binding with membrane and 

soluble IL-6R.Both of them play essential role during the process of IL-6 signal transduction. 

To identify the antagonist of IL-6R from natural products, we developed a novel screening 

model with recombinant hsIL-6R from insect cells as target based on the competitive 

receptor-ligand interactions in this study. The hsIL-6R gene were cloned into pAcGP67B vector 

and co-transfected with wild type AcMNPV DNA into insect cells. The recombinant baculovirus 

was purified by plaque assay and end-point dilution and was amplified up to 10 8 pfu/ml. A 

47kDa protein was confirmed by ligand-receptor binding assay and western-blot. The purified 

hsIL-6R was used to sceen its antagonist. Ligand was first coated in the microplate, the sIL-6R 

and natural products and antibody were subsequently added. Using this sandwich-like 

screening model, we have screened more than 1000 microorganisms metabolic products and 

found 10 positive strains. The positive ratio is 1%. The hsIL-6R antagonist screening model is 

new one, the work based on it is going further. This work will be useful for the new drug study 

of the diseases linked to IL-6. 

P91 

Decreased Types I and III Collagen Levels in Rodent Experimental Aortic 

Aneurysms in Males Compared to Females 

Brenda S Cho, Karen Roelofs, Peter K Henke, Matthew J Eagleton, James C Stanley, Gilbert 

R Upchurch, Jr.; Univ of Michigan, Ann Arbor, MI 

Objective: The objective of this study was to examine differences in collagen subtype levels in 

males compared to females during rodent experimental abdominal aortic aneurysm (AAA) 

formation. Methods: Infrarenal abdominal aortas of adult male and female Sprague Dawley rats 

(n 6 per group) were isolated and perfused with pancreatic elastase (6 units/ml) or saline 

(control). Aortic diameters were measured on post-operative day 7 prior to aortic explantation. 

Aortic tensiometry was performed on whole tissue segments to determine aortic tensile 

strength in newtons per square millimeters (N/mm 2 ) using an instron machine. Collagen was 

extracted from aortic tissue using pepsin solubilized in acetic acid. Collagen subtypes I, III, and 

IV protein levels were determined by Western Blotting. Aortic sections were stained for total 

collagen with Masson’s Trichrome. Results were analyzed by repeated measures ANOVA. 

Results: Seven days following aortic perfusion, mean (SEM) aortic diameter increased 

significantly in elastase-perfused aortas of males compared with females (178 23% male 

vs. 107 7% female; P0.005). Type I collagen levels decreased 72% (P0.001) and type 

III decreased 58% (P0.001) in males compared to females. No statistically significant 

differences in collagen type IV levels (P0.131) were detected. By Masson’s Trichrome, less 

adventitial collagen was present in the elastase-perfused aortic sections of males compared 

with females. Conclusions: This study documents a decrease in collagen types I and III in 

experimental aortic aneurysms of male rats compared to females. Strategies aimed at 

promoting collagen synthesis reduce the risk of aortic rupture associated with diminished 

collagen in patients with aortic aneurysms. 

P92 

Role of Endothelin, Nitric Oxide and Matrix Metalloproteinases in Inward 

Arterial Remodeling in Response to Decreased Blood Flow 

Dorota Dajnowiec, B. L Langille; Univ of Toronto, Toronto, Canada 

Arterial remodeling in response to altered blood flow/shear forces greatly influences development, 

adaptation, and the progression of pathologies in the vascular system. We hypothesized 

that inward remodeling of arteries in response to decreased blood flow is initiated by 

up-regulation of endothelin (ET) and down-regulation of nitric oxide (NO) production. To test this 

hypothesis, ligation of the left external carotid artery was performed on adult male NZW rabbits, 

which reduced left common carotid artery blood flow by 70%. Arteries were examined at 3 days 

(acute), when significant narrowing was entirely vasoconstrictor, and at 14 days (chronic), 

when remodeling was entrenched. The roles of ET and NO in narrowing of arteries were 

assessed by the administration of the ET receptor antagonist, TAK-044, and/or the NO synthase 

inhibitor, L-NAME. TAK-044 significantly suppressed acute (P0.05) and chronic (P0.02) 

vessel narrowing. Acute inhibition of NO production failed to influence vessel narrowing; 

however, L-NAME administration for 14 days significantly inhibited the vascular narrowing as 

compared to controls (P0.02). The combined inhibition of ET and NO system did not produce 

additive effects. We also tested the hypothesis that matrix metalloproteinases (MMPs) 

participate in inward remodeling following blood flow reduction by administering the 

non-specific MMP inhibitor, doxycycline. Similar to ET receptor block, doxycycline significantly 

reduced (P0.003) chronic inward remodeling even at early time points when narrowing was 

entirely due to vasoconstriction. MMP-2 has beenDownloaded shown to process from 

big endothelin to produce 

P90 



a novel vasoconstrictor, ET[1–32], rather than ET[1–21], and our data are consistent with a role 

for this pathway in initiating inward remodeling after flow reduction. 

The Adaptator Molecule Lnk Controls Tnfa-Induced Endothelial Cell 

Activation via a Mechanism Implicating Heme Oxygenase-1 and the 

PI3-K/Akt Pathway 

Juliette Fitau, Gwenola Boulday, Flora Coulon, Beatrice Charreau; INSERM, Nantes, France 

Lnk belongs to the adaptator molecule family, including APS and SH2-B, that participates in 

intracellular signalling by protein-protein interactions. Lnk has no enzymatic activity but is 

implicated in the differentiation pathway of B lymphocytes, hematopoietic stem cells and 

platelets as well as in the activation pathway of T lymphocytes. Previously, we have shown that 

Lnk is expressed in endothelial cells (ECs) and regulated by the pro-inflammatory cytokine, 

TNFa. The aim of this study was to evaluate the role of the Lnk protein in the TNFa-mediated 

pathway in ECs. We overexpressed Lnk in human primary ECs derived from umbilical cords 

(HUVECs) using a recombinant adenovirus. Quantitative RT-PCR, flow cytometry and western 

blot analysis were used to analyse the effect of Lnk on endothelial activation and to identify the 

signalling pathway and mechanism involved. Our results indicate that Lnk overexpression did 

not induce the adhesion molecules VCAM-1 and E-selectin in ECs but, in contrast, significantly 

reduced TNFa-mediated expression of VCAM-1 and E-selectin (at both the mRNA and protein 

levels) while ICAM-1 expression was unmodified. We also showed that Lnk had no effect on 

phosphorylation of IkB, the natural inhibitor of the transcription factor NFkB, or on it’s 

degradation. However, Lnk induced the transcriptional and protein expression of heme 

oxygenase-1 (HO-1) in ECs, suggesting that the anti-inflammatory properties of HO-1 are 

responsible for the down-regulation of E-selectin and VCAM-1. Furthermore, Lnk induced Akt 

phosphorylation (at Ser 473) in ECs. The treatment of ECs expressing Lnk with the specific 

PI3-Kinase inhibitor, Wortmannin, decreased Akt activation as well as HO-1 expression. These 

results show that Akt activation in ECs expressing Lnk is dependent on PI3-K and that the 

PI3-K/Akt pathway plays a key role in HO-1 regulation. In conclusion, altogether, our results 

suggest that Lnk modulates the inflammatory response in ECs via a mechanism involving the 

molecule HO-1 and the PI3-kinase/Akt pathway. 

P94 

Expression of Thermolysin-Like Zinc Metalloendopeptidases in Vascular 

Endothelial Cells is Regulated by Shear Stress and Reactive Oxygen 

Species 

Paul A Fitzpatrick, BSc, Eoin J Cotter, BSc, Yvonne A Birney, PhD, Dublin City Univ, Dublin, 

Ireland; Marc J Glucksman, PhD, Univ of Medicine and Science, Chicago, IL; Paul A Cahill, 

PhD, Philip M Cummins, PhD, Dublin City Univ, Dublin, Ireland; Rosalind Franklin; Univ of 

Medicine and Science, Chicago, IL 

Introduction: Numerous studies have demonstrated biochemical and functional modulation of 

the vascular endothelium by hemodynamic forces. In this study, we have examined the effect 

of pulsatile laminar shear stress on expression of Thermolysin-Like Zinc Metalloendopeptidases 

or TLZMs (e.g. EP24.15, ACE, ECE etc.), a pivotal family of vasopeptidases, in bovine aortic 

endothelial cells (BAECs). Furthermore, much recent evidence supports the recruitment of 

reactive oxygen species (ROS) by mechanotransduction signaling pathways. In this regard, 

recent studies highlight the ROS-dependent nature of ECE shear sensitivity. As such, we have 

also examined the putative effect of ROS (i.e. H 2O 2) on TLZM expression in static BAECs. 

Methods: A CELLMAX In Vitro Artificial Capillary System (Spectrum Biolabs, Ca) was employed 

to expose BAECs to low (0.5 dynes/cm 2 ) and high (25 dynes/cm 2 ) levels of pulsatile shear stress 

for 24 h. Following shear, cells were harvested for analysis of TLZM gene expression by 

Real-Time PCR. BAECs were also cultured under static conditions and treated with H 2O 2 (0 –100 

M, 1.5 h), and again subsequently harvested for analysis of TLZM gene expression. Results: 

Chronic shear stress significantly increased EP24.15, EP24.16 and ECE mRNA levels [4.41.2, 

5.21.4 and 1.70.0 fold, respectively, n3, P0.05], whilst ACE expression was attenuated 

[0.70.2 fold, n3, P0.05]. Moreover, 50 ìM H 2O 2 treatment of BAECs for 1.5 h increased 

EP24.15, ACE and ECE expression by 1.7, 1.4 and 1.1 fold, respectively. Interestingly, higher 

concentrations of H 2O 2 decreased expression of these enzymes. Discussion: Our results 

indicate that chronic pulsatile shear stress modulates TLZM gene expression in vascular 

endothelial cells both positively (EP24.15, EP24.16 and ECE) and negatively (ACE). Moreover, 

we have also shown that TLZM expression is dose-dependently ROS-sensitive. These findings 

confirm a role for hemodynamic factors in regulating TLZM expression and function within 

blood vessels. Furthermore, considering their ability to modulate TLZM expression levels under 

static conditions, a putative role for endogenous ROS in shear-dependent regulation of TLZM 

expression is proposed. 

P95 

Connective Tissue Growth Factor is Involved in the Angiotensin Ii Induced 

Collagen Synthesis in the Vascular Adventitial Fibroblast 

Pingjin GAO, Zaiqian Che, Dingliang Zhu; Shanghai Institute of Hypertension, Shanghai, 

China 

Connective tissue growth factor (CTGF) is a 38 kDa cysteine-rich, heparin-binding peptide that 

has the ability to produce more collagen and increase deposition of extracellular matrix 

components. Previous studies in our lab showed distribution of collagen type I and III were 

mainly located in vascular adventitia. This study tested whether CTGF could be expressed and 

participate collagen synthesis in vascular adventitial fibroblasts. The adventitial fibroblasts were 

isolated and cultured from Wistar-Kyoto rat (WKY) thoracic aorta. The expression of CTGF in 

fibroblasts and its conditional media was measured by Western blot or real time PCR. Collagen 

synthesis was assessed by the incorporation of [ 3H]proline into cultured adventitial fibroblasts. 

Our results by showed guest that on CTGF June is 29, expressed 2013in 

adventitial fibroblasts and its conditional media 

P93

in mRNA or protein levels. Ang II-induced expression of CTGF in a time- and dose-dependent 

manner with a maximal induction after 24 h and a dose of 10 –7mol/L. Ang II-induced 

expression of CTGF was suppressed by the AT 1 antagonist valsartan., whereas no effect was 

observed with AT 2 antagonist PD123319. Ang II-induced the incorporation of [ 3 H] proline into 

cultured adventitial fibroblasts in a dose-dependent manner. Meanwhile CTGF antisense 

oligodeoxynucleotide inhibited Ang II-induced the incorporation of [ 3 H]proline into adventitial 

fibroblasts. This study for the first time demonstrates that CTGF is present in cultured vascular 

adventitial fibroblasts and play a role in Ang II-induced collagen synthesis. 

Increased Expression of Cyclooxygenase-2 and the Prostanoid EP4 

Receptor in a Mouse Model of Abdominal Aortic Aneurysms 

Jonathan M Gitlin, Darshini B Trivedi, Victoria L King, Charles D Loftin; Univ of Kentucky, 

Lexington, KY 

Abdominal aortic aneurysms (AAA) involve inflammatory responses throughout the course of 

their development. Inflammatory cells infiltrate the vessel wall and begin the process of 

remodelling, which may cause eventual rupture of the vessel. Increased activity of 

cyclooxygenase-2 (COX-2) has been proposed to contribute to the inflammation associated with 

AAA formation by increasing production of the lipid mediator prostaglandin E2 (PGE2). PGE2 

binds cell surface receptors to increase MMP production and induce apoptosis of smooth 

muscle cells, pathologies often associated with AAAs. Recently, an animal model of AAA has 

been identified which utilizes chronic angiotensin II (AngII) infusion in mice. In the current study, 

we used the AngII-induced AAA model to examine the mRNA expression of COX-2 and the PGE2 

receptor EP4 in the abdominal aortas of mice. Twenty male mice (age 6 – 8 weeks) were 

implanted with osmotic pumps containing either saline or AngII (1000 mg/kg/day). The animals 

were treated with AngII for 21 days, at which point they were sacrificed and the aortas removed 

for total RNA extraction and gene expression analysis by real-time PCR. Expression of COX-2 

and EP4 were normalised to HPRT mRNA. COX-2 expression was significantly increased in the 

mice treated with AngII compared to saline controls (ratio COX-2:HPRT, AngII 0.30 /- 0.06, 

n10 vs. saline 0.13 /- 0.01, n9, p0.022 by Mann Whitney test). When subdivided into 

groups of animals which display AAAs, the difference was more pronounced (ratio COX-2:HPRT, 

AngII 0.39 /- 0.09, n5, vs. saline 0.13 /- 0.01, n9, p0.001 by Mann Whitney test). 

Expression of the EP4 receptor trended towards an increase following AngII treatment 

compared to saline, although this was not significant (ratio EP4:HPRT, AngII 0.79 /- 0.15, 

n10 vs. saline 0.45 /- 0.11, n9, p0.053 by Mann Whitney test). When the AngII treated 

animals were subdivided into those that displayed AAAs, the increase in EP4 expression was 

significantly higher compared to saline (ratio EP4:HPRT, AngII 0.91/- 0.25, n5 vs. saline 

0.45 /- 0.11, n9, p0.042 by Mann Whitney test). These data suggest a role for 

COX-2-derived PGE2 acting at the EP4 receptor in the development of AAA, and a possible 

therapeutic avenue for their treatment. 

P97 

Regulation of Endothelial Cell Nitric Oxide and Superoxide Generation by 

PECAM-1 

Reema Goel, Michelle Stapleton, Carmen Bergom, Peter J Newman, Blood Rsch Institute, 

Milwaukee, WI; Balaraman Kalyanaraman, Yanping Liu, David Gutterman, Med College of 

Wisconsin, Milwaukee, WI; Debra K Newman; Blood Rsch Institute, Milwaukee, WI 

In the process of flow-mediated dilation (FMD), increased fluid shear stress causes vascular 

endothelial cells (ECs) to produce nitric oxide (NO), prostacyclin (PGI 2) and endothelium-derived 

hyperpolarizing factor (EDHF), which together induce smooth muscle cell relaxation and blood 

vessel dilation. Recent studies suggest that FMD may be influenced by the vascular cell 

adhesion and signaling receptor, PECAM-1, as FMD was found to be impaired in coronary 

arteries derived from PECAM-1-deficient (KO) relative to wild-type (WT) mice. Reduced FMD in 

KO vessels was shown to be due, at least in part, to excess production of peroxynitrite, which 

impairs vessel responses to EDHF. Peroxynitrite is the product of reaction of NO with O 2 , both 

of which are elevated in KO relative to WT coronary arteries. To explore the mechanism 

underlying increased production of NO and O 2 in KO endothelial cells, we stably transduced 

endothelioma cells derived from the lungs and brains of KO mice with a lentivirus encoding 

wild-type murine PECAM-1 to obtain PECAM-1 reconstituted (RC) cell lines. NO and O 2 levels, 

assessed in cells loaded with the NO- and O 2 - sensitive fluorescent dyes, 

diaminofluoresceine-2 and dihydroethidium, respectively, were constitutively elevated in KO 

endothelioma cells. These data suggest that PECAM-1, when present, functions to negatively 

regulate production of NO and O 2 in endothelial cells. One potential source of NO and O2 is 

endothelial cell nitric oxide synthase (eNOS), which produces both NO and O 2 under 

pathophysiological conditions. Since PECAM-1 has recently been reported to associate with 

eNOS to downregulate its activity (Dusserre, N. et al., Arterioscler. Thromb. Vasc. Biol. 

24:1796 – 802, 2004), we looked for evidence of PECAM-1/eNOS complexes by both 

co-immunoprecitipation and immunofluorescence microscopy. In contrast to Dusserre et al., 

we found no consistent evidence for PECAM-1/eNOS interactions, suggesting that PECAM-1 

might regulate eNOS activity via indirect mechanisms. Future studies will explore candidate 

signaling pathways by which PECAM-1 regulates NO and O 2 generation in endothelial cells. 

P96 


investigates the role of the antioxidant proteins heme oxygenase-1 (HO-1) and ferritin as 

targets for, and potential mediators of the action of rosuvastatin and simvastatin. In cultured 

human endothelial cells (ECV 304), rosuvastatin increased HO-1 mRNA and protein in a 

concentration- and time-dependent fashion. Similar results were observed with simvastatin, 

suggesting a class effect for statins. HO-1 induction was accompanied by a marked increase 

in ferritin protein expression, a secondary antioxidant protein that is often induced in tandem 

with HO-1. The HMG-CoA product mevalonate and L-NAME had no influence on HO-1 induction 

by statins, demonstrating that isoprenoid- and NO-dependent pathways were not involved. 

HO-1 mRNA induction was abrogated in the presence of actinomycin D and cycloheximide. In 

cells transfected with a reporter gene construct containing the proximal 4 kB of the HO-1 gene 

promoter 5’-flanking region, significant upregulation of promoter activity was detected, 

indicating that regulatory elements binding to this region were involved in transcriptional HO-1 

induction by statins. Increased transcriptional expression of HO-1 by rosuvastatin was followed 

by a reduction of NADPH-dependent free radical formation. Blockade of free radical formation 

by rosuvastatin or simvastatin was rescued in the presence of the HO inhibitor SnPP suggesting 

a causal inference between HO-1 induction and antioxidant protection. Our results show that 

HO-1 and ferritin are regulatory targets for rosuvastatin and simvastatin activity in endothelial 

cells. Statins lead to promoter activation, transcription and protein accumulation of HO-1. This 

novel mechanism may contribute to, and explain the pleiotropic antioxidant, anti-inflammatory 

and antiatherogenic actions of statins. 

P99 

Controlled Release of Basic Fibroblast Growth Factor from Gelatin Hydrogel 

Sheet Inhibits Neointimal Hyperplasia and Improves the Patency of Vein 

Graft in Rat 

Tomonori Haraguchi, Kenji Okada, Kobe Univ Graduate Sch of Medicinem, Kobe, Japan; 

Yasuhiko Tabata, Kyoto Univ Institute for Frontier Med Science, Field od Tissue Engineering, 

Kyoto, Japan; Yoshimasa Maniwa, Yutaka Okita; Kobe Univ Graduate Sch of Medicinem, 

Kobe, Japan 

Background Saphenous vein graft (SVG) is still of much use in CABG. However, its long-term 

patency is insufficient. Collapse of the intimal layer of vein graft by the mechanical forces of 

blood flow is one of the factors of smooth muscle cell (SMC) migration leading to SVC occlusion. 

It is recognized that basic fibroblast growth factor (bFGF) of an angiogenic factor is a survival 

factor of endothelial cells (ECs) by Raf-1 phosphorylation. The objective of this study is to 

confirm whether bFGF released from gelatin hydrogel sheet (GS) not only provides external 

mechanical supports for the vein graft, but also pharmacologically preserves the ECs and 

inhibits SMC migration. Methods Infra renal abdominal aorta was interposed with autologous 

femoral vein in 77 rats. The rats were divided into 3 groups as follows; no treated grafts (Group 

1n23), wrapped with bFGF free GS (Group 2 n26), wrapped with GS incorporating bFGF 

(Group 3 n28). The blood flow of graft, graft dilation, and the state of neointimal formation 

were assessed at 2, 4, and 8 weeks later. Pathological investigations involving immunohistochemistry 

with antibody specific for rat phosphorylated Raf-1 at Serines 338 (p-Raf-1) were 

also performed. Results GS was hydrolyzed completely within 3 weeks. The mean blood flow 

of grafts was higher and the neointimal formation was more suppressed in Group3 than that 

of the other groups significantly. The graft dilation was significantly inhibited in Group3 and 2 

compared with Group1. (Table ref.) The intimal layers in Group 1 and 2 were disrupted at 2 and 

4 weeks later, respectively, which was substituted by thicker-layered neointima. On the 

contrary, in Group 3 the intimal layer was kept intact with positive staining for p-Raf-1antibody. 

The neointima was thinner even at 8 weeks later. Conclusion This study demonstrates that the 

controlled release of bFGF from the GS inhibits the dilation and neointimal hyperplasia of vein 

graft by Raf-1 phosphorylation and could improve its patency. 

Group1 Group2 Group3 

Flow (ml/min) 9.23.3 9.83.9 13.62.6 * 

Dilation ratio (%) 104.970.9 55.778.7 59.658.7** 

% Neointima (%) 50.726.7 48.525.1 25.213.4*** 

*p0.003 vs Group 1 p0.017 vs Group 2 **p0.045vsGroup1***p0.015 vs Group 1 p0.047 vs Group 2 

P100 

Environmental Pollutants Induce Inflammatory Signaling: Implications in 

Atherosclerosis 

Bernhard Hennig, Zuzana Majkova, Gudrun Reiterer, Elizabeth Oesterling, Michal Toborek; 


Environmental pollutants, such as polychlorinated biphenyls (PCBs) are ubiquitous environmental 

contaminants that can activate endothelial cells thus leading to atherogenic events. In 

order to explore the mechanism of PCB-induced inflammation, various transcription factors and 

associated enzymes involved in PCB signaling were considered. Vascular endothelial cells were 

exposed to the coplanar PCB 77, and the non-coplanar PCB 153. Both PCBs significantly 

induced inflammatory transcription factors such as NF-B and signal transducers and 

activators of transcription-3 (STAT-3) as well as the downstream inflammatory genes COX-2 

and IL-6 at concentrations reported after acute exposure. Many of these pro-inflammatory 

genes are associated with or localized into lipid rafts such as caveolae and as such can 

P98 contribute to inflammatory events induced by environmental contaminants. There is evidence 

Heme Oxygenase-1 and Ferritin Induction by Rosuvastatin: A Protective 

that caveolae, a subset of lipid rafts characterized by proteins called caveolins, play a critical 

Antioxidant Stratagem in Endothelial Cells 

role in the pathology of atherosclerosis. Treatment of primary endothelial cells with PCB 77 (3.4 

M) resulted in a significant increase in gene expression of both caveolin-1 and the 

Nina Grosser, Anke Hemmerle, Kati Erdmann, Urte Hinkelmann, Martin Luther Univ, Halle, caveolae-associated signaling molecule annexin-2. It is possible that the transfer of PCBs to 

Germany; Nastiti Wijayanti, Stephan Immenschuh, Justus Liebig Univ, Giessen, Germany; 

these lipid rafts is linked to co-transport with albumin to albumin-binding glycoprotein gp60 

Graham Smith, Astra Zeneca, Macclesfield, United Kingdom; Henning Schröder; Martin 

localized in caveolae. Protection from PCB-induced inflammation was also studied using 

Luther Univ, Halle, Germany 

agonists of the anti-inflammatory transcription factors PPAR and . For example, pretreatment 

of cells with a PPAR agonist (fenofibrate, 10 M) or PPAR agonist (thiazolidinedione, 

HMG-CoA reductase inhibitors (statins) lead to cholesterol-independent (pleiotropic) anti- 25 M) prevented the PCB-induced DNA-binding activity of NF-B and STAT-3. In addition, 

inflammatory and antioxidant actions by asDownloaded yet unidentified from 

mechanisms. http://atvb.ahajournals.org/ 

This study PPAR agonists by guest protected on against June the 29, PCB 2013 77 and PCB 153-induced mRNA expression of COX-2 


E-70 Vol 25, No 5 May 2005 

and IL-6, suggesting that PPARs can down-regulate inflammatory signaling pathways induced 

by PCBs. Furthermore, PCB 77 inhibited PPAR DNA binding and transcriptional activity. In 

summary, we demonstrated a possible caveolae-dependent mechanism of PCB-mediated 

inflammation and the role of PPAR agonists in preventing PCB-induced endothelial activation 

and atherosclerotic events. Supported in part by grants from NIH/NIEHS (ES 07380). 

Molecular Mechanisms in the Progression of Aortic Valve Stenosis: 

Upregulation of Apoptosis, Osteopontin, and TGF- 1 in Calcified Valve 

Cusps Correlate with Severity of Stenosis 

Theresa Jacob, Geetanjli Sangwan, Sunil Abrol, Deepak Thekkoott, Hemavathy Kirugaval, 

Barbara Paris, Enrico Ascher; Maimonidas Med Cntr, Brooklyn, NY 

P101 

Purpose: This study investigates the hypothesis that expression of osteopontin (OPN), 

transforming growth factor - beta1 (TGF- 1) and the degree of apoptosis in calcified aortic 

valves correlate to the severity of aortic stenosis (AS). METHODS: Stenotic tricuspid aortic 

valves were retrieved from patients undergoing aortic valve replacement. Controls were valves 

from aortic regurgitation patients. The patient ages ranged 53 to 88 years (mean SD, 75.3 

8.5 yrs) and gender distribution was even in both groups. Principal risk factors, co-morbidities, 

and clinical symptoms were also compared. The valve area and peak gradient of flow for each 

patient was determined and the valves were stratified into 3 classes based on severity of 

stenosis. Levels of OPN, TGF- 1, and actin transcripts were quantified with the use of real-time 

RT-PCR. Western blotting with chemiluminiscent detection followed by densitometry was used 

to quantify OPN/ TGF- 1 expression. Immunolocalization of these proteins was also performed. 

Detection of apoptosis was by TUNEL assay. RESULTS: The AS valves demonstrated a 

significantly high expression of OPN and TGF- 1 transcripts. In AS specimens, there was more 

than a 200-fold increase in OPN expression as compared with controls (p0.001) and over a 

2-fold increase in TGF- 1 transcripts as compared with that in controls (p0.01). Further, the 

upregulation of OPN and TGF- 1, inversely correlated with valve area (p0.01), and positively 

correlated to the flow gradient in AS valves at mRNA levels (p0.01) and with the amount of 

extracellular matrix present in valve cusps. Although apoptosis was rarely observed in control 

valves, apoptotic bodies were frequent in AS specimens (p0.01). Apoptosis was observed in 

endothelial cells, vascular smooth muscle cells, fibroblasts, and interstitial cells and significantly 

correlated with disease severity. CONCLUSIONS: These results demonstrate for the first 

time a link between the magnitude of apoptosis, cellular dysfunction, cell turnover, OPN 

upregulation, TGF- 1 overexpression, and severity of AS disease. The increased expression of 

OPN /TGF- 1, linked to overproduction of extracellular matrix, and apoptosis, may be 

associated with progression of AS and deserves further study. 

P102 

Flow-Mediated Gab1 Tyrosine Phosphorylation is Required for Akt and 

Enos Activation in Endothelial Cells 

Zheng-Gen Jin, Chelsea Wong, Univ of Rochester, Rochester, NY; Jie Wu, H.Lee Moffit 

Cancer Cntr and Rsch Institute, Tampa, FL; Bradford C Berk; Univ of Rochester, Rochester, 

NY 

Fluid shear stress generated by blood flow modulates endothelial cell function via specific 

intracellular signaling events. Physiologically, fluid shear stress is the most important stimulus 

for the continuous formation of nitric oxide from endothelial nitric oxide synthase (eNOS) in 

vessels. Previously we have showed Src kinases-dependent transactivation of vascular 

endothelial growth factor receptor 2 (VEGFR2) is required for flow-mediated the phosphatidylinositol 

3-kinase (PI3K), Akt and eNOS activationin inendothelial cells (Jin et al, Circ Res 2003, 

93: 354 – 6). However, the exact mechanisms by which flow stimuated PI3K/Akt/eNOS signal 

pathways remain not well understood. The scaffold adaptor Gab1 (Grb2-assocated binder 1) 

plays an important role in receptor tyrosine kinase-mediated signal transduction. Here we 

found that laminar flow (shear stress 12 dynes/cm2) rapidly stimulated Gab1 tyrosine 

phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial 

cells, in parallel with Akt and eNOS activation. Gab1 phosphorylation as well as activation of 

Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 and VEGFR2 kinase 

inhibitors SU1498 and VTI, suggesting flow-mediated Gab1 phosphorylation is Src kinasedependent 

and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally 

important because flow stimulated association of Gab1 with the PI3K subunit p85 in a 

time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites 

inhibited flow-mediated Akt and eNOS activation. Finally, knowndown of endogenous Gab1 by 

siRNA attenuated flow-mediated Akt and eNOS activation. These data demonstrate a critical 

role for Gab1 in flow stimulation of PI3K/Akt/eNOS Downloaded signaling pathway from 

in endothelial cells. 

P103 

Angiotensin II Activated Integrins Alpha-v Beta-3 and Alpha-v Beta-5 of 

Vascular Smooth Muscle Cells 

Seung-Jae Joo, Ki-Seok Kim; Cheju National Univ College of Medicine, Jeju, Republic of 

Korea 

Background: Integrins mediate the migration and proliferation of cardiac fibroblasts and 

vascular smooth muscle cells. We assessed the hypothesis that angiotensin (AT) II activates 

integrins alpha-v beta-3 and alpha-v beta-5 in human aortic smooth muscle cells (HASMC) 

Method: The expression of integrins alpha-v beta-3 and alpha-v beta-5 on HASMC were 

analysed by flow cytometry. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium 

bromide) assay was used to evaluated the proliferation of HASMC. Activations of integrins and 

the underlying signal transduction pathway were evaluated by the adhesion assay using 

prothrombin as an activation-dependent ligand. Results: AT II did not increase the expressions 

of integrins alpha-v beta-3 and alpha-v beta-5 and the proliferation of HASMC. HASMC had 

more integrin alpha-v beta-5 than alpha-v beta-3. AT II increased the adhesion of HASMC to 

prothrombin in a dose-dependent manner, which was inhibited by [Sar1, Thr8]-angiotensin II, 

an AT 1 receptor antagonist. The adhesion was prevented partially by LM609, which blocks 

integrin alpha-v beta-3 (182.8% inhibition; n5, p0.05) and markedly by P1F5, which 

blocks integrin alpha-v beta-5 (891.2% inhibition; n5, p0.05). U73122, an inhibitor to 

phospholipase C, attenuated the AT II-stimulated adhesion of HASMC in a dose-dependent 

manner (5 M, 563.2% inhibition; 25 M, 952.0% inhibition; n5, p0.05). Conclusions: 

AT II had no effects on the expressions of integrins alpha-v beta-3 and alpha-v beta-5 and the 

proliferation of HASMC, but it activated integrins alpha-v beta-3 and alpha-v beta-5 in HASMC 

through phospholipase C pathway after AT 1 receptor binding. The adhesion of HASMC to 

prothrombin appeared to be mediated mainly by integrin alpha-v beta-5. 

WITHDRAWN 



Apoe Expression Promotes Sterol Efflux in the Absence of Abca1 

Expression 

Zhi H Huang, Theodore Mazzone; Univ of Illinois at Chicago, Chicago, IL 

P104 

P105 

Apolipoprotein E (apoE) and the ATP-binding cassette transporter A1 (ABCA1) are two key 

molecules in the regulation of cellular lipid trafficking and prevention of atherosclerosis. The 

aim of this study was to investigate the relationship between endogenous apoE and 

ABCA1-mediated lipid efflux. We isolated mouse dermal fibroblasts (MDF) from ABCA1 -/- mice 

and increased apoE expression by adenoviral transduction. Increased apoE expression 

promoted both cholesterol (0.89 0.04 vs. 0.54 0.08 g/mg protein, p0.01) and 

phospholipid (0.77 0.07 vs. 0.54 0.02 g/mg protein, p0.05) efflux compared to cells 

infected with control adenovirus. The absolute magnitude of the effect of apoE on sterol efflux 

in the absence of ABCA1 was even greater when ABCA1 -/- MDF were loaded with cholesterol 

prior to apoE expression (39.19 2.93 vs. 19.78 3.12 g/mg protein, p0.01 in 

apoE-expressing vs. non-expressing cells respectively). Endogenous apoE expression also 

increased sterol efflux in the presence of extracellular phospholiposomes in an ABCA1independent 

manner. To differentiate between the effects of endogenous and exogenous apoE, 

we investigated the ability of apoE-containing conditioned medium collected from apoE 

adenovirus infected ABCA1 -/- MDF for promoting sterol efflux. The addition of conditioned 

medium containing apoE increased sterol efflux from wild-type MDF but not from ABCA1 -/- MDF. 

To confirm these results in macrophages, we knocked down ABCA1 expression using siRNA in 

apoE-expressing macrophages. This resulted in decreased sterol efflux to exogenous apoA1, 

but the incremental sterol efflux mediated by endogenous apoE was not affected. On the other 

hand, when we reduced apoE expression in ABCA1 -/- and WT peritoneal macrophages, sterol 

efflux was significantly decreased in both cell types (25.8% and 31.1% decrease respectively). 

In summary, endogenous apoE expression can promote cellular sterol and phospholipid efflux 

from cells independently of ABCA1 expression. These results indicate that macrophages 

express redundant and independent pathways for defending cellular sterol homeostasis. 

P106 

Identification of the cAMP-Responsive Elements in the Murine ABCA1 Gene 

Wilfried Le Goff, Jonathan D Smith; Cleveland Clinic Foundation, Cleveland, OH 

We first demonstrated that cholesterol efflux to apoAI is inducible by cAMP analogs in 

RAW264.7 mouse macrophages. We and other then reported that ABCA1 is the cAMP-inducible 

apolipoprotein receptor that mediates cholesterol efflux from mouse macrophages, and that 

ABCA1 mRNA is markedly induced by cAMP. Using quantitative real-time PCR experiments in 

the presence of Actinomycin D, we now report that the cAMP induction of ABCA1 occurs at a 

transcriptional level. To identify cAMP-response elements on the murine ABCA1 gene 

responsible for the induction of ABCA1 expression by cAMP analogs, we generated a set of 

constructs by shotgun cloning using a BAC containing 118.1 kB of the mouse ABCA1 locus. 

Each DNA fragment was then cloned upstream the minimal mouse ABCA1 promoter and a 

luciferase gene. Using this set of constructs in transient transfections of RAW cells in the 

presence or absence of 8Br-cAMP permitted us to identify three DNA regions that mediate 

cAMP responsiveness. These 3 regions A, B and C were localized in the promoter, 1st intron and 

5th intron, respectively. Among the 3 regions, the B DNA fragment mediated the strongest cAMP 

induction, whereas the contribution of both the A and C fragments were modest. Deletional and 

mutational by analysis guest on on theJune B fragment 29, 2013 led us to identify a cAMP-responsive binding protein

(CREB) site that was essential for the cAMP induction. Gel shift experiments confirmed that the 

activated form of CREB1, phospho-CREB1, binds this site in the presence of nuclear extracts 

from cells treated with 8Br-cAMP. Finally using a CREB dominant negative, we demonstrated 

that CREB was required for the cAMP induction through the A, B and C fragments and for 

induction of ABCA1 expression by cAMP in RAW264.7 cells. Thus, we demonstrated that cAMP 

induction of murine ABCA1 gene expression occurs through 3 regions (BCA) and requires 

CREB-1. Finally the A, B and C fragments are not conserved in the human ABCA1 gene, thus 

explaining why the human ABCA1 expression is not strongly stimulated by cAMP analogs. 

Cellular Localization, Trafficking, and Function of the Human ABCG1 

Transporter 

Edward B Neufeld, Katherine G O’Brien, John A Stonik, Stephen J Demosky, Jr., Steven 

Sabol, Alan T Remaley, Silvia Santamarina-Fojo, H. B Brewer, Jr.; NHLBI, NIH, Bethesda, 

MD 

P107 

The ABCG1 transporter has been shown to play a role in the removal of cellular cholesterol by 

HDL and other lipoprotein acceptors. However, to date, little is known of the intracellular 

localization or site(s) of function of ABCG1. In the present study, we show that the 

carboxy-terminal GFP-tagged human ABCG1 transporter stably expressed in HeLa cells is 

functional insofar as it enhances efflux of cellular cholesterol to HDL, HDL2 and HDL3. 

ABCG1-GFP traffics from its site of synthesis in the ER, to the cell surface, and then to 

cholesterol-enriched late endocytic vesicles. Time lapse fluorescence confocal microscopy 

reveals a vesicular trafficking pathway that links ABCG1-containing perinuclear late endocytic 

vesicles back to the cell surface. ABCG1 generates a pool of detergent-resistant cholesterol on 

the cell surface, and then dissociates from the lipid domain. We show that ABCG1, unlike the 

ABCA1 transporter, does not support the uptake or lipidation of apoA-I. Taken together, these 

studies suggest that ABCG1 generates a distinct pool of cholesterol that is available for 

HDL-mediated efflux at the cell surface. These studies establish that the ABCG1 and ABCA1 

transporters generate separate pools of cellular cholesterol that are available for removal from 

the cell by distinctive mechanisms, involving HDL, and, poorly-lipidated apoA-I, respectively. 

P108 

Apolipoprotein A-I Activates Cellular Cdc42 Signaling through the ABCA1 

Transporter 

Jerzy-Roch Nofer, Renata Feuerborn, Institute for Clinical Chemistry, Münster, Germany; 

Thomas Engel, Institute for Arteriosclerosis Rsch, Münster, Germany; Alan T Remaley, 

National Institutes of Health, Bethesda, MD; Gerd Assmann; Institute for Clinical Chemistry, 

Münster, Germany 

It has been suggested that the signal transduction initiated by apolipoprotein A-I (apo A-I) 

activates key proteins involved in cholesterol efflux. ATP binding cassette protein A1 (ABCA1) 

has been shown to serve as a binding partner for apo A-I, but its participation in the apo 

A-I-induced signal transduction remains controversial. We here show that the exposure of 

human dermal fibroblasts to apo A-I or other amphipatic apolipoproteins known as ligands for 

ABCA1 (apo A-II, apo C-III, apo E) results in generation of intracellular signals including 

activation of small G-protein Cdc42, protein kinases located downstream to Cdc42 (PAK-1 and 

p54 JNK ), as well as actin polymerisation. A similar sequence of events was registered in cells 

exposed to amphipatic helical peptides, which interact with ABCA1. The apo A-I-induced signal 

transduction was abrogated by glyburide, an unspecific inhibitor of the ABCA1 transporter 

family, and in fibroblasts from Tangier disease, which do not express functional ABCA1. 

Conversely, induction of ABCA1 expression with liver X receptor (LXR) agonist, T0901317, 

potentiated apo A-I-induced signal transduction. Similar effects were observed in HEK293 cells 

overexpressing ABCA1. We conclude that ABCA1 is involved in transducing signal from apo A-I 

and, therefore, acts as a full apo A-I receptor. 

Differential Effects of Pioglitazone on Cholesterol Efflux in Human 

Macrophages and HepG2 Cells 

Sarah Ramirez, Annabelle Rodriguez; Johns Hopkins Univ Sch of Medicine, Baltimore, MD 

P109 

As a high affinity ligand for peroxisome proliferator-activated receptors (PPAR), pioglitazone 

(pio) increases HDL-C, which could be linked to increased expression of the ABCA1 transporter. 

In this study we compared the effects of pio on ABCA1 and apoA-I expression and function in 

primary human macrophages and the human hepatoma cell line, HepG2. Despite an increase 

in ABCA1 RNA expression in human macrophage foam cells, pio did not increase cholesterol 

efflux, whether in the presence or absence of HDL or apoA-I. We next examined cholesterol 

efflux in HepG2 cells (n3) by first radiolabeling cells with [ 3H]cholesterol for 24 h and then 

incubating cells with DMEM alone, pio (10 uM), HDL (50 g protein/ml), apoA-I (25 g 

protein/ml), HDLpio or apoA-Ipio for an additional 24 h. Cholesterol efflux increased in cells 

exposed to HDL (107.6%), apoA-I (95.7%), HDLpio (111.6%) and apoA-Ipio (92.2%) 

compared with control cells. In cells exposed to pio alone, cholesterol efflux increased 

significantly (88.8%) compared to control cells (p0.04) and the effects were comparable to 

HDL and apoA-I. As compared with control, cellular ABCA1 and apoA-I protein expression in 

cells exposed to pio alone increased (47% and 62% respectively) as well as secreted apoA-I 

measured by immunoprecipitation. While pio induced ABCA1 RNA expression in macrophages 

and hepatocytes, cholesterol efflux was significantly increased only in HepG2 cells. This likely 

was from the stimulatory effect of pio on apoA-I in HepG2 cells, which was absent in 

macrophages due to the lack of apoA-I in these cells. Therefore, pio exerts differential effects 

on ABCA1 and apoA-I expression and function in primary human macrophages and HepG2 

cells, suggesting that its favorable effect on HDL-C is significantly greater at the site of the liver 

than at the periphery. 





P110 

Molecular Mechanisms Involved in the Cytokine-Regulated Expression of 

Genes in Macrophages Implicated in Foam Cell Formation and 


Pelagia Foka, Nishi N Singh, Scott A Irvine, Kirsty Greenow, Sandra Evans, Sarah Rogers, 

Elizabeth Harvey, Saira Ali, Konstantinos Arnaoutakis, Dipak P Ramji; Cardiff Univ, Cardiff, 

United Kingdom 

The transformation of macrophages into foam cells is a key step in the pathogenesis of 

atherosclerosis. Several genes have been implicated in the regulation of foam cell formation. 

As atherosclerosis is considered to be a form of chronic inflammation, the action of cytokines 

on the expression of such genes will have a major impact on the initiation and the development 

of the disease. The purpose of this study was to investigate the mechanisms by which 

cytokines, particularly interferon- (IFN-) and transforming growth factor- (TGF-), modulate 

the expression of such key genes, including lipoprotein lipase (LPL), apolipoprotein E (apoE) 

and ATP-binding cassette transporter-A1 (ABCA1). IFN- reduced the expression of LPL, ABCA1 

and apoE mRNA in macrophages. The corresponding mechanisms were investigated using LPL 

as a model gene. IFN- decreased LPL gene expression at the transcriptional level. A 

combination of promoter dissection experiments and DNA-protein interaction studies showed 

that the action of IFN- was mediated through a novel pathway that involved a cytokinemediated 

decrease in the binding of the transcription factors Sp1 and Sp3 to regulatory 

sequences present in the LPL gene. Analysis of the signal transduction pathway showed a 

critical role for casein kinase 2 in the response. TGF- also inhibited LPL gene transcription 

through Sp1 and Sp3 but via a different mechanism as no changes in the binding of the factors 

was seen. In addition, a key role for the c-Jun NH 2-terminal kinase/stress-activated protein 

kinase (JNK/SAPK) was identified. In contrast to LPL, TGF- induced the expression of apoE 

and ABCA1. The mechanisms by which this occurs are currently being investigated and will be 

presented. These studies provide novel insights into the mechanisms by which cytokines 

regulate the expression of key genes in macrophages implicated in the regulation of foam cell 

formation and atherosclerosis. 

Inhibition of Rho Kinase Suppresses Atherosclerosis Related T Cell 

Function 

Qingyu Lian, Chie Nakajima, Nobuhiko Kubo, Hongwei Wang, James K Liao, William A 

Boisvert; Brigham and Women’s Hosp, Cambridge, MA 

P111 

Inflammation plays an important role in the pathogenesis of atherosclerosis. T helper 

lymphocytes regulate host inflammatory response through cytokine production. Recent studies 

have shown that inhibition of Rho kinase (ROCK) suppresses various atherosclerotic process, 

however, mechanisms of this inhibition have been unclear. We hypothesized that inhibition of 

ROCK may attenuate the atherosclerotic process by altering the inflammatory response in T 

cells. In our studies, we measured the cytokine production profiles of T lymphocytes treated 

with ROCK inhibitors, Fasudil and Y27632. In the in vitro study, IFN- and IL-2, two cytokines 

that are key regulators of the pro-inflammatory Th1 pathway, were decreased by about 50% 

in the Fasudil (10 uM) and Y27632 (10 uM) treated mouse spleen T cells (p0.05), whereas 

IL-4 and IL-10, two of the key regulators of the Th2 pathway, were inhibited to a lesser extent 

with Fasudil or Y27632 treatment (p0.05). Treatment of mice with Fasudil for 3 days also led 

to similar cytokine profiles. Other cytokines such as IL-3, 5, 9, 12, and 18 were not altered by 

ROCK inhibition. The IgG1 to IgG2a ratio in mice treated with 30 mg/kg/day of Fasudil for 3 days 

was significantly increased compared to that in control mice (p0.05), indicating a Th2 

polarization. Treatment of mouse splenocytes with anti-CD3 antibody and Fasudil (10 uM) 

resulted in transcriptional inhibition of IL-2, IL-4, IL13 and IFN-. Real-time PCR study indicated 

that the observed change of cytokine production may caused by the decrease of the mRNA 

levels of t-bet and GATA3, two master transcription factors that regulate the Th1 and Th2 

response respectively. These studies indicate that inhibition of ROCK polarizes the immune 

system toward an anti-inflammatory Th2 response, which contributes to the antiatherosclerotic 

effect of ROCK inhibition. 

Plasminogen Inhibits Monocyte Apoptosis via a PAR1-Dependent 

Mechanism 

Jennifer W Mitchell, Nagyung Baik, Scripps Clinic Rsch Inst, La Jolla, CA; Francis J 

Castellino, Univ of Notre Dame, Notre Dame, IN; Lindsey A Miles; Scripps Clinic Rsch Inst, 

La Jolla, CA 

P112 

Monocytes serve as major mediators of the inflammatory response and apoptosis provides a 

mechanism for regulating their activity. Furthermore, apoptotic monocytoid cells express a 

higher plasminogen binding capacity than viable cells. Therefore, we investigated the ability of 

cell-surface plasminogen to modulate apoptosis in monocytes. Here we report that plasminogen 

inhibits monocyte apoptosis and that the cytoprotective effect requires the proteolytic 

activity of plasmin. Monocytic cells (fresh human monocytes, U937 cells or THP1 cells) were 

cultured in plasminogen-deficient serum and induced to undergo apoptosis using either TNF 

or cycloheximide. The viable, early apoptotic and late apoptotic/necrotic cell populations were 

identified using dual color quantitative fluorescence activated cell sorting with annexin V-FITC 

and propidium iodide. When apoptosis was induced in the presence of plasminogen, apoptosis 

was inhibited in a dose-dependent manner with full inhibition achieved at 2 M plasminogen. 

In time course experiments, plasminogen treatment inhibited apoptosis even when added up 

to 3 hours following induction of apoptosis. Plasminogen treatment also markedly reduced the 

intranucleosomal DNA fragmentation and caspase 3 activity induced by TNF and CHX. 

Because monocytoid cell synthesize plasminogen activators, we examined the role of plasmin 

proteolytic activity in the anti-apoptotic effects of plasminogen. A plasminogen active site 

mutant, [D(646)E]Plg, failed to recapitulate the cytoprotective effect of wild-type plasminogen. 

In addition, by theguest anti-apoptotic on June activity 29, of 2013 plasminogen was blocked by increasing concentrations

E-72 Vol 25, No 5 May 2005 

of 2-antiplasmin with full reversal at 2 M 2-antiplasmin, suggesting that the cytoprotective 

effect of plasminogen requires its activation to plasmin. Furthermore, antibodies against PAR1 

blocked the anti-apoptotic effects of plasminogen. Our results suggest that plasminogen 

protects monocytic cells from apoptosis, via a mechanism requiring the proteolytic activity of 

plasmin and mediated by PAR1. Because monocyte apoptosis regulates inflammation and 

atherosclerosis, these results provide insight into a novel role for plasminogen in these 

processes. 

CD36 Initiates the Host Response to Staphylococcus Aureus and the 

Toll-Like Receptor Ligand, Lipoteichoic Acid 

P113 

Lynda M Stuart, Jessica M Silver, Kazue Takahashi, Jiusheng Deng, R A Ezekowitz, Kathryn 

J Moore; Massachusetts General Hosp, Boston, MA 

The macrophage scavenger receptor CD36 plays an essential role in the innate immune 

response to modified host ligands such as oxidized lipoproteins and fibrillar -amyloid. CD36 

has been proposed to mediate inflammatory signaling in response to these ligands, contributing 

to the chronic inflammation associated with both atherosclerosis and Alzheimer’s disease. 

However, the mechanism of CD36-signaling remains unknown. Recently, we showed that 

CD36 is a receptor for the Gram positive bacterium, Staphylococcus aureus, and its cell wall 

component lipoteichoic acid, a ligand for the Toll-like receptors TLR2 and TLR6. In transfection 

assays, expression of CD36 conferred binding and phagocytosis of Gram-positive and, to a 

lesser extent, Gram-negative bacteria. Macrophages deficient in CD36 demonstrate a reduced 

capacity to bind and internalize S. aureus, but not E. coli, a specificity that is conserved in the 

Drosophila CD36 paralogue, Croquemort. Using structure-function analysis, we demonstrate 

that CD36-mediated internalization of S. aureus was dependent on the C-terminal cytoplasmic 

portion of the receptor, which has been proposed to interact with signaling molecules. Mutation 

of these cytoplasmic residues, specifically tyrosine 463, abolished CD36-mediated phagocytosis. 

Importantly, CD36 null macrophages showed a marked defect in TNF and IL-12 

production in response to both S. aureus and lipoteichoic acid, and CD36 null mice failed to 

efficiently clear S. aureus in vivo resulted in a profound bacteraemia. Together, these data 

show that CD36 is required to mount an effective host response to S. aureus. These data raise 

the intriguing possibility that CD36 may function as an accessory receptor to present these 

bacterial ligands, and potentially other molecules, to Toll-like receptors at the cell surface in 

a manner analagous to the LPS receptor CD14. Furthermore, through its ability to phagocytose 

S. aureus and LTA, CD36 may deliver ligands to the phagosome where they can also engage 

Toll-like receptor signaling pathways. 

P114 

M-csf Stimulates SR-A Expression in Macrophages via the Ptx-Sensitive 

Activation of P38 Kinase 

Steve R Post, Liqin Du, Dejan M Nikolic; Univ of Kentucky, Lexington, KY 

Macrophage-Colony-Stimulating-Factor (M-CSF) is a cytokine that is essential for macrophage 

survival and differentiation from bone marrow progenitor cells. M-CSF binding to its surface 

receptor stimulates multiple signaling cascades that regulate expression of proteins that may 

be involved in atherosclerosis, including Class A Macrophage Scavenger Receptors (SR-A). 

SR-A mediates the uptake of modified lipoproteins by macrophages and can promote excessive 

cholesterol accumulation that is characteristic of macrophage foam cells. Because M-CSF can 

be produced locally in atherosclerotic lesions, MCSF-mediated increases in SR-A expression 

might affect the progression of atherosclerotic disease. In this study, we examined the 

signaling pathways required for M-CSF-mediated increase in SR-A expression and function. We 

found that M-CSF (25 ng/ml; 24 hrs) increased SR-A expression and modified lipoprotein 

association in mouse peritoneal macrophages (MPM). Pretreating cells with pertussis toxin 

(PTX) reduced the effect of M-CSF on both SR-A expression and modified lipoprotein uptake 

indicating a role for Gi/o activation in regulating SR-A expression. Because of their importance 

in regulating gene expression, we examined the role of the mitogen activated protein kinase 

(MAPK) family members in mediating the effect of M-CSF on SR-A expression. Treating MPM 

with M-CSF stimulated the phosphorylation of p38-kinase, extracellular signal-regulated kinase 

(ERK1/2), and cJun N-terminal kinase (JNK). However, only M-CSF-mediated p38-kinase 

phosphorylation was inhibited by PTX. To confirm that p38-kinase activation is required for the 

effects of M-CSF on SR-A expression and function, we assessed M-CSF-stimulated SR-A 

expression and modified lipoprotein uptake in cells treated with the specific p38-kinase 

inhibitor, SB203580. Pretreating macrophages with SB203580 inhibited M-CSF-induced SR-A 

expression and modified lipoprotein association. In contrast, inhibitors of ERK1/2 and JNK did 

not affect the ability of M-CSF to enhance SR-A expression. In summary, we have shown that 

M-CSF enhances SR-A expression and function by a pathway that involves the Gi/o-mediated 

activation of p38-kinase. 

P115 

Triglyceride-Rich Very low Density Lipoproteins Induce an Inflammatory 

Response in Macrophages via Activation of Mitogen Activated Protein 

Kinases 

Viswanathan Saraswathi, Alyssa H Hasty; Vanderbilt Univ, Nashville, TN 

Elevated levels of very low density lipoproteins (VLDL) associated with obesity, the metabolic 

syndrome, and post-prandial hyperlipidemia, are thought to contribute to increased risk for 

vascular disease. As macrophages are one of the primary cell types involved in atherogenesis, 

we sought to determine the impact of VLDL on macrophage lipid accumulation and 

inflammatory processes. VLDL treatment of mouse peritoneal macrophages for 6 h resulted in 

a significant 17.5-fold increase in triglyceride and 2.7-fold increase in fatty acid accumulation. 

Incubation of macrophages with VLDL (50 –150 g/ml) for 6 h induced the inflammatory 

cytokine, tumor necrosis factor (TNF-) byDownloaded up to 12-fold from 

and intracellular adhesion 



molecule-1 (ICAM-1) by greater than 6-fold. The upregulation of these inflammatory genes 

occurred after 1 h and their expression remained elevated after 24 h of exposure. Release of 

free fatty acids from VLDL may be at least partially responsible for its effects, as incubation of 

macrophages with stearic acid caused a 8.2-fold induction of TNF mRNA. A profound increase 

in phosphorylation of MAPK pathway members ERK1/2, p38 MAPK and JNK/SAPK was detected 

in VLDL-treated cells and addition of U0126, a specific inhibitor of ERK1/2 pathway completely 

abolished VLDL-stimulated TNF- gene expression. In conclusion, VLDL promotes inflammatory 

cytokine production in macrophages, and this phenomenon occurs in part, via release of 

free fatty acids and activation of MAPK pathways. These findings have implications for 

understanding the role of hypertriglyceridemia in the pathology of inflammatory diseases such 

as atherosclerosis. 

P116 

Serine Palmitoyltransferase Deficiency Causing Sphingolipid Reduction in 

Mice 

Mohammad R Hojjati, Zhiqiang Li, Xian C Jiang; SUNY Downstate Med Cntr, Brooklyn, NY 

Sphingolipids play very important role on cell membrane formation, signal transduction, and 

plasma lipoprotein metabolism. All these functions might have impacts on atherosclerosis 

development. Serine palmitoyl-CoA transferase (SPT) is the key enzyme for sphingolipid 

biosynthesis. Researches on Saccharomyces cerevisiae and CHO cells showed that SPT 

contains at least two subunits, long chain base1 and long chain base2 (LCB1 and LCB2). To 

evaluate the in vivo SPT activity and its role in sphingolipid metabolism, we used homologous 

recombination in embryonic stem cells and produced mice with long chain base (LCB1 and 

LCB2), two subunits of SPT, gene deficiency. Homozygous LCB1 or LCB2 mice are embryonic 

lethal, whereas, heterozygous of both animals (LCB1/-, LCB2/-) are healthy. Analysis of 

LCB1/- and LCB2/-mice showed that, comparing to WT mice, 1) decreased liver LCB1 and 

LCB2 mRNAs (p0.001, respectively); 2) decreased liver LCB1 and LCB2 mass (p0.001, 

respectively), moreover, LCB1 mass decreased in LCB2/- mouse liver (p0.001), while LCB2 

mass decreased in LCB1/- mouse liver (p0.001); 3) decreased liver SPT activity (p0.001); 

4) decreased plasma ceramide (p0.01) sphingosine-1-phosphate (p0.01) and sphingosine 

(p0.01) levels; 5) dramatically decreased plasma lysosphingomyelin (p0.0001); and 6) 

there was no change of plasma sphingomyelin, triglyceride, total cholesterol and total 

phospholipid levels. These results indicate that LCB1 and LCB2 make a multimeric protein with 

similar phenotypes in both heterozygote mice and LCB1 is unstable in the absence of LCB2 and 

vice versa. Manipulation of SPT activity might affect the process of diseases, such as 

atherosclerosis. 

P117 

Multiple Complementary Pathways for Efficient Cholesterol Absorption in 

Mice 

Jahangir Iqbal, M. Mahmood Hussain; SUNY Downstate Med Cntr, Brooklyn, NY 

ApoB-dependent and apoB-independent pathways for cholesterol transport have been described 

in cultured cells. Here, we show that the apoB-independent pathway involves 

apoAI-containing high-density lipoproteins (HDL). Cholesterol secretion by the HDL, but not by 

the apoB, pathway was significantly reduced in primary enterocytes isolated from chow and 

cholesterol fed apoAI -/- mice. In apoAI -/- mice, the absorption of a bolus of cholesterol over 48 h 

was similar in control and apoAI -/- mice fed chow or high cholesterol diet. However, short-term 

(2 h) studies revealed that cholesterol absorption was occurring over longer lengths of the 

intestine, and cholesterol, but not triglyceride, transport to the plasma and liver in chow and 

cholesterol fed apoAI -/- mice was significantly reduced. These studies indicate that, in apoAI 

deficiency, there is a delay in cholesterol absorption, but a bolus of cholesterol is eventually 

absorbed. Long-term studies involving multiple feedings showed significant reduction in 

cholesterol absorption after 4 days. We propose that multiple pathways complement each other 

to ensure efficient cholesterol absorption in mice. 

Dual Inhibitor against Low-Density Lipoprotein (LDL) and Acyl-CoA: 

Cholesterol Acyltransferase (ACAT) from Torreya Nucifera 

Woo-Song Lee, Ju-Ryoung Kim, So-Jin An, Kyung-Soon Kim, Kyung-Hyun Cho, Tae-Sook 

Jeong; Korea Rsch Institute of Bioscience & Biotechnology, Daejeon, Republic of Korea 

P118 

LDL-oxidation and ACAT have been known to play a crucial role in the development of 

atherosclerosis and hypercholesterolemia. Six abietane diterpenoids 1–6, isolated from the 

leaves of T. nucifera, and compound 7, derived from 3, exhibited significant low-density 

lipoprotein (LDL)-antioxidant activity in the thiobarbituric acid-reactive substance (TBARS) 

assay (IC50 0.43–15.20 M) and also were confirmed to be potent LDL-antioxidants by 

various assay systems. Also, the compounds exhibited inhibitory activities against hACAT-1 

with IC50 values of 37–229 M or hACAT-2 with IC50 values of 42–309 M, respectively. AC29 

cells stably expressing African green monkey ACAT-1 or ACAT-2, kindly gifted from Dr. L. L. 

Rudel of the Wake Forest University School of Medicine (Winston-Salem, NC), were used to test 

the effects of compounds. Compounds 1–3 showed more potent inhibitory activity against 

ACAT-1 with IC50 1.5–3.6 M compared to ACAT-2 (IC50 27.4–48.3 M). The compounds 

showed more specificity of ACAT-1 than ACAT-2 by the level of lipophilicity. Interestingly, the 

ethanolic extracts from T. nucifera exhibited strong cholesterol-lowering effect in high 

cholesterol-fed C57BL/6J mice. Furthermore, the hexane extracts containing compounds 1–6 

from T. nucifera showed cholesterol lowering and anti-atherosclerotic effects in high 

cholesterol-fed by guest New Zealand on June White 29, rabbits. 2013

P119 

Clusterin / Apolipoprotein J Protection Begins at day 1 and Persists to day 

56 in the Vascular Response to Injury 

Eddy S Konaniah, Kari L Theurer, Jon R Cook, Scott E Street, David Y Hui, Norm A 

Granholm; Univ Cincinnati, Cincinnati, OH 

Clusterin / Apolipoprotein J is an enigmatic protein. Although its function is incompletely 

defined, clusterin expression at sites of injury is associated with enhanced cell survival. We 

hypothesized that clusterin provides a protective effect in the vascular response to injury. We 

tested this hypothesis with FVB/N clusterin null (Clu -/-) and wild type (wt) male mice in a model 

of vascular injury to the left common carotid artery that avoids injury to the elastic laminæ. The 

right common carotid artery serves as uninjured control. Mice are sacrificed at 1, 5, 10, 14, 28, 

and 56 days (D) after injury. Whole neck sections are prepared from 5 levels and stained for 

elastin. Injured and control vessels are measured to digitally quantify vessel wall thickness, 

lumen area, and areas enclosed by the internal and external elastic laminæ. The data yield 

calculated values for media area, neointima area, % stenosis, and intima-media (I/M) ratio. 

From D5 to D56, neointima area, wall thickness, % stenosis, and I/M ratio are larger for injured 

vessels of Clu -/- mice compared to injured vessels of wt mice; media areas of injured vessels 

are not different within any time point. The data suggest that clusterin mitigates the vascular 

response to injury in wt mice. This suggestion is strengthened by the significant increase of 

neointima in injured vessels of Clu -/- mice at D10, D14, and D28. The enlarged neointima 

contributes to significantly increased wall thickness in injured vessels of Clu -/- mice at D14, 

D28, and D56 and to significant increases in both % stenosis and I/M ratio in injured vessels 

of Clu -/- mice at D5, D10, D14, and D28. Remarkably, the impact of clusterin is observed in 

control vessels as well. Media thickness and area are significantly greater at D28 in control 

vessels of Clu -/- compared to clusterin wt mice. We know that clusterin is present at injury 

sites in the vessel wall at D1, is detectable at low levels at D56 in wt mice, and is 

immunohistochemically undetectable in walls of control vessels at all times studied. Clusterin 

persistence at injury sites, clusterin genotype effect at remote yet responsive control vessels, 

and the foregoing metric data support the hypothesis that clusterin protects, directly and/or 

indirectly, the vessel wall in the vascular response to injury. 

Modulation of Triglyceride Metabolism by Citrus Polymethoxylated 

Flavones in Hamster Model of Dyslipidemic Insulin Resistance 

Elzbieta M Kurowska, KGK Synergize Inc., London, Canada; Rachel W Li, Adele Casaschi, 

Teresa Douglas, Andre G Theriault; Univ of Hawai’i at Manoa, Honolulu, HI 

P120 

Citrus polymethoxylated flavones (PMFs) reduced blood cholesterol and triacylglycerol (TG) 

concentrations in hamsters with diet-induced hypercholesterolemia. The most prevalent PMF, 

tangeretin, also acted as potent inhibitor of apo B secretion in human hepatoma HepG2 cells. 

This was associated with i) substantial reduction in TG synthesis and mass, ii) inhibited transfer 

of TG to apo B and iii) activation of nuclear peroxisome proliferator-activated receptors (PPAR) 

positively implicated in regulation of sugar, fatty acids and lipoprotein metabolism. To evaluate 

TG-lowering and anti-diabetic potential of PMFs in vivo, we used hamsters with dyslipidemic 

insulin resistance (IR) induced by feeding 60% (60 of 100) fructose diet. Male Golden Syrian 

hamsters were fed fructose-rich diet for 2 weeks, after which they continued on the same diet 

with or without PMFs (either 62.5 or 125 mg/kg) for 4 wks. During the initial 2 wks, animals 

developed hypercholesterolemia and hypertriglyceridemia. After the subsequent 4 wks of PMF 

treatment plasma cholesterol and TG concentrations were normalized (dose-dependently for 

cholesterol) indicating hypolipidemic action. PMF-rich diets also dose-dependently reversed 

fructose-induced increases in serum insulin concentrations, suggesting antidiabetic potential. 

In tissues, high PMF dose significantly reduced epididymal fat mass (after correction for body 

weight) and significantly reduced TG concentration in liver and heart, but not in skeletal muscle. 

High dose of PMF was also associated with increased expression of PPAR- and- in the liver 

(2.8-fold and 2.4-fold, respectively) but not PPAR- in adipose tissue, indicating improvement 

of hepatic TG and glucose metabolism. In addition, PMF supplement dose-dependently 

increased serum concentration of adiponectin, the adipose tissue-derived hormone negatively 

associated with obesity, and decreased serum levels of another adipose hormone, leptin, which 

often predisposes to obesity. In conclusion, PMF supplementation was proven to be effective 

in preventing hypertriglyceridemia and hyperinsulinemia in hamster model of IR. Dietary PMFs 

altered TG and insulin metabolism via multiple mechanisms. 

Pcsk9 Inhibits Ldl Clearance but Does not Affect Apob Containing 

Lipoprotein Production in Mice 

Gilles Lambert, Philippe Costet, Michel Krempf, Florent Lalanne; Inserm U539, Nantes, 

France 

P121 

Mutations in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) have recently been associated 

with Autosomal Dominant Hypercholesterolemia (ADH). In vivo kinetic studies indicate that LDL 

catabolism is impaired and that apolipoprotein (apo) B containing lipoprotein synthesis is 

enhanced in two patients presenting with the S127R mutation on PCSK9. Here we show that 

PCSK9 dramatically impairs the expression of the LDL receptor (LDLr) in mouse models. 

Compared to adenovirus (Ad) Ad-Null injected controls, Ad-PCSK9 injected C57BL6/J mice have 

delayed plasma clearance of 125I-apoB-LDL (FCR 5.40.2 vs. 4.50.2 day-1 ,p0.03). In 

contrast, 125I-apoB-LDL catabolism is similar in LDLr deficient (KO) mice infused with either 

Ad-Null or Ad-PCSK9 (FCR 4.00.3 vs. 4.10.4 day-1 ,p0.9). As a consequence the 

plasma cholesterol is increased upon Ad-PCSK9 infusion in C57BL6/J (17623 vs. 748 

mg/dL, p0.05) but not in LDLr-KO mice ( 22221 vs. 23913 mg/dL, p0.8). Plasma 

apoB100 levels follow accordingly. By FPLC, the cholesterol distribution in Ad-PCSK9 infused 

C57BL/6 exhibit a dramatic increase only within the LDL sized lipoproteins. In contrast to data 

obtained in PCSK9-S127R patients presenting with increased apoB production, wild-type 

PCSK9 does not alter the production and/or secretion Downloaded of VLDL apoBfrom in mice. Finally, we show 




that unlike PCSK9 overexpression in mice, the S127R mutation in patients lead to increased 

LDL but also VLDL apoB levels. In summary our study demonstrates a definitive role for PCSK9 

in the modulation of the LDLr metabolic pathway and suggests a potential gain of function for 

S127R PCSK9 in humans. 

P122 

Discoidal Complexes of Apolipoprotein A-I with Different Phospholipids 

Share a Common Model for Size Heterogeneity 

Jianguo Chen, James C Patterson, Andrea Catte, Jere P Segrest, Ling Li; Univ of Alabama 

at Birmingham, Birmingham, AL 

It is well known that discoidal apolipoprotein A-I (apoA-I): phospholipid complexes exist as 

multiple discrete-sized particles containing a constant number of apoA-I. Recently, we have 

shown that apoA-I complexed with dimyristoyl phosphatidylcholine (DMPC) forms six discretesized 

discoidal particles containing two molecules of apoA-I, each with a unique diameter. Also, 

we have developed well substantiated models to explain the molecular bases for size 

heterogeneity of apoA-I:DMPC discs. To investigate whether discs of apoA-I with other 

phospholipids adopt the same structure as discs with DMPC, discoidal complexes of apoA-I 

were prepared with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), dipalmitoyl phosphatidylcholine 

(DPPC), and distearoyl phosphatidylcholine (DSPC), respectively, by the cholate 

dialysis method. The complexes were analyzed by non-denaturing gradient gel electrophoresis 

(NDGGE) and compared with discs containing DMPC. The results show that size distributions 

of discoidal complexes with different lipids follow a similar pattern, although the extent of 

heterogeneity of discrete-sized particles in the complexes varies from lipid to lipid. The rank 

order of band broadness on NDGGE is: DMPC DPPC POPC DSPC. We hypothesized that: 

1) the particles exist in discrete sizes corresponding to energetic minima; and 2) the broadness 

of the bands would be inversely related to the exchange rate of lipids between particles 

distributed around each minimum. A slower rate of lipid exchange would be expected to 

produce a broader distribution of kinetically trapped particles. To confirm this possibility, 

apoA-I:POPC complexes were prepared and immediately analyzed, or incubated for 4 weeks 

and analyzed weekly by NDGGE. The results show that the bands of apoA-I:POPC complexes 

became sharper with time, while their peak positions stayed constant. These results 

demonstrate that discoidal complexes of apoA-I with different phospholipids share common 

structural properties and suggest that structural models proposed for discs with one lipid apply 

to discs with other lipids. 

P123 

Decreased ApoA1 Synthesis and Increased HDL-Cholesterol Clearance are 

Major Determinants for the HDL Decrease in Hyperhomocysteinemia 

Dan Liao, Hongmei Tan, Baylor College of Medicine, Houston, TX; Rutai Hui, Fuwai Hosp, 

Beijing, China; Xiaohua Jiang, Fan Yang, John Gaubatz, Baylor College of Medicine, 

Houston, TX; Zhaohui Li, Fuwai Hosp, Beijing, China; William Durante, Baylor College of 

Medicine, Houston, TX; Andrew I Schafe, Univ of Pennsylvania Sch of Medicine, 

Philadelphia, PA; Lawrence Chan, Henry J Pownall, Xiaofeng Yang, Hong Wang; Baylor 

College of Medicine, Houston, TX 

Hyperhomocysteinemia (HHcy) is an important non-lipid risk factor for cardiovascular disease 

and is associated with increased aortic lesions and decreased plasma HDL-cholesterol (HDL-C) 

in mice (1) . The mechanism for this association is unknown. In this study, we found that HHcy 

is significantly and negatively correlated with plasma HDL-C and apoA1 protein levels in 

patients with coronary heart disease (CHD). To study the underlying mechanisms, we employed 

mice with targeted deletions of the genes for apolipoprotein E (apoE) and cystathionine 

-synthase (CBS). HDL-C decrease in severe HHcy was caused by the loss of large HDL 

particles. HDL composition analysis revealed an increase in the free cholesterol ratio and a 

decrease in the total protein ratio. Mouse plasma from CBS/apoE deficient mice attenuated 

cholesterol efflux from cholesterol-loaded macrophages. ApoAI protein levels were decreased 

in the liver and plasma of HHcy mice, and this is correlated with reduced plasma 

lecithin:cholesterol acyltransferase fractional activity against endogenous substrates. In 

addition, severe HHcy resulted in faster clearance of HDL-cholesterol ester that correlated with 

elevated hepatic SR-B1 protein levels. These results suggest that decreased apoA1 synthesis 

and increased HDL-cholesterol clearance contribute to HDL reduction in HHcy. (1). Wang H, 

Jiang XH, Yang F, Gaubatz JW, Ma L, Magera MJ, Yang XF, Berger PB, Durante W, Pownall HJ, 

Schafer AI, (2003) Hyperhomocysteinemia accelerates atherosclerosis in cystathionine 

-synthase and apolipoprotein E double knockout mice with and without dietary perturbation. 

Blood 101:3901–3907 

P124 

Lipoprotein (a) with Small Size Apo (a) has Higher Arterial Wall 

Accumulation Rate than LDL and Localizes to the Endothelial Cell Laye 

Guijing Lu, Andrew F Powers, Bernard Ormsby, John C Rutledge, Lars Berglund; Univ of 

California,Davis, Davis, CA 

Background: Lipoprotein(a) [Lp(a)] is an emerging risk factor for development of atherosclerosis, 

and consists of a LDL-like moiety in addition to one copy of apolipoprotein(a) [apo(a)]. 

Lp(a) levels are inversely related to apo(a) size. Small apo(a) size (22 K4 repeats) has been 

associated with cardiovascular disease independent of Lp(a) levels. However, the nature of the 

interaction between Lp(a) and the arterial wall is largely unknown. We tested the hypothesis 

that Lp(a) with small size apo(a) accumulates in perfused arteries and compared the 

accumulation rate to that of LDL. Methods: Lp(a) and LDL isolated from the same subjects 

were labeled with a fluorescent hydrocarbon probe (DiI). Carotid arteries from young male rats 

were removed and perfused with fluorescently labeled Lp(a) or LDL (20 mg/dL). Arterial 

lipoprotein flux was measured using quantitative fluorescence microscopy. Results: The apo(a) 

size used was 19 K4 repeats. The rate of baseline DiI-Lp(a) accumulation was 70% greater than 

that of DiI-LDL (7.31.0 vs. 4.30.7 ng cholesterol/min/cm2 by guest on June 29, 2013 

, n10 and 8 vessels,

E-74 Vol 25, No 5 May 2005 

respectively; P0.05). The artery wall was injured by perfusion of TNF (50 ng/ml), resulting 

in increased accumulation of DiI-LDL and DiI-Lp(a) relative to control. Post-perfusion 

histological analysis of cross-sectioned vessels demonstrated greater baseline endothelial cell 

deposition of DiI-Lp(a) compared to DiI-LDL. After treatment with TNF, both LDL and Lp(a) 

accumulated extensively in the subendothelial space. Summary: Lp(a) with small size apo(a) 

accumulated on the lumenal surface of the arterial endothelium at a significantly higher rate 

than LDL. Furthermore, Lp(a) penetrated the endothelium and localized in the subendothelial 

space in response to endothelial injury. Conclusion: These results support the hypothesis that 

Lp(a) with small size apo(a) is atherogenic and demonstrates its potential to accumulate in the 

arterial wall in response to inflammatory injury. 

P125 

Regulation of Hepatic Apolipoprotein B Secretion by Dietary 

Polyunsaturated Fatty Acids Involves Apolipoprotein B Aggregation and 

Direct Oxidative Modification 

Meihui Pan, Vatsala Maitin, NYU Sch of Medicine, New York, NY; Kevin J Williams, Thomas 

Jefferson Univ, Philadelphia, PA; Edward A Fisher; NYU Sch of Medicine, New York, NY 

Background: The dietary polyunsaturated fatty acids (PUFAs) have lipid-lowering properties in 

vitro and in vivo. We reported earlier that this is partially related to their ability to attenuate 

apolipoprotein B (apoB) secretion by hepatocytes through activation of post-ER, pre-secretory 

proteolysis (PERPP). This process is activated by intracellular conversion of PUFAs to lipid 

peroxides. We also observed that PUFAs, including docosahexaenoic acid (DHA) and eicosapentaenoic 

acid (EPA), induce the intracellular formation of high molecular weight (HMW) 

aggregates precipitated by anti-apoB antiserum. Objectives: (a) To identify the molecular 

species present in these HMW aggregates, including oxidative modifications, and (b) to 

investigate the degradative pathway stimulated by PUFAs. Results: Aggregates contained 

essentially only apoB by mass spectrometry. When apoB aggregates were immunoprecipitated 

from DHA-treated hepatocytes and subjected to Western blot analysis, they were highly 

reactive to anti-malondialdehyde (MDA) antibody, suggesting covalent oxidative modification. 

This modification was dependent on lipid peroxidation, as it was prevented by co-treatment 

with desferrioxamine, an inhibitor of iron-dependent peroxidation. We previously showed that 

PUFA-induced apoB degradation was non-proteasomal. Autophagosomes, however, are known 

to degrade proteins damaged by oxidative modifications. Thus, we tested the participation of 

autophagosomes in the degradation of the modified aggregates. Stimulation of the autophagosome/lysosome 

system with rapamycin decreased, and inhibition with 3-methyladenine 

(3-MA), increased apoB aggregate levels, but did not affect their formation. Conclusions: 1) 

Dietary PUFA stimulates formation of lipid peroxides and subsequent oxidative damage and 

aggregation of hepatic apoB, and, 2) this form of apoB likely is degraded by the autophagosomes, 

consistent with the role of this pathway in the turnover of similarly modified proteins. 

P126 

Lipid Acquisition by Apolipoprotein A-i in Endoplasmic Reticulum and Golgi 

Compartments of Primary Mouse Hepatocytes 

Jovana Maric, Yves L Marcel; Univ of Ottawa Heart Institute, Ottawa, Canada 

The main site of apolipoprotein A-I (apoA-I) synthesis and secretion is the hepatocyte. The aim 

of this study is to determine the localization and significance of intracellular lipidation of newly 

synthesized apoA-I in ER and Golgi, by labeling primary mouse hepatocytes with 3H-choline, 

LDL-3H-cholesterol or 3H-mevalonate to label de novo synthesized cholesterol. Phospholipidation 

of apoA-I is significant and most evident in ER and medial Golgi. The lipidation occurs 

to lumenal apoA-I, although it may also occur on the inner leaflet of ER and medial Golgi 

membrane. In the presence of cycloheximide, endogenous apoA-I is almost fully phospholipidated 

intracellularly and only slightly after export out of the cell. In cells labeled with 

LDL-3H-cholesterol, intracellular cholesterol lipidation of apoA-I is entirely absent, but the 

exported apoA-I rapidly accumulates cholesterol in the media. On the other hand, de novo 

cholesterol is able to lipidate apoA-I intracellularly. This implies that HDL formation begins with 

intracellular apoA-I phospholipidation in the ER, followed by modest cholesterol lipidation in the 

Golgi, which later promotes significant cholesterol accumulation at the plasma membrane. In 

hepatocytes lacking ABCA1, lipidation by LDL-cholesterol was significantly reduced at the 

plasma membrane. Phospholipidation and lipidation by de novo cholesterol were both reduced 

in Golgi compartments, while ER lipidation remained mostly unchanged, suggesting that the 

early, lipidation in ER is ABCA1 independent while the lipidation in Golgi as well as at the 

plasma membrane seems to require ABCA1. Thus, we have supporting evidence to 

demonstrate that apoA-I is lipidated intracellularly, partially dependent on ABCA1, with the bulk 

of cholesterol lipidation occurring at the plasma membrane. 

VLDL ApoB Kinetics in HIV Patients Treated with Protease Inhibitors: 

Lipodystrophy is Associated with Reduced VLDL ApoB Clearance 

John S Millar, Ian Frank, Jennifer Dykhouse, Phyllis L May, Ruth Brower, Daniel J Rader; 

Univ Pennsylvania, Philadelphia, PA 

P127 

Background: Protease inhibitors (PI) effectively interrupt viral replication when used in patients 

with HIV. One side-effect of treatment with these agents is the development of lipodystrophy, 

frequently accompanied by dyslipidemia, which places these patients at increased risk for 

developing cardiovascular disease. Aim: We studied apoB kinetics in 18 HIV patients 

undergoing antiretroviral treatment with PI to determine the mechanism(s) responsible for 

PI-associated dyslipidemia. Methods: Patients were classified as being lipodystrophic (LD) or 

non-lipodystrophic (non-LD) based on self-reported changes in body fat distribution following 

initiation of PI therapy. Patients underwent a primed-constant infusion of a deuterated leucine 

tracer, apoB and the incorporation of tracer into VLDL apoB measured in samples taken over 

a 72 hour period. Tracer data were then fit toDownloaded a multicompartmental from 

model to determine 



production and clearance rates of VLDL apoB. Results: Eight patients were reported as being 

LD as a result of PI therapy. LD patients had levels of total cholesterol, triglyceride and HDL that 

were similar to non-LD patients. LD and non-LD patients also had similar levels of total apoB 

but LD patients had higher levels of VLDL apoB (16.8 5.1 vs. 10.8 6.5 mg/dl, p.059). 

VLDL apoB kinetic studies showed LD patients had a significantly reduced VLDL apoB100 

fractional catabolic rate (4.51.5 vs. 6.52.4 pools/d, p.03). Despite significantly higher 

free fatty acid levels (0.300.06 vs. 0.210.11 meq/L, p.03) there was no significant 

difference in the VLDL apoB100 production rate of LD patients when compared to non-LD 

patients (32.2 9.1 vs. 27.1 10.8 mg/kg/d, p.70, LD vs. non-LD). Conclusion: We 

conclude that HIV patients developing lipodystrophy in response to PI treatment have reduced 

VLDL apoB clearance which may result in accumulation of atherogenic lipoproteins. Furthermore, 

LD patients under treatment with PI have similar VLDL apoB production rates to non-LD 

patients under treatment with PI despite elevations in plasma free fatty acid levels. 

P128 

Nobiletin is a Potent Inhibitor of Hepatocyte apoB Secretion Mediated 

through Activation of the MAPKerk Signaling Pathway, Independent of 

Insulin Receptor Phosphorylation 

Erin E Mulvihill, Justin K Lee, Emma M Allister, Robarts Rsch Institute, London, Canada; 

Elzbieta M Kurowska, KGK Synergize Inc., London, Canada; Murray W Huff; Robarts Rsch 

Institute, London, Canada 

Citrus derived flavonoids have been described as potent cholesterol lowering agents but little 

is known of their potential activation of insulin signaling cascades, which may partially mediate 

this effect. Recently, we demonstrated that naringenin markedly inhibits hepatocyte apoB 

secretion by activating two pathways of the insulin signaling cascade, namely phosphoinositide 

3-kinase (PI3K) and mitogen activated protein kinase/extracellular regulated kinase (MAPK erk ). 

In the present study, our objective was to determine if a related polymethoxy flavonoid, 

nobiletin, isolated from tangerines, inhibits apoB secretion and elucidate the mechanism(s) 

involved. Incubation of HepG2 cells with nobiletin (24 hr; 100nM to 25M) significantly 

decreased apoB accumulation in the media up to 80%, with an IC 50 of 10M: 10-fold more 

potent than naringenin. Insulin also inhibits apoB secretion from HepG2 cells, which we have 

shown is dependent on activation of both PI3K and MAPK erk signaling. Co-incubation of cells 

with nobiletin (10M) and the PI3K inhibitor wortmannin (1M), had no significant effect on the 

nobiletin-induced reduction in apoB secretion. In contrast, inhibition of MEK1/2 with UO126 

(10M) completely blocked the nobiletin-induced decrease in apoB100 secretion, suggesting 

nobiletin activates the MAPK erk pathway. The activity of MAPK p38 , which normally attenuates 

MEK1/2 signaling, was inhibited by SB203580 (10M) and this enhanced the inhibitory effect 

of nobiletin on apoB secretion, thus confirming activation of the MAPK erk pathway. Activation of 

MAPK erk signaling in HepG2 cells is known to inhibit the expression of MTP, suggesting that the 

effect of nobiletin involves reduced MTP expression. Initiation of MAPK erk activation by nobiletin 

was independent of tyrosine phosphorylation of both the insulin receptor subunit and insulin 

receptor substrate-1/2. We conclude that downstream signaling of MAPK erk is essential for the 

inhibition of apoB secretion by nobiletin. Therefore, nobiletin has the potential to attenuate the 

increased hepatic apoB secretion, characteristic of insulin resistant states. 

P129 

Molecular Dynamics of Apolipoprotein A-I: The Complementary Roles of the 

N and C-Terminal Regions 

Michael N Oda; Children’s Hosp Oakland Rsch Institute, Oakland, CA 

During lipid association apolipoprotein A-I (apoA-I) undergoes a dramatic conformational 

transition. A majority of this conformational transition occurs at apoA-I’s C-terminal residues 

(188 - 210) in a manner analogous to the conformational trigger found in viral fusion proteins. 

To determine whether the stimulus derived regulation observed in viral fusion proteins is 

present in apoA-I, we examined its N-terminal residues (1 - 81) through a combination of 

electron paramagnetic resonance spectroscopy and fluorescence resonance energy transfer 

studies. Our results suggest that this region of apoA-I may stabilize the lipid-free conformation 

of the apoA-I C-terminus and participate in the regulation of apoA-I’s conformational transition. 

P130 

HDL Kinetics in Overweight-Obese Subjects with Stable Isotopy: Relative 

Significance of Catabolism and Production in Determining Apolipoprotein 

A-I Plasma Concentration 

Esther M Ooi, Gerald F Watts, Dick C Chan, P. Hugh R Barrett; Univ of Western Australia, 

Perth, Australia 

Objective: Low plasma concentrations of high-density lipoprotein (HDL)-cholesterol and 

apolipoprotein A-I (apoA-I) are powerful, independent predictors of coronary artery disease and 

often associated with obesity and the metabolic syndrome. However, the underlying kinetic 

determinants of HDL metabolism are poorly understood. Methods: We pooled data from 13 

stable isotope studies to investigate the kinetic determinants of apoA-I concentrations in lean 

and overweight - obese individuals. We also examined the associations of HDL kinetic 

parameters with age, sex, BMI, fasting plasma glucose, fasting insulin, HOMA score, HDL 

particle size and concentrations of apoA-I, triglyceride, HDL-cholesterol and LDL-cholesterol. 

Results: Compared with lean subjects, overweight - obese individuals had significantly higher 

HDL apoA-I fractional catabolic rate (FCR) (0.21 0.01 vs. 0.33 0.01 pools/day, p0.001) 

and production rate (PR) (11.3 4.4 vs. 15.8 2.77 mg/kg/day, p 0.001). In the lean 

group, HDL apoA-I PR was significantly associated with apoA-I concentration (r 0.455, 

p0.004) while in the overweight - obese group, both HDL apoA-I FCR (r-0.396, p0.050) 

and HDL apoA-I PR (r0.399, p0.048) were significantly associated with apoA-I concentration. 

After adjustment for fasting insulin or HOMA score, HDL apoA-I PR was shown to be an 

independent by predictor guest on of apoA-I June 29, concentration 2013 in the overweight - obese group of subjects.

Conclusions: Our findings suggest that overweight - obese subjects have apo A-I hypercatabolism, 

which is paralleled by an increased synthesis of apoA-I. Although HDL apoA-I FCR 

contributes to regulating apoA-I concentration, HDL apoA-I PR is the stronger determinant of 

apoA-I concentration. We suggest that in both these groups of subjects the plasma 

concentration of apoA-I is chiefly determined by HDL apoA-I PR, a notion that could have a 

significant therapeutic implication for the management of cardiovascular disease in the 

metabolic syndrome. 

P131 

Effects of Phytosterols on Lipoprotein Metabolism in Subjects with the 

Metabolic Syndrome 

Esther M Ooi, Gerald F Watts, P. Hugh R Barrett, Univ of Western Australia, Perth, Australia; 

Peter M Clifton, CSIRO, Health Sciences and Nutrition, Adelaide, Australia; Paul J Nestel; 

Baker Heart Rsch Institute, Melbourne, Australia 

Objective: To investigate the effects of dietary plant sterols supplementation on lipoprotein 

metabolism in men with the metabolic syndrome. Subjects: Nine men with the metabolic 

syndrome as defined by the following: BMI 30kg/m 2 or waist:hip ratio 0.9, triglycerides 

(TG) 1.7mmol/L, high-density lipoprotein (HDL)-cholesterol 1.10 mmol/L and fasting 

glucose 6.1 mmol/L, while consuming an ad libitum weight-maintenance diet. Study 

Designs: Randomised, double-blind, cross-over study of 2x4weeks therapeutic periods with 

placebo or plant sterols (2g/day), and 2 weeks placebo wash-out between therapeutic periods. 

Methods: VLDL-, IDL- and LDL-apoB and HDL apoA-I kinetics were measured using 

intravenous, bolus D 3-leucine (4mg/kg), GCMS and compartmental modelling (SAAMII). 

Results: Plant sterols did not have a significant effect on total cholesterol, TG, LDL-cholesterol, 

HDL-cholesterol and plasma concentrations of apoB, apoA-I or apoA-II. There were no 

significant changes to VLDL-, IDL-, LDL-apoB or HDL apoA-I fractional catabolic rate (FCR) or 

production rate (PR) between treatment and placebo phases. Campesterol was significantly 

increased on plant sterols treatment (2.53 0.35 vs 4.64 0.59 l/ml, p0.05). Lathosterol, 

a marker of endogenous cholesterol synthesis did not increase significantly during plant sterols 

treatment. Conclusions: There appears to be no significant advantage gained by addition of 

phytosterols to the daily diet of subjects with the metabolic syndrome with respect to 

cholesterol lowering or improvement in lipoprotein profile or metabolism. The lack of response 

may be attributable to the high rates of endogenous cholesterol synthesis relative to cholesterol 

absorption observed in subjects with the metabolic syndrome. Further studies with a higher 

dose of phytosterols or other therapeutic agents that reduce endogenous cholesterol synthesis 

may confer benefits in managing the dyslipidaemia observed in these individuals. 

P132 

Paradoxical Expansion of the Liver-TG Pool with Fasting in the Txnip -/- 

Mouse 

Elizabeth J Parks, Kerry L Peterson; Univ of Minnesota, St. Paul, MN 

The impact of the Txnip mutation on the postprandial expansion of liver-TG stores was 

investigated in HcB-19 mice, a mouse model of hyperlipidemia. Txnip -/- and their wild-type 

littermates (WT), n35 for both groups, were meal-fed 6 h/d chow containing glyceryl 

tri(hexandecanoate-d 31) for 13 d. Animals were killed after fasting for 16 h, or feeding for 3 h. 

Total liver-TG content was determined by enzymatic assay, and the portion of TG derived from 

dietary sources determined by GC/MS. Although total body weights were similar (23 g), inguinal 

adipose depots were greater in Txnip -/- compared to WT (358 0.102 vs 215 0.045 mg, 

P0.0003). Furthermore, total liver weights were greater in both fasted (10%, P0.05) and 

fed states (12%, P0.0001, Txnip -/- , compared to controls). With feeding, the Txnip -/- liver-TG 

pool size was reduced (see figure) and the amount of TG derived from the diet did not increase. 

By contrast, with feeding, WT liver-TG increased 42% from fasting values, and the amount from 

the diet doubled (figure). Over the labeling period, the daily deposition of dietary-TG was greater 

in fasted compared to fed Txnip -/- mice (62 to 47 g/d). Conversely, in WT mice dietary-TG 

deposition was higher with feeding compared to fasting (20 to 33 g/d). The expected 

expansion of the liver-TG pool with feeding in normal mice was absent in Txnip -/- and could be 

due to greater postprandial liver fatty acid oxidation of fatty acids and/or greater flux of dietary 

fatty acids to periphery. 

The Role of the Apolipoprotein A-IV N-Terminus in Lipid Binding 

Kevin Pearson, Univ of Cincinnati, Cincinnati, OH; Richard B Weinberg, Wake Forest Univ 

Health Sciences, Winston Salem, NC; W. S Davidson; Univ of Cincinnati, Cincinnati, OH 

P133 

Apolipoprotein (apo) A-IV is a 376 amino acid member of the family of exchangeable 

apolipoproteins that includes apoA-I and apoE. It has been proposed to play a number of roles 

including chylomicron assembly, reverse cholesterol transport and protection from inflammation. 

In vivo, apoA-IV exists in both a lipid-poor and a lipid associated form and the balance 

between these two states may modulate its function. We have examined the structural 

elements of apoA-IV that modulate its binding to lipid. Using deletion mutagenesis on bacterially 

expressed apoA-IV, we made a series of C-terminal deletion mutants. The result of these 

studies indicated that removal of only a 10 amino acid region between residues 333 and 343 

strongly increased the ability of apoA-IV to bind and reorganize DMPC liposomes (8.5% 0.8% 

/min) vs. full length apoA-IV (3.5% 0.8% /min). Thus, this domain appears to inhibit the 

lipid-binding potential of WT apoA-IV. Interestingly, a double mutant lacking both the N-terminal 

20 amino acids and this ‘inhibitory’ domain failed to exhibit the increased lipid binding effect 

(4.2 0.2% /min). Thus, the N-terminus is required for apoA-IV rapid lipid binding. Our 

working hypothesis is that there is a complex interaction between residues 333–343 and the 

N-terminus that puts human apoA-IV in an unfavorable conformation for lipid binding. Current 

studies are focused on narrowing down these regions with the overall goal of engineering 

mutant forms of apoA-IV that vary widely in their ability to interact with lipids. These forms will 

be useful for future studies designed to understand the role of lipid affinity in the functionality 

of apoA-IV. 

P134 

Detergent Treatment and Removal Emulates Plasma Phospholipid Transfer 

Protein 

Henry J Pownall; Baylor College of Medicine, Houston, TX 





Background: Reverse cholesterol transport (RCT) is the putative cardioprotective process by 

which cholesterol that is synthesized in peripheral tissues, including the arterial wall, is 

transferred to early forms of high density lipoproteins (HDL), and esterified by lecithin:cholesterol 

acyltransferase (LCAT). After additional remodeling in the plasma compartment, the 

HDL-cholesteryl esters (CE) are selectively removed by hepatic SR-BI. Phosphatidylcholine (PC) 

is the essential cholesterol-binding component of native HDL and the donor of the acyl chain 

that forms CE in HDL. Many laboratories have used detergent treatment and removal (DTR) with 

sodium cholate to prepare model HDL from apo A-I and PC. We have applied this method to 

the total lipoproteins (TLP) of human plasma. Hypothesis: DTR of total human plasma 

lipoproteins (TLP) in the absence and presence of added PC might be cardioprotective through 

effects on PC redistribution among lipoprotein subclasses. Results: According to size exclusion 

chromatography, DTR of TLP redistributes PC among plasma lipoproteins, increases the particle 

size of LDL and HDL, and releases apo A-I but not apo A-II as a lipid-free species. This effect 

is dose dependent with respect to cholate and more profound above its critical micelle 

concentration (CMC15 mM); similar results were obtained with the TLP of eleven normal 

volunteers. DTR by dialysis, desalting by gel filtration chromatography, and dilution below the 

CMC of cholate gave similar results. Whereas DTR of HDL alone gives rise to a larger particle 

with the concurrent release of lipid-free apo A-I, DTR of LDL alone had no effect on its size. 

Thus, DTR emulates human plasma PLTP, which transfers PC among lipoproteins with 

subsequent dissociation of apo A-I, and HDL fusion (Lusa et al 1996). DTR of TLP in the 

presence of added PC increases the PC content of LDL and HDL by 80 and 150%, 

respectively. Conclusion: DTR emulates the activity of plasma phospholipid transfer protein 

and in the presence of exogenous PC, increases the PC content of LDL and especially HDL, an 

effect that should enhance the cholesterophilicity of plasma lipoproteins, potentiate RCT, and 

enhance the cardioprotective properties of HDL. 

Suppression of Endothelial Lipase Expression Decreases Lipoprotein 

Uptake and Cholesterol Efflux in Human Macrophages 

Guosong Qiu, John S Hill; Univ of British Columbia, Vancouver, Canada 

P135 

Macrophages play a critical role in the development and progression of atherosclerosis and 

have received increasing attention as a potential therapeutic target. Investigations utilizing 

mouse models of atherosclerosis have indicated that the expression and secretion of several 

lipases by macrophages promotes atherosclerosis. However, the specific role of macrophagederived 

endothelial lipase (EL) has not been investigated. The purpose of the present study is 

to determine the extent to which EL may influence the atherogenic capability of human 

macrophages as it relates to lipoprotein binding, uptake, and cholesterol efflux. Methods and 

Results: RNA interference was mediated by transduction with a lentivirus containing a vector 

sequence of short-hairpin RNA corresponding to a specific target sequence within the human 

EL gene. A vector containing a scrambled sequence with no known homology to human genes 

was used as a control. THP-1 monocytes were differentiated by the addition of phorbol 

12-myristate 13-acetate (PMA) after transduction with the lentivirus. The extent of LDL binding 

and cell association was assessed by incubations with DiI-LDL at 4°C and 37°C, respectively. 

Cholesterol efflux was measured following loading with 3H-cholesterol and with apo A-I as the 

acceptor. We achieved a transduction efficiency of 100% at a multiplicity of infection (MOI) of 

20 as analyzed by flow cytometry of EGFP expression in THP-1-derived macrophages 

transduced with the lentivirus. EL mRNA expression after PMA stimulation was suppressed by 

75% at MOI of 20 as analyzed by real-time quantitative PCR. In comparison to cells transduced 

with the lentivirus containing the control vector, EL suppressed macrophages exhibited a 65% 

and 61% decrease in native LDL binding and association, respectively. The suppression of EL 

in THP-1 macrophages was also associated with a 33% decrease in cholesterol efflux. The 

mechanism by which EL mediates these effects is currently being investigated. Conclusions: 

The expression by guest of ELon in human June macrophages 29, 2013 appears to modify both lipoprotein uptake and

E-76 Vol 25, No 5 May 2005 

cholesterol efflux and thus is likely to influence the formation of macrophage-derived foam cells 

within atherosclerotic lesions. 

P136 

Mechanisms of Glucosamine-Induced Impairment in Hepatic Assembly and 

Secretion of Apolipoprotein B100-Containing Lipoproteins 

Wei Qiu, Rita Kohen-Avramoglu, Angela Rutledge, Julie Tsai, Khosrow Adeli; Hosp for Sick 

Children, Toronto, Canada 

Glucosamine-induced endoplasmic reticulum (ER) stress, as measured by increases in the level 

of Grp78 and Grp 94, is associated with increased proteasomal degradation of apoB100 in 

cultured hepatocytes (Arterioscler Thromb Vasc Biol. 2004 Dec 23; [Epub]). In the present 

study, we have examined the mechanisms linking glucosamine-induced ER stress and 

co-/post-translational apoB100 assembly and degradation. Trypsin sensitivity studies of 

radiolabeled apoB100 showed different fragmentation patterns for untreated and glucosaminetreated 

HepG2 cells, suggesting glucosamine-induced changes in apoB100 conformation. 

Endoglycosidase H studies of newly-synthesized apoB100 suggested that glucosamine induced 

N-linked glycosylation defects resulted in reduced apoB100 secretion. We also examined 

glucosamine-induced changes in VLDL assembly and secretion. Following 16 hour glucosamine 

treatment, there was a dose-dependent decrease in VLDL-apoB100 secretion in 

primary hepatocytes (24.2– 89.2% at 1–10 mM doses) and rat McA-RH7777 cells (23.2- 67.3% 

at 1–10 mM doses). Glucosamine also inhibited the assembly of larger VLDL and IDL particles, 

but did not affect HDL-size particles. The formation of apoB48-containing lipoproteins in 

McA-RH7777 cells was unchanged by glucosamine treatment, suggesting that the glucosamine 

effect was specific to the assembly of larger apoB molecules. Cultured hepatocytes were also 

radiolabeled and then treated with 5 mM glucosamine only during the chase period. This short 

term (posttranslational) glucosamine treatment led to a 24% reduction in apoB100 secretion 

(p0.01, n4) but did not induce intracellular apoB100 accumulation suggesting that 

glucosamine may promote post-ER apoB degradation. Interestingly, the glucosamine-induced 

reduction in apoB100 secretion (posttranslationally) could be partially reversed following 

treatment with vitamin E or desferrioxamine. Taken together these data suggest that long-term 

glucosamine treatment may cause defects in apoB100 folding and glycosylation resulting in 

enhanced proteasomal degradation. Posttranslationally, glucosamine may either directly or 

indirectly interfere with VLDL assembly processes leading to degradation via non-proteasomal 

pathways. 

P137 

Robust Triacylglycerol Transfer Activity of Microsomal Triglyceride Transfer 

Protein is not Critical for Primordial ApoB-Particle Assembly and Secretion 

Paul Rava, SUNY Downstate Med Cntr, Brooklyn, NY; Gregory S Shelness, Wake Forest Univ 

Sch of Medicine, Winston-Salem, NC; M. Mahmood Hussain; SUNY Downstate Med Cntr, 

Brooklyn, NY 

Microsomal triacylglycerol (TAG) transfer protein (hMTP) is essential for human apoBlipoprotein 

assembly and secretion. We showed previously that Drosophila melanogaster MTP 

(dMTP) lacked TAG transfer and yet supported the secretion of apoB34 and apoB41 (Sellers et 

al. JBC (2003) 278:20,367). To understand how dMTP achieves apoB secretion, we first asked 

whether dMTP-mediated apoB secretion responds to oleic acid supplementation. ApoB45 

secretion was induced when coexpressed with either hMTP (20-fold) or dMTP (7-fold) in 

COS cells. Oleic acid supplementation further augmented apoB45 secretion in these cells by 

20.4 7.0% and 73.5 21.0% for hMTP and dMTP, respectively. The particles secreted in 

all these conditions had similar densities of 1.1–1.2 g/ml. We next determined the ability of 

dMTP to rescue longer apoB polypeptides. dMTP facilitated the secretion of all apoB 

polypeptides in the range of B48-B72, but it was 50% as efficient as the hMTP. Finally, 

various lipid transfer activities were measured using equal amounts of the purified hMTP and 

dMTP. The activities of hMTP and dMTP for each lipid class (%transfer/h) were as follows: TAG 

(97.6 6.4 and 5.5 0.8); cholesterol esters (4.4 0.5 and 7.14 0.6); and phospholipids 

(5.4 0.3 and 6.3 0.1). Thus, while phospholipid lipid transfer activity was similar in these 

homologues, cholesterol ester and triglyceride transfer activities in dMTP were 62 and 6% of 

the hMTP value. We conclude, based on these data, that robust TAG transfer activity of hMTP 

is not essential for the assembly and secretion of primordial apoB-lipoprotein particles, but may 

play a role in their second step core expansion. 

Essential Role of ABCA1 in HDL Catabolism in Mice 

P138 

Franz Rinninger, May Brundert, Martin Merkel, Joerg Heeren, Univ Hosp Eppendorf, 

Hamburg, Germany; Michael Hayden, Roshni Singaraja; Univ of British Columbia, Vancouver, 

Canada 

Objective: The role of ABCA1 in HDL biogenesis is established. However, the function of this 

protein in HDL catabolism has not been addressed. Here the effect of ABCA1 on plasma HDL 

metabolism and on HDL uptake by tissues was investigated. Methods: ABCA1 knockout (KO, 

homozygous) and ABCA1-KO/ABCA1-BAC-transgenic (humanized ABCA1-KO) mice were one 

model. Besides, ABCA1-BAC-transgenic (TG) mice were compared with wildtype (WT) 

littermates. Murine HDL was labeled in the protein ( 125I-TC) and in the CE ([ 3H]CEt) moieties. 

After HDL injection, plasma decay of both tracers was analyzed (24 hours) for FCR calculation. 

Finally tissue sites of HDL uptake were investigated. Results: In WT mice, plasma cholesterol 

was 61 /-4, in ABCA1-KO cholesterol decreased to 18 /- 2 and in humanized ABCA1-KO, 

cholesterol was restored to normal levels (45 /- 7, n 6). Compared to WT, ABCA1-KO mice 

had an elevated FCR for both HDL tracers in plasma and a higher selective CE removal from 

plasma. In humanized ABCA1-KO, HDL plasma FCR and selective CE removal were close to WT. 

With respect to tissue sites of HDL uptake, compared to WT, ABCA1-KO mice had an increased 

selective CE uptake by liver (86 versus 779 pools x h-1 x103 ,n 5, p 0.005) and adrenals 

(1.5 versus 46.3 pools x h-1 x103 ,n 5, p 0.05). Downloaded In humanizedfrom ABCA1-KO, HDL selective 



CE uptake by liver (261, n 6, p 0.05) and adrenals (6.7, n 6, p 0.05) again was close 

to WT. HDL metabolism by kidneys, spleen, stomach, intestine, brain, heart, lungs and carcass 

was investigated. Compared to WT, ABCA1-BAC-TG mice had an increase in plasma cholesterol 

(61 /- 4 versus 85 /- 2 mg/dl, n 5, p 0.001). Compared to controls, FCR’s for both 

HDL tracers and selective CE removal from plasma (0.0963 versus 0.0852 pools/h) decreased 

in ABCA1-BAC-TG. Compared to WT, HDL selective CE uptake by the liver decreased (86 versus 

74 pools x h -1 x10 3 ). ABCA1 expression had no significant effect on HDL tracer uptake and on 

HDL selective CE uptake by 8 remaining tissues. Conclusions: The role of ABCA1 in cellular 

cholesterol efflux is established. Here we show that ABCA1 expression has a substantial effect 

on HDL catabolism in plasma and HDL selective CE uptake by liver and adrenals. 

The Role of the Beta5 Loop of Endothelial Lipase in the Hydrolysis of 

Reconstituted High-Density Lipoprotein Phospholipids 

P139 

Chatri Settasatian, Anita van der Meer, The Heart Rsch Institute, Sydney, Australia; Daniel J 

Rader, Univ of Pennsylvania Sch of medicine, Philadelphia, PA; Philip J Barter, Kerry-Anne 

Rye; The Heart Rsch Institute, Sydney, Australia 

Endothelial lipase (EL) is a member of triglyceride lipase gene family that exhibits high 

phospholipase and low triglyceride lipase activity. High-density lipoproteins (HDL) are the main 

substrates of EL. The putative beta5 loop of EL, as indicated by sequence alignment with 

well-established structure of pancreatic lipase (PL), is between amino acid residues isoleucine71 

and tryptophan83. To investigate the role of the beta5 loop in EL-mediated phospholipid 

hydrolysis, site-directed mutagenesis was used to substitute cysteine (C) for the conserved 

residues, isoleucine71 (I71C), glycine73 (G73C), glycine78 (G78C), and glutamic acid81 (E81C). 

The ability of these mutants to hydrolyse phospholipids was assessed using as the substrate 

discoidal reconstituted HDL containing apolipoprotein (apo) A-I and 1-palmitoyl-2-oleoyl 

phosphatidylcholine, (A-I)rHDL. Compared to wild type EL, EL-I71C, EL-G73C, EL-G78C, and 

EL-E81C respectively hydrolyzed 46.7, 1.1, 53.1 and 21.0% of the discoidal (A-I)rHDL 

phospholipids. This is consistent with mutation of these conserved residues, especially G73, to 

cysteine altering the conformation of beta5 loop and reducing EL catalytic activity. The 

influence of apolipoproteins on the ability of the EL mutants to hydrolyse HDL phospholipids 

was also investigated using spherical rHDL containing either apoA-I, (A-I)rHDL, apoA-II, 

(A-II)rHDL, or apoA-I as well as apoA-II, (A-I/A-II)rHDL, as the substrate. EL-I71C, showed the 

similar pattern of hydrolysis to that of wild type EL, with the rate of phospholipid hydrolysis in 

the (AI/AII)rHDL(AI)rHDL(AII)rHDL. Phospholipid hydrolysis in the (A-I/A-II)rHDL and 

(A-I)rHDL was comparable for EL-G78C and EL-E81C mutations and approximately 40 – 60% 

lower than that was observed for wild type EL. These findings demonstrate that the beta5 loop 

of EL regulates phospholipid hydrolysis in discoidal and spherical rHDL. 

Role of Rho-Kinase in Vascular Abnormalities Associated with Insulin 

Resistance 

Takeshi Kanda, Koichi Hayashi, Shu Wakino, Koichiro Homma, Kyoko Yoshioka, Satoru 

Tatematsu, Kazuhiro Hasegawa, Ichiro Takamatsu, Naoki Sugano, Takao Saruta; Sch of 

Medicine, Keio Univ, Tokyo, Japan 

P140 

The impairment of insulin signaling in vascular tissue contributes to insulin resistance and 

subsequent development of hypertension. Rho-kinase plays an important role in hypertension 

and is reported to interfere with insulin signaling through serine-phosphorylation of insulin 

receptor substrate-1 (IRS-1) in cultured vascular smooth muscle cells. Nevertheless, whether 

Rho-kinase is activated in obesity and insulin resistance has not been elucidated. Here, the role 

of Rho-kinase was investigated in obese Zucker rats. 4-week treatment with fasudil, a selective 

Rho-kinase inhibitor, lowered the blood pressure to the level of lean rats (obese rats fasudil 

20 mg/kg/day, 1043 mmHg vs. control obese rats, 1223 mmHg, p0.01, n6). Treatment 

with fasudil improved glucose and lipid metabolism in a dose dependent manner. The activity 

of Rho-kinase was markedly increased in the aorta from obese rats, and was suppressed by 

the treatment with fasudil in a dose-dependent manner. Furthermore, fasudil reduced the 

enhanced serine-phosphorylation of IRS-1, and rescued the diminished insulin-stimulated 

Akt-phosphorylation. The phosphorylation level of Erk1/2 in the aorta from obese rats was 

2.1-fold higher than that from the lean control, and was suppressed by fasudil treatment. By 

using CCD camera to directly visualize the arteriolar response within the skeletal muscle, we 

found that both endothelium-dependent and -independent vasodilation were markedly impaired 

in obese rats and this blunted responses were restored by subdepressor dose of fasudil 

(3mg/kg/day). In vitro analysis showed that TNF- activated Rho-kinase in endothelial cells. 

Both fasudil and Y-27632, another Rho-kinase inhibitor, restored the downregulation and the 

decreased phosphorylation of endothelial nitric oxide synthase by TNF-. From these results, 

we conclude that Rho-kinase plays an important role in the impairment of insulin signaling and 

hemodynamic abnormalities in insulin resistance and that the inhibition of Rho-kinase would 

serve to reduce the risks of cardiovascular disease associated with insulin resistance. 

Thrombin Inhibition with Melagatran Prevents Plaque Rupture in 

Apolipoprotein E Knockout Mice 

Sharada Karanam, Bristol Heart Institute, Univ of Bristol, Bristol, United Kingdom; Regina 

Fritsche-Danielson, AstraZeneca, Molndal, Sweden; Christopher L Jackson; Bristol Heart 

Institute, Univ of Bristol, Bristol, United Kingdom 

P141 

Atherosclerotic plaque rupture and subsequent thrombogenicity cause the majority of acute 

coronary events. Therefore, the effect of the oral direct thrombin inhibitor melagatran on 

atherosclerotic plaques was investigated in the fat-fed apolipoprotein E (apoE) knockout mouse, 

in which plaque ruptures occur in the brachiocephalic artery after 8 weeks on a high fat diet. 

Eighty male apoE knockout mice, 6–8 weeks old, were fed diet containing 21% fat and 0.15% 

cholesterol by for guest 8 weeks. on June Forty 29, mice2013 were treated with 500 mol/Kg bodyweight/day of

melagatran throughout the 8 weeks. The brachiocephalic artery was removed following 

perfusion fixation with formalin at a constant pressure of 100 mmHg and embedded in paraffin. 

Computerised morphometry of serial sections was used to measure the areas of the plaque, 

the media and the lumen. The number of buried fibrous layers within the plaque and presence 

or absence of acute plaque rupture was also recorded. Immunostaining was used to detect the 

expression of markers of inflammation and thrombogenicity.Melagatran treatment at 500 

mol/Kg bodyweight/day significantly reduced plaque size from 38.5 5.6 to 7.3 1.3x10 3 

m 2 (-81%; p0.001) and the incidence of plaque rupture from 0.43 0.10 to 0.10 0.05 

ruptures/mouse (-77%; p0.05). Immunohistochemical analysis of serial sections showed 

reduced staining for various inflammatory and thrombogenic markers. This study supports the 

hypothesis that serial accumulation of thrombus through repeated episodes of non-fatal plaque 

rupture contributes to atherosclerotic plaque growth in the apoE knockout mouse and also that 

inhibition of thrombin activity may be a useful strategy for inhibiting plaque rupture. 

P142 

Phospholipase A2 Levels are Associated with Coronary Artery Disease and 

Reduced with Statin Therapy 

Sang-Hyun Kim, Joo-Hee Zo, Myung-A Kim; Div of Cardiology, Dept of Internal Medicine, 

Seoul National Univ Boramae Hosp, Seoul National Univ College of Medicine, Seoul, 

Republic of Korea 

Objectives : The association of lipoprotein-associated phospholipase A2(PLA2) with coronary 

artery disease risk factors, and with the incidence of major adverse events have been reported 

in several studies. But the associations of phospholipase A2 with angiographic coronary artery 

disease and the effect of statin therapy have not been reported yet. Methods : PLA2 levels were 

measured in 64 patients undergoing coronary angiography who admitted due to chest pain. 

Demographic findings and the PLA2 levels were measured in the patients with angiographically 

proven coronary artery disease and/or taking statin pills. Results : Mean age was 63 years and 

20% were women. The mean PLA2 level was 214 ng/mL. PLA2 levels were correlated with 

LDL, total cholesterol, fibrinogen, and serum glucose level. PLA2 levels correlated with the 

extent of angiographic CAD. During the median follow-up of 1 year, statin therapy significantly 

reduced the PLA2 level and the incidence of major coronary events by 25 % (p 0.01). Higher 

PLA2 levels were associated with a greater risk of events and this association was significant 

after adjusting C-reactive protein. Conclusion : Higher PLA2 levels were associated with 

angiographically proven coronary artery disease and a higher incidence of major adverse 

events. Statin therapy significantly reduced PLA2 level and the clinical outcomes. 

P143 

Combined Effects of Lovastatin and Estrogen Therapy on the Development 

of Atherosclerosis in the Hyperlipidemic Mice 

Eun-Young Kim, Young M Lee, Jae-Hoon Choi, Jong-Gil Park, Seung-Phil Park, Young-Han 

Ryu, Hye Kyoung Song, Mi-Ni Lee, Mi-Ran Lee, Goo Taeg Oh; EWHA Womans Univ, Seoul, 

Republic of Korea 

OBJECTIVES Post-menopausal women have a high risk of atherosclerosis. The additional 

effects of hormone replacement therapy (HRT) and statins on the serum lipid profile have been 

reported in post-menopausal women. However, combined effects on atherosclerotic lesions in 

postmenopausal model has not been defined well. Thus, the aim of this study is to determine 

the effect of continuous combined HRT with lovastatin on atherogenesis using ovariectomized 

atherosclerotic mice model. METHODS & RESULTS Ovariectomized (OVX) female low density 

lipoprotein receptor-deficient (LDLR-/-) mice were fed an atherogenic diet (1.25% cholesterol, 

15% fat, 0.5% Na-cholate) for 8 weeks. Mice were divided into four groups: group I - 

atherogenic diet only; group II - mice receiving HRT (implanted subcutaneously with 60-day 

release 17-estradiol (E2) pellets); group III - supplemented with lovastatin (0.1%) in the same 

atherogenic diet; group IV-HRT lovastatin (0.1%). Combination of estrogen and lovastatin 

significantly decreased fatty streak lesions in aortic valve compared to single treatment of 

estrogen or lovastatin. However, treatments of estrogen and/or lovastatin were not associated 

with any plasma lipid level (plasma total cholesterol, triglyceride, HDL-C, LDL-C level) changes 

in three treatment group. In summary, combination of HRT and statins significantly decreased 

fatty streak lesion compared to HRT or lovastatin alone in OVX atherosclerotic (LDLR-/-) mice. 

CONCLUSIONS Based on our results, we can conclude the combination of lovastatin and 

continuous combined HRT seems to be more effective than lovastatin only in the treatment of 

atherosclerosis in post-menopausal women in the abscence of lipid-lowering effect. 

P144 

Enzyme-Linked Immunosorbent Assay for Molecular Characterization of 

Antibody-Targeted Echogenic Liposomes 

Melvin E Klegerman, EchoDynamics, Inc., Chicago, IL; Shaoling Huang, Northwestern Univ, 

Evanston, IL; Devang Parikh, Amanda Adeleye, Northwestern Univ, Chicago, IL; Robert C 

MacDonald, Northwestern Univ, Evanston, IL; David D McPherson; Northwestern Univ, 

Chicago, IL 

Antibody-conjugated echogenic liposomes (Ab-ELIP) have been developed for atheroma 

characterization. The purpose of the present study was to determine whether an enzyme-linked 

immunosorbent assay (ELISA) method is suitable for determination of thermodynamic 

parameters for antibody/peptide conjugation. Anti-human fibrin(ogen) Ab were conjugated to 

ELIP through a thioether linkage as previously described (Klegerman et al.: Anal. Biochem 

300:46 –52, 2002). For the ELISA, primary incubations with free Ab or Ab-ELIP were performed 

at 4, 23, and 37° C; all other incubations were at 37° C. Secondary incubations were performed 

with anti-rabbit or anti-mouse immunoglobulin G-alkaline phosphatase conjugates. Substrate 

incubations were carried out with paranitrophenyl phosphate (4 mg/ml). Antibody affinities 

were calculated based on the hyperbolic relation between absorbance at 405 nm and 

conjugated Ab concentration, with dissociation constant Ab concentration at half-maximal 

absorbance. Thermodynamic parameters, H°, G°, Downloaded and S°, werefrom calculated from equations 

of state after determination of H° from van’t Hoff plots of 1/T vs. log association constant. 

Both the affinities and patterns of association thermodynamics of unconjugated Ab determined 

by ELISA were similar to those determined by radioimmunoassay. Specific binding was 

characterized by moderate negative H° (2–9 kcal/mole) and high positive S° (11–30 

cal/mole°). Both ELISA and RIA revealed significant changes in these parameters after Ab 

conjugation, consisting of an increase in H°, large increases in S° (14–37 cal/mole°), and 

up to a 100-fold increase in affinity. These data validate the ELISA method for determination 

of Ab- and Ab-ELIP-fibrin(ogen) binding kinetics. Further studies are needed to determine 

whether ELIP enhancement of Ab binding involves recruitment of acyl hydrocarbon chains in 

hydrophobic interactions with the antigen (Ag), straining the specific Ab-Ag association, or 

coupling of phospholipid phase transitions to Ab-Ag association kinetics. Results of these 

studies will have implications for all targetable phospholipid contrast agents undergoing 

conjugation of antibodies/peptides. 

P145 

Macrophage-Specific Expression of Carboxyl Ester Lipase Modifies 

Cholesterol and Ceramide Metabolism and Increases Atherosclerosis in 

Apo E null Mice 

Ahmer Kodvawala, David Y Hui; Univ of Cincinnati, Cincinnati, OH 


Carboxyl Ester Lipase (CEL) is a lipolytic enzyme with multiple substrate reactivity. We have 

previously demonstrated that CEL is expressed in human but not mouse macrophages. Human 

macrophage expression of CEL is stimulated by oxLDL, suggesting a possible role of this 

enzyme in atherosclerosis. This hypothesis was tested by generating macrophage-specific CEL 

transgenic mice to examine the role of CEL in macrophage cholesterol metabolism and 

diet-induced atherosclerosis. Macrophage-specific CEL transgenic mice were generated using 

visna virus long terminal repeats as a promoter for the expression of rat CEL cDNA. 

Macrophages from wild type and transgenic mice were obtained and acLDL uptake was 

measured by incubation with [ 125 I]acLDL. Cholesteryl ester mass was measured by gas 

chromatography. The ceramide contents of basal and cholesterol loaded macrophages were 

measured by diacylglycerol kinase reaction. Results showed no significant difference in the 

acLDL uptake by CEL transgenic and wild type macrophages. However, when cholesteryl ester 

mass was quantified using gas chromatography, CEL transgenic macrophages displayed 

elevated intracellular cholesteryl ester level under basal as well as acLDL-loaded conditions 

compared to wild type macrophages(p0.05). In addition, CEL transgenic macrophages were 

found to have less ceramide contents than wild type macrophages(p 0.05). The macrophagespecific 

CEL transgenic mice were then crossbred with apoE null mice to generate CEL-tg/apoE 

null. The animals were fed a western type diet containing 21% milk fat and 0.15% cholesterol 

for 8 weeks prior to obtaining their aorta to quantify atherosclerosis lesions. The CEL-tg/apo E 

null mice were found to have significantly higher lesion area in the aortic arch compared to the 

apoE KO mice without the CEL transgene. These results underscore the importance of CEL in 

atherosclerosis. CEL appears to confer susceptiblity to atherosclerosis by hydrolyzing ceramide 

which allows continuous esterification of membrance bound choleterol. These results, along 

with earlier reports of localization of CEL in human atherosclerotic lesions suggest that this 

enzyme affects the pathogenesis and severity of atherosclerosis. 

P146 

The Vasculo-Protective Enzyme Heme Oxygenase-1 is a Novel and Direct 

Target Gene for Peroxisome Proliferator Activated Receptors (PPARs) in the 

Vascular Wall 

Gerhard Kronke, Alexandra Kadl, Univ of Virginia, Charlottesville, VA; Elena Ikonomu, Stefan 

Bluml, Alexander Furnkranz, Univ of Vienna, Vienna, Austria; Laszlo Nagy, Univ of Debrecen, 

Debrecen, Hungary; Bernd R Binder, Univ of Vienna, Vienna, Austria; Norbert Leitinger; Univ 

of Virginia, Charlottesville, VA 

Activation of the ligand-activated transcription factors peroxisome proliferator activated 

receptors (PPARs) alpha and gamma inhibits the development of atherosclerosis, which has 

been attributed to direct vasculo-protective and local anti-inflammatory effects on cells of the 

vascular wall. These beneficial effects are poorly understood and little is known about the 

regulation of anti-inflammatory or tissue-protective genes by PPARs. We found that treatment 

of mice with the PPAR agonist rosiglitazone induced expression of the highly antiinflammatory 

enzyme heme oxygenase-1 (HO-1) in endothelial cells (ECs) and vascular smooth 

muscle cells (VSMCs) in the wall of the carotid artery. In vitro analysis revealed a differential 

regulation of HO-1 expression both by ligands for PPAR and PPAR in ECs, VSMCs and 

macrophages and the enzymatic activity of HO-1 was involved in the anti-inflammatory 

properties exerted by a PPAR ligand on VSMCs. Subsequent analysis of the human HO-1 

promoter using transient-transfection assays as well as electrophoretic mobility shift assays 

confirmed that HO-1 is a direct PPAR target gene regulated by 2 functional PPAR-responsive 

elements. Finally we demonstrate that a common polymorphism within the human HO-1 

promoter critically influences its transcriptional regulation by both PPAR and PPAR. The 

discovery of HO-1 as a novel PPAR target gene will lead to a better understanding of the 

beneficial effects exerted by PPARs on the vascular wall and provides an additional rationale 

for the use of PPAR-activating drugs in the treatment of atherosclerosis and other inflammatory 

diseases. 

P147 

HMG-CoA Reductase Inhibition by Rosuvastatin Inversely Regulates 

Expression of LOX-1 and eNOS in Cytokine-Treated Human Endothelial 

Cells 

Martin Landsberger, Franziska Jantzen, Stephan B Felix; Ernst Moritz Arndt Univ, 17487 

Greifswald, Germany 

BACKGROUND: The expression of LOX-1, a major endothelial receptor for oxidized LDL 

http://atvb.ahajournals.org/ (ox-LDL), by is increased guest on in endothelial June 29, dysfunction 2013 and atherosclerosis, and regulated by several 


E-78 Vol 25, No 5 May 2005 

inflammatory mediators, e.g. TNF-, and ox-LDL. Inhibition of HMG-CoA reductase by statins 

may directly influence initiation and progression of atherosclerosis. The aim of this study was 

to analyze the effects of rosuvastatin (RSV) on the expression of LOX-1 in TNF--treated human 

venous endothelial cells (HUVEC). As Nitric Oxide deficiency leads to an up-regulation of LOX-1 

expression, we additionally analyzed the effects of RSV on eNOS mRNA and protein expression. 

METHODS: Total RNA and protein extracts were isolated from HUVEC after 8 hours with 

standard procedures; concentrations of RSV tested were 10 -8 to 10 -3 mol/l. LOX-1 and eNOS 

mRNA expression was determined by TaqMan ® . LOX-1 and eNOS protein expression was 

determined by Western blotting with monoclonal antibodies against eNOS and polyclonal 

antibodies raised against the C-type lectin-like domain of LOX-1. Expression was calculated 

relative to the expression in cells treated with TNF- in the absence of RSV. RESULTS AND 

CONCLUSIONS: Untreated cells exhibited 12.47.5% LOX-1 mRNA and 81.56.9% protein 

expression of TNF--treated cells. Co-incubation with RSV decreased the TNF--stimulated 

LOX-1 protein expression (61.017.8%, P0.05 for 10 -7 mol/l RSV), but higher concentrations 

of RSV trended to a non-significant increase in mRNA expression of LOX-1 above control levels. 

RSV increased the expression of eNOS mRNA and protein expression both in non-treated and 

in TNF--treated cells. The effects on eNOS expression mediated through RSV could be 

abolished through incubation of the cells with 10 -4 mol/l mevalonate. Treatment with 

geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate reversed the increase of eNOS 

mRNA and protein expression induced by RSV. We conclude that rosuvastatin reverses the 

detrimental effects of TNF--induced changes in eNOS and LOX-1 protein expression by the 

inhibition of HMG-CoA reductase and subsequent blocking of isoprenoid synthesis. 

P148 

ASCL002, 5-(4-Hydroxy-2,3,5-Trimethylbenzylidene) Thiazolidin-2,4-Dione, 

Ameliorates Atherosclerosis by the Inhibition of Monocyte Recruitment 

Young M Lee, Jae-Hoon Choi, Mi-Ran Lee, Eun-Young Kim, Jong-Gil Park, Mi-Ni Lee, Hye 

Kyoung Song, Young-Han Ryu, EWHA Womans Univ, Seoul, Republic of Korea; Tae-Sook 

Jeong, Woo Song Lee, Korea Rsch Institute of Bioscience and Biotechnology, Daejon, 

Republic of Korea; Goo Taeg Oh; EWHA Womans Univ, Seoul, Republic of Korea 

Objective ASCL002, 5-(4-hydroxy-2,3,5-trimethylbenzylidene)thiazolidin-2,4-dione, is a derivative 

of thiazolidinedinones (TZDs) which exhibit anti-diabetic and anti-inflammatory activities 

as dual 5-lipoxygenase and cyclooxygenase inhibitors. Here we investigated whether this 

compound reduces atherogenesis in low density lipoprotein receptor deficient mice (Ldlr-/-). 

Methods and Results We first investigated whether ASCL002 affected atherogenesis in Ldlr-/mice 

by quantifying areas of lesions formed in the aortic sinus of mice treated and untreated 

with the compound. Mice in the test groups were fed an atherogenic diet (1.25% cholesterol, 

15% fat, and 0.5% Na-cholate) supplemented with 0.1% ASCL002. The sections of aortic roots 

from Ldlr-/- mice in each group were stained with Oil-red O, and quantified positiveatherosclerotic 

lesions by the image analysis. After 8 weeks of the atherogenic diet, Ldlr-/mice 

developed the fatty streak lesion in the aortic sinus, whereas this lesion was dramatically 

reduced by ASCL002, as well as the macrophage infiltration into the lesion. These results 

indicate that ASCL002 potentially inhibited atheroma formation in high cholesterol fed Ldlr-/mice. 

To elucidate the potential action mechanism of ASCL002 on the atherogenesis, 

leukotriene B 4 (LTB 4) was measured in the plasma of Ldlr-/- mice. The Ldlr-/- mice treated with 

ASCL002 reduced serum levels of leukotriene B 4. However, plasma total cholesterol, LDL, and 

HDL levels were not changed in ASCL002 treated group compared to control group. In addition, 

ASCL002 down-regulated the aortic expression of pro-atherogenic molecules including TNF-, 

IL-1, IL-6, MCP-1 and VCAM-1 involved in the recruitment of monocyte into the aortic wall. 

Conclusions ASCL002 inhibit the release of arachidonic metabolite LTB4 into the plasma of diet 

induced atherogenic mice, that leading to down-regulation of pro-atherogenic molecules 

involving monocyte recruitment such as TNF-, IL-1, IL-6, MCP-1 and VCAM-1. Thus, we can 

conclude that ASCL002 ameliorates atherosclerotic lesion formation by the inhibition of 

monocyte recruitment into the aortic wall. 

P149 

Hypercholesterolemia Results in Impairment of Inwardly-Rectifying K 

(Kir) Channels in Aortic Endothelial Cells in Vitro and in Vivo 

Yun Fang, Univ of Pennsylvania, Philadelphia, PA; George H Rothblat, Children’s Hosp of 

Philadelphia, Philadelphia, PA; Emile R Mohler, Irena Levitan; Univ of Pennsylvania, 


Hypercholesterolemia-induced dysfunction of the vascular endothelium occurs at an early stage 

of atherosclerosis and is a strong predictor of CVD development. Recently, we have shown that 

enriching aortic endothelial cells with cholesterol using methyl- cyclodextrin carrier system 

strongly suppresses the activity of endothelial inwardly-rectifying K (Kir) channels, a major 

class of endothelial membrane proteins, which are responsible for maintaining endothelial 

membrane potential and play a key role in endothelium-dependent vasorelaxation. Here we 

extend these studies to show that Kir channels in aortic endothelium are suppressed by 

hypercholesterolemic lipoprotein levels in vitro and by serum hypercholesterolemia in vivo. To 

test the effect of lipoprotein hypercholesterolemia on endothelial Kir channels in vitro, human 

aortic endothelial cells (HAECs) were exposed to acetylated LDL (acLDL), a modified LDL with 

a high-affinity for endothelial scavenger receptors, showing similar effect on endothelial Kir 

channels. Exposure of HAECs to 10 or 50 g/ml acLDL for 1, 6, 12 or 24 hours resulted in a 

time- and concentration-dependent increase in cellular cholesterol accompanied with a gradual 

decrease in Kir current. Importantly, acLDL-induced Kir suppression was rescued by reducing 

cellular cholesterol or by replacing cholesterol by its optical isomer, epicholesterol, indicating 

that the effect is due to an increase in cellular cholesterol. Furthermore, the ability of 

epicholesterol to rescue the activity of the channels suggests that it may have a potential for 

alleviating hypercholesterolemia induced endothelial dysfunction. To determine the effect of 

dyslipidemia on endothelial Kir in vivo, the activities of endothelial Kir channels were measured 

in pig aortic endothelial cells freshly isolated from healthy, hypercholesterolemic and 

hypercholesterolemic/diabetic Juvenile Yorkshire Pigs maintained on high cholesterol diet 

and/or streptozocin treatment for 6 months. Downloaded Our data show strong from 

inhibition effect of 

endothelial K activity (60%) in hypercholesterolemic and hypercholesterolemic/diabetic pig 

endothelium, indicating that hypercholesterolemia impairs endothelial Kir channels in vivo. 

P150 

Chronic Stress Induces Rapid Occlusion of Angioplasty-Injured Carotid 

Artery with an Atherosclerotic-Like Lesion 

Lijun Li, Georgetown Univ Med Cntr, Washington, DC; Ann-Cathrine Jonsson-Rylander, 

AstraZeneca, Mondal, Sweden; Zofia Zukowska; Georgetown Univ Med Cntr, Washington, 

DC 

Why in some cases angioplasty results in restenosis while in others it does not - remains 

unknown. Our work suggests that stress and neuropeptide Y (NPY) may play a role. NPY is a 

sympathetic neurotransmitter, vasoconstrictor and vascular growth factor acting via its multiple 

G-protein coupled receptors (Rs), Y1, Y2 and Y5. It is released from nerves during intense stress 

but the extent of its release and vascular activities shows genetic variability. Plasma 

NPY-immunoreactivity (ir) responses to stress are exaggerated in rodents with additional source 

of NPY in platelets, and in humans with NPY signal peptide polymorphism, where they associate 

with accelerated restenosis and atherosclerosis. Here, we tested the hypothesis that in rats, 

which express platelet NPY, stress accelerates post-angioplasty restenosis by activating the 

NPY-Y1/Y5 receptor system. Rats were subjected to carotid artery balloon angioplasty, and 

were either stressed by cold water (4°C) exposure (2hr/day/14days, the first being 2 hours 

before angioplasty) or not (NS). In NS rats, angioplasty alone increased plasma NPY-ir levels but 

only in the platelet fraction (PRP, 282ng/mL), and this associated with neointima formation 

(.08.01mm 2 ) and vascular up-regulation of Y1 and Y5R expression (mRNA and protein). In 

stressed rats, PRP NPY levels (658ng/mL), Y1/Y5 receptor expression and neointima 

formation (.22.02mm 2 ) increased even further. In addition, stress induced adventitial 

thickening and totally occluded the vessel with a lesion which contained cd68() macrophages, 

lipid deposition, thrombus and microvessels. Immunostaining for NPY, and Y1 and 

Y5Rs showed up-regulation/induction of the peptide and/or its Rs in the areas of smooth 

muscle cells and macrophages. Stress-induced changes were mimicked by administration of 

a slow release NPY pellet (10 g/14 days) next to the injured artery, and both effects were 

completely prevented by specific Y1R antagonist (.02mol/kg/min/14 days, iv), which also 

60% reduced neointima induced by angioplasty alone. Thus, stress, by activating sympathetic 

nerves and releasing NPY, may be an underappreciated risk factor for restenosis and 

atherosclerosis, and Y1R antagonists potentially useful as drugs inhibiting them. 

Coronary Artery Progressive Stenosis Relates to Vascular Intimal 

Macrophage Infiltration and Plaque Formation 

Jing Li, Lei Sun, Lianhong Li, Jianwu Tang, Dalian Med Univ, Dalian, China; Eiketsu Sho; 

KAIpharmaceuticals, Int, South San Francisco, CA 

P151 

Macrophages play an important role in atherosclerotic plaque formation, progression and 

rupture, which are responsible for the majority of acute coronary syndromes. This study was 

to assess the relationship between intimal macrophage infiltration and the progressive stenosis 

in coronary arteries. Thirty-nine left anterior descending coronary arteries in 39 autopsied 

cases were enrolled. The arteries were analyzed and divided into 3 groups according to the 

degree of stenosis (group A: 50% stenosis; group B: 50% 75% stenosis; group C: 75% 

stenosis). Inflammatory response was graded from G-0 to G-4 according to inflammatory cell 

infiltration in adventitia, media and intima. Immunohistochemistry was used to visualize the 

presence of macrophages (CD68). Vascular progressive stenosis with significant atherosclerotic 

plaque formation was recognized, showing plaque area in group C larger than in group B 

(p0.001), while there was no plaque in group A. Inflammatory response was related to the 

degree of vascular stenosis and plaque formation. Significant macrophage infiltration in intima 

was observed in group B and C, mainly appearing in the plaque area (Table). We concluded that 

the degree of luminal stenosis much more related to the formation of atherosclerotic plaque, 

which are suggested to relate to the degree of macrophage infiltration. CSAi: cross sectional 

area; CSAm: cross sectional area of media; MØ/IT: macrophages in intima/cross sectional area; 

MØ/Media: macrophages in media/cross sectional area. 

CSAi plaque area CSAm MØ/IT MØ/Media 

Group A 5.551.80* 00 4.791.09 13.336.44* 0.780.67 

Group B 11.854.55† 3.722.44† 4.650.70 17581.21† 6.387.68 

Group C 15.416.34 13.705.55 4.192.04 19571.93 6.094.95 

*p0.01 vs. Group B and C; †p0.01 vs. group C. 



P152 

Platelet-Derived Growth Factor (PDGF) Enhances mRNA Stability in Rat 

Aortic Smooth Muscle Cells by Down-Regulating a Ribnuclease Activity 

Bin liu, Univ of Rochester, Rochester, NY; Micheal Poon, Mount Sinai Sch of Medicine, New 

York, NY; yingqian Xu, Mark B Taubman; Univ of Rochester, Rochester, NY 

Platelet-derived growth factor (PDGF) has protean manifestations, including the regulation of 

growth and migration, in many cell types. We have previously reported that PDGF induces the 

accumulation of monocyte chemoattractant protein (MCP)-1 mRNA in smooth muscle cells 

(SMC), in large part due to an increase in mRNA stability. To elucidate the mechanism by which 

PDGF stabilizes MCP-1 mRNA, we have employed in vitro RNA gel mobility shift and decay 

assays. Cytoplasmic extracts from PDGF-treated SMC increased the half life of in vitro 

transcribed MCP-1 mRNA from ? 45 min to 2 h. PDGF-inhibitable degradation was not 

dependent on specific regions of the MCP-1 mRNA and was equally effective on in vitro 

transcribed tissue factor and c-fos mRNAs. Angiotensin II had a similar effect on MCP-1 mRNA 

stability, whereas tumor necrosis factor- and basic fibroblast growth factor did not. The 

PDGF-inhibitable by guest RNAse on activity June was 29, active 2013at 

pH 6.6 and heat stable, but was sensitive to

proteinase K. Extracts from PDGF-treated cells inhibited the RNAse activity of control extracts, 

suggesting that the effect of PDGF is due to activation of a soluble inhibitor of the RNAse. The 

effect of PDGF was blocked by inhibitors of tyrosine phosphorylation, but not by inhibitors of 

phosphatidylinositol 3-kinase, the Src family of kinases, or mitogen activated protein kinases. 

These studies suggest that PDGF has a generalized effect in prolonging mRNA stability in SMC. 

This effect was seen only with extracts from cells treated for at least 2 hrs with PDGF. 

Therefore, the contribution of enhanced mRNA stability to PDGF-mediated mRNA accumulation 

would not be seen with genes, such as c-fos, whose transcriptional activation are particularly 

short-lived and whose mRNAs have intrinsically short half-lives. A review of PDGF-inducible 

genes in SMC suggests that there are several patterns of gene induction, all consistent with the 

above findings. These studies should provide novel insights into PDGF-mediated mRNA 

accumulation. 

P153 

Loss of the Heparan Sulfate N-deacetylase/n-sulfotransferase-1 in Diabetic 

Liver: Role of Angiotensin Ii 

Ming-Lin Liu, Yanqing Zhu, Peter McCue, Kumar Sharma, Kevin J Williams; Thomas 

Jefferson Univ, Philadelphia, PA 

Diabetes mellitus increases the risk for atherosclerotic cardiovascular disease, but the basis is 

unclear. Diabetes is associated with loss of heparan sulfate (HS) from the liver, which may 

impede lipoprotein clearance and thereby worsen atherosclerosis. To study hepatic HS loss in 

diabetes, we studied regulation of the HS N-deacetylase/ N-sulfotransferase-1 (NDST), a key 

enzyme in hepatic HS biosynthesis. To induce type 1 diabetes, rats were injected with 

streptozotocin or saline (controls), and two weeks later, livers were harvested for measurements 

of NDST mRNA by real-time quantitative PCR, NDST protein by Western blot using an 

anti-NDST peptide antibody, and heparan N-sulfotransferase activity by cellulose thin-layer 

chromatography. Diabetes caused a substantial (50%) suppression of hepatic NDST mRNA, 

protein, and enzymatic activity. Importantly, treatment of diabetic rats with enalapril, an 

angiotensin converting enzyme (ACE) inhibitor, had no effect on hyperglycemia or hepatic NDST 

mRNA levels, yet significantly increased hepatic NDST enzymatic activity by 40% over the value 

in untreated diabetic animals. Enalapril also increased the amount of hepatic NDST protein. 

Similar results were obtained in diabetic animals treated with losartan, which blocks the type 

1 receptor (AT1) for angiotensin II. To study the mechanism, we found that diabetic livers 

exhibited increased ACE expression, which should enhance local angiotensin II action. 

Moreover, addition of angiotensin II to cultured McArdle hepatoma cells reduced NDST activity 

and protein. We conclude that diabetes substantially suppresses hepatic NDST mRNA, protein, 

and enzymatic activity. Angiotensin II contributes to the suppression of NDST protein and 

enzymatic activity, while mRNA suppression occurs independently. Suppression of hepatic 

NDST may contribute to diabetic dyslipidemia, and stimulation of NDST activity by angiotensin 

II inhibitors may provide cardiovascular protection. 

Non Esterified Fatty Acids Upregulate Cyclooxygenase-2 and Convert 

Macrophages to Triglyceride-Rich Foam Cells 

Eric E Lloyd, John W Gaubatz, Henry J Pownall; Baylor College of Medicine, Houston, TX 

P154 

Objective- Type 2 diabetes, a major risk factor for atherosclerosis, is associated with elevated 

plasma non-esterified fatty acids (NEFA). The objective of this study was to test the hypothesis 

that elevated NEFA contributes to atherogenesis by dysregulating lipid metabolism at the levels 

of lipid synthesis, gene expression, and uptake of modified low-density lipoproteins by 

macrophages. Methods and Results- Cellular NEFA uptake and esterification by THP-1 

macrophages, a cellular model for human monocyte-derived macrophages, was based on 

cell-associated radioactivity following incubation with [3H]oleic acid (OA), and on Oil Red O 

staining (ORO) and thin layer chromatography of the intracellular products. OA uptake increased 

following PMA-mediated differentiation of THP-1 macrophages to foam cells, was dosedependent 

with respect to OA, and was positively correlated with ORO staining of intracellular 

neutral lipids, the majority of which was triglyceride. Cells incubated with 1.8 mM OA exhibited 

higher uptake of Ac-LDL than those incubated with 0.2 mM OA, and showed increased 

expression of CD36, adipocyte fatty acid binding protein, and cyclooxygenase-2. Conclusions- 

These observations support a mechanistic link between elevated plasma NEFA and atherogenesis 

that is mediated by glycerol lipid synthesis and uptake of modified LDL. 

Insulin-Like Growth Factor-I Alterations in Mixed Hyperlipidemia 

Jan Malik, Tomas Stulc, General Univ Hosp and 1st Sch of Medicine, Charles Univ, Prague 

2, Czech Republic; Vojtech Melenovsky, The Johns Hopkins Hosp, Baltimore, MD; Zdena 

Lacinova, Dan Wichterle, Jan Simek, Richard Ceska; General Univ Hosp and 1st Sch of 

Medicine, Charles Univ, Prague 2, Czech Republic 

P155 

Total plasma total insulin-like growth factor-I (IGF-I) has been found decreased in both coronary 

artery disease and type 2 diabetes mellitus patients. This study was aimed at comparing total 

and free IGF-I levels between subjects with mixed hyperlipidemia and healthy controls. 

METHODS: We included 59 otherwise healthy subjects with mixed hyperlipidemia (total 

cholesterol 6.0 mmol/L and triglycerides 2.0 mmol/L) and 26 healthy normolipidemic 

controls (cholesterol 6.0 mmol/L and triglycerides 2.0 mmol/L). Plasma levels of total 

IGF-I and of IGF-binding protin-3 (IGFBP-3) were analyzed radioimmunoanalytically. Molar ratio 

of IGF-I and IGFBP-3 was calculated as a measure of free IGF-I. Differences between groups 

were analyzed using ANCOVA with the adjustment for age. RESULTS are shown in the Table 

as mean SD. CONCLUSIONS: As expected, mixed hyperlipidemia was associated with the 

decrease of total IGF-I. However, the levels of free IGF-I were increased. The increase of free 

IGF-I in mixed hyperlipidemia probably reflects an “IGF-I resistant” condition that parallels the 

hyperinsulinemia due to insulin resistance. Downloaded from 

Controls Mixed hyperlipidemia 

Age (years) 47.16.3 48.79.4 

Total cholesterol (mmol/L) 5.00.6 7.61.48*** 

Triglycerides (mmol/L) 1.10.6 5.43.7*** 

Insulin (mU/L) 5.73.9 26.111.3*** 

IGF-I (ug/ml) 20973 15354*** 

IGFBP-3 (ug/ml) 4.30.6 2.70.6*** 

IGF-I/IGFBP-3 ratio 0.170.04 0.250.06*** 

*p0.05; **p0.01; ***p0.0001 

Recombinant Heat Shock Protein-70 Prevents Augmented Intimal 

Thickening in Mice Exposed to Cigarette Smoke 

P156 

Michiaki Matsumoto, Charles Wang, Miha Cercek, Juliana Yano, Kuang-Yuh Chyu, Prediman 

K Shah, Bojan Cercek, Paul C Dimayuga; Cedars-Sinai Med Cntr, Los Angeles, CA 

Background: Gene chip analysis showed a significant reduction in Hsp70 expression in arteries 

of mice exposed to cigarette smoke (CS). Reports show that heat preconditioning results in the 

induction of Hsp70 and reduced intimal thickening. Hsp70 modulation of cell signaling is 

mediated by sequestering the Raf-1-activator, Bag-1. We investigated the role of Hsp70 and 

Bag-1 in intimal thickening of mice exposed to CS. Methods: Carotid artery Hsp70 expression 

was assessed in a cuff model of arterial injury by RT-PCR. Intimal thickening was assessed 21 

days after injury. Hsp70 and Bag-1 expression in aortas and injured arteries were analyzed by 

Western blot and immunostaining. Recombinant Hsp70 was injected i.v. to mice exposed to CS 

on the day of injury and 3, 7, 11, and 15 days after. Smooth muscle cells (SMC) were 

pulse-treated for 1 hour with cigarette smoke condensate (CSC) and were harvested after 6 and 

24 hours. Results: Cuff-injury resulted in increased Hsp70 mRNA expression in carotid arteries 

after 1 day and persisted for 21 days. CS exposure was associated with decreased expression 

of aortic Hsp70 (1.210.55 vs. 7.43.13 densitometric units, n3 each; p0.05) and 

percent Hsp70 stained area (5.12.3% vs. 10.84.9%, n4 each; p0.05). Percent Bag-1 

stained area in injured arteries was significantly higher in mice exposed to CS compared with 

air (8.22.9%, n6 vs. 3.62.3%, n7; p0.05). Intimal thickening and Bag-1 protein 

expression 21 days after injury were significantly increased in mice exposed to CS compared 

with air and were significantly reduced by Hsp70 injection (Table). Pulse-treatment with CSC 

also resulted in increased Bag-1 protein expression in SMC in vitro. Conclusion: Hsp70 

expression is induced by arterial injury. CS decreased Hsp70 but increased Bag-1 expression 

and intimal thickening. Exogenous Hsp70 decreased intimal thickening in mice exposed to CS. 

Reduced Hsp70 modulation of Bag-1 may be one mechanism by which CS increases intimal 

thickening. 

Group 

Room air 

(n9) 


CS 

(n7) 

CSSaline 

(n6) 

CSHsp70 

(n7) 

Intimal area (mm 2 x10 -2 ) 1.21.3 2.61.5* 2.40.5 1.00.3** 

Aortic Bag-1 Protein # 2.021.01 4.351.35* 6.001.75 1.571.28** 

*p0.05 vs. Room air, **p0.05 vs. Room air, CSSaline; ANOVA. # Relative densitometric units, n4 each 

Regulation of Foam Cell Formation in Type 2 Diabetic Db/db Mice 

P157 

Jeremy P Mauldin, Univ of Virginia, Charlottesville, VA; Abraham K Gebre, Wake Forest Univ 

Sch of Medicine, Winston-Salem, NC; David T Bolick, Suseela Srinivasan, Univ of Virginia, 

Charlottesville, VA; John S Parks, Wake Forest Univ Sch of Medicine, Winston-Salem, NC; 

Catherine C Hedrick; Univ of Virginia, Charlottesville, VA 

Atherosclerosis development is accelerated several-fold in patients with Type 2 diabetes. 

Monocyte transmigration and foam cell formation are critical steps in atherosclerotic plaque 

formation. In the current study, we examined foam cell formation in macrophages freshly 

isolated from control C57BL/6J and Type 2 diabetic Lepr db/db (db/db) mice, a well-established 

genetic model of Type 2 diabetes. We observed increased foam cell formation, with 4-fold 

higher cholesterol ester content, in db/db compared to control macrophages. Since cholesterol 

content in macrophages is regulated by both influx and efflux pathways, we examined 

expression of key candidate molecules involved in cholesterol transport. We found significant 

2.5- and 4-fold higher expression of scavenger receptor CD36 mRNA and protein, respectively, 

in db/db macrophages, suggesting that CD36 transcription was modulated in diabetic 

macrophages. There was no change in expression of the scavenger receptor SR-A. We also 

examined expression of key ABC transporters on macrophages that regulate cholesterol efflux. 

Interestingly, in db/db macrophages, we found 85% less mRNA and 50% less ABCG1 protein 

expression; however, there was no change in ABCA1 expression. To determine whether 

glucose regulated CD36 and ABCG1 expression, we examined J774a.1 macrophages cultured 

for 14d in either 5.5mM (NG) or 25mM (HG) glucose. We found several-fold higher CD36 mRNA 

and 30% higher CD36 protein in HG-cultured macrophages and a dramatic 80% reduction in 

ABCG1 mRNA and a 50% reduction in ABCG1 protein in HG vs. NG-cultured macrophages. 

Thus, our data indicate that macrophage cholesterol homeostasis in db/db mice is regulated, 

at least in part, by elevated glucose. 

The Role of Osteopontin in the Development of Angiotensin II-Induced 

Atherosclerosis in Apo E Deficient Mice 

J I Mendez, Heather Prayor, D Patrick Cowan, Daiana Weiss, W R Taylor; Emory Univ, Atlanta, 

GA 

Background. Osteopontin (OPN) is a phosphorylated glycoprotein that functions both as a 

cell-matrix adhesion molecule and an inflammatory cytokine. Our study evaluated the role of 

http://atvb.ahajournals.org/ osteopontin by inguest the development on June of29, atherosclerosis 2013 in ApoE deficient mice. Methods and Results. 


P158

E-80 Vol 25, No 5 May 2005 

OPN null mice on a C57BL/6 background were crossed with ApoE null mice to obtain ApoE/OPN 

null animals. Angiotensin II (AngII) (0.7 mg/kg/d SC) was infused through osmotic mini-pumps 

into ApoE/OPN null mice and ApoE controls. The mice were placed on either an atherogenic or 

standard chow diet. After 8 weeks of treatment, the descending thoracic and abdominal aorta 

was analyzed en face. The luminal atherosclerotic lesion surface area was modestly decreased 

in ApoE/OPN null female mice but not in males when compared to their ApoE controls. Lesion 

surface area for female ApoE/OPN null animals vs. controls was 37.34.5% vs. 72.77.0% 

(p0.001) in mice treated with AngII and atherogenic diet, 18.26.2% vs. 26.04.2% (pns) 

in mice treated with AngII, 4.10.4% vs. 8.72.6% (pns) in mice on an atherogenic diet, 

and 4.41.5% vs. 3.80.7% (pns) in untreated mice. There was no significant difference 

in lesion surface area between ApoE/OPN null males and ApoE controls throughout all treatment 

groups. Similar results were observed in the ascending aorta. Blood pressures among the 

ApoE/OPN null mice and ApoE controls were similar at baseline, and increased in a similar 

fashion with exposure to AngII. In a parallel experiment, ApoE/OPN null mice and ApoE controls 

were fed standard chow or western diet for 6 months. No significant difference was observed 

in the atherosclerotic surface lesion area of the descending aorta between ApoE/OPN null mice 

and ApoE controls at 6 months. Conclusions. We observed that OPN deficiency causes a 

modest decrease in atherosclerosis only in female ApoE deficient mice when infused with AngII 

and fed an atherogenic diet. These findings confirm the contribution of OPN to atherosclerosis 

in the ApoE knock-out model, but to a more limited extent than previously reported. Earlier 

studies used OPN knock-out mice in a mixed C57BL/6x129 background and the 129 phenotype 

is known to be atherosclerosis resistant. This could account for the larger difference observed 

in previous studies. 

WITHDRAWN 

Activation of Heterodimers of Toll-Like Receptor 2 and 1 Exacerbates 

Atherosclerosis in Hyperlipidemic LDLR-/- Mice 

Adam E Mullick, Peter S Tobias, Linda K Curtiss; The Scripps Rsch Institute, La Jolla, CA 

P159 

P160 

Emerging data suggest a link between atherosclerosis and innate immunity. As receptors for 

a variety of microbial components, toll-like receptors (TLR) could provide a mechanistic link 

between innate immunity and atherosclerosis. We sought to determine the atherosclerotic 

effect of TLR2/TLR1 activation in a LDLR-/- mouse fed a high fat diet (HFD). Twelve week old 

female mice received weekly intraperitoneal injections (i.p.) of Pam3CSK4 (Pam3), a synthetic 

Toll agonist that mimics the triacylated amino terminus of bacterial lipoproteins and activates 

TLR2/TLR1 heterodimers. Twenty-one mice were split into 3 groups receiving i.p. injections of 

vehicle (PBS), 25 (25-Pam) or 50 ug Pam3 (50-Pam). After 12 weeks of the HFD and weekly 

injections, animals were sacrificed and aortae harvested for en face quantitation of 

atherosclerosis. Blood was collected at 4 week intervals after an overnight fast. Throughout the 

study, all three groups experienced similar rates of weight gain. After 4 weeks of the HFD and 

injections, total plasma cholesterol increased in all groups, from 260 to 1330 (PBS), 1750 

(25-Pam) and 1340 mg/dl (50-Pam). By the eighth and twelfth weeks, the Pam3 groups had 

similar plasma cholesterol levels, which were reduced relative to PBS: 1110 (PBS) vs. 870 

mg/dl (Pam3); p0.05. Averaging each group’s plasma cholesterol during the HFD feeding 

revealed a slight cholesterol lowering effect of Pam3. The acute phase reactant, serum amyloid 

A (SAA) was measured in plasma samples to monitor inflammatory status. 24 hours after the 

initial 25 or 50 ug Pam3 injections, SAA levels rose 9- and 26-fold relative to the PBS, 

respectively. By comparison, HFD feeding alone results in 5-fold increase in SAA. Throughout 

the study, SAA levels were slightly elevated in the Pam3 groups: 136 (PBS), 386 (25-Pam), 851 

ug/ml (50-Pam). Analysis of aortic atherosclerosis demonstrated a significant increase in lesion 

areas. A dose response effect of Pam3 and extent of lesion was observed, with the 50-Pam 

group having the largest lesion burden: 9% (PBS), 28% (25-Pam), 52% lesion coverage 

(50-Pam); p0.05. Therefore, our data demonstrates that TLR2/TLR1 activation increases 

atherosclerotic lesion development concomitant with a slight lowering effect on plasma 

cholesterol levels. 

P161 

Induction of Nuclear PDE1A Correlates with Increased Vascular Smooth 

Muscle Cell Growth 

David J Nagel, Toru Aizawa, Heng Wei, Bradford C Berk, Chen Yan; Univ of Rochester, 

Rochester, NY 

It is well known that vascular smooth muscle cells (VSMC) in the medial layer of intact vessels 

have a contractile phenotype. However, cultured and neointimal VSMCs have a synthetic 

phenotype. Abnormal VSMC growth has been implicated in a multitude of cardiovascular 

diseases, including stroke, atherosclerosis, and restenosis. Numerous studies have shown that 

cyclic nucleotides, cAMP and cGMP, inhibit VSMC proliferation. Phosphodiesterases (PDEs), 

through the breakdown of cyclic nucleotides, play critical roles in controlling intracellular cAMP 

and cGMP levels. Because cGMP is a key mediator of VSMC growth and gene expression, we 

hypothesized that a nuclear isozyme of PDE might downregulate cGMP and promote VSMC 

growth. PDE1A is a Ca2/Calmodulin-stimulated PDE, which hydrolyzes both cAMP and cGMP, 

but has a higher affinity for cGMP. In both rat balloon-injured carotid arteries and human 

coronary arteries with atherosclerotic lesions, we observed that PDE1A localized to the 

cytoplasm in the majority of VSMC (contractile) in the medial layer. In contrast, in the neointimal 

VSMC (synthetic), PDE1A was primarily in the nucleus. In cultured rat aortic smooth muscle 

cells (RASM), we found cytosolic PDE1A in primary cultured RASMs at passage 0. We also 

observed this same localization in human coronary smooth muscle cells (hCASM) cultured in 

differentiating medium that is able to induce markers of contractility. These markers include 

smooth muscle calponin and smooth muscle alpha actin. In contrast, we observed nuclear 

PDE1A in sub-cultured RASM, and in hCASM cultured in growth-promoting medium. In addition, 

we demonstrated that VSMC growth was significantly Downloaded reduced from 

in the presence of a 

pharmacological inhibitor of PDE1A, as well as when treated with a siRNA that selectively 

blocked PDE1A expression. Together, these observations suggest that nuclear PDE1A may play 

an important role in regulating VSMC growth by modulating the levels of cGMP. Therefore, 

specific inhibitors directed at nuclear PDE1A may prove to be a novel therapy in reducing the 

occurrence of restenosis and atherosclerosis. 

Markers of Endothelial Apoptosis in Carotid Artery Disease 

P162 

Krista Nuotio, Jani Saksi, Petra Ijäs, Biomedicum Helsinki, Helsinki, Finland; Mikko 

Mäyränpää, Wihuri Rsch Institute, Helsinki, Finland; Tiina Sairanen, Biomedicum Helsinki, 

Helsinki, Finland; Lauri Soinne, Dpt of Neurology, Helsinki, Finland; Eija Saimanen, Dpt of 

Surgery, Lappeenranta, Finland; Petri Kovanen, Wihuri Rsch Institute, Helsinki, Finland; 

Markku Kaste, Dpt of Neurology, Helsinki, Finland; Perttu J Lindsberg; Biomedicum Helsinki, 

Helsinki, Finland 

Objectives We have previously observed denudation of endothelium in symptomatic carotid 

plaques (CP). This study examined cell proliferation and apoptosis, Fas signaling and 

apoptosis-related gene expression in high-degree symptomatic (SCP) and asymptomatic (ACP) 

carotid plaques using morphological and molecular approaches. Methods Ninety-two consecutive 

patients underwent carotid endarterectomy (CEA). CEA specimens were studied by 

scanning electron microscopy (SEM), TUNEL assay, immunostaining against the proliferation 

marker Ki-67, active caspase 3 (aCASP3) and Fas receptor (FasR), and DNA microarray. 

Findings SEM studies revealed morphological changes suggestive of endothelial denudation. 

However, the expression of endothelial aCASP3 was increased in ACPs compared to SCPs (4.57 

% 0.72 % vs. 3.30 % 0.71 %, mean SE, P0.049) and the expression of endothelial 

FasR correlated positively to enhanced balance towards apoptosis (r s0.268, P0.042). There 

was a trend towards increased TUNEL-positivity in ACPs. A positive correlation was observed 

also between Ki-67 and aCASP3 (r s0.275, P0.040). Twenty-two genes associated with 

apoptosis (both pro-apoptotic and anti-apoptotic genes) were differentially expressed between 

symptom groups. Intrestingly two genes upregulated in SCPs were inhibitors of apoptosis 

proteins: BIRC1 (fold change 1.41, P0.049) and BIRC2 (fold change 1.30, P0.018). 

Conclusions Our observations suggest that the endothelial denudation present in SCPs does not 

solely occur by apoptotic cell death. In addition to damaging mechanisms such as necrotic and 

apoptotic cell death, detachment of endothelial cells as a result of shear stress and weakening 

of pericellular adhesions may be important. We raise the possibility that constant endothelial 

cell turnover marked by co-expression of aCASP3 and Ki-67, may contribute to maintained 

cell-cell contacts and better preservation of endothelial lining in ACPs. 

P163 

Macrophage Low-Density Lipoprotein Receptor Related Protein Deletion 

and Atherosclerosis 

Cheryl D Overton, Patricia Yancey, Amy S Major, MacRae F Linton, Sergio Fazio; Vanderbilt 

Univ, Nashville, TN 

Low density lipoprotein receptor related protein (LRP) is a multifunctional protein with 

significant roles in lipoprotein clearance and cholesterol homeostasis. Because complete 

genetic ablation of LRP is not compatible with embryonic development, we used Cre/lox 

recombination to specifically delete LRP in murine macrophages. To generate transgenic mice 

with macrophage-specific LRP deletion (MLRP -/- ), engineered mice with loxP sites flanking 

the LRP gene were crossed with mice expressing Cre recombinase under the lysozyme 

promoter. To establish the physiological consequence of macrophage LRP deletion in 

atherogenesis, we generated chimeric mice by transplanting bone marrow from either 

MLRP -/- or WT (C57BL6) mice into LDLR -/- mice (12–15, 8-w/o female mice per group). The 

recipient mice were placed on a Western diet for 8 weeks and atherosclerosis was analyzed 

in the aortic sinus using oil-red O lipid staining. Macrophage deletion of LRP resulted in a 4-fold 

increased expression of MCP-1 and 3-fold increase of matrix metalloproteinases MMP-9 and 

proMMP-2. These changes would be expected to be pro-atherogenic. However, LRP-null 

macrophages also had a 3-fold increased expression of apoE, an effect expected to reduce 

lesion size. Proximal aorta lesion area in MLRP -/- recipients was increased significantly by 

40% compared to WT recipients. These data suggest that the deletion of macrophage LRP has 

a pro-atherogenic effect. This is the first report to show an increase in atherogenesis by 

deletion of a receptor that mediates lipoprotein endocytosis in macrophages. 

P164 

Loss of the Lysophosphatidylcholine Effector, G2A, Promotes Lesional 

Macrophage Accumulation in Low-Density Lipoprotein Receptor-Deficient 

Mice 

Brian W Parks; Univ of Alabama, Birmingham, Birmingham, AL 



Lysophosphatidylcholine (LPC) is a bioactive lysophospholipid produced during low-density 

lipoprotein (LDL) oxidation by patelet activating factor-acetylhydrolase (PAF-AH) hydrolysis of 

oxidized phosphatidylcholine. LPC mediates multiple effects on vascular and inflammatory 

cell-types with key roles in atherosclerotic lesion development. The G protein-coupled receptor, 

G2A, is a specific effector of LPC expressed in inflammatory and endothelial cells. LPC 

stimulates chemotaxis of macrophages and T cells and induces apoptosis of inflammatory cells 

via G2A. To determine how LPC modifies atherogenesis via G2A we bred G2A-deficient mice 

onto the LDL receptor knockout (LDLR-/-) background. G2A deficiency did not affect the size 

of developing atherosclerotic lesions in the aortic root of LDLR-/- mice and morphological 

analyses revealed no significant differences in lesional T cell content or distribution. However, 

loss of G2A function increased, rather than decreased, macrophage content of lesions and this 

was associated with reduced lesional collagen deposition. Our data show that despite 

recognized G2A-mediated effects of LPC on chemotactic responses of macrophages and T cells 

in vitro, they are not manifested in vivo in terms of lesional composition. Rather, loss of 

G2A-mediated by guest actionson of June LPC promotes 29, 2013 the accumulation of macrophages in atherosclerotic

lesions of LDLR-/- mice, suggesting that pro-apoptotic rather than chemotactic effects of G2A 

are most penetrant during atherosclerotic lesion development. Furthermore, increased lesional 

macrophage content in the absence of G2A is associated with reduced collagen deposition, 

suggesting that by promoting macrophage death via G2A, LPC may attenuate the progression 

of atherosclerotic lesions and promote their stabilization due to reduced macrophage-derived 

metalloproteinase activity. 

P165 

Unilateral Renal Artery Stenosis Markedly Increases the Progression of 

Aortic Atherosclerosis in ApoE -/- Mice 

Alok Pathak, Mauricio Rojas, Jianhua Huang, Renyi Zhao, David Jack, George Stouffer; 

UNC-CH, Chapel Hill, NC 

Background- Several studies have shown that angiotensin II (Ang II) infusion in hyperlipidemic 

mice results in formation of atherosclerotic lesions and abdominal aortic (AA) aneurysms. We 

developed a highly reproducible in-vivo mice model to investigate the correlation between 

unilateral renal artery stenosis (RAS) and atherosclerotic lesions in the aorta. Methods- Apo 

E-/- mice backcrossed into a C57/B6 background were randomly assigned to sham surgery 

(n5) or partial ligation of the right renal artery (RAS group; n12). In the RAS group, blood 

flow through the right renal artery was assessed using a 0.5 PSB flowprobe in the artery. 

Systolic blood pressure (BP) was measured (in conscious mice utilizing BP analysis tail-cuff 

system) before and 15, 30, 45 and 90 days after surgery. Mice were sacrificed 90 days after 

surgery. The proximal and distal aortas were collected for histological analysis (trichrome 

staining) and Oil red O staining (en face evaluation of atherosclerotic deposits). Results- The 

average (mean SD) systolic BP just prior to surgery and then at 30, 60 and 90 days was 

99.4 3.5, 102.5 2.6, 96.6 1.2 and 99.1 2.5 mm Hg in the sham operated group 

and 94.1 1.5, 110.2 3.2, 110.8 4.2 and 106.2 4.3 mm Hg in the RAS group. There 

was no significant change in body weight (29.4 1.3 gms at baseline and 30.4 1.4 gms 

at 45 days; p0.05). Lipid deposition, as detected by Oil red O staining, was present in 2.6 

1.0% of the area of the aortic arch in sham-operated mice and 34.9 10.4% of the aortic arch 

in RAS mice. In the distal aorta, lipid deposition was detected in 5.6 1.2% of the area of the 

descending aorta in sham-operated mice and 19.3 5.6% of the descending aorta in RAS 

mice. Trichrome staining of serial sections of the aorta revealed that RAS mice had increased 

medial degeneration, collagen deposition and intimal lipid deposits with foam cells. Conclusions 

- Unilateral RAS in Apo E-/- mice results in development of hypertension and markedly 

increases the extent and progression of atherosclerotic plaques in the aorta. 

P166 

Cyclooxygenase-2-Derived Prostacyclin Restrains Platelet Thromboxane 

Biosynthesis in Toll -Like Receptor 4 Polymorphisms 

Paola Patrignani, Concetta Di Febbo, Stefania Tacconelli, Valeria Moretta, Gioavanna 

Baccante, Maria G Sciulli, Emanuela Ricciotti, Marta L Capone, Ivana Antonucci, Liborio 

Stuppia, Ettore Porreca; “G. d’Annunzio” Univ, Chieti, Italy 

The mechanisms implied in the apparent cardiovascular protection observed in people with 

Toll-like Receptor 4(TLR4) polymorphisms Asp299Gly and Thr399IIe are uncertain but may 

include dampened inflammatory response to gram-negative endotoxins and/or endogenous 

ligands. We addressed whether the biosynthesis of platelet thromboxaneA2 and vascular 

prostacyclin - which play opposite roles in the initiation and progression of atherosclerosis - 

were altered in 19 subjects with TLR4 polymorphisms Asp299Gly and Thr399Ile versus 19 

wild-type subjects, matched for age, sex and cardiovascular risk factors, untreated with aspirin. 

The 2 groups had comparable levels of systemic markers of inflammation, i.e. soluble CD40L, 

monocyte chemoactractant protein-1, C-reactive protein and fibrinogen, and oxidant stress. 

Carriers of TLR4 polymorphisms presented reduced endothelial activation as evidenced by 

decreased circulating levels of soluble vascular cell adhesion molecule-1 and systemic 

prostacyclin versus wild-type subjects [2,3-dinor-6-keto-prostaglandinF 1: 122(50 –577) versus 

188(86 – 436) pg/mg creatinine, P0.041, respectively, median(range)]. We found a 

coincident reduction of systemic thromboxane A 2 biosynthesis in carriers versus noncarriers of 

TLR4 polymorphisms [11-dehydro-thromboxaneB 2: 174(63– 462) versus 540(211–836), 

P0.0001] which was reversed by pharmacological inhibition of COX-2-dependent prostacyclin. 

In fact, the COX-2 inhibitor rofecoxib caused a similar inhibition of the urinary excretion 

of 2,3-dinor-6-keto-prostaglandinF 1 (60%) in the two groups that was associated with 

increased urinary excretion of 11-dehydro-thromboxaneB 2 in carriers of TLR4 polymorphisms, 

but not in wild-type [174(136 –354) versus 71(67– 84)% of predosing values, respectively, 

P0.002]. This finding reveals a restrainable effect of prostacyclin on platelet thromboxane 

biosynthesis in this setting. We presume that reduced platelet activation and thromboxane A2 

biosynthesis contribute to the protective cardiovascular phenotype of TLR4 polymorphism 

carriers. Importantly, TLR4 polymorphisms might enhance the consequences of vascular 

prostacyclin inhibition by cyclooxygenase-2 inhibitors on platelet function. 

Expression of the Lyst beige Mutation is Atheroprotective in Chow-Fed 

Apolipoprotein E-Deficient Mice 

Ramona J Petrovan, Linda K Curtiss; The Scripps Rsch Institute, La Jolla, CA 

P167 

LDL receptor-deficient mice (LDLr-/-) crossed with Lystbeige mice (Bg,LDLr-/-) display exacerbated 

atherosclerosis compared to LDLr-/- mice. To verify that the beige phenotype is not 

unique to LDLr-/- mice, we examined atherosclerosis in beige, apolipoprotein E-deficient 

mutant mice (Bg,ApoE-/-). No differences in fasting plasma cholesterol levels were observed 

between ApoE-/- and Bg,ApoE-/- mice fed a chow diet for 12 and 16 weeks, and only minimal 

aortic lesions were observed in both genotypes. In contrast to our earlier results with Bg,LDLr-/mice, 

the Bg,ApoE-/- mice developed less aortic valve lesion areas than the ApoE-/- animals 

at both time points. Because the disease and likely the rates of atherosclerosis progression 

differ in these two mouse models of atherosclerosis, Downloaded we performed from 

two bone marrow 




transplantation (BMT) studies to evaluate the role of macrophage-specific expression of the 

beige mutation in ApoE-/- mice. Cohorts of 8–10 weeks old male mice were irradiated with 

1000 Rad -radiation and reconstituted with bone marrow (BM) from ApoE-/- or Bg,ApoE-/mice. 

Mice were allowed to recover for four weeks after BMT, then were fed a chow diet for 

additional 20 weeks. In study #1, irradiated ApoE-/- mice were used, so that only BM-derived 

cells expressed the beige mutation. Total cholesterol levels of the mice receiving Bg,ApoE-/- 

BM were significantly lower than mice reconstituted with control BM. These mice developed 

less severe atherosclerotic lesions, both in the aorta and in the heart valve. This indicates that 

BM-derived cells are sufficient for full expression of the atheroprotection conferred by the beige 

mutation in ApoE-/- mice. The BMT recipients in study #2 were Bg,ApoE-/- mice, so that all 

cells expressed the beige mutation except the BM-derived cells. No significant differences in 

fasting plasma cholesterol levels were observed at 4, 8, 16 and 20 weeks. Lesion areas of en 

face aortas were minimal but similar in both groups of mice. However, heart valve lesion areas 

in mice receiving Bg,ApoE-/- BM were significantly smaller compared to mice reconstituted 

with ApoE-/- BM. These results confirm that in ApoE-/- mice fed a chow diet the Lyst beige 

mutation expressed only by macrophages plays a key role in the less severe disease 

phenotype. 

Retinoid Receptor-Specific Agonists Reduced Atheroma Formation in 

Hyperlipidemic-Diabetic Rats 

Jun PU, Sr., Ben HE, Renji Hosp, Shanghai Second Med Univ, Shanghai, China; Yi JIANG; 

Shanghai East Hosp, Tong Ji Univ, Shanghai, China 

P168 

Background: It is well recognized that retinoids have potent anti-proliferative and antiinflammatory 

effects. The biological activities of the retinoids are mediated by two nuclear 

hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). In this 

study, we examined the effect of TTNPB, an RAR-selective retinoid, and LGD1069, an 

RXR-selective retinoid, in a rat model that combines diabetes and atherosclerosis. Methods: 

Male Wistar rats were made diabetic by intraperitoneal injection of alloxan (40 mg/kg), and then 

were fed a high fat diet (4% cholesterol 1% cholic acid) for 10 wks. They were randomized 

into 3 groups (n10 per group): hyperlipidemic-diabetic control group (HD), TTNPB (3 

g/kg/d)-treated group and LGD1069 (100 mg/kg/d)- treated group. Non-diabetic rats received 

standard diet were used as normal controls (NC, n8). At the end of the trial, lipids, glucose 

and malondialdehyde (MDA) levels in serum, superoxide dismutase (SOD) activity in plasma, 

and atheroma formation in aortic arch were examined. Results: Serum levels of total 

cholesterol (HD, 48.7 2.5; NC, 2.0 0.2 mmol/l; P 0.001), triglycerides (HD, 13.8 1.7; 

NC, 0.9 0.3 mmol/l; P 0.001), fasting glucose (HD, 26.4 3.1; NC, 5.8 0.5 mmol/l; 

P 0.001), and MDA (HD, 9.2 1.3; NC, 5.1 1.8 mol/l; P 0.01) were increased, 

whereas plasma SOD activity (HD,112.1 16.3 ; NC, 171.4 39.7 U/ml; P 0.01) was 

reduced in hyperlipidemic-diabetic control compared with normal control rats. LGD1069, but 

not TTNPB, reduced serum glucose (-43%) and triglycerides (-31%) levels in hyperlipidemicdiabetic 

rats (P 0.05). Both treatments significantly elevated serum total cholesterol (P 

0.01). Animals treated with TTNPB or LGD1069 showed a 32% and 40% decrease in serum 

MDA content, and a 1.28-fold and 1.37-fold increase in plasma SOD activity, respectively, 

compared with control animals(P 0.01). TTNPB and LGD1069 treatment significantly 

(P0.01) reduced the intimal thickness to 63% and 54%, and lowered the number of 

infiltrating macrophages to 58% and 47%, resectively of hyperlipidemic-diabetic control values. 

Conclusion: Our data indicate that both RAR agonist and RXR agonist ameliorate atheroma 

formation in hyperlipidemic-diabetic rats by antioxidant mechanisms. 

P169 

Defibrase-Derived Fibrin and Thrombin-Derived Fibrin Downregulate 

Endothelial Nitric Oxide Synthase Expression in Human Endothelial Cells 

Jun Pu, Ben He, Cardiovascular Dept, Renji Hosp, Shanghai Second Med Univ, Shanghai, 

China; Yi Jiang; Cardiovascular Dept, Shanghai East Hosp, Tong Ji Univ, Shanghai, China 

Background: Hyperfibrinogenemia has emerged as an independent risk factor for atherosclerosis 

disease. This study was designed to examine the effects of defibrase-induced fibrin and 

thrombin-induced fibrin on nitric oxide (NO) release and endothelial NO synthase activity and 

expression in cultured human umbilical vein endothelial cells. Methods: Fibrin was prepared 

with thrombin, which cleaves both fibrinopeptide A (FPA) and fibrinopeptide B (FPB) from 

fibrinogen, and prepared with defibrase, a thrombin-like enzyme from snake venom, which 

cleaves only FPA. Human umbilical vein endothelial cells were incubated with thrombin-derived 

fibrin, defibrase-derived fibrin or fibrinogen for 24h. NO release was examined by detecting 

nitrite generation by the Griess assay. Functional endothelial NO synthase activity was 

determined by the conversion of [ 3H]-L-arginine to [ 3H]-L-citrulline. Messenger RNA expression 

for endothelial NO synthase was determined by real-time reverse transcription-polymerase 

chain reaction, and protein expression was determined by Western blot analysis. Results: 

Addition of fibrinogen in given concentrations (0.25–1.0mg/ml) to cultured human umbilical 

vein endothelial cells did not significantly alter the release of NO in the medium (PNS). 

However, both thrombin-derived fibrin and defibrase-derived fibrin induced a significant timeand 

concentration-dependent decrease in the NO liberation (P0.05). Thrombin-derived and 

defibrase-derived fibrins, but not fibrinogen, also decrease endothelial NO synthase activity and 

messenger RNA and protein expressions in a concentration-dependent manner in cultured 

endothelial cells after incubation for 24 h (P0.05). Conclusions: Our results demonstrate that 

both thrombin-derived and defibrase-derived fibrins decrease the bioavailability of endothelial 

NO by down-regulating the activity and expression of endothelial NO synthase in human 

umbilical vein endothelial cells, which suggests that the transition of fibrinogen to fibrins could 

contributeby to guest NO-mediated on June endothelial 29, 2013 dysfunction in atherosclerotic disease.

E-82 Vol 25, No 5 May 2005 

Atorvastatin Inhibits Mitral Regurgitation in an Experimental 

Hypercholesterolemia Rabbit Model 

Nalini M Rajamannan, Jason Gocek, Frank Caira, Northwestern Univ, Chicago, IL; 

Malayannan Subramaniam, Thomas C Spelsberg; Mayo Clinic, Rochester, MN 

P170 

Degenerative mitral regurgitation is the most common cause of mitral regurgitation in the 

United States. Recent epidemiologic studies have linked degenerative mitral valve disease and 

risk factors for atherosclerosis. We studied a model of hypercholesterolemia with and without 

atorvastatin to determine the cellular mechanisms of the mitral leaflet abnormalities. Methods: 

48 Watanabe rabbits were assigned to one of 3 groups: cholesterol (1%) diet, cholesterol (1%) 

plus atorvastatin (2.5 mg/kg), and normal diet for six months, the rabbits were echoed and then 

the mitral valves were studied. Electron Microscopy, Immunohistologic staining and Real time 

PCR was performed. The following atherosclerotic markers were tested including: macrophage(RAM11), 

-actin smooth muscle, and Proliferating Cell Nuclear Antigen (PCNA). Bone 

matrix expression was determined by staining for osteopontin, osteocalcin, staining for alizarin 

red and measuring alkaline phosphates gene expression. A 4-point grading system was used 

to visually describe the staining on each of the slides (1no staining, 4high staining). Real 

Time PCR was performed to determine alkaline phosphatase gene expression. Results: Mitral 

regurgitation, Macrophage, -actin, osteopontin, osteocalcin and PCNA were increased the 

mitral valves from the cholesterol treated rabbits. Alkaline phosphatase was unchanged in the 

control versus cholesterol treatment groups. Atorvastatin decreased the amount of mitral 

regurgitation, atherosclerosis, proliferation and alkaline phosphatase expression in these 

treated valves. Conclusion: Experimental hypercholesterolemia induces proliferation and bone 

matrix expression in the mitral valve that may potentially be modified with the use of a 

lipid-lowering agent. 

TABLE 1 

Mitral 

Regurgitation 

Severity RAM-11 -actin 

Osteopontin 

Osteocalcin 

PCNA 

Alkaline 

Phosphatase 

Control 0 10 1.50.7 10 1.50.7 1.50.7 0.9970.620 

Cholesterol 3 3.50.7 40 403.50.7 40 0.7270.033 

Cholesterol 

Atorvastatin 

1 2.50.7 2.50.7 1.50.7 2.50.7 20 0.4790.286 

P171 

Atorvastatin Inhibits Hypercholesterolemia-Induced Calcification in the 

Aortic Valves via the Lrp5 Receptor Pathway 

Nalini M Rajamannan, Northwestern Univ, Chicago, IL; Malayannan Subramaniam, Mayo 

Clinic, Rochester, MN; Frank Caira, Stuart R Stock, Northwestern Univ, Chicago, IL; Thomas 

C Spelsberg; Mayo Clinic, Rochester, MN 

Introduction: Calcific aortic valve disease is the most common indication for surgical valve 

replacement in the USA. The cellular mechanisms of valve calcification are not well understood. 

We have previously shown that cellular proliferation and osteoblastogenesis are important in 

the development of valvular heart disease. Lrp5, a known low-density receptor-related protein, 

plays an essential role in cellular proliferation and osteoblastogenesis via the -catenin 

signaling pathway. We hypothesize that Lrp5 also plays a role in aortic valve (AV) calcification 

in experimental hypercholesterolemia. Methods: We examined the effects of cholesterol (chol) 

and atorvastatin (atorv) in Watanabe rabbits (n54). Group I (n18) normal diet, Group II 

(n18) 0.25% chol diet, and Group III (n18) 0.25% (w/w) chol dietatorv for the 

development of calcification. The AVs were examined for cellular proliferation, Lrp5/-catenin 

and bone matrix markers. Bone formation was assessed by micro Computed Tomography 

(microCT), calcein injection and osteopontin expression. Low-density lipoprotein with and 

without atorvastatin was also tested in AV myofibroblasts for cellular proliferation and 

regulation of the Lrp5/-catenin pathway. Results: The cholesterol diet induced complex bone 

formations in the calcified AVs with an increase in the Lrp5 receptors, osteopontin and p42/44 

expression. Atorvastatin reduced bone formation, cellular proliferation and Lrp5/-catenin 

protein levels in the AVs. In vitro analysis confirmed the Lrp5/-catenin expression in 

myofibroblast cell proliferation. Conclusion: Hypercholesterolemic AV calcification is attenuated 

by atorvastatin and is mediated in part by Lrp5/-catenin pathway. This developmental 

pathway may be important in the signaling pathway of this disease. 

P172 

Natural Killer Cell Deficiency Impairs Early-Stage Lesion Development in 

Apolipoprotein E Null Mice 

Tanya A Ramsamy, Leah Rogers, Mirela Hasu, Monjur M Siddiqui, Thien Huynh, Nancy L 

Tam, Univ of Ottawa Heart Institute, Ottawa, Canada; Wayne M Yokoyama, Howard Hughes 

Med Institute, St.Louis, MO; Stewart C Whitman; Univ of Ottawa Heart Institute, Ottawa, 

Canada 

Objective: Natural killer (NK) cells are key components of innate immunity and have been 

identified in both human and mouse atherosclerotic lesions. To determine the temporal role of 

NK cells in lesion development, we created atherosclerotic-susceptible apolipoprotein E null 

(apoe-/- ) mice that were specifically deficient in functional NK cells through expression of a 

transgene encoding the MHC class I protein Ly49A. Methods/Results: Ly49A transgenic and 

non-transgenic sex-matched littermates were placed on a diet enriched in saturated fat and 

cholesterol for 4 or 13 weeks to induce early- and late-stage lesions, respectively. After 4 and 

13 weeks, no differences were observed in total serum cholesterol concentrations or lipoprotein 

cholesterol distribution in the Ly49A mice compared to non-transgenic sex-matched littermates. 

Deficiency of functional NK cells reduced the size of the atherosclerotic lesions, as 

determined by cross-sectional analysis of the aortic root, by 65.3% (7.6x10-3 1.1x10-3 Downloaded from 

vs 

2.2x10 -2 4.7x10 -3 mm 2 , mean lesion area SEM; p0.006) and 64.4% (0.011 1.6x10 -3 

vs 0.031 5.6x10 -3 mm 2 , mean lesion area SEM; p0.001) at 4 weeks in male and female 

mice, respectively. At 13 weeks, reduction in lesion size was found to be significant in female 

but not male mice (23.1% [0.285 0.023 vs 0.371 0.031 mm 2 , mean lesion area SEM; 

p 0.039] versus 25.6% [0.296 0.035 vs 0.399 0.033 mm 2 , mean lesion area SEM; 

NS]). Using immunohistochemistry, less MHC class II positive cells were detected in the 

atherosclerotic lesions of male mice (62.8% (2.9 0.4 vs 7.7 2.5, value represents mean 

cell number SEM; p0.016) but not female mice at 4 weeks. Furthermore, by coupling the 

techniques of laser capture microdissection with quantitative real time RT-PCR, we found that 

expression of the proatherogenic cytokine interferon- within the atherosclerotic lesions did not 

differ between control and experimental mice regardless of the gender of the mice at either 4 

or 13 weeks. Conclusion - These studies suggests for the first time that NK cells participate 

in the process of atherogenesis in a temporal-specific manner by promoting early- but not 

later-stage lesion development within the vessel wall of apoe -/- mice via an interferon- 

independent pathway. 

P173 

Role of AT1a Receptors on Arterial versus Infiltrating Cells in the 

Development of Angiotensin II-Induced Vascular Diseases 

Debra L Rateri, Qingwei Zhao, Lisa A Cassis, Alan Daugherty; Univ of Kentucky, Lexington, 

KY 

Objective: Infusion of angiotensin II (AngII) into hyperlipidemic mice leads to several vascular 

pathologies including acceleration of atherosclerosis, and formation of aneurysms in ascending 

arch and abdominal regions of the aorta. Previously, we demonstrated that AT1a receptor 

(AT1ar) deficiency prevented AngII-induced vascular pathologies. The purpose of this study was 

to determine the role of AT1ar on selected cell types present in AngII-induced vascular 

diseases. Methods and Results: LDL receptor-/- mice that were either AT1ar / or -/- were 

irradiated and repopulated with donor cells of these genotypes. Therefore, 4 groups of chimeric 

mice were created to define the effect of recipient versus donor phenotype. Six weeks after 

irradiation, mice were fed a diet enriched in saturated fat (21% wt/wt) and cholesterol (0.15% 

wt/wt). Osmotic mini pumps infusing AngII (1000 ng/kg/min) were implanted 1 week later. 

AT1ar genotype of either recipient or donor did not influence the number of cells in blood, 

plasma cholesterol concentration, or lipoprotein-cholesterol distributions. Blood pressure was 

increased in AT1ar / recipients by AngII infusion, but not in AT1ar -/- recipients; 

irrespective of donor genotype. The extent of atherosclerosis was dependent on the AT1ar 

genotype of the recipient, not donor cells, with AT1ar -/- mice having markedly reduced lesion 

size. AngII-induced aneurysms in both the ascending arch and abdominal aorta of AT1ar/ 

recipients were modestly attenuated by absence of AT1ar in donor cells. However, both forms 

of aortic aneurysms were ablated in AT1ar -/- recipients, irrespective of the donor cell 

genotype. Conclusion: The AT1ar genotype of the recipient determined the development of 

AngII-induced vascular pathologies, but there was a contribution of bone marrow derived cells 

to aneurysm formation. 

P174 

Resveratrol Downregulates CC Chemokine Receptor 2 Binding and 

Expression on THP-1 Monocytes 

John P Cullen, Ying Jin, Nicholas von Offenberg Sweeney, James V Sitzmann, Univ of 

Rochester Med Cntr, Rochester, NY; Paul A Cahill, Dublin City Univ, Dublin, Ireland; Eileen 

M Redmond; Univ of Rochester Med Cntr, Rochester, NY 

A lower incidence of cardiovascular disease in people who drink red wine, compared to both 

drinkers of other alcoholic beverages and teetotalers, has been reported. Resveratrol 

(3,5,4“-trihydroxystilbene), a polyphenolic phytoalexin present in red wine, inhibits a number of 

pro-atherogenic events, both in vitro and in vivo. Much evidence supports a role for monocyte 

chemotactic protein-1 (MCP-1) in the pathogenesis of atherosclerosis. MCP-1 mediates its 

biological activity through interaction with its G protein-coupled receptor, CCR2, on the surface 

of its target cells. The aim of our study was to determine the effect of Resveratrol on CCR2 

binding activity and expression on monocytes. Methods: CCR2 protein expression in THP-1 

cells (a human monocytic cell line) was determined by Western blot analysis. Equilibrium 

binding assays were performed by incubating (90 min, RT) monocytes with 125 I-MCP-1 (specific 

activity 2200 Ci/mmol) in the absence or presence of 100 nM unlabelled MCP-1 to determine 

nonspecific binding. The reaction was terminated by filtration through a GF/B filter using a 

Brandel cell harvester. Results: Binding of 125 I-MCP-1 to THP-1 cells was saturable and 

specific. Nonspecific binding was 5–20% of the total binding. Estimates of Kd (binding affinity) 

and Bmax (number of binding sites) values were 0.12 nM and 3.52 fmol/10 6 cells, respectively. 

CCR2 binding characteristics were similar in THP-1 cells and in freshly isolated peripheral blood 

monocytes. Resveratrol (24h) inhibited CCR2 protein expression, and dose-dependently 

inhibited 125 I-MCP-1 binding to THP-1 cells; 31%, 56%, 84% decrease for 10, 50 and 100 M 

Resveratrol, in the absence of any effect on receptor affinity. Co-culture of THP-1 cells with 

endothelial cells, but not CaCo-2 cells, potently increased (from 1.50.1 to 3.10.2 fmol 

bound/10 6 cells, n8) 125 I-MCP-1 binding on THP-1 cells, an effect inhibited by resveratrol. 

Conclusions: Resveratrol inhibited CCR2 expression, and basal and endothelial cell-stimulated 

125 I-MCP-1 binding to THP-1 cells. Modulation of the expression and/or activity of the CCR2 

receptor through which MCP-1 mediates its biological activity represents an additional potential 

mechanism of resveratrol’s cardioprotection. 

Increased CD36 Scavenger Receptor Expression in THP-1 Human 

Monocytes Exposed to SLE Patient Serum 

Allison B Reiss, Winthrop Univ Hosp, Mineola, NY; David W Wan, Edwin S Chan, Bruce N 

Cronstein, NYU Sch of Medicine, New York, NY; Hong Wei Zhang, Louis Ragolia, Steven 

Carsons; Winthrop Univ Hosp, Mineola, NY 

Premature ASCVD is a common and devastating complication of SLE, a chronic, inflammatory 

autoimmune disease that mainly affects young women. It is likely that immunologic 

http://atvb.ahajournals.org/ derangements by guest contribute on June to premature 29, 2013 ASCVD in these patients, possibly by disrupting 


P175

homeostatic mechanisms that orchestrate cholesterol balance in the vessel wall. Monocytes/ 

macrophages play a crucial role in the atherosclerotic process. CD36 is a class B macrophage 

scavenger receptor that is expressed on the cell surface of monocyte/macrophages where it is 

the major receptor responsible for high-affinity recognition of OxLDL.CD36 participates in 

atherosclerotic foam cell formation. To determine whether CD36 protein expression is 

modulated by lupus serum, we studied expression of CD36 protein in THP-1 cells, a human 

monocytoid cell line. THP-1 (10 6 cells per ml), were incubated (6h, 37 0 C, 5% C0 2) in medium 

containing increasing concentrations (0 –50%) of either SLE patient serum or normal human 

serum. Cellular protein was isolated and immunoblotting performed. The primary antibody was 

a mouse monoclonal IgM anti-peptide antibody raised against human CD36 protein. There was 

a general positive direct correlation between increasing percentages of lupus serum and CD36 

protein expression. The presence of 50% lupus serum upregulated CD36 protein expression by 

482.3 76.2%, whereas the presence of 50% normal healthy serum increased expression by 

239.8 61.9%. We have demonstrated that lupus serum disproportionately upregulates CD36 

expression. To our knowledge, this is the first demonstration that CD36 expression is regulated 

by serum from patients with an autoimmune disorder. Premature atherosclerosis is common 

in SLE patients and myocardial infarction is a leading cause of death in these patients. The 

upregulation of CD36 may contribute to this pathological process by increasing vulnerability to 

cholesterol overload. Demonstration of disrupted cholesterol homeostasis in this select group 

of patients provides further evidence of the involvement of the immune system in atherogenesis 

and may inform us of the role of CD36 in the general atherogenic process. 

Low Bone Mineral Density as a Novel Risk of Atherosclerosis in 

Postmenopausal Women 

Ryoko Sato, Kouji Kajinami, Hironobu Akao, Hiroaki Uenishi, Akihiro Fukuda, Michihiko 

Kitayama; Kanazawa Med Univ, Kahoku-gun, Japan 

P176 

Atherosclerotic vascular calcification is a regulated process resembling bone formation. The 

object of this study was to examine the potential link between vascular atherosclerosis and 

bone metabolism, both of which develop rapidly after menopause. We studied consecutive 101 

postmenopausal women receiving coronary angiography with multi-slice computed tomography 

to quantify coronary calcification (CS), with ultrasonography to assess carotid plaque, and 

with DXA scanner to measure radial bone mineral density (R-BMD). Each carotid plaque was 

scored as 1 (soft), 2 (mixed), and 3 (hard) according to image intensity, and plaque score (PS) 

was defined as the sum of the score multiplied by total plaque number. No subjects received 

hormone replacement, nor associated condition influencing bone metabolism. CS (logtransformed) 

and PS values in 27 patients with significantcoronary narrowing (50% stenosis) 

(Patient) were significantly higher than 74 subjects free from lesions (Control); 1.982.18 vs. 

5.561.59 (p0.0001) and 1.172.21 vs 4.963.02 (p0.0001), respectively. R-BMD was 

significantly lower in Patient relative to Control (0.430.08 vs 0.380.08, p0.0004). 

Furthermore, R-BMD showed significant and negative associations with both CS (r0.592, 

p0.0001) and PS (r0.629, p0.0001). Our results suggest that the low bone mineral 

density may be a novel risk factor of vascular atherosclerosis in postmenopausal women. 

Free Fatty Acids and Vascular Smooth Muscle Cells: Implications for 


Irene E Schauer, Jane E Reusch; Univ of Colorado Health Sciences Cntr, Aurora, CO 

P177 

A component of metabolic syndrome likely to contribute to increased cardiovascular disease 

risk is dyslipidemia, including elevation of free fatty acid (FFA) levels. Exposure of vascular 

smooth muscle cells (VSMC) to FFA has been shown to stimulate proliferation and migration via 

ERK, PKC, and reactive oxygen species (ROS). Other literature supports the existence of FFA 

that are beneficial to the vasculature. This group has established that cAMP response element 

binding protein (CREB) plays a key role in the maintenance of the quiescent SMC phenotype. 

Various toxic exposures lead acutely to CREB activation (presumed cytoprotective) and 

chronically to downregulation of CREB function (pathological response) in rodent models and 

SMC culture. We hypothesize that CREB participates in the activation of VSMC by FFA and have 

used bovine aortic VSMC primary cultures to define the effects of different classes of FFA on 

CREB. Results: Exposure of aortic VSMC to palmitic (saturated), oleic (monounsaturated), or 

linoleic acid (n-6 polyunsaturated) leads to a rapid 3– 6-fold increase in CREB phosphorylation. 

Preliminary experiments indicate that linolenic acid (n-3 polyunsaturated) does not have this 

effect. Activation of CREB is transient, with return to basal levels within 1–3 hours. These FFA 

also causes transient, acute activation of P38 MAPK, ERK, P54-JNK, and Akt. Repeated 

challenges with FFA cause increasing degrees of ERK and CREB activation and repeated 

transient activation of JNK, Akt, and P38 MAPK. Inhibitors of P38 MAPK, MEK, and PKC and an 

antioxidant (N-acetyl cysteine) were used to define the pathways involved in regulation of CREB 

phosphorylation. Activation of CREB by oleic acid is blocked by PKC inhibition, partially blocked 

by MAPK inhibition and, in contrast to ERK phosphorylation, unaffected by anti-oxidant. 

Summary: Acute exposure to high physiological levels of the most abundant saturated, 

monounsaturated, or n-6 polyunsaturated FFA causes acute, transient activation of CREB 

protein involving PKC, but independent of ROS. CREB may be important for the early 

physiological vascular response to FFA injury. 

Circulating Adhesion Molecules and Atherosclerosis Susceptibility in 

Apoe-Deficient Mouse Strains 

P178 

examine plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and sP-selectin 

in two apoE-/- strains C57BL/6 (B6) and BALB/c with early or advanced lesions. Mice were fed 

chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. On either 

diet, BALB/c.apoE-/- mice developed much smaller atherosclerotic lesions and displayed 

significantly lower levels of sVCAM-1 and sP-selectin than B6.apoE-/- mice. The Western diet 

significantly elevated sVCAM-1 levels in both strains and sP-selectin levels in B6.apoE-/- mice. 

BALB/c.apoE-/- mice exhibited a 2-fold increase in HDL cholesterol levels on a chow diet and 

a 16-fold increase on the Western diet compared with B6.apoE-/- mice, although the two 

strains had comparable levels of total cholesterol and triglyceride. Thus, increased atherosclerosis 

is accompanied by increases in circulating VCAM-1 and P-selectin levels in the two 

apoE-/- mouse strains, and the high HDL level may protect against atherosclerosis by inhibiting 

the expression of adhesion molecules in BALB/c.apoE-/- mice. 

Reduction of Arteriosclerotic Nanoplaque Formation by Garlic Extract 

Günter Siegel, Werner Schneider, Charité, Campus Benjamin Franklin, Berlin, Germany; 

Eckhart Buddecke, Univ of Münster, Münster, Germany; Martin Malmsten; Uppsala Univ, 

Institute of Physical Chemistry, Uppsala, Sweden 

P179 

Objective: In an in vitro biosensor model (patent PCT/EP 97/05212), the interplay between 

different lipoproteins in arteriosclerotic nanoplaque formation, as well as aqueous garlic extract 

(0.2–5.0 g/l from LI 111 powder) as a possible candidate drug against arterio/atherosclerosis 

were tested within the frame of a high throughput screening. Methods: The processes 

described below were studied by ellipsometric techniques quantifying the adsorbed amount 

(nanoplaque formation) and layer thickness (nanoplaque size). A thorough description of the 

experimental setup has been given previously. Results: Proteoheparan sulfate (HS-PG) 

adsorption to hydrophobic silica was monoexponential and after approximately 30 min 

constant. The addition of 2.52 mmol/l Ca 2 led to a further increase in HS-PG adsorption 

because Ca 2 was bound to the polyanionic glycosaminoglycan (GAG) chains thus screening 

their negative fixed charges. Incubation with 0.2 g/l aqueous garlic extract (GE) for 30 min did 

not change the adsorption of HS-PG. However, the following addition of Ca 2 ions reduced the 

increase in adsorption by 50.8% within 40 min. Having detected this inhibition of receptor 

calcification, it could be expected that the build-up of the ternary nanoplaque complex is also 

affected by garlic. The LDL plasma fraction (100 mg/dl) from a healthy probationer showed 

beginning arteriosclerotic nanoplaque formation already at a normal blood Ca 2 concentration, 

with a strong increase at higher Ca 2 concentrations. GE applied acutely in the experiment, 

markedly slowed down this process of ternary aggregational nanoplaque complexation at all 

Ca 2 concentrations used. In a normal blood Ca 2 concentration of 2.52 mmol/l, the garlic 

induced reduction of nanoplaque formation and molecular size amounted to 14.8% and 3.9%, 

respectively, as compared to the controls. After ternary complex build-up, GE similar to HDL, 

was able to partly dissolve nanoplaques. Conclusions: These experiments clearly proved that 

garlic extract strongly inhibits Ca 2 binding to HS-PG. In consequence, the formation of the 

ternary HS-PG/LDL/Ca 2 complex, initially responsible for the ’nanoplaque’ composition and 

ultimately for the arteriosclerotic plaque generation, is decisively blunted. 

Prospective Study of Atherosclerosis and Venous Thromboembolism 

Laura M Reich, Aaron R Folsom, Nigel S Key, Lori L Boland, Univ of Minnesota, 

Minneapolis, MN; Susan R Heckbert, Univ of Washington, Seattle, WA; Wayne Rosamond, 

Univ of North Carolina, Chapel Hill, NC; Mary Cushman; Univ of Vermont, Burlington, VT 

P180 

Background: Atherosclerotic disease and venous thrombosis are considered to have distinctly 

different risk factors. However, in as many as one-third of patients with venous thromboembolism 

(VTE), the precipitating cause is unknown. Many investigations have demonstrated that 

atherosclerosis is associated with activation of the coagulation system, leading to the question 

of whether this prothrombotic state could contribute to development of VTE. One case-control 

study reported a higher prevalence of carotid plaques in patients with VTE compared with 

thrombosis-free controls. However, no confirmation or prospective evaluation of this question 

is available. Objective: To determine whether atherosclerosis, manifest as increased carotid 

intima-media thickness (IMT), predicts VTE incidence. Methods: The Atherosclerosis Risk in 

Communities (ARIC) study is a prospective cohort study of 15,792 adults aged 45– 64 years, 

examined at baseline (1987–1989) and followed for cardiovascular events for a mean of 12.6 years. 

VTE was validated using abstracted medical records. Bilateral carotid ultrasound for IMT 

measurements was performed at baseline for the common and internal carotid arteries and the 

carotid bifurcation. Exclusion criteria included anticoagulant use, prevalent CHD, stroke, or VTE, and 

incomplete data. Results: Among 13,113 individuals with no previous VTE, 235 incident VTE cases 

were identified. Compared with those in the lowest quartile of mean baseline IMT, the 3 rd and 4 th 

quartiles of IMT were associated with a greater crude risk of VTE. However, this association 

disappeared after adjustment for age, gender, and ethnicity, and remained null with further 

adjustment for BMI, diabetes, and CVD during follow up. Conclusion: Increased carotid IMT is not 

independently associated with an increased incidence of VTE in this middle-aged cohort. 

HAZARD RATIOS (95% CI) FOR VTE BY QUARTILES OF CAROTID IMT 


Weibin Shi, Jing Tian, Hong Pei, Jessica C Jessica C. James, Yuhua Li, Alan H Matsumoto,, 

Gregory A Helm; Univ of Virginia, Charlottesville, VA 

IMT Quartile Crude Multivariate adjusted 

0.37–0.62 1 1 

Recruitment of inflammatory cells in the arterial wall by vascular adhesion molecules plays a 0.62–0.70 1.16 (0.77–1.74) 

0.70–0.81 1.69 (1.16–2.46) 

key role in development of atherosclerosis. Apolipoprotein E-deficient (apoE-/-) mice have 

0.81–2.28 1.57 (1.07–2.31) 

spontaneous hyperlipidemia and develop all phases Downloaded of atherosclerotic from 

lesions. http://atvb.ahajournals.org/ 

We sought to 

by guest on June 29, 2013 


0.96 (0.63–1.45) 

1.24 (0.84–1.84) 

0.93 (0.61–1.43)

E-84 Vol 25, No 5 May 2005 

Molecular Analysis of the Regulation of the Human TAFI Promoter 

Mathieu Garand, Michael B Boffa, Marlys L Koschinsky; Queen’s Univ, Kingston, Canada 

P181 

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, 

once activated, attenuates fibrinolysis. Plasma levels of TAFI, which may contribute to the risk 

for thrombotic disorders, are under strong genetic control but are also affected by age (in 

women), oral contraceptive use, pregnancy, renal disease, and inflammation. Within the 

proximal promoter region of the TAFI gene, we have identified 10 potential transcription factor 

(TF) binding sites using DNaseI footprinting. Of these, we have reported that Site A (53 to -40) 

binds to C/EBP and Site C (92 to -78) binds to the glucocorticoid receptor (GR). Indeed, we have 

reported that glucocorticoids increase TAFI promoter activity in cultured human hepatoma 

(HepG2) cells. We now report further analysis of the TFs binding to Sites B (-76 to -59) and C. 

Using transient transfection of HepG2 cells, we found that Site B is critical for basal TAFI 

promoter activity. Using electrophoretic mobility shift assays, we determined that Site B binds 

to NF-Y. Using a similar approach, we found that Site C binds to hepatic nuclear factor-1 

(HNF-1). We postulate that HNF-1 may be required for liver-specific expression of the TAFI gene 

and may contribute to regulation of the TAFI promoter by glucocorticoids through synergistic 

interactions with GR. We also found a role for sex hormones in modulation of TAFI gene 

expression in HepG2 cells: progesterone and 17-estradiol were found to significantly 

decrease both TAFI mRNA abundance and TAFI promoter activity. Furthermore, progesterone 

was found to abolish glucocorticoid-induced activation of the TAFI promoter. We hypothesize 

that ligand-activated progesterone receptor affects TAFI gene expression by competing with 

ligand-activated GR for the binding to Site C. The role of sex steroids in regulation of TAFI gene 

expression is complex, as our findings are consistent with the observation that TAFI levels rise 

after menopause and are decreased by some types of hormone therapy; on the other hand, 

TAFI levels rise during pregnancy and are increased by hormone replacement therapy in 

premenopausal women and by oral contraceptive use. The regulation of TAFI gene expression 

by sex steroids clearly requires extensive further analyses. 

Characterizing the Thrombin-Independent Activation of Factor XIII 

Kathryn C Gersh, Robert Blue, Oleg V Gorkun, Susan T Lord; Univ of North Carolina at 

Chapel Hill, Chapel Hill, NC 

P182 

In the traditional view of factor XIII (FXIII) activation, thrombin cleaves the activation peptide 

from the A-subunit, and in the presence of fibrinogen and CaCl2, this peptide dissociates from 

the enzyme followed by the B-subunits, leaving the active form of factor XIII (FXIIIa). However, 

in 2001 Siebenlist and coworkers showed that FXIII alone, uncleaved by thrombin, can become 

active in the presence of low concentrations of CaCl2. Our experiments were performed to 

determine the cofactors necessary for this activation, and to probe the structure of 

thrombin-independently activated FXIII. Preliminary work showed that at least a one-hour 

incubation period with activation cofactors 1 mM CaCl2 and 0.3 mg/mL fibrinogen was 

necessary for cross-linking to occur. When either one of these cofactors was omitted during 

the activation period but added back and incubated for just enough time to allow cross-linking 

but not activation to occur, no cross-linking took place, indicating that both fibrinogen and 

CaCl2 are necessary cofactors for thrombin-independent activation of FXIII. We probed the 

regions on fibrinogen responsible for this cofactor activity by using recombinant mutant 

fibrinogens and the dansylcadaverine incorporation assay. FXIII was incubated with CaCl2 and 

fibrinogen to allow for activation, and then dansylcadaverine and N,N-dimethylcasein were 

added. Fluorescence emission was monitored at 495 nm and quantified by the slope of 

fluorescence increase over time. The fibrinogens studied were normal recombinant fibrinogen, 

g’/g’ fibrinogen which has two alternatively spliced extended gamma prime chains, and Aa251 

fibrinogen which is missing the C-terminus of the Aa chain after residue 251. Thrombinindependent 

activation of FXIII was reduced by 39% at physiological concentrations of CaCl2 

for g’/g’ fibrinogen (p 0.05) compared to normal fibrinogen, but was no different for Aa251 

fibrinogen. These results suggest that the g’ chain, thought to contain a binding site for FXIII 

zymogen, affects the rate of thrombin-independent activation, while the Aa chain which has 

been suggested as a binding site for FXIIIa, has no effect. Studies are underway to compare 

the structure of thrombin-activated FXIII with thrombin-independently activated FXIII. 

Peptidergic Regulation of Plasminogen Activator Inhibitor-1 Gene 

Expression in Vivo 

Neill Gingles, Robert J Parmer; VA San Diego Healthcare System, and Univ of California, 

San Diego, La Jolla, CA 

P183 

Plasminogen activator inhibitor-1 (PAI-1) is a major inhibitor of fibrinolysis, and is implicated in 

thrombosis and atherosclerosis. Stress-induced increases in PAI-1 may contribute to the 

altered fibrinolytic activity, hypercoagulability and prothrombotic potential associated with 

stress and aging; however, the mechanisms by which PAI-1 biosynthesis is altered during 

stress have not been fully elucidated. Recent studies suggest a major role for neuro-peptidergic 

modulation of the stress response by PACAP (pituitary adenylate cyclase-activating polypeptide), 

a member of the VIP/secretin/glucagon family. Here, we tested the hypothesis that PACAP 

regulates PAI-1 biosynthesis during stress in vivo. PAI-1 gene expression was monitored by 

specific quantitative RT-PCR in adrenals harvested from C57Bl/6 mice that were either 

unstressed or subjected to restraint stress for 2 hours. PAI-1 mRNA expression was markedly 

increased in adrenals from stressed mice (10.51.8 fold, n5, compared to unstressed 

controls, n5, P0.001; data normalized to 18S RNA). The observed increases in PAI-1 mRNA 

during stress were substantially blunted (by 552%, P0.001) by pretreatment with the 

specific PACAP-1 receptor antagonist, PACAP6 –38 (6 mg/kg, IP), compared to pretreatment 

with vehicle. Administration of PACAP 38 agonist (0.006 to 0.6 mg/kg IP) resulted in a 

dose-dependent increase in adrenal PAI-1 mRNA (up to 14.73.3 fold induction versus 

1.00.3, n5, for vehicle treated control mice, P0.001). Restraint stress resulted in much 

smaller increments in adrenal tPA mRNA (3.00.7 Downloaded fold induction), suggesting from 

that local adrenal 



tPA/PAI-1 biosynthetic balance is markedly altered by stress. We conclude that adrenal PAI-1 

mRNA expression is markedly increased by stress, and that the PACAP peptidergic signaling 

pathway plays a major role in mediating the stress-induced increase in PAI-1 biosynthesis. 

P184 

Autologous Fibrin Coated Small Caliber Vascular Prostheses Improve the 

Antithrombogenicity by Less Immunological Response 

Tomomi Hasegawa, Kenji Okada, Yutaka Okita; Kobe Univ Graduate Sch of Medicine, Kobe, 

Japan 

Introduction: Development of small caliber vascular prostheses is still challenging. Recently 

we have developed a new technique of fibrin coatings for vascular grafts. We hypothesized that 

autologous fibrin coatings could improve the antithrombogenicity of grafts by less immunological 

response. We also examine the graft healing characteristics after implantation. 

Methods: Knitted polyester fabric vascular prostheses, 2mm in internal diameter, were coated 

with autologous (rabbit, A-graft) or xenologous (human, X-graft) fibrin by modified ethanol 

method from plasma. Fifty Japanese white rabbits were implanted with the grafts in bilateral 

carotid arteries and divided into three groups by the selection of grafts in the individual rabbit: 

A&X-grafts (Group I), A&A-grafts (Group II), X&X-grafts (Group III). To evaluate graft patency, 

anti-graft serum antibody, 111 Indium platelet scintigraphy and histology, the grafts in Group I 

were explanted on postoperative days (PODs) 1, 3, 7, 10, 14, 30, 60 and 180. Serum levels of 

IgG and IgM, tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 

(PAI-1) in Group II and III were measured serially to compare immunological, coagulative and 

fibrinolytic reactions. Rsults: All of grafts except one X-graft on POD 10 were patent without 

stenosis and thrombus. In Group I, the maximal anti-graft serum antibodies (0.170.02 optical 

density at 490 nm [OD 490]) and the maximal platelet deposition (10.31.610 6 /mm 2 )in 

A-grafts were significantly less than those in X-grafts (0.460.06 OD 490 and 17.41.410 6 / 

mm 2 , respectively; p0.0001 in both). Although serum levels of IgG, IgM, tPA and PAI-1 were 

increased at the time of fibrin degradation of grafts during POD 7 to 14 in Group II and III, those 

in A-grafts were significantly less than those in X-grafts (p0.01, ANOVA). The PAI-1/tPA ratio 

in both groups was decreased as platelet deposition of the grafts was increased. In both grafts, 

each level of IgG, IgM, tPA and PAI-1 was correlated with platelet deposition significantly 

(p0.01 for each). Conclusion: These findings suggest that autologous fibrin coatings improve 

the antithrombogenicity by less immunological response and have a potential use for hybrid 

small caliber vascular prostheses. 

Study of Platelet Signaling System and Hemodynamic Response in 

Hypercholesterolemia-Induced Atherosclerosis in Rabbits 

Mahdi O Garelnabi, Emory Univ Sch of Medicine, Atlanta, GA; Jayashree Bhattacharjee, 

Anita Kotwani, Usha Gupta, Vinod Gupta, Mallika V; G B Pant Hosp and M A Med College, 

New Delhi, India 

P185 

Introduction: Pathological and clinical studies have indicated that platelets play an important 

role in the pathogenesis and progression of cardiovascular diseases. Objectives: The current 

study was designed to investigate the platelet nitric oxide signaling system and hemodynamic 

response in hypercholesterolemic rabbit models. Methods: Platelet aggregation, Nitric oxide 

(NO), Nitric oxide synthase (NOS), Ca2 and cGMP were studied in two groups of 

hypercholesterolemia-induced atherosclerosis rabbits (12 in each group), and a group of 

normal control rabbits (n11). The hypercholesterolemic rabbits received either NO donor 

(sodium nitroprusside) or NO inhibitor (L-NMMA). Arterial systolic and diastolic blood pressure 

and heart rate were measured before and after administration of drugs in all rabbit models. 

Student t-test was used to determine the significance of differences between the means, p 

value of 0.05 or less is considered significant. Results: Platelet aggregation increased 

significantly (P0. 001) in hypercholesterolemic groups compared to normal controls, whereas 

the increase in Ca2 was not significant. NO-cGMP system showed a diminished activity, with 

significant decrease in NO (P0. 02), and cGMP (P0. 001), however the decrease in NOS 

activity was not significant; the hemodynamic measurements didn’t show a significant 

response to NO donor or inhibitor in hypercholesterolemic models. Conclusion: This study 

indicates that hypercholesterolemia-induced atherosclerosis in rabbits results in reduced NOS 

activity and NO release. Diminished NO bioavailability, increases platelet activity mediated 

through the enhanced Ca2 influx. 

P186 

The Platelet PlA1/A2 (Glycoprotein IIIa) and HPA2 (Glycoprotein Ib alpha) 

Polymorphisms and the Relationship between Carotid Artery Intima-Media 

Thickness and Platelet Activation 

Maria Lukasik, Univ Sch of Med Sciences, Poznan, Poland; Marcin Rozalski, Magdalena 

Boncler, Cezary Watala, Med Univ of Lodz, Lodz, Poland; Wojciech Kozubski; Univ Sch of 

Med Sciences, Poznan, Poland 

Background: The relationship between platelet glycoproteins polymorphisms and platelet 

reactivity as the association between common carotid intima-media thickness (IMT) and 

platelet activation markers were confirmed previously. The purpose of the study was to assess 

the potential effect of the PlA1/A2 polymorphism in platelet GP IIIa and HPA2 polymorphism in 

platelet GP Ib alpha on the relation between IMT and platelet activation. Matherial and Methods: 

The PlA1/A2 and HPA2 polymorphisms were studied in a group of 84 subjects (41 after stroke 

patients and 43 healthy volounteers) aged 45–75 years (medial: 58,7years, SD10,6 years). 

The maximal, avarage carotid artery IMT and maximal carotid artery bulb IMT were evaluated 

with B-Mode ultrasonography according to the standardized protocol. The whole blood 

microplatelets and platelet aggregates fraction, platelets glycoproteins CD62p and active 

GPIIb/IIa expression were assessed with flow cytometry in the rest platelets and after 8uM 

TRAP, 5uMby ADP guest and thrombin on June (0,15 29, IU/L) 2013 stimulation. The statistical evaluation was performed

using non-parametric ANOVA tests. Results: The following genotype frequencies of the GPIIIa 

polymorphism were found: PlA1/A1 - 86,9% (73 of 84), PlA1/A2 - 13,1% (11 of 84) and the 

following genotype frequencies of the GP Ib alpha HPA2 polymorphism were detected: Thr/Thr 

- 84,5% (71 of 84), Thr/Met - 15,5% (13 of 84). No differencies in allels frequency between 

stroke nand non-stroke group were found. The positive statistically significant correlation 

between IMT and microplatelet fraction after thrombin stimulation (Rs 0,45; p0,00003) and 

negative significant association between IMT and active GPIIb/IIIa expression (Rs -0,41; 

p0,0003) and between IMT and CD62p expression (Rs -0,28; p 0,018) in PlA1/A1 and 

Thr/Thr genotype subgroups were established. No significant correlations between IMT and 

platelet activation markers were found in subjects with PlA1/A2 and Thr/Met genotypes. 

Conclusions: In PlA1/A2 or Thr/Met genotype carriers the platelets role in the vascular wall 

remodelling may differ from the relations platelets-arterial wall in PlA1/A1 and Thr/Thr 

genotype subjects 

P187 

Rosuvastatin-Dependent Anti-Oxidant and Anti-Platelet Effects in a Syrian 

Hamster NIDDM-Model 

Bulent Mutus; Univ of Windsor, Windsor, Canada 

Objective: Recently, we showed that the ability of human platelets to release NO (or denitrosate) 

S-nitrosothiols (RSNOs) is dependent on cell surface protein-disulfide isomerase (csPDI). We 

also observed both in NIDDM humans and fructose-fed hamsters that platelet NAPDH oxidase 

(NOX) is elevated and NOX-dependent ROS attenuates csPDI activity, thus promoting a 

proaggregatory response. The objective of this study was to determine whether rosuvastatin 

could normalize NOX-dependent ROS production and csPDI activity in platelets and aortic 

endothelial cells of fructose-fed Syrian hamsters. Methods: Both control and fructose-fed 

hamsters were treated with rosuvastatin for seven days prior to collection of cells and tissues. 

Platelets were analyzed for 1) aggregatibility 2) csPDI-denitrosation activity and 3) NOX activity. 

Hamster aortic endothelial cells cultivated with or without rosuvastatin were also analyzed for 

NOX activity. Results: Platelet PDI NO-releasing activity was 75% (P .05) higher in control 

than fructose-treated hamsters. Rosuvastatin resulted in a 16% (P .005) decrease in 

control animals and 60% (P .005) increase in fructose-treated animals. Platelet NOX 

Activity of fructose-fed animals was 100% higher than controls (P .05). Rosuvastatin had 

no effect in control but in fructose-fed it decreased the NOX activity by 20%. Initial rate of 

Platelet Aggregation was 1.7 fold higher in fructose-fed than controls (P .035). Rosuvastatin 

attenuated the fructose-fed rate by 2.0 fold thus reversing the fructose effect. 1o Culture 

Aortic Endothelial Cell NOX Activity was 1.6-fold higher in fructose-fed than controls. 

Treatment with 50 nM rosuvastatin lowered NOX levels to 3-fold less than that of controls 

(P .0005). Conclusion: Rosuvastatin attenuated fructose-induced increases in NOX-mediated 

ROS in platelets and endothelial cells and normalized platelet function, demonstrating that 

rosuvastatin has vasculoprotective effects in this Syrian hamster NIDDM model. 

P188 

High Glucose Concentrations Alter Hypoxia-Induced Cell Fate Decisions in 

Bovine Aortic Smooth Muscle Cells 

Wei Gao, Ronan P Murphy, Gail Ferguson, Paul A Cahill; Dublin City Univ, Dublin, Ireland 

Diabetes, a metabolic disorder characterised by chronic hyperglycaemia due to relative insulin 

deficiency, insulin resistance or both, is associated with micro- and macro-vascular complications. 

Both diabetes and hypertension have been shown to produce tissue hypoxia - a 

reduction in the normal level of tissue oxygen - that can occur from direct decreases in blood 

supply or from venous/arteriole occlusion. Cellular responses to hypoxia include proliferation, 

angiogenesis, metabolism, apoptosis and migration. In this study, we examined the effects of 

hyperglycemia on vascular smooth muscle cell growth (proliferation and apoptosis) under 

normoxic and hypoxic conditions. Bovine aortic smooth muscle cells (BASMC) were exposed to 

normoxic (5% CO2, 95% air) and hypoxic (2% O2, 5% CO2, 93% N2) conditions in the absence 

or presence of high glucose (HG, 25mM) before the effects on BASMC proliferation and 

apoptosis were assessed. Under normoxic HG conditions, there was no change observed in 

either proliferation or apoptosis of BASMC when compared to normoxic controls. FACS analysis 

using carboxyfluorescein diacetate succinimidyl ester (CFDA SE) - a cell tracing agent that is 

retained throughout cell division - showed that hypoxia caused a significant increase in cell 

proliferation in the presence of glucose when compared to hypoxic controls in the absence of 

glucose. Subsequent western blot analysis of concurrent cell lysates for proliferating cell 

nuclear antigen (pCNA) confirmed this increase in cell proliferation (3.84 0.45 fold (hypoxia 

HG) vs 1.26 0.10 (hypoxia); n3, p0.05). Further investigation by FACS analysis using a 

Vybrant Apoptosis Assay Kit revealed a concomitant decrease in the number of apoptotic cells 

under the same conditions (12.98 1.31 fold vs 26.98 1.24 fold; n3, p0.05). 

Colorimetric based caspase assays carried out on cell lysates demonstrated a significant 

decrease in caspase 3 activity (1.83 0.39 fold vs 2.96 0.39; n4, p0.05) concomitant 

with an increase in the expression of the anti-apoptotic protein Bcl-xL (5.61 0.29 vs 4.12 

0.44 fold; n2, p0.05) under hypoxic HG conditions. These results provide the first clear 

evidence that hypoxia-induced BASMC growth can be altered by the presence of high glucose 

concentrations. 


Endothelial Nitric Oxide Synthase Inhibition Does not Alter Endothelial 

Progenitor Cell Colony Forming Capacity or Migratory Activity 

Greta L Hoetzer, Heather M Irmiger, Kimber M Westbrook, Rebecca S Keith, Christopher A 

DeSouza; Univ of Colorado, Boulder, CO 

P189 

Endothelial nitric oxide synthase (eNOS) activity has been shown to play a pivotal role in the 

mobilization of endothelial progenitor cells (EPCs) into the circulation from the bone marrow. 

Indeed, in eNOS deficient mice exercise-induced EPC mobilization is severely diminished. 

Moreover, in vitro studies have demonstrated that colony forming capacity and migratory 

function of circulating EPCs are impaired in conditions associated with reduced nitric oxide 

bioavailability, suggesting an involvement of eNOS in circulating EPC regulation. We determined 

ex vivo whether circulating EPC colony-forming capacity and migratory activity are influenced 

by eNOS activity. Peripheral blood samples (20 mL) were collected from 20 healthy adults (13 

men, 7 women) ranging in age from 27 to 67 years. Peripheral-blood mononuclear cells were 

isolated, preplated for 2 days and non-adherent cells were further cultured for 7 days in the 

presence and absence of N G -nitro-L-arginine methyl ester (L-NAME, 300 M), a specific eNOS 

antagonist. Colony formations consisting of multiple thin, flat cells emanating from a central 

cluster of rounded cells were counted in 4 random wells. Endothelial cell lineage was confirmed 

by FACS analysis with endothelial-specific antibodies recognizing PE-conjugated CD34 and 

vascular endothelial growth factor receptor 2 as well as APC conjugated CD133. Migratory 

activity of EPCs was determined by the fluorescence of migrated cells through a modified 

Boyden chamber. The number of EPC colony forming units was not significantly different when 

cultured in the absence or presence of L-NAME (215 vs185). Moreover, eNOS inhibition 

did not alter EPC migratory activity, mean fluorescence was similar in samples cultured with 

(983126 RFUs) and without (962105 RFUs) L-NAME. The lack of an effect of eNOS 

inhibition on EPC colony formation and migration was consistent regardless of the age and sex 

of the subject. These in vitro results suggest that, in contrast to EPC mobilization from the bone 

marrow, eNOS does not exert a modulatory influence on the functional capacity of circulating 

EPCs to either form colonies or migrate. 

WITHDRAWN 


P190 

P191 

Streptozotocin-Induced Diabetes Induces Endothelial Transcriptional 

Responses in Vivo: Key Alterations in Transcripts Regulating Angiogenesis, 

Lipid-Metabolism, and Inflammatory-Cell Adhesion 

John G Maresh, Huaxia Xu, Nan Jiang, Ralph V Shohet; UT Southwestern, Dallas, TX 

We hypothesized that hypoinsulinemic hyperglycemia would alter gene expression within the 

vascular endothelium. We obtained a mouse model of insulin deficiency by high dose (180 

mg/kg) streptozotocin injection. We utilized transgenic mice expressing green fluorescent 

protein under the control of an endothelial-specific promoter, which allows the rapid isolation 

of endothelial cells by fluorescent activated cell sorting (FACS). Two weeks post-injection, those 

animals determined to be frankly hyperglycemic by urine and blood glucose analysis were 

chosen for cell isolation. In parallel, age-matched mice received sham intraperitoneal 

injections. Aortae from the root to the iliac bifurcation were rapidly processed by proteolytic 

digestion followed by FACS to yield populations of 95% purity. RNA was isolated from 

100,000 endothelial cells and subjected to oligo-dT/T7 amplification prior to transcriptional 

analysis using long oligo microarrays created from the Operon V3‰ set. As shown in the 

figure, dysregulated transcripts were confirmed by real-time PCR, and included glycam1, 

angptl4, slc36a2, cidea, adam5, ces3, adipsin and adiponectin. Several of these transcripts 

were thought to be adipocyte-specific. Our study suggests that they are expressed in 

endothelial cells in response to the altered metabolic milieu of insulin deficiency. By 

comprehensively examining cellular gene responses in vivo in a whole animal model of type I 

diabetes, we have identified novel regulation of key endothelial transcripts that likely contribute 

to the metabolic, angiogenic and pro-inflammatory responses that accompany diabetes. 



E-86 Vol 25, No 5 May 2005 

P192 

Hedgehog Regulation of Notch Signaling in Vascular Smooth Muscle Cells 

in Vitro 

David Morrow, Shaunta T Guha, Yvonne Birney, Catherine Sweeney, Phillip Cummins, 

Vascular Health Rsch Cntr, Dublin, Ireland; Dermot Walls, Biotechnology, Dublin, Ireland; 

Eileen Redmond, Univ of Rochester, Rochester, NY; Paul A Cahill; Vascular Health Rsch 

Cntr, Dublin, Ireland 

Vascular cell fate decisions are hallmarks of the vascular cell response to injury and play a 

crucial role in the pathogenesis of vascular disease. Notch receptor-ligand interactions and the 

Hedgehog (hh) signaling pathway have been strongly implicated in vascular morphogenesis and 

remodeling of the embryonic vasculature, with hh activation upstream of Notch signaling during 

development. We therefore tested the hypothesis that hh and Notch pathway interact to 

promote changes in vascular cell fate in adult vascular smooth muscle cells (SMC) in vitro. 

Using real-time PCR, western blot , immunocytochemistry and luciferase reporter assays, we 

identified hh ligands, sonic (Shh), indian (Ihh), the hh receptor patched-1, the transmembrane 

protein, smoothened (smo) and hh target gene promoter gli2 activity in SMC in vitro. Activation 

of hh with Shh recombinant protein (3ug/ml) resulted in significant fold increase in cell number 

(cell counts, 1.3 .03 and proliferating cell nuclear antigen expression, pCNA 2 .05) 

concomitant with a significant decrease in SMC apoptosis (apoptotic nuclear stains with 

Annexin V/propidium iodide and acridine orange/ethidium bromide, bax/bcl-xL ratio). In parallel 

studies, recombinant Shh increased Notch target gene protein and mRNA expression (hrt-1, 

hrt-2 and hrt-3) concomitant with a significant fold increase in gli2 mRNA levels and promoter 

activity (5 .04 and 6 .07, respectively). Inhibition of hh signaling with cyclopamine (20uM) 

resulted in a significant fold decrease (1.8 .02) in cell proliferation concomitant with a 

significant fold increase in SMC apoptosis (8 .02), while concurrently decreasing hrt-1, hrt-2 

and hrt-3 mRNA and protein expression by 2 .05, 2 .05 and 1.5 .05, respectively. 

Furthermore, over expression of constitutively active Notch 3IC resulted in a marked 

upregulation of hh signaling (18-fold and 3-fold increase in smo and gli2 mRNA levels, 

respectively, 2-fold increase in Shh and Ihh protein levels). Moreover, inhibiting Notch signaling 

following expression of the CBF-1/RBP-Jk inhibitor, RPMS-1, significantly attenuated the 

Notch-induced increase in hh signaling. We conclude that hh and Notch signaling pathways 

interact in adult SMC to promote SMC growth. 

Src Kinases Promote Interactions between Adherent Platelets and 

Circulating Neutrophils 

P193 

Zehra N Pamuklar, UNC, Chapel hill, NC; Licia Totani, Laborotory of Vascular Biology and 

Pharmacology, Santa Maria Imbaro, Italy; Mauricio Rojas, UNC, Chapel hill, NC; Antonio 

Piccoli, Labarotory of Vascular Biology and Pharmacology, Santa Maria Imbaro, Italy; Virgilio 

Evangelista, Laborotory of Vascular Biology and Pharmacology, Santa Maria Imbaro, Italy; 

Susan S Smyth; UNC, Chapel hill, NC 

At sites of vascular damage, platelet-PMN interactions contribute to intimal hyperplasia, 

atherosclerosis, and ischemic/reperfusion injury. We previously reported that Src kinase activity 

is necessary to sustain 2 integrin-dependent platelet-PMN mixed conjugates under high 

rotatory shear. The present study used human and mouse cells to determine the role of Src 

kinases in promoting PMN recruitment to adherent platelets. Phase-contrast video microscopy 

was used to study initial PMN interactions with surface adherent platelets at 2 dynes/cm 2 , then 

the shear stress was stepped up to 5 dynes/cm 2 and chamber perfused with cell-free media 

to measure firm adhesion. Initial PMN recruitment was abolished by monoclonal antibodies that 

block P-selectin and by the use of platelets from P-selectin-/- mice. At 5 dynes/cm 2 ,95 5% 

and 59 12 % of interacting PMNs remained firmly adhered in the human and mouse system, 

respectively. Like 2-integrin blocking monoclonal antibodies, the Src kinase inhibitor PP2 

barely affected the total number of interacting cells but reduced the percent of firmly adherent 

PMNs to 40 5% in the human (n5) and 11% 4% (n3) in mouse systems.Firm adhesion 

of PMNs from Lyn/Fgr-/- (n5) and Lyn/Fgr/Hck-/- (n3) mice was also reduced to 68 11% 

and 36 24% of controls, respectively. To confirm these observations , we examined the 

effect of Src kinase inhibition in a mouse model of arterial injury in which endothelial 

denudation is followed by platelet deposition and PMN recruitment. Src kiase inhibitor PP 1 ( 1.5 

mg/kg i.v) did not alter P-selectin expression on adherent platelets one hour after injury. 

However, fewer PMNs were recruited by the platelets in PP1-treated mice (2.4 .06; n 10) 

than in control mice (20 3.3; n7; p0.05). Additionally, the platelet layer appeared less 

compact and extended further into the lumen in PP1-treated animals. These results confirm 

that Src kinase activity contributes to physiologic platelet-PMN interactions and suggest that 

PMN interactions may be required for platelet passivation in the setting of arterial injury. 

The Role of Metal Ions and Their Binding Sites in 3 Integrins 

Michelle M Kish, Cleveland Clinic Foundation, Cleveland, OH; Czeslaw S Cierniewski, Med 

Univ, Lodz, Poland; Edward F Plow; Cleveland Clinic Foundation, Cleveland, OH 

P194 

The two 3 integrins, llb3 and v3, share the same 3 subunit and have subunits that are 

36% identical. Both 3 integrins bind fibrinogen but there is a difference in ligand binding 

mechanisms; llb3 binds the carboxyl terminus on the fibrinogen -chain while v3 binds a 

RGD sequence on the A-chain. Also, high Ca2 suppresses ligand binding to v3 but not to 

llb3. The main site of ligand interaction lies between the seven-bladed propeller of the 

subunit and the A like-domain from the 3 subunit. The 3 A-domain contains 3 metal sites 

for binding ligands: a metal ion-dependent adhesion site (MIDAS), a site adjacent to the MIDAS 

(ADMIDAS), and a ligand-induced metal binding site (LIMBS). The MIDAS is involved directly 

with the ligand and the ADMIDAS has been suggested to regulate ligand binding, but the roles 

of the ADMIDAS and LIMBS sites are still unresolved. Here we show that, in the presence of 

low calcium, fibrinogen binds, but at higher levels of calcium or EDTA, binding affinity 

decreases. These data are derived from both surface Downloaded plasmon resonance from 

studies and microtiter 

plate assays with immobilized 3 A-domain. We also show that point mutations that selectively 

inactivate the metal binding sites in the A-domain can block the suppressive effects of high 

calcium on fibrinogen binding. Our results support the hypothesis that occupancy of the 

ADMIDAS may be responsible for the suppressive effect of Ca 2 . When fibrinogen is bound 

through its -chain ( llb 3), the MIDAS is not influenced by the ADMIDAS; but, when bound 

through its RGD ( v 3), the ADMIDAS now becomes regulatory in a divalent cation specific 

manner. Either the ADMIDAS or LIMBS has a preference for Ca 2 over Mg 2 . Taken together, 

we propose that our observed fibrinogen binding to the isolated 3 A-domain mimics the 

response of v 3 with respect to specificity and divalent ion dependence. 

P195 

Gene Expression Profiling of Human Endothelial Cells Overexpressing Nrf2 

Anna-Liisa Levonen, Henna-Kaisa Jyrkkänen, Sanna-Kaisa Häkkinen, Suvi Jauhiainen, 

Seppo Ylä-Herttuala; Univ of Kuopio, Kuopio, Finland 

Background: Reactive oxygen species (ROS) play a major role in the pathogenesis of many 

cardiovascular diseases such as atherosclerosis and restenosis after angioplasty. The 

transcription factor NF-E2-related factor-2 (Nrf2) is a central regulator of a number of genes 

involved in antioxidant defense and detoxification, thus balancing the potential adverse effects 

of ROS in the vasculature. Recent gene array studies in the liver, lung and brain of Nrf2 

knock-out animals has revealed that Nrf2 has widespread effects on gene expression; however, 

the global expression profile of Nrf2 dependent genes in endothelial cells is unknown. Our aim 

was therefore to assess the transcriptional profile of human endothelial cells transduced with 

Nrf2 expressing adenovirus. Methods: Human umbilical vein endothelial cells (HUVEC) were 

infected with either human Nrf2-overexpressing adenovirus (MOI 50), or CMV-promoter 

containing empty adenovirus (control). Triplicate samples of cells were collected 36 h and 72 h 

post-transduction. The RNA was extracted, amplified and cRNA probes prepared according to 

Affymetrix protocol. Probes were hybridized to Affymetrix HG U133 Plus 2.0 oligonucleotide 

microarrays. Data analysis was performed by dCHIP 1.3 software. Results: Gene array analysis 

revealed more than 1.5-fold changes in 883 genes at 36 h and 3031 genes at 72 h after 

transduction (p0.05). Nrf2 over-expression induced clusters of genes involved in antioxidant 

defense, detoxification, NADPH regeneration, ubiquitination and proteasomal degradation. In 

addition, several transcription factors and other proteins involved in Nrf2 signaling were 

regulated by Nrf2. Induction of antioxidant genes heme oxygenase-1, NAD(P)H quinone 

oxidoreductase-1, as well as glutamate-cysteine ligase modifier and catalytic subunit was also 

verified by real-time PCR and Western Blotting. Conclusion: Adenoviral gene transfer of Nrf2 

induces a large battery of cytoprotective genes in the endothelium. Concerted induction of 

Nrf2-regulated genes by pharmacological means or gene therapy may have therapeutic 

potential in ROS related vascular diseases. 

WITHDRAWN 



P196 

P197 

The Calcium-Dependent Protease Calpain Controls Frizzled-7 Receptors 

Turnover in Endothelial Cells 

Ian Struewing, Derek Adams, Catherine D Mao; Univ of Kentucky, Lexington, KY 

Endothelial cell (EC) polarity and directed migration are crucial for vascular development and 

angiogenesis. In drosophila, cell polarity is controlled in part by the Frizzled (Fz) receptors via 

the activation of the planar cell polarity (PCP) pathway. The activation of the PCP pathway is 

associated with an asymmetric localization of numerous polarity complexes including Fz 

complexes. We have found that EC express Fz7 gene by RT-PCR and herein we report the 

subcellular localizations of the Fz7 receptors in BAEC using transient and stable expression of 

dually N- and C-termini-tagged Fz7 proteins as well as the regulation of their turnover at the 

plasma membrane. In confluent BAEC, the localization of mature Fz7 receptors at the plasma 

membrane was only observed at sites of early cell-cell adhesion and cell-cell contacts, where 

the Fz7 receptors colocalized with actin but not with -catenin, a marker of mature adherens 

junctions. In addition, the accumulation of the Fz7 receptors in filipodia was also observed. 

During the polarization and migration of BAEC in response to either cell monolayer wounding 

or cell attachment and spreading onto fibronectin, the Fz7 proteins were asymmetrically 

localized at the leading edge of the cells during lamellipodium formation. This polarization of 

the mature Fz7 receptors preceded the polarization of the actin cytoskeleton. In addition, we 

have identified a 10 kDa C-terminus proteolytic fragment of the Fz7 receptor that was 

associated with intracellular vesicle membranes. Interestingly, the formation of Fz7 C-terminus 

cleaved fragment was increased upon cell density as well as after short treatment with the 

Ca 2 ionophore ionomycin and was inhibited by calpeptin and Z-LL-CHO, two specific inhibitors 

of calpain activities. Altogether, these results suggest that cell adhesion events regulate the 

asymmetric localization of the Fz7 receptors at the plasma membrane as well as the turnover 

of the Fz7 receptors via a Ca 2 and calpain-dependent mechanism. Furthermore, the 

asymmetric localization of Fz7 suggests that the planar cell polarity pathway is activated during 

EC migration. 

P198 

SRC- and EGF Receptor-Independent Activation of Extracellular Regulated 

Kinase (ERK) 1/2 in Thrombin- and Angiotensin II-Stimulated VSMC 

Xing Yin, Evelyne Polidano, Claude Faverdin, Pierre Marche; CNRS UMR 7131, Paris, France 

Differentiation, migration and proliferation of VSMC, key events in the pathogenesis of 

atherosclerosis, are under the control of a variety of signaling enzymes including ERK 1/2, EGF 

receptor (EGFR) and Src. Under physiological conditions, thrombin and angiotensin II (AII) trigger 

ERK1/2 activation by guest through on June the sequential 29, 2013 activation of Src family and EGFR kinases. However

we recently demonstrated that under restriction of intracellular Ca2 ([Ca2]i) elevation by 

BAPTA, agonist-triggered ERK1/2 activation could occur independent on EGFR. Since calcium 

channel blockers (CCBs) also impair agonist-induced [Ca2]i elevation, we investigated the 

signaling pathways involved in ERK1/2 activation of VSMC treated by amlodipine, isradipine or 

verapamil. Cultured VSMC, isolated from rat aortae, were pretreated or not with CCB and with 

inhibitors specific of Src family kinase (PP2, PP1) or of EGFR kinase (AG1478, PD153035) prior 

to stimulation by thrombin or AII. The phosphorylation/activation status of Src, EGFR and 

ERK1/2 was determined by Western blots using phospho-specific antibodies. By themselves, 

CCBs did not alter the phosphorylation level of Src, EGFR and ERK1/2. In this respect, CCBs do 

not behave like BAPTA. However when EGFR kinase or Src family kinases or both kinases were 

inhibited by AG1478, PP2 or the combination of both, respectively, CCBs dose-dependently 

protected agonist-induced ERK1/2 phosphorylation against the inhibitory action of drugs, as 

does BAPTA pre-treatment. A clinically relevant concentration of amlodipine as low as 10 nM 

was significantly efficient. The effect of CCBs could not be reproduced by treatment of VSMCs 

with the stereoisomer amlodipine R, which is devoided of Ca2 channel blocking property. 

The results indicated that the prevention or the reduction of [Ca2]i elevation, together with 

the inhibition of Src or EGFR kinase or of both, unmasked new signaling pathways allowing the 

agonist-elicited activation of ERK1/2 in a Src- and EGFR-independent manner. These results 

might be of pathophysiological importance in hypertensive patients with cancer since EGFR 

kinase inhibitors are used in cancer treatment and since CCB are commonly used for the 

treatment of hypertension. 

Endothelin-1 Stimulation of the ET-A Receptor Promotes Migration of 

Pulmonary Artery Smooth Muscle Cells 

P199 

David F Meoli, Mark B Taubman, R J White; Univ of Rochester Sch of Medicine, Rochester, 

NY 

Pulmonary arterial hypertension (PAH) is a devastating disease that may result from a variety 

of causes, all sharing the common pathology of smooth muscle cell (SMC) hypertrophy and 

even more complex vascular lesions composed of disorganized endothelial and SMC. A large 

body of evidence suggests an important role for the potent vasoconstrictor endothelin-1 (ET-1) 

in the pathophysiology of PAH. Patients with PAH have elevated circulating ET-1 levels, and 

ET-1 receptor antagonists improve functional class and probably reduce mortality. While earlier 

paradigms of PAH pathophysiology assigned a central role to excessive vasoconstriction, recent 

evidence highlights a more complicated pattern of vascular dysfunction, including inappropriate 

SMC migration and proliferation. Therefore we tested the hypothesis that ET-1 promotes 

migration of SMC in a clonal line of pulmonary artery SMC (PAC). Using a modified Boyden 

chamber we found that ET-1 induced SMC migration in a dose dependent fashion. This effect 

was inhibited in a dose dependent manner by an ET-A selective (BQ-123) and a non-selective 

ET-A/ET-B (PD 142,893) antagonist, but not by an ET-B antagonist (BQ-788). ET-1 was as 

efficacious as PDGF-BB in promoting migration, and the ET receptor antagonists had no effect 

on PDGF-induced migration. Therefore ET-1 induces migration of PAC that is specifically 

inhibited by ET-A receptor blockade. This finding expands our understanding of ET-1’s role in 

the development of the complex vascular lesions characteristic of advanced PAH. Since ET-1 

is as efficacious as PDGF in inducing migration, it will be important to elucidate the signaling 

mechanisms downstream of the ET-A receptor. 

P200 

Interleukin-1 Beta and Early Vascular Degeneration in Arterialized Human 

Saphenous Vein 

Ayumi A Miyakawa, Thaiz F Borin, Luciene C Campos, Bruno B Ctenas, Luís A Dallan, 

Sérgio A de Oliveira, José E Krieger; Heart Institute (InCor), Sao Paulo, Brazil 

The vein graft is subjected to increased tensile stress and the complex adaptive vein response 

to the arterial hemodynamic condition may predispose to bypass failure in some individuals. In 

this work, we investigated the early effect of arterialization on the expression of IL-1 gene in 

human saphenous vein. Human saphenous vein segments were cultured ex vivo under both 

venous (flow: 5 mL/min; no pressure) or arterial hemodynamic condition (flow: 50 mL/min; 

pressure: 80 mmHg) for 24 hours. Exposure of saphenous vein to arterial condition resulted in 

significant induction of IL-1 expression (venous: 1.0 0.2 vs. arterial: 1.95 0.3, N16, 

p0.04). Immunohistochemical analysis showedDownloaded a tendency of IL-1 from 

staining upon arterial- 

ization compared to venous condition (venous: 1.1 0.3 vs arterial: 2.1 0.4, N9, p0.09). 

These changes were accompanied by increased apoptosis assessed by TUNEL in arterialized 

vein segments. Consistent with these findings, primary culture of human saphenous vein 

smooth muscle cells treated with IL-1 (48 hours) showed a dose dependent inhibition in [3H] 

timidine incorporation (IL-1 in ng/mL - 0: 100 4.5%; 0.1: 112 0.7%; 1: 83.8 4.7%; 10: 

69.1 3.8%; 100: 67.3 10.9%; p0.01). Together, these data suggest that IL-1 is 

up-regulated in arterialized human saphenous vein, which may be related to early events of 

vein graft degeneration including cellular losses and induction of vascular apoptosis. 

P201 

Apolipoprotein E Recruitment and its Activation of iNOS is Required for 

Inhibition of Vascular Injury-Induced Smooth Muscle Cell Proliferation 

Zachary W Moore, David Y Hui; Univ of Cincinnati, Cincinnati, OH 



Apolipoprotein E (apoE) has been shown previously to be recruited to the medial layers of 

carotid arteries after vascular injury in vivo. In addition, apoE directly induces expression of 

inducible nitric oxide synthase (iNOS) in smooth muscle cells in vitro. This investigation 

explores the relationship between medial apoE recruitment, iNOS activation, and their 

protection against neointimal hyperplasia. Wildtype mice with FVB/N and apoE-null C57BL/6 

phenotypes were used as the neointimal hyperplasia-susceptible strains. Wild type C57BL/6 

and apoE-overexpressing transgenic FVB/N mice were used as the resistant strains for these 

studies. In addition, iNOS knockout mice on a neointimal hyperplasia resistant background 

were also used. Immunohistochemical detection of apoE was observed in both wildtype strains 

twenty-four hours after carotid denudation, but detection of iNOS was seen only in the 

neointimal hyperplasia-resistant C57BL/6 strain. In the absence of apoE, however, iNOS was 

not observed in the apoE knockout C57BL/6 mice. In contrast to the FVB/N wildtype mice, iNOS 

expression in the injured vessels was seen in apoE overexpressing animals where neointimal 

hyperplasia was also suppressed. In the relevant strains, double-fluorescent labeling indicated 

colocalization of apoE and iNOS in medial smooth muscle layers. Morphometric analysis of 

iNOS knockout mice 14 days after carotid denudation indicated no difference in neointimal 

hyperplasia compared to wildtype, but a significant increase in medial thickness and area. 

These data indicate that the injury-induced activation of iNOS requires apoE recruitment. Both 

apoE and iNOS are necessary for suppression of the proliferative, but not migratory response 

to vascular injury in vivo. However, apoE suppression of medial smooth muscle cell migration 

does not require iNOS expression. 

Signaling Pathways Regulating Class A Macrophage Scavenger 

Receptor-Mediated Cell Adhesion 

Dejan M Nikolic, Mr, Steven R Post; Univ of Kentucky, Lexington, KY 


P202 

Macrophage adhesion to modified extracellular matrix (ECM) is an essential component of many 

inflammatory conditions including atherosclerosis. Unlike integrins, which are the main cell 

surface receptors involved in adhesion to native ECM, macrophage class A scavenger receptors 

(SR-A) bind to modified ECM, but not native ECM. The ability of SR-A to mediate adhesion to 

modified ECM indicates that SR-A may play an important role in macrophage retention at sites 

of inflammation. In this study, we used isolated peritoneal macrophages and SR-A-expressing 

human embryonic kidney (HEK) cells to examine the regulatory mechanisms involved in 

SR-A-mediated adhesion. In both cell types, SR-A enhanced cell attachment and subsequent 

cell spreading to an immobilized SR-A ligand, malondialdehyde bovine serum albumin 

(MDA-BSA). To define the role of intracellular signaling pathways in regulating SR-A-mediated 

adhesion, we examined the effects of inhibiting specific SR-A-generated signals on cell 

attachment and subsequent spreading on MDA-BSA. We found that inhibiting Gi/o proteins 

decreased the SR-A-dependent attachment of macrophages and SR-A expressing HEK cells by 

40%. However, Gi/o inhibition did not affect the spreading of attached cells on MDA-BSA 

suggesting that the spreading response is regulated by additional signals. To assess this 

possibility, we examined the role of PI3-kinase and src in SR-A-dependent cell adhesion. We 

found that both PI3-kinase and src kinase inhibitors prevented SR-A-mediated cell spreading 

without decreasing cell attachment. However, inhibiting PI3-kinase and src after cells were 

allowed to adhere for 16 hrs had no effect on the spread morphology indicating that these 

kinases are required for initiating but not maintaining SR-A-mediated cell spreading. Similar to 

SR-A, integrin-mediated attachment to fibronectin was not affected by PI3-kinase or src 

inhibitors. Integrin-mediated cell spreading was prevented by inhibiting src kinase, but was 

independent of PI3-kinase activation. Overall, our results suggest that, similar to integrins, 

SR-A mediates cell adhesion by a dynamic process involving multiple signals that differentially 

regulate cell attachment and spreading. 

P203 

Enhancement of Vascular Mineralization by Extracellular Transglutaminase 

Maria Nurminskaya, Jennifer Johnson, Lidia Faverman; Tufts Univ, Boston, MA 

Cardiovascular calcification (mineralization) is a common pathologic condition associated with 

ageing, diabetes and chronic renal insufficiency. It often leads to stroke, amputation and 

ischemic heart disease. Vascular smooth muscle cells (SMCs) induced to mineralize in cell 

cultures, trans-differentiate to osteoblast phenotype. Previously, we showed acceleration of 

osteoblast differentiation by an extracellular enzyme transglutaminase (TGase) {Nurminskaya et 

al, Dev. Biol., 2003, v. 263, p.139–152}. Here, we analyzed the potential of TGases to regulate 

vascular calcification. First, we analyzed distribution of TGases in the areas of warfarin/vitamin 

K(1)-induced medial calcification in a rat model system. TGase isoform FXIIIa is accumulated 

in the calcified arterial areas, suggesting a role of this TGase in regulation of vascular 

calcification. Next, we established a cell culture system for in vitro analysis of the molecular 

mechanisms that regulate matrix mineralization in SMCs. Matrix mineralization in high density 

cultures (5x105cells/cm2 ) of rat SMCs was induced in the presence of beta-glycerophosphate 

and ascorbic by guest acid byon addition June 29, of the 2013 conditioned medium of hypertrophic chondrocytes.

E-88 Vol 25, No 5 May 2005 

Mineralization in rat SMCs at the 5 th passage occurs rapidly (6 –7 days), while the cells at 10 th 

passage deposit mineralized matrix at a slower rate (up to 4 weeks). Recombinant TGase 

(0.01U/ml) enhanced matrix mineralization by 25–30% in both rat and mouse SMC cultures. 

Further we employed the gene microarray analysis using the 32K Oligonucleotide mouse 

GeneChip (Qiagen) to identify the TG-regulated genes in mouse vascular SMCs. The“patternrecognition” 

analysis of the primary gene-expression data by GeneMap software package 

identified statistically reliable coordinate changes in the expression of activators and inhibitors 

of several signaling and metabolic pathways. For example, glycolysis pathway and Wnt/betacatenin 

signaling are activated by extracellular TG2 in mouse SMCs, while TGF-beta signaling 

is down-regulated. Current analysis is aimed at identification of TG-binding proteins in SMCs 

for better understanding of the mechanisms that promote vascular calcification. 

P204 

Role of Glutaredoxin in Tumor Necrosis Factor Alpha-Induced Cell Death 

Shi Pan, Bradford C Berk; Univ of Rochester, Rochester, NY 

Glutaredoxin (Grx) is a ubiquitous redox molecule that regulates protein function through its 

thioltransferase activity, similar to thioredoxin. Characterization of Grx function in the 

cardiovascular system is quite limited. Previously we showed that TNF-induced signaling in 

endothelial cells (EC) was inhibited by thioredoxin. Therefore we investigated the effect of Grx 

on TNF-mediated signaling, focusing on EC death. Grx overexpression enhanced TNFinduced 

death signaling as shown by increased JNK and caspase-3 activation, and inhibited 

TNF-mediated survival signaling as shown by decreased IB degradation. In contrast, 

TNF-induced death signaling and EC apoptosis were reduced in Grx siRNA knockdown cells 

compared to control siRNA. Furthermore, our results showed that the thioltransferase activity 

of Grx was important for its effect on TNF-signaling, since transfection of a cysteine to serine 

mutant of Grx, that abolished thioltransferase activity, almost completely reversed the effect of 

wild type Grx. These data establish the critical role of Grx in TNF-mediated EC death, and 

suggest that Grx may play a pro-inflammatory role in cardiovascular disease. 

Hic-5 Promotes Endothelial Cell Migration 

Christie Avraamides, Michael E Bromberg, Tracee S Panetti; Temple Univ, Philadelphia, PA 

P205 

Hic-5, a paxillin homolog, localizes to focal adhesions and regulates cell spreading. Prior 

studies show that Hic-5 localization to the focal adhesions correlates with bovine pulmonary 

artery endothelial (BPAE) cell migration to lysophosphatidic acid (LPA). These studies address 

the role of Hic-5 in endothelial cell migration. BPAE cells recruit more Hic-5 to the focal 

adhesions at the wound edge, compared to diffuse, cytoplasmic Hic-5 in non-migrating cells 

by confocal microscopy. Hic-5 is recruited to the pseudopodia or cellular protrusions through 

3 micron pores, after stimulation with LPA or sphingosine 1-phosphate (S1P). After stimulation 

of BPAE cells with LPA or S1P, Hic-5 is immunoprecipitated with anti-phosphotyrosine, but the 

reverse immuno-precipitation of Hic-5 shows no detectable phosphotyrosine suggesting that 

Hic-5 may bind a phosphorylated protein after stimulation with LPA or S1P. Next we transduced 

BPAE cells with Hic5-GFP using retrovirus resulting in similar expression levels to the 

endogenous Hic-5 by Western blot. On fibronectin, endogenous Hic-5 and Hic5-GFP localize to 

the focal adhesions of BPAE cells in a similar pattern by fluorescence microscopy. On collagen, 

Hic-5 is localized to the focal adhesions of BPAE cells only after stimulation with LPA or S1P 

while Hic5-GFP localizes to the focal adhesion in the absence of stimulation. BPAE cells 

expressing Hic5-GFP show a 50% increase in cell spreading on collagen, not fibronectin, 

compared to BPAE cells. Expression of Hic5-GFP in BPAE cells increases migration on a 

collagen- and fibronectin-coated filters by 3-fold in the presence of LPA in the modified-Boyden 

chamber. GFP expression in BPAE cells has no effect on migration. There is a two-fold increase 

in Rac in Hic5-GFP-BPAE cells, compared to BPAE cells, attached to collagen as detected by 

Western blot of cell extracts. Therefore, increased Hic-5 in the focal adhesions through 

over-expression or recruitment at the wound edge promotes cell migration potentially through 

changes in cell spreading and Rac. 

P206 

Mevastatin Inhibits Interleukin-6-Stimulated Pregnancy Associated Plasma 

Protein-A Secretion via Regulation of Rho GTPases 

Zachary Resch, C. D Link, Univ of Missouri, Columbia, MO; Laurie Bale, Mayo Foundation, 

Rochester, MN; James Sowers, Univ of Missouri, Columbia, MO; Cheryl Conover; Mayo 

Foundation, Rochester, MN 

Pregnancy associated plasma protein-A (PAPP-A), an insulin-like growth factor binding protein 

(IGFBP) protease, mediates local insulin-like growth factor (IGF-I) bioavailability. PAPP-A 

expression is increased in experimental intimal hyperplasia, culprit plaques, and appears to be 

a possible diagnostic and prognostic factor for those individuals suffering an acute coronary 

syndrome. Elevated circulating inflammatory cytokines, including interleukin-6 (IL-6), have 

been associated with those individuals susceptible to suffering ACS. We therefore examined 

whether IL-6 regulates PAPP-A production in vascular tissue (coronary artery smooth muscle 

cells (CASMCs)). Indeed, CASMCs treated with IL-6 (25 to 75 ng/ml) and the soluble form of the 

IL-6 receptor (sIL-6R, 50 to 150 ng/ml) significantly increased PAPP-A secretion in a 

dose-related manner. Mevastatin, a HMG-CoA reductase inhibitor, has been shown to have 

many beneficial affects independent of lowering circulating lipid levels. We investigated 

whether one of the pleiotropic effects of mevastatin could be the inhibition of IL-6- stimulated 

PAPP-A secretion. Pretreatment with mevastatin significantly inhibited IL-6-stimulated PAPP-A 

secretion. Our group has previously reported that statins inhibit the activation of Rho family of 

GTPases by preventing their geranylgeranylation and membrane localization. Furthermore, 

previous reports have shown that Rho GTPases can directly phosphorylate signal transducer 

and activator of transcription-3 (STAT-3), the major signaling pathway associated with IL-6R 

activation. Indeed, mevastatin treatment almost completely inhibited the phosphorylation of 

STAT-3 in response to IL-6/sIL-6R activation. Using Downloaded an inhibitor of from 

the Rho family GTPases, 



Clostridium difficile-derived Toxin B, we were able to show that Toxin B (1 ng/ml) inhibited 

IL-6-stimulated PAPP-A secretion from CASMCs. Taken together, these data suggest that 

IL-6-stimulated PAPP-A production is inhibited by mevastatin in a vascular tissue previously 

shown to express PAPP-A at high levels following vascular injury and cardiovascular diseases. 

PAPP-A may be a useful target of both current (statins) and novel strategies for the treatment 

of cardiovascular complications. 

P207 

TGF-Beta Inhibits Cyclin a Gene Expression in Vascular Smooth Muscle 

Cells through Regulation of CREB Phosphorylation 

Kenji Sakakibara, R. P Hom, Evan Ryer, K. C Kent, Bo Liu; Weill Med College of Cornell 

Univ, New York, NY 

Abnormal vascular smooth muscle cell (SMC) proliferation contributes to the development of 

artherosclerostic and restenotic lesions. We have previously shown that TGF-beta, a cytokine 

that is abundantly released during vascular injury, potently inhibits SMC proliferation at least 

in part by suppressing cyclin A gene expression. The underlying mechanism through which 

TGF-beta regulates cyclin A expression has been previously explored in non-SMCs cells, 

however, controversial results were reported. In this study, we first confirmed that TGF-beta 

induced a 50% reduction in the level of cyclin A protein, mRNA as well as promoter activity 

in A10 SMCs. Using a deletion analysis, we determined that the TGF-beta effect was mediated 

by a DNA-cis element located at the -79 to -54 region of the cyclin A promoter, and therefore, 

we defined this region as the TGF-beta responsive element (TRE). Interestingly, this TRE 

contains a putative binding site for the CREB family of transcription factors. Mutations to the 

CREB-binding site abolished the TGF-beta response, suggesting that TRE-CREB interaction is 

a critical target downstream of the TGF-beta signaling. We then examined protein/TRE 

interaction in a gel-shift assay. When incubated with nuclear extract isolated from control A10 

SMCs, the cyclin A TRE formed a protein/DNA complex containing CREB but not Smad3. 

TGF-beta significantly diminished the abundance of protein/TRE complex. To understand how 

TGF-beta influences the protein/TRE interaction, we examined the phosphorylation status of 

CREB. TGF-beta treatment led to a rapid but transient increase in the level of CREB 

phosphorylation without affecting total CREB protein. We then blocked CREB phosphorylation 

using a dominant negative mutant of CREB (DNCREB) that bares a S133A mutation. Expressing 

DNCREB in A10 SMCs significantly increased the level of cyclin A promoter activity and gene 

expression, suggesting that CREB phosphorylation at S133 may play an inhibitory role in cyclin 

A expression. In summary, our data demonstrate that TGF-beta inhibited cyclin A gene 

expression by interrupting interactions between cyclin A promoter and the CREB family of 

transcription factors through a mechanism that may involve TGF-beta-induced CREB phosphorylation. 

Insulin Reduces Inflammation in a Mouse Model of Endotoxemia by 

Activation of Phosphoinositide-3 Kinase 

Gernot Schabbauer, Michael Tencati, Todd Holscher, Nigel Mackman; The Scripps Rsch 

Institute, La Jolla, CA 

P208 

In endotoxemia, bacterial lipopolysaccharide (LPS) in the bloodstream induces a systemic 

inflammatory response. The pathophysiology of endotoxemia is still poorly understood. We are 

interested in studying protective signaling pathways that regulate host inflammatory response 

during endotoxemia. One of these protective pathways is the phosphoinositide-3 kinase (PI3K) 

pathway. This pathway has been shown by our group and others to suppress LPS induction of 

pro-inflammatory cytokines in vitro and in vivo. Insulin is a potent activator of the PI3K pathway. 

In addition, it has been suggested that insulin has anti-inflammatory properties. In this study, 

we investigated the effects of insulin in a mouse endotoxemia model. We found that very low 

doses of insulin administered via subcutaneous mini-osmotic pumps significantly improved 

survival of endotoxemic mice. These low levels of insulin did not affect plasma glucose levels. 

LPS induction of TNF and IL-6 was also reduced by insulin. In addition, levels of soluble 

E-selectin, a marker of endothelial cell dysfunction, were decreased by insulin. Administration 

of the PI3K inhibitor, wortmannin, in insulin-pump treated, endotoxemic mice completely 

abrogated the protective effect of insulin and abolished the insulin-mediated suppression of 

pro-inflammatory cytokine expression. Taken together, our data indicate that insulin suppresses 

the inflammatory response in a PI3K-dependent manner. 

Induction of Endothelial Dysfunction by the Chemokine Fractalkine 

P209 

Andreas Schäfer, Univ of Würzburg, Würzburg, Germany; Christian Schulz, Technical Univ of 

Munich, Munich, Germany; Daniela Fraccarollo, Univ of Würzburg, Würzburg, Germany; 

Steffen Massberg, Technical Univ of Munich, Munich, Germany; Johann Bauersachs; Univ of 

Würzburg, Würzburg, Germany 

Background: The chemokine fractalkine activates platelets and induces leukocyte adhesion to 

the endothelium. The fractalkine receptor has been described on endothelial and smooth 

muscle cells. We assessed whether fractalkine would induce endothelial dysfunction. Methods 

and Results: Incubation of isolated rat aortic rings with fractalkine for 2 hours impaired 

acetylcholine-induced, nitric oxide-mediated relaxation (maximum relaxation: control 863%, 

fractalkine 404%, p0.001) after preconstriction with phenylephrine. Endotheliumindependent 

relaxation remained unaltered. Treatment with the radical scavenger tiron 

normalized acetylcholine-induced relaxation as did inhibition of the chemokine domain by 

antibody or blocking of fractalkine receptor in vascular tissues by an inhibiting antibody. Aortic 

rings which were slightly preconstricted to 15–20% of their maximum contraction displayed 

less vasoconstriction following NOS inhibition with NG-nitro-L-arginine (L-NNA, 100 mol/L; 

final contraction in % of maximum: control 912, fractalkine 718, p0.05) indicating less 

basal NO bioavailability after incubation with fractalkine. Fractalkine stimulated aortic 

superoxide by formation guest on (lucigenin June [5 29, M]-enhanced 2013 chemiluminescence[arbitrary units] control

1264110, fractalkine 3697257, p0.01), which was normalized by tiron. Superoxide 

formation detected by ethidiumbromide staining was prominent throughout all layers of the 

vascular wall. Expression of NAD(P)H oxidase subunits remained unaltered after stimulation 

with fractalkine. Conclusion: In addition to its role as a chemokine and adhesion molecule, 

fractalkine induces endothelial dysfunction by stimulating vascular superoxide generation 

resulting in reduced NO bioavailability. 

Measuring Cholesterol Ester and Phospholipid Transfer Activities of 

Microsomal Triglyceride Transfer Protein using Fluorescent Lipids 

Paul Rava, Humra Athar, Caroline Johnson, M. Mahmood Hussain; SUNY Downstate Med 

Cntr, Brooklyn, NY 

P210 

Microsomal triglyceride transfer protein (MTP) activity is classically measured using radioactive 

lipids. We presented a simple fluorescence assay to measure triacylglycerol (TAG) being 

transferred by MTP. Here, we show that phosphatidylethanolamine and phosphatidylcholine 

vesicles were better donors for measuring phospholipid (PL) and cholesterol ester being 

transferred, respectively. Both activities increased with time as well as the amount of MTP. 

MTP antagonist, BMS197636, inhibited both activities, but was a less potent inhibitor for PL 

transfer. We also describe a method to measure the net transfer of lipids. In this procedure, 

negatively charged donor vesicles are incubated with MTP and acceptor vesicles, donor 

vesicles and MTP are removed by adding DE52, and lipids transferred to acceptors are 

quantified. Net lipid transfer was dependent on the time and amounts of MTP. Cholesterol ester 

and PL transfer activities in purified MTP were 62–73% and 6 –9% of the TAG transfer activity 

using both methods. Although both assays are sensitive, the method determining lipids being 

transferred is recommended for MTP activity measurements for its simplicity. Availability of 

these methods may help in the identification of specific inhibitors for individual lipid transfer 

activities and in the characterization of mutants that bind but cannot transfer lipids. 

SR-BI Mediated HDL Trafficking in Wif-B Cells 

P211 

Shoba Shetty, Erik R Eckhardt, Lei Cai, Nancy R Webb, Deneys R van der Westhuyzen; Univ 

of kentucky, Lexington, KY 

The HDL receptor SR-BI mediates selective cholesterol ester uptake from HDL. We are studying 

the mechanism of this process in Wif-B cells, a polarized hepatocyte model system. Wif-B cells 

form bile canalicular spaces on differentiation in majority of cells with well defined basolateral 

and apical surfaces. Wif-B cells express endogenous Class B scavenger receptors, SR-BI and 

CD36. These cells mediated selective uptake from HDL and selective uptake was significantly 

increased when SR-BI was over-expressed through adenoviral mediated gene transfer. Studies 

using fluorescently labeled HDL revealed endocytosis of HDL and SR-BI followed by a 

transcytosis of a fraction of HDL (both lipid and protein) to the bile canalicular membrane. 

Studies tracking fluorescently labeled HDL uptake over shorter time periods during which ligand 

was continuously present in the medium at 37 o C, revealed a loss of surface bound ligand within 

10 minutes followed by a recovery of surface bound HDL over the next 10 minutes. This 

suggests a ligand dependent trafficking of SR-BI into the cell followed by a recycling to the cell 

surface. We conclude that selective uptake from HDL by SR-BI in Wif-B cells follows an 

endocytic route similar to that described previously in primary hepatocytes. Thus Wif-B cells 

may represent a useful model for studying SR-BI mediated HDL and cholesterol trafficking and 

transport. 

P212 

ABCG1 Redistributes Cellular Cholesterol to Plasma Membrane Domains 

Accessible for Removal by HDL but not by Lipid-Free Apolipoproteins 

Ashley M Vaughan, John F Oram; Univ Of Washington, Seattle, WA 

ATP binding cassette transporter G1 (ABCG1) mediates the efflux of cholesterol from cells to 

high density lipoprotein (HDL). It is therefore thought to play a role in reverse cholesterol 

transport, whereby HDL transports excess cellular cholesterol from the periphery to the liver for 

excretion in the bile. We show here that over-expression of ABCG1 in baby hamster kidney cells 

causes a significant increase in efflux of cellular cholesterol to HDL particles but not to purified 

apoA-I. A cell-surface biotinylation assay showed that ABCG1 is transported to the cell surface. 

Interestingly, an ATP binding domain mutant of ABCG1 having no significant HDL-mediated 

cholesterol efflux activity was expressed but not localized to the cell surface, demonstrating 

that the interaction of ATP with ABCG1 is required for its transport to the plasma membrane. 

This is in contrast to ABCA1, where similar mutations do not affect its transport to the plasma 

membrane. Using a cholesterol oxidase assay, we found that induction of ABCG1 causes the 

movement of cholesterol to the outside of the plasma membrane. This cholesterol is 

subsequently removed by HDL but not by apoA-I. Therefore, like ABCA1, ABCG1 translocates 

cholesterol to cell-surface domains that are accessible for removal from the cell. These lipid 

domains appear to have distinct properties, however, as lipid-free apolipoproteins remove 

cholesterol and phospholipids from domains formed by ABCA1, and lipidated lipoproteins 

remove only cholesterol from ABCG1-generated domains. These studies show that ABCA1 and 

ABCG1 can act in concert to transport excess cellular cholesterol from tissue macrophages into 

the HDL pathway and provide a coordinated defense Downloaded against atherosclerosis. from 




P213 

Modulation of Sterol Transport in ABCG1 x ABCG5/G8 Transgenic Mice 

Elke M Wagner, Federica Basso, Marcelo J Amar, Boris Vaisman, Lita Freeman, H B 

Brewer, Jr, Silvia Santamarina-Fojo; NIH, Bethesda, MD 

ABCG5, ABCG8 and ABCG1 are ATP-binding cassette (ABC) half-transporters involved in sterol 

transport. Whereas ABCG5 and ABCG8 have a well-established role in secretion of sterols from 

liver into bile and from enterocytes into the gut lumen, the function of ABCG1 is not as well 

understood. ABCG1 has been reported to enhance cholesterol efflux to HDL but also has been 

hypothesized to participate in bile sterol secretion. To investigate possible interactions between 

the ABCG1 and ABCG5/G8 pathways, we generated different lines of transgenic that 

overexpress human ABCG1 in liver, macrophages and brain (1.5–2 fold; all) and crossed them 

with hABCG5/G8-Tg mice which overexpress human ABCG5 and ABCG8 in both liver and 

intestine (2–3-fold; both) and which have increased hepatobiliary cholesterol secretion rates. 

On a regular chow diet, plasma total cholesterol and HDL-C levels were similar (mean SEM 

in mg/dL; p0.05; all) in C57Bl/6 (n6; 101 8 and 98 5), G5/G8-Tg (n5; 97 9 and 

91 8) and ABCG1 x ABCG5/G8-Tg mice (n4; 95 9 and 90 4). However, ABCG1 

significantly increased biliary cholesterol secretion in ABCG5/G8-Tg mice. The biliary cholesterol 

concentration and secretion rates, measured after cannulation of the common bile duct 

were increased (p0.04; all) in hABCG5/G8-Tg (1491 194 uM and 5407 524 

nmoles/hr/kg) compared to C57Bl/6 mice (704 74 uM and 3220 509 nmoles/hr/kg) and 

were further enhanced in ABCG1 x ABCG5/G8-Tg mice (2285 266 uM and 7702 690 

nmoles/hr/kg). Overexpression of ABCG1 alone did not alter fractional intestinal cholesterol 

absorption suggesting that most of the increased cholesterol secreted into the intestine via bile 

is reabsorbed. Consistently, liver cholesterol content was not altered in ABCG1 x ABCG5/G8-Tg 

vs G5/G8-Tg or C57Bl/6 mice (n2; 2.4 ug/mg; all) and no compensatory changes in hepatic 

expression of either HMGCoA-reductase or LDL-r were evident. These combined results 

indicate that hepatic overexpression of ABCG1 can enhance ABCG5/G8 dependent hepatobiliary 

cholesterol transport providing new in vivo evidence for a potential coordinate role of ABCG1 

and ABCG5/G8 in modulating hepatic cholesterol homeostasis. 

P214 

Pitavastatin Differently Modulates ATP Binding Cassette A1-Mediated Lipid 

Efflux in Macrophages and Hepatic Cells 

Ilaria Zanotti, Elda Favari, Maria P Adorni, Francesca Zimetti, Bernini Franco; Univ of Parma, 

Parma, Italy 

The beneficial effects of statins on the treatment of cardiovascular disease are in part related 

to pleiotropic effects, whose underlying mechanisms are not fully understood. ABCA1 exerts 

antiatherosclerotic properties by promoting the formation of HDL in the liver and by inducing 

lipid efflux from macrophages in the arterial wall. Objective: we investigated the ability of 

pitavastatin to modulate ABCA1-mediated efflux from hepatic cells and macrophages. Methods: 

cholesterol and phospholipid efflux were quantified in Fu5AH rat hepatoma cells and J774 

macrophages. Cells were radiolabeled with 3 H-cholesterol or 3 H-choline, equilibrated in 

presence or absence of 22OH 5g/ml-cRA 10M or cAMP 0,3mM and exposed to apoA-I 

20g/ml. To analyze mRNA and protein content of ABCA1, RT-PCR and western blot were 

performed. Results: pitavastatin dose-dependently increased cholesterol efflux from Fu5AH 

both in basal conditions and when ABCA1 was stimulated by 22OH/cRA. This effect was fully 

recovered by mevalonate and geranyl geraniol. In J774 macrophages pitavastatin significantly 

reduced cAMP ability to stimulate cholesterol and phospholipid efflux and to up-regulate ABCA1 

mRNA and protein. Mevalonate, but not geranyl geraniol, reversed these inhibitions. No effect 

of pitavastatin was detected when ABCA1 was induced by 22OH/cRA. Importantly, pitavastatin 

up to 50M did not affect lipid efflux or ABCA1 expression in cholesterol-loaded macrophages, 

a cellular model of foam cells. Conclusions: pitavastatin promotes ABCA1-mediated efflux from 

hepatic cells; this effect could partially explain the improvement in HDL plasma profile observed 

in patients treated with statins. Pitavastatin inhibits ABCA1-mediated efflux in macrophages 

only when the protein expression is induced by cAMP, suggesting that depletion of cellular 

sterols is potentially involved in cAMP-mediated activation of ABCA1. The lack of influence in 

foam cells is likely to exclude a potential negative pleiotropic effect by statins. 

Measurement of Net Cholesterol Flux between Cells and Lipoproteins 

Francesca Zimetti, Ginny K Weibel, Children’s Hosp of Philadelphia, Div of GI Nutrition, 

Philadelphia, PA; George H Rothblat; Children’s Hosp of Philadelphia, Div of GI nutrition, 


P215 

Many studies have quantitated efflux of cellular cholesterol to extracellular acceptors. If these 

acceptors are lipoproteins containing cholesterol, there is bidirectional flux consisting of efflux 

of free cholesterol (FC) and influx of lipoprotein FC and esterified cholesterol (EC). Net 

movement of cholesterol between cellular and extracellular compartments reflects the balance 

between efflux and influx. Measurement of bidirectional flux is more complicated than the 

quantitation of just efflux. We have developed an assay that provides an estimate of cholesterol 

mass movement in and out of cells and allows an estimate of the net flux when cells are 

exposed to either isolated lipoproteins or whole serum. In this assay cellular FC pools are 

labeled with 3H-FC in the presence of an ACAT inhibitor. Following an equilibration period the 

specific activity of cellular FC is determined (time 0). Monolayers are incubated for 8h with the 

acceptors and efflux is measured by the release of 3H-cholesterol. After the efflux period 

specific activity of cell cholesterol is determined again, and influx of extracellular cholesterol 

is estimated from the reduction in cholesterol specific activity between time 0 and 8h. Based 

on these measurements an influx to efflux ratio can be calculated. We used this approach to 

establish the influx/efflux ratio when Fu5AH hepatoma cells, that highly express SR-BI, are 

exposed to reconstituted apo AI/phospholipid discs (50 g protein/ml), human HDL (25 g 

protein/ml), LDL (100 g protein/ml) and a pool of human serum at 2.5%. Influx / efflux 

ratios 1 reflect net cholesterol efflux, whereas ratios 1 indicated net influx. With 

cholesterol-free by guest apo AI/PL on June discs the 29, ratio 2013 was 0.03 demonstrating essentially only efflux, as is

E-90 Vol 25, No 5 May 2005 

expected. With HDL the ratio was 1.06 consistent with exchange without significant net flux. 

Addition of LDL resulted in a large net influx with a ratio of 2.35. Finally, whole serum produced 

net influx (ratio 1.55). Similar studies have been done using the human fibroblast cells 

WI38VA13 stably transfected with SR-BI. The results with these cells are consistent with those 

from Fu5AH. This assay will provide an approach that can be used with a variety of cells 

exposed to isolated lipoproteins or whole serum. 

Interferon- Could Stimulate Expressions of Many Pro-Inflammatory 

Proteins and Scavenger Receptors in U 937 Monocyte-Like Cells 

Katsuya Tashiro, Eiichi Ishii, Yuhei Hamasaki; Saga Univ, Faculty of Medicine, Saga, Japan 

P216 

(Background) Monocytes and macrophages are important cells in the progression of various 

inflammatory diseases, including infectious diseases, auto-immune diseases, and atherosclerosis. 

Interferon-gamma (INF-) would be one of the cytokines which play important roles in 

these diseases. In this study, we examined the effect of INF- on U937 monocyte-like cells, 

using immunocytochemistry technique. (Results) We revealed that INF- significantly increased 

the expression of inflammation-related proteins such as, Monocyte chemoattractant protein-1 

(MCP-1), C-reactive protein (CRP), Ferritin, Cyclooxyganase-2 (COX-2) and inducible nitric oxide 

synthase (iNOS) simultaneously. Furthermore, expressions of scavenger receptor A, scavenger 

receptor B and oxidized LDL receptor (LOX-1) were also markedly amplified in these cells by 

stimulation of INF-. However, TNF- did not show such marked effects on U937 cells. 

(Conclusion) These results demonstrate that INF- could be a strong activator against 

monocytes and macrophages and make the cells produce various inflammation-related 

proteins, which might advance inflammatory process further. In addition, INF- could 

upregulate expressions of scavenger receptors which play important roles on the formation of 

foam cells. 

Rho Kinase Inhibition Impairs Macrophage Migration 

Hong-Wei Wang, Jonathan Gitlin, Nobuhiko Kubo, Qingyu Lian, James K Liao, William A 

Boisvert; Brigham and Women’s Hosp, Harvard Med Sch, Boston, MA 

P217 

Rho-kinase (ROCK) participates in diverse cellular signaling functions such as cytoskeleton 

rearrangement, cell migration and proliferation. There is evidence from in vitro and in vivo 

studies suggesting that ROCK inhibitors have beneficial effects on cardiovascular diseases such 

as arteriosclerosis and coronary vasospasm. However, the atheroprotective mechanism(s) of 

ROCK inhibition remains to be clarified. The purpose of this study was to determine the role of 

ROCK activity in regulating macrophages functions. Mouse bone marrow derived macrophages 

were generated and the cells were subjected to chemotaxis assay in response to MCP-1. 

Addition of ROCK inhibitors, Fasudil and Y27632, resulted in abrogation of cell migration 

stimulated by MCP-1. Furthermore, treatment with either Fasudil or Y27632 caused an 

impairment in macrophage’s ability to invade through a synthetic basement membrane called 

Matrigel. We also examined the ROCK production and activity in the THP-1 cells. These cells 

were treated with 100ng/ml phorbol myristate acetate (PMA) for 2 days to differentiate from 

monocyte-like cells into macrophage-like cells. Both the expression and activity of ROCK I and 

ROCK II were reduced in the differentiated cells, suggesting that ROCK activity is more 

prominent in circulating monocytes perhaps because of their migratory activity. Immunohistochemical 

examination of atherosclerotic lesions from apoE-/- mice revealed that ROCK I and 

ROCK II as well as one of their substrates was colocalized in the lesion macrophages. Our 

results indicate that ROCK plays a critical role in regulating macrophage migration, and that 

inhibition of ROCK may have a protective effect on the development of atherosclerosis. 

P218 

Role for Alkenals in Glutathione Depletion and Macrophage Injury Induced 

by OxLDL 

Yanmei Wang, Jim G. Begley, Li Xu, Jason Stevens, Reto Asmis; Univ of Kentucky, 

Lexington, KY 

The development and progression of atherosclerotic lesions are associated with the formation 

of OxLDL and, in vitro, OxLDL was shown to induce cell death in human macrophages and 

macrophage-derived foam cells. Previously, we demonstrated that OxLDL-induced macrophage 

injury is mediated by the increased formation of intracellular peroxyl radicals, but increased 

reactive oxygen species (ROS) formation alone is not sufficient to promote macrophage death 

(Circ.Res. (2003) 92, e20-e29). Here we show that OxLDL-induced macrophage death is 

preceded by decreases in both total glutathione levels and the glutathione/glutathione disulfide 

ratio (GSH/GSSG). Depletion of intracellular glutathione with the alkylating agent 

N-ethylmaleimide and glutathione synthesis inhibitor L-buthionine-sulfoximine alone did not 

decrease the GSH/GSSG ratio and did not promote cell death, but potentiated OxLDL-induced 

macrophage injury. The peroxyl radical scavenger Trolox blocked OxLDL-induced cell death, 

but restored neither intracellular glutathione levels nor the GSH/GSSG ratio, indicating that 

glutathione depletion induced by OxLDL occurs independent of peroxyl radical formation. 

4-Hydroxynonenal (4-HNE) is one of the oxidation products found in OxLDL known to react with 

thiols. Here we show that 4-HNE, like OxLDL, depletes intracellular glutathione, decreases the 

GSH/GSSG ratio and promotes macrophage lysis. Our data suggest that glutathione depletion 

induced by alkenals found in OxLDL contribute to Downloaded its toxicity in human from 

macrophages. 

P219 

The Influence of Apolipoprotein E Isoforms on High Density Lipoprotein 

Remodelling by Phospholipid Transfer Protein 

Nongnuch Settasatian, Khon Kaen Univ, Faculty of Associated Med Sciences, Khon Kaen, 

Thailand; Philip J Barter, Kerry-Anne Rye; The Heart Rsch Institute, Sydney, Australia 

The apolipoprotein (apo) E in human plasma exists as three isoforms, with cysteine/arginine 

interchanges at residues 112 and 158. In normolipidemic plasma approximately 70% of apoE 

is associated with HDL. Phospholipid transfer protein (PLTP) is a plasma factor that remodels 

apoA-I-containing HDL into large and small particles, and mediates the dissociation of apoA-I. 

The aim of this project was to determine if these events also occur when PLTP remodels HDL 

that contain apoE. Discoidal reconstituted HDL (rHDL) containing either apoE2, apoE3, apoE4 

or apoA-I as a sole apolipoprotein were prepared by the cholate dialysis method and converted, 

by incubation with LCAT and LDL, into spherical particles with respective diameters of 10.2, 

10.5, 10.5 and 9.4 nm. Large and small particles were formed when the spherical rHDL were 

incubated with PLTP. The apoE-containing rHDL were remodelled more rapidly than the 

(A-I)rHDL. By 24 h all of the (E2)rHDL, (E3)rHDL and (E4)rHDL had been converted into large 

particles 12.1, 12.5 and 13.0 nm in diameter, and into small particles (diameter 7.9 nm). Under 

the same conditions approximately 24% of the (A-I)rHDL did not change in size. The remaining 

(A-I)rHDL were converted into large particles (diameter 10.9 nm) and small particles (diameter 

7.8 nm). Remodelling of the (A-I)rHDL was accompanied by dissociation of lipid-free or 

lipid-poor apoA-I. ApoE did not dissociate from the (E2)rHDL, (E3)rHDL or (E4)rHDL. The 

composition of the large and small apoE-containing conversion products was determined. 

These results were consistent with the large particles being fusion products with six apoE 

molecules/particle. The composition of the small conversion products was consistent with 

particles containing two molecules of apoE/particle. These were probably formed by 

rearrangement of the large fusion product. It is concluded that apoE enhances the 

PLTP-mediated remodeling of spherical rHDL into large and small particles by a process that 

involves particle fusion without the dissociation of apoE. 

P220 

Dimeric Apolipoprotein A-I in Discoidal High Density Lipoproteins Adopts a 

“Double Belt” Conformation 

R. A Silva, Univ of Cincinnati, Cincinnati, OH; George M Hilliard, Univ of Tennessee Health 

Science Cntr, Memphis, TN; Ling Li, Jere P Segrest, UAB Med Cntr, Birmingham, AL; W. S 

Davidson; Univ of Cincinnati, Cincinnati, OH 

Apolipoprotein (apo) A-I, a 243 residue 28 kDa protein is the main protein constituent of high 

density lipoprotein (HDL) particles. Discoidal forms of HDL are shown to be critical 

intermediates between lipid-poor apoA-I and mature spherical HDL that comprise the bulk of 

the circulating particles. Thus, many studies have focused on understanding apoA-I structure 

in discs reconstituted in vitro. Recent experimental as well as theoretical work supports a “belt” 

molecular arrangement for apoA-I in which repeating amphipathic helical domains of the 

protein run parallel to the plane of the lipid disc. However, disc-associated apoA-I can adopt 

several tertiary arrangements that are consistent with a belt orientation. To distinguish different 

belt models, we cross-linked near-neighbor Lys groups in homogeneous 96 Å discs containing 

exactly two molecules of apoA-I. After delipidation and tryptic digestion, high resolution mass 

spectrometry was used to identify 9 intermolecular and 11 intramolecular cross-links. The data 

strongly suggest a “double belt” molecular arrangement for apoA-I in which two apoA-I 

molecules wrapped around the lipid bilayer forming two stacked rings in an antiparallel 

orientation with helix 5 of each apoA-I in juxtaposition (LL5/5 orientation). The data also 

suggests the presence of an additional double belt orientation with a shifted helical registry 

(LL5/2 orientation). Furthermore, a 78 Å particle containing two molecules of apoA-I fit the 

same double belt motif with evidence for conformational changes localized to the N-terminus 

and the region near helix 5 of each apoA-I. A comparison of this work to a previous study is 

suggestive that a third molecule of apoA-I can form a hairpin in larger discoidal particles that 

contain three molecules of apoA-I. 

WITHDRAWN 



P221 

P222 

Class B Scavenger Receptor-Mediated High Density Lipoprotein Trafficking 

Bing Sun, Deneys R van der Westhuyzen, Nancy R Webb; Univ of Kentucky, Lexington, KY 

Closely related class B scavenger receptors CD36 and scavenger receptor class B type I (SR-BI) 

play important and distinct roles in lipoprotein metabolism. CD36 is thought to play a role in 

the formation of lipid laden macrophage foam cells by mediating the uptake and degradation 

of oxidized LDL (oxLDL). SR-BI is considered to be the principal receptor that mediates the 

selective uptake of HDL cholesterol ester (CE), a process in which cellular uptake of CE exceeds 

that of protein. Although CD36 mediates high affinity HDL binding, selective uptake by CD36 

is considerably less efficient compared to SR-BI. We hypothesized that the difference in HDL 

metabolism by CD36 and SR-BI may be due to differences in intracellular trafficking of the HDL 

ligand. To investigate this possibility, transfected COS-7 cells expressing SR-BI or CD36 were 

incubated with fluorescent-labeled HDL, and the cellular distribution of HDL was monitored by 

confocal microscopy. We also quantitatively determined the intracellular accumulation of HDL 

in SR-BI or CD36-expressing cells. Our experiments provide the following findings: 1) After 

incubation for 1 hour at 37°C, the majority of Alexa-HDL appeared to be on or near the cell 

surface in SR-BI-expressing cells and distributed in a punctate pattern throughout CD36expressing 

cells. 2) Using trypan blue to quench surface-bound fluorescent ligand, analysis by 

flow cytometry showed that the rate of HDL intracellular accumulation in CD36-expressing cells 

is approximately 2-fold faster compared to SR-BI-expressing cells. 3) Using a biotin-conjugated 

ligand, weby have guest determined on June that 75% 29, 2013 of HDL remains on the cell surface in SR-BI-expressing

cells after 1 hour incubation. These results indicate that SR-BI and CD36 mediate HDL 

internalization at different rates, possibly through distinct internalization and trafficking 

pathways. Future studies will investigate the relationship between intracellular trafficking and 

selective CE uptake by class B scavenger receptors. 

P223 

A Role of Phospholipid Transfer Protein and Cholesteryl Ester Transfer 

Protein in Cholesterol Efflux 

Urbain Tchoua, Genevieve Escher, Baker Heart Rsch Institute, Melbourne, Australia; Cyrille 

Maugeais, La Roche-Hoffman, Basel, Switzerland; Dmitri Sviridov; Baker Heart Rsch 

Institute, Melbourne, Australia 

CETP and PLTP have well-established roles in lipoprotein metabolism in plasma. There are 

however unconfirmed reports that they might also have intracellular role. Future use of CETP 

and PLTP inhibitors requires that these roles to be fully investigated and effects on plasma and 

cellular levels dissected. Two cell types are known to have PLTP/CETP and play a key role in 

the pathogenesis of atherosclerosis, hepatic cells and macrophages. Here we demonstrated 

that overexpression of PLTP alone in RAW 264.7 macrophages slightly increased cholesterol 

efflux to plasma. This increase was boosted (4 fold) when PLTP was co-over expressed in 

combination with several other genes involved in cholesterol efflux (CYP27A1, SR-B1) on the 

background of activated LXR. Overexpression of PLTP did not affect cholesterol efflux from 

HepG2 cells. CETP overexpression did not affect cholesterol efflux from either RAW 2634.7 or 

HepG2 cells. The effect of PLTP was further tested in an animal model of cholesterol efflux. 

RAW 264.7 cells were transfected with PLTP alone or in combination with several other genes 

involved in cholesterol efflux. Cells were radiolabelled with 3 H-cholesterol and injected into 

mice. We found that macrophage specific overexpression of PLTP caused an enhanced removal 

of cholesterol to faeces. Overexpression of combination of genes caused an early (6 hours) and 

dramatic enhancement of removal of cholesterol to blood, faeces and liver. Thus, although 

PLTP alone has limited impact on the rate of cholesterol efflux it may be involved in a 

rate-limiting step of cholesterol efflux from macrophages when other rate-limiting steps are 

eliminated. It might require ABCA1, SR-B1 and CYP27A1 activated to affect cholesterol efflux. 

Green Tea Leaf Extract Improves Lipid and Glucose Homeostasis in a 

Fructose-Fed Insulin Resistant Hamster Model 

Rachel W Li, Teresa D Douglas, Univ of Hawaii, Honolulu, HI; Khosrow Adeli, The Hosp for 

Sick Children, Toronto, Canada; Andre G Theriault; Univ of Hawaii, Honolulu, HI 

P224 

The present study was designed to evaluate the effect of a herbal supplement, green tea leaf 

extract, on lipid and glucose homeostasis in a fructose-fed hypertriglyceridemic, insulin 

resistance hamster model. Syrian golden hamsters were fed a fructose-enriched diet for two 

weeks to induce hypertriglyceridemia and insulin resistance, and then continued on a 

fructose-enriched diet supplemented with or without 150 or 300 mg/Kg/day of a green tea 

epigallocatechin gallate-enriched extract for four weeks. Fructose feeding in the initial two 

weeks caused a significant increase in plasma cholesterol and triglyceride levels. There was 

a significant decrease in plasma triglyceride levels following supplementation of the extract 

(42% to 62%, n7, P0.05) with cholesterol levels remaining essentially unchanged. 

Compared to baseline, the fructose control group at the end of the study showed elevated 

serum insulin and apolipoprotein B levels, and decreased serum adiponectin levels. The 

fructose/green tea extract group showed a reversal in all of these metabolic defects, including 

an improvement in glucose levels during the glucose tolerance test. Triglyceride content was 

also examined in various tissues. Compared to the control fructose group, supplementation of 

the green tea extract (300 mg/Kg) reduced triglyceride content in liver and heart tissues. There 

was molecular evidence of improved lipid and glucose homeostasis with green tea extract 

treatment based on peroxisome proliferator-activated receptor (PPAR) protein expression. 

Compared to the control fructose group, supplementation of the green tea extract (300 mg/Kg) 

significantly increased PPAR and PPAR protein expression. In summary, the data suggest 

that green tea extract can ameliorate the hypertriglyceridemia and the insulin resistant state in 

part through PPAR activation. 

P225 

Hypotriglyceridemic Property of Cruciferous Indole-Based Compounds in 

HepG2 Cells: Inhibition of DGAT Activity 

Adele Casaschi, Geoffrey K Maiyoh, Andre G Theriault; Univ of Hawaii, Honolulu, HI 

The purpose of the present study was to examine the role of plant indolic compounds, found 

mainly in cruciferous vegetables, on diacylglycerol acyltransferase (DGAT) activity, triglyceride 

(TG) synthesis, and apolipoprotein B (apoB) secretion in HepG2 cells. Initially, three plant indoles 

(indole-3-carbinol, 3,3’-diindolylmethane, brassinin) and three non-plant indoles (melatonin, 

indomethacin, and carvedilol) were screened for their inhibitory effects on DGAT activity with 

an in vitro assay using HepG2 microsomes as the source of DGAT. All indoles inhibited DGAT 

activity in a dose-dependent manner with indole-3-carbinol (I-3-C) showing the greatest effect 

(IC50 of 50 mol/L). We further confirmed the data with cell culture experiments. HepG2 cells 

were incubated with the IC50 dose of I-3-C for 24 hours and microsomal DGAT activity was 

assayed. The results indicated that DGAT activity, notably DGAT-1 (-51%) and DGAT-2 (-59%), 

decreased significantly in the presence of I-3-C confirming our in vitro data. Additional studies 

on the mechanism of action revealed that I-3-C inhibited DGAT activity non-competitively. I-3-C 

was also shown to significantly decrease TG synthesis Downloaded (-25%) and from 

secretion (-50%). Since 




availability of TG is widely accepted as a major contributing factor in the regulation of 

apoB-containing lipoprotein (apoB-Lp) secretion in HepG2 cells, we examined the effect of 

I-3-C on apoB secretion. Interestingly, I-3-C significantly reduced apoB secretion into the media 

(-31%). The data suggest that the reduction in DGAT activity and hepatic TG synthesis by I-3-C 

may have an important role in I-3-C-mediated decrease in apoB-Lp secretion. In summary, 

indoles represent a new class of DGAT inhibitors and were shown to be effective agents in 

lowering the secretion of apoB-Lp. 

Secretory Phospholipase A 2 Expression in Transgenic Mice Results in 

Increased Hepatic Selective HDL Cholesteryl Ester Uptake without 

Influencing Biliary and Fecal Sterol Excretion 

P226 

Uwe J Tietge, Rick Havinga, Juul F Baller, Fjodor van der Sluijs, Folkert Kuipers; Groningen 

Univ Med Cntr, Groningen, The Netherlands 

The acute phase protein secretory phospholipase A 2 (sPLA 2) is a major mediator of decreased 

plasma HDL cholesterol levels in inflammatory states. The aim of the present study was to 

investigate the metabolic effects of sPLA 2 overexpression in transgenic (tg) mice on biliary and 

fecal sterol excretion as indicators of reverse cholesterol transport. Compared with controls 

sPLA 2 tg mice had decreased plasma HDL cholesterol levels (by 27%, p0.01) and increased 

in vivo selective uptake of HDL cholesteryl esters into the liver (by 38%, p0.001) and the 

adrenals (by 94 %, p0.001), organs with high expression of SR-BI. Despite increased 

selective uptake, biliary cholesterol, phospholipid and bile salt secretion as well as fecal bile 

salt and neutral sterol excretion remained unchanged in sPLA 2 tg mice. Livers of sPLA 2 tg were 

significantly larger and enriched in free cholesterol (each p0.001). Consistent with increased 

cholesterol uptake, hepatic expression of HMG-CoA reductase and the LDL receptor were 

significantly decreased in livers from sPLA 2 tg mice (each p0.001), while expression of SR-BI 

and ABCG5/G8 were not different between the groups. Hepatic VLDL production in sPLA 2 tg was 

significantly increased (p0.01), whereas the composition of VLDL particles secreted by the 

liver did not differ between groups. In summary, sPLA 2 expression decreases plasma HDL 

cholesterol levels and increases selective uptake via SR-BI into the liver. However, these 

changes do not translate into increased biliary and fecal sterol excretion. These data suggest 

that, in contrast to modulating hepatic SR-BI expression levels, modification of HDL as the 

ligand for the SR-BI receptor does not impact on reverse cholesterol transport. 

The N- and C-Terminal Domains of Apo A-I Contain Alpha-Helical 

Segments that Promote ABCA1-Mediated Lipid Efflux 

Charulatha Vedhachalam, Hiroyuki Saito, Padmaja Dhanasekaran, David Nguyen, Sissel 

Lund-Katz, Michael C Phillips; Children’s Hosp of Philadelphia, Philadelphia, PA 

P227 

Lipid free apolipoprotein (apo) A-I is postulated to have two structural domains: an N-terminal 

-helix bundle and a less organized C-terminal domain. The contributions of these domains to 

ABCA1-mediated lipid efflux are not well understood. This study is focused on the N- and 

C-terminal segments of apo A-I (residues 1–43 and 223–243) that are the most hydrophobic 

regions in the molecule and the role of these putative -helices in the lipid-free structure and 

in ABCA1-mediated efflux have been examined. Measurements of helicity by Circular Dichroism 

demonstrate that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the 

-helix disrupt the tertiary organization of the C-terminal domain without affecting the stability 

of the N-terminal helix bundle. Similarly, proline insertion into the N-terminus (Y18P) disrupted 

the bundle structure in the N-terminal domain, indicating that the -helical segment in this 

region contributes to the formation of the helix bundle. Disruption or deletion of these N- or 

C-terminal helices reduced the affinity of apo A-I for ABCA1-mediated cholesterol efflux from 

mouse J774 macrophages. Removal or disruption of the C-terminal domain (223–243, L230P 

and L230P/L233P/Y236P) resulted in low-affinity non-saturable binding as indicated by a linear 

dependence of ABCA1-mediated cholesterol efflux to apo A-I concentration. Similar experiments 

with the N-terminal mutants (1–43 and Y18P) demonstrated a two-fold reduction in 

K m values compared to WT apo A-I implying that this domain contributes significantly to the 

lipid efflux process mediated by ABCA1. These results indicate that there are stable -helical 

structures in residues 1–43 and 223–243 and both helices are important for the proper tertiary 

organization of apo A-I and promoting efficient ABCA1-mediated efflux. 

P228 

Residues 333 to 344 in Human Apolipoprotein A-IV Determine its Unique 

Behavior at Hydrophobic Lipid Interfaces 

Richard B Weinberg, Victoria R Cook, Wake Forest Univ Health Sciences, Winston Salem, 

NC; Kevin Pearson, W. S Davidson; Univ of Cincinnati, Cincinnati, OH 

Background: Apolipoprotein (apo) A-IV is expressed in the mammalian intestine, where its 

synthesis is regulated by fat absorption. ApoA-IV binds slowly to hydrophobic interfaces, but 

displays high interfacial elasticity, properties we propose enable it to facilitate expansion and 

lipidation of nascent chylomicrons. Indeed, overexpression of apoA-IV in polarized IPEC1 cells 

results in increased lipid transport and secretion of larger particles. Here we have examined the 

structural elements of apoA-IV that mediate its unique interfacial behavior. Methods: Human 

apoA-IV C-terminal truncation mutants, containing enzyme cleavable N-terminal His-tags, were 

expressed in E.Coli and purified by Ni-affinity chromatography. His-tags were removed before 

study. Binding by guest rate and on elasticity June 29, at 2013 the triolein/water interface were determined using a

E-92 Vol 25, No 5 May 2005 

pulsating oil drop tensiometer. Results: Full length (376 AA) apoA-IV bound to the triolein/water 

interface with a rate constant of 3.3 0.3 msec-1 and an elasticity of 36.4 1.3 mN/m. The 

352–376 (lacking the distinctive EQQQ-rich domain) and 344 –376 (“chicken-like”) mutants 

displayed slight decreases in binding rate and elasticity. However, further truncation by only 11 

residues (333–376) resulted in a 4-fold increase in binding rate to 27.2 0.5 msec-1, and 

a sharp drop in elasticity to 22.4 0.9 mN/m. Conclusions: These data suggest that residues 

333–344 in human apoA-IV, a region that is highly conserved in mammals, is a critical 

determinant of its unique interfacial behavior. As this region is not predicted to possess ordered 

secondary structure, its impact is likely mediated via an effect on the global conformation of 

apoA-IV. 

P229 

Apolipoprotein CI Deficiency Reduces Hyperlipidemia and Atherosclerosis 

in Apolipoprotein E-Knockout Mice 

Marit Westerterp, Willeke de Haan, Jimmy F Berbée, Louis M Havekes, Patrick C Rensen; 

TNO-Quality of Life and LUMC, Leiden, The Netherlands 

We have previously demonstrated that human apoCI-overexpressing mice develop severe 

hypertriglyceridemia in addition to elevated cholesterol levels, mainly caused by apoCI-induced 

inhibition of LPL. The hypertriglyceridemia was even aggravated on an apoe -/- background, 

correlating with higher VLDL-apoCI levels. The aim of the present study was to assess whether 

endogenous apoCI levels are sufficient to modulate plasma lipid levels. Lipid levels were not 

affected upon apoCI deficiency on the wild-type (C57Bl/6) background, which is in line with our 

previous results, yet on the apoe -/- background (i.e. a condition with high VLDL levels) apoCI 

deficiency gene-dose-dependently reduced VLDL-TG (58%; p0.001) and VLDL-cholesterol 

(23%; p0.05) as compared to apoe -/- littermates. The mechanisms underlying this finding 

were elucidated. Whereas intestinal [ 3 H]TG absorption was not affected, VLDL-TG and 

VLDL- 35 S-apoB production were decreased by 26% and 28%, respectively (p0.05). In 

addition, the postprandial TG response to an intragastric olive oil load was decreased (59%; 

p0.05), and the uptake of i.v. injected [ 3 H]TG derived from VLDL-like emulsion particles by 

gonadal and perirenal white adipose tissue (WAT) was increased (52% and 33%, respectively; 

p0.05). As LPL is the main enzyme involved in the clearance of TG-derived free fatty acids 

by WAT, and total post-heparin LPL levels were unaffected, these data demonstrate that the 

endogenous expression of apoCI suffices to attenuate the lipolytic activity of LPL on the apoe -/background. 

These effects of apoCI-deficiency were accompanied by a reduced atherosclerotic 

lesion area (46%; p0.05) in the aortic root, as assessed in apoe -/- apoc1 -/- vs apoe -/- mice on 

chow diet at 26 weeks of age. We thus conclude that apoCI-deficiency reduces plasma TG 

levels in apoe -/- mice resulting from decreased VLDL-particle production and/or relieved 

LPL-inhibition, which is accompanied by reduced atherogenesis. 

Impaired Endocytosis of LRP1 Minireceptors in Transfected Cells is 

Associated with Abnormal Cell Growth and Foci Formation 

Hongyu Zhang, Zemin Yao; Univ of Ottawa and Univ of Ottawa Heart Institute, Ottawa, 

Canada 

P230 

The LDL receptor-related protein 1 (LRP1) acts as a co-receptor in the metabolism of 

lipoproteins and other ligands that are important in various cell functions. We hypothesized that 

the two tyrosine residues (Y29 and Y63) of the respective NPxY motifs within the LRP1 

cytoplasmic domain play a role in regulating cell surface presentation of the receptor and the 

function of ligands. To test this hypothesis, we created LRP1 minireceptors (composed of the 

cluster IV ligand-binding domain, and the transmembrane and cytoplasmic domains, designated 

LRP1-IVwt) in which the respective Y29 and Y63 residues were substituted by alanine 

(designated Y29A and Y63A). The minireceptors, when stably expressed in the LRP1-null 

13–5-1 CHO cells, showed normal ER-to-Golgi trafficking and post-translational processing 

(cleaved into - and -chains). However, cells expressing Y63A mutant exhibited enhanced 

cell surface presentation, spontaneous foci formation, increased cell growth rate, and 

enhanced anchorage-independent cell growth. These phenotypes were absent in cells 

expressing LRP1-IVwt or Y29A mutant. While increased cell surface presentation of Y63A is 

most likely attributable to impaired endocytosis by disruption of the Y63XXL motif, the 

transformation phenotype suggests altered signal transduction leading to cell transformation. 

Indeed, cells expressing Y63A mutant displayed drastically altered caveolin-1 solubility in 

detergent, elevated intracellular cholesterol (both free and esterified), and markedly increased 

activity of MMP-9 in conditioned medium. Thus, Downloaded impaired LRP1 internalization from 

is associated 



with altered lipid homeostasis, disregulated caveolae function, and abnormal activity of 

extracellular matrix proteins. 

A Role for Adipocyte Cholesterol Efflux in Lipidation of High Density 

Lipoprotein in Vivo 

Yuzhen Zhang, Univ of Pennsylvania, Philadelphia, PA; George Rothblat, Children’s Hosp of 

Philadelphia, Philadelphia, PA; Jane M Glick, Christine C Hinkle, Daniel J Rader, Muredach 

P Reilly; Univ of Pennsylvania, Philadelphia, PA 

P231 

Adipose tissue is one of the body’s largest pools of free cholesterol and is a potential source 

for HDL lipidation. ABCA1 regulates cholesterol efflux and plasma HDL-C levels. However, the 

tissue sources of cholesterol for lipidation of circulating HDL remain unclear. We examined the 

regulation of adipocyte cholesterol efflux in vitro and determined whether adipocytes 

contributed to HDL lipidation in vivo. ABCA1 and SR-B1 expression increased during 3T3-L1 

adipocyte differentiation. In 3T3-L1 adipocytes, treatment (24hrs) with a combination of LXR 

(22r-hydroxycholesterol; 22r-HC 10 M) and RXR (9-cis-retinoic acid; 9-CRA 10 M) agonists 

upregulated ABCA1 (mRNA 2.0 0.6 x fold), but not SR-BI. Efflux of 3 H-cholesterol (4 hours) 

from differentiated 3T3-L1 adipocytes, was 1.54 0.12% to ApoA-I (20 g/ml) and 5.80 

0.26% to HDL 3 (50 g/ml). Efflux to apoA-I increased by 65% with the combination of 22r-HC 

and 9-CRA coincident with upregulation of ABCA1. Following intra-peritoneal injection of 

3 H-cholesterol-labeled 3T3-L1 adipocytes into C57BL/6 mice, 3 H-Cholesterol was detected in 

the plasma (over 70% in HDL fraction), liver and feces over 48 hours. Mouse embryonic 

fibroblasts (MEF), differentiated into mature adipocytes, effluxed cholesterol to acceptors in a 

similar manner to 3T3-L1. In MEF adipocytes derived from ABCA1 deficient embryos, 

3 H-cholesterol efflux was reduced to apoA-I (0.26 0.01% vs 2.75 0.07%; p0.001), and 

to 5% mouse serum (4.73 0.05% vs 7.67 0.10%; p0.01) compared MEF adipocytes 

derived from wild-type litter mate embryos. Plasma and fecal 3 H-cholesterol was reduced by 

22% and 38% respectively (p0.05 for both) following intra-peritoneal injection into C57BL/6 

mice of 3 H-cholesterol labeled ABCA1 deficient MEF adipocytes compared to wild-type control 

MEF adipocytes. These findings support a role for adipocyte cholesterol efflux in HDL lipidation 

and suggest ABCA1 dependent and independent regulation of this process to mature HDL in 

vivo. 

Adipose Tissue Specific CETP Expression in Mice: Impact on Plasma 

Lipoprotein Metabolism 

Hongwen Zhou, David W Jang, Zhiqiang Li, Xian C Jiang; SUNY Downstate Med Cntr, 

Brooklyn, NY 

P232 

CETP is a hydrophobic plasma glycoprotein that mediates the transfer and exchange of 

cholesteryl ester (CE) and triglyceride (TG) between plasma lipoproteins, and plays an important 

role in HDL metabolism. Adipose tissue appears to be a highly conserved site of CETP 

expression across species. However, its function in adipose tissue is still unknown. In order to 

investigate the relationship between adipose tissue CETP and plasma CETP activity, and also 

between adipose tissue CETP and plasma lipoprotein levels, we created adipose tissue specific 

CETP transgenic mice by using the aP2 promoter and enhancer. Out of five founders, we 

established 1–3 and 1–9 lines. CETP mRNA was highly expressed in the adipose tissue of lines 

1–3 and 1–9, and was expressed at lower levels in the heart, muscle and lungs of line 1–9. 

Plasma lipoprotein analysis showed a marked reduction of HDL cholesterol and phospholipids, 

as well as an induction of non-HDL lipids in both lines 1–3 and 1–9. The distribution of lipids 

was also determined by FPLC of pooled plasma samples. This confirmed that HDL cholesterol 

was dramatically decreased in aP2-CETP transgenic mice while non-HDL cholesterol was 

increased when compared to wild-type mice. Assessment of apolipoprotein composition of 

centrifugally isolated lipoproteins by SDS-PAGE revealed a marked decrease in apoAI and an 

increase in apoB48 and apoB100. Moreover, aP2-CETP mice showed an increased VLDL 

particle size, but decreased LDL and HDL particle sizes. In summary, we established the 

aP2-CETP transgenic mice, which express human CETP mRNA predominantly in adipose tissue. 

Adipose tissue CETP can be secreted into the circulation and make major contributions to 

plasma lipoprotein levels. 

P233 

Hif 1 and 2 Expression in Vulnerable Human Atherosclerotic Plaques 

Judith C Sluimer, Ann P Bijnens, Univ Hosp Maastricht, Maastricht, The Netherlands; 

Jean-Marie Gasc, INSERM, Paris, France; Luc H van den Akker, Maasland Hosp, Sittard, 

The Netherlands; Pierre Corvol, INSERM, Paris, France; Mat J Daemen; Univ Hosp 

Maastricht, Maastricht, The Netherlands 

Hypoxia has been detected in rabbit atherosclerotic plaques in inflammatory, macrophage-rich 

regions (Bjornheden, ATVB 1999). In addition, hypoxia is observed in diverse inflammatory 

responses, inducing hypoxia-inducible factors (HIF) 1, 2 and HIF responsive genes. Since 

inflammation is critical in atherogenesis, we hypothesize that these pathways are also involved 

in atherosclerosis. Therefore, we investigated the expression of HIF1, 2 and HIF responsive 

genes involved in carbohydrate metabolism, i.e. Glucose Transporter (GLUT) 1 and 3, Carbonic 

Anhydrase (CA) IX, Hexokinase (HK) I, II and III in human carotid arteries with early or stable 

atherosclerotic lesions or lesions with a thrombus. Microarray analysis showed a 2.1 fold 

upregulation of HIF1 mRNA in lesions with a thrombus versus early lesions. Strikingly, HK III 

was 18.9 fold upregulated, whereas HK II and GLUT1 only 1.5–2.5 fold. HIF2, GLUT3 and HK 

I were not differentially expressed and CA IX was downregulated 0.6 fold. Furthermore, HK III 

was the only gene differentially expressed between early and stable plaques (1.6 fold). In situ 

hybridization was used to determine the cell types expressing HIF1 and 2 RNA in human 

atherosclerosis. RNA was strongly expressed in adventitial endothelial cells of the vasa 

vasorum (VV), in intimal macrophages and smooth muscle cells (SMC), but little expression was 

detected by in the guest mediaon or in June intimal 29, endothelial 2013 cells (EC). Since the HIF1 and 2 pathway is

egulated through protein stabilization, we also validated the RNA expression pattern using 

immunohistochemistry. HIF1 and 2 protein were strongly expressed in adventitial VV and 

macrophage foam cells and modestly in intimal SMC and EC. Expression was more pronounced 

in advanced lesions compared to early lesions and absent in non-diseased vessels. Moreover, 

we demonstrated that the protein expression of various HIF responsive genes in human 

atherosclerosis was similarly distributed. Thus, in human atherosclerosis HIF1, 2 and their 

responsive genes are principally expressed in macrophage foam cell regions. Since these 

inflammatory regions are associated with plaque vulnerability, we postulate that expression of 

HIF1, 2 and their responsive genes induce a vulnerable plaque phenotype. 

Novel Loci Detected for Atherosclerosis Susceptibility from a Strain 

Intercross of ApoE-Deficient Mice 

P234 

Jonathan D Smith, Julie Baglione, Megan Settle, Daoquan Peng, Wilfried Le Goff; Cleveland 

Clinic Foundation, Cleveland, OH 

ApoE-deficient mice were bred onto the AKR/J and DBA/2J genetic backgrounds. Sixteen-week 

old chow-diet fed DBA/2 mice had 14-fold larger lesions in the aortic root than the AKR mice 

for both genders. The DBA/2 apoE-deficient mice have the largest lesions in the aortic root 

compared to all of the other inbred strains that we have bred apoE-deficient mice onto. In order 

to identify the genes that are responsible for this large strain effect on lesion area, we 

performed a strain intercross and bred 213 F 2 mice. Mice were maintained on a chow diet and 

aortic root lesion areas were determined at 16 weeks of age. Lesions in the male, but not 

female, F 2 mice had a markedly bimodal distribution. In the male, but not female, F 2 mice lesion 

areas and log lesion areas had a weak but significant correlation with total plasma cholesterol 

values (R 2 0.06 and 0.08, respectively). A genome scan of each F 2 mouse was performed 

using 100 strain polymorphic markers. Quantitative trait locus (QTL) analysis was performed 

using the r/qtl software package. Using log lesion area as the phenotype, one major locus was 

found on chromosome 17 for male mice, and a different major locus was found on 

chromosome 3 for female mice. For both of these loci, the DBA/2 allele was associated with 

larger lesions, in agreement with the lesion phenotype of the parental strains. Mouse 

atherosclerosis susceptibility loci have not previously been described on either of these 

chromosomes. Combining both genders and using gender as an interactive covariate, these 

two loci were even stronger and additional loci were uncovered on chromosomes 5 and 15. We 

are now poised to perform fine mapping to narrow these QTL intervals and identify the 

atherosclerosis susceptibility genes that reside in these loci. 

Oxidative Stress Produced with Cell Migration Increases Synthetic 

Phenotype Properties of Vascular Smooth Muscle Cells 

Hak-Joon Sung, Suzanne G Eskin, Yumiko Sakurai, Andrew Yee, Noriyuki Kataoka, Larry V 

McIntire; Georgia Institute of Technology, Atlanta, GA 

P235 

Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species 

(ROS) are important in vascular pathogenesis. Understanding how these factors relate to cell 

migration can improve design of therapeutic interventions to control vascular disease. We 

hypothesized that ROS-induced VSMC phenotypic change was enhanced by cell migration. We 

compared the proliferation, protein content and migration of cultured aortic VSMC from wild 

type (WT) vs. transgenic mice (Tg p22phox ), in which overexpression of p22phox was targeted to 

VSMC. Also, we compared H 2O 2 generation and expression of specific phenotypic marker of 

non-migrating with migrating WT vs. Tg p22phox VSMC in an in vitro wound scratch model. We 

found that Tg p22phox expressed markers of synthetic VSMC more and showed an increase in 

proliferation and protein content over WT VSMC. Tg p22phox showed higher expression of TM4 and 

SMemb, but lower expression of smooth muscle -actin than WT VSMC. Tg p22phox produced 

higher intracellular H 2O 2 and had a greater migration rate than WT VSMC. Suppression of ROS 

with ebselen decreased the migration of both cell types. These results showed a strong 

correlation between ROS production and VSMC migration. More importantly, we found that 

VSMC migrating across the wound edge produced significantly more H 2O 2 than non-migrating 

VSMC of both cell types, which we postulate resulted in higher expression of TM4 and SMemb, 

and lower expression of -actin. These results indicate that increased ROS produced during 

migration correlated with the synthetic phenotype in both VSMC types. Our results provide 

evidence to support an important role for oxidative stress in phenotypic modulation of VSMC. 

We also demonstrate the strong possibility that VSMC migration into the wound area can trigger 

synthetic properties through an increase in ROS production. This may elucidate the role of 

VSMC phenotypic modulation in the progress of intimal hyperplasia. 

P236 

HIV Impairs Reverse Cholesterol Transport from Macrophages: A Possible 

Mechanism of HIV-Induced Atherosclerosis 

Zahedi Mujawar, The George Washington Univ Med Cntr, Washington, DC; Honor Rose, 

Baker Heart Rsch Institute, Melbourne, Australia; Tatiana Pushkarsky, The George 

Washington Univ Med Cntr, Washington, DC; Anthony Dart, Baker Heart Rsch Institute, 

Melbourne, Australia; Michael Bukrinsky, The George Washington Univ Med Cntr, 

Washington, DC; Dmitri Sviridov; Baker Heart Rsch Institute, Melbourne, Australia 

Both asymptomatic HIV-1 infection and AIDS are associated with increased risk of coronary 

artery disease and development of atherosclerosis. A characteristic feature of atherosclerosis 

is accumulation of foam cells in the walls of arteries. The reason for accumulation of 

cholesterol in the vessel wall and formation of foam cells may be dyslipidemia, impairment of 

intracellular cholesterol metabolism or a combination of the two. Here we demonstrate that 

HIV-1 infection of human monocyte-derived macrophages leads to severe impairment of 

apolipoprotein A-I -dependent cholesterol efflux. The HIV-1 protein Nef mediates this effect, as 

Nef-deficient HIV-1 did not impair cholesterol efflux. Transient transfection of RAW 264.7 

murine macrophages with the Nef-expressing construct Downloaded resulted infrom reduction of efflux, while 




mutated Nef2GA was inactive. One of the mechanisms responsible for this effect of Nef is 

reduction of expression and abundance of ATP-binding cassette transporter A1, a main 

transporter of cholesterol to apolipoprotein A-I. Transfection of RAW 264.7 cells with Nef also 

resulted in redistribution of ABCA1 from equal partitioning between cytosol and plasma 

membrane to predominantly plasma membrane localization. Infection of monocyte-derived 

macrophages with HIV or transfection of RAW264.7 cells with Nef resulted in higher rates of 

cholesteryl ester synthesis and significant accumulation of cholesteryl esters in these cells. 

These results suggest a mechanism by which HIV-infected macrophages may initiate 

atherosclerotic plaque formation. Combination of enhanced cholesterol uptake due to 

HAART-induced hypercholesterolemia and impairment of cholesterol efflux due to HIV infection 

may therefore be a key combination resulting in sharply increased risk of CAD in HIV patients. 

Hyperhomocysteinemia Inhibits Post-Injury Reendothelialization in 

Cystathionine -Synthase Knockout Mice 

Hongmei Tan, Xiaohua Jiang, Fan Yang, Dan Liao, Chuantao Jiang, Baylor College of 

Medicine, Houston, TX; Mark J Magera, Mayo Clinic, Rochester, MN; William Durante, 

Baylor College of Medicine, Houston, TX; Andrew Shafer, Univ of Pennsylvania Sch of 

Medicine, Philadelphia, PA; Xiaofeng Yang, Hong Wang; Baylor College of Medicine, 

Houston, TX 

P237 

Hyperhomocysteinemia (HHcy) is a risk factor for cardiovascular disease and has been reported 

to inhibit endothelial cell (EC) growth. The effect and mechanisms by which HHcy regulates EC 

growth in vivo are largely unknown. In this study, we established a mouse carotid artery air-dry 

endothelium denudation and reendothelialization model, and evaluated the effect of HHcy on 

post-injury reendothelialization in mice with the gene deletion of cystathionine--synthase 

(CBS). Severe HHcy was induced in CBS -/ mice with a high methionine diet. Post-injury 

reendothelialization was impaired, correlating with increased neointima formation in hyperhomocysteinemic 

mice. To illustrate the underlying mechanism, we examined circulating 

endothelial progenitor cells (EPC) in hyperhomocysteinemic mice, and studied the effect of Hcy 

on proliferation and migration of human umbilical vein endothelial cells (HUVEC), and on 

adhesion in mouse EPC and HUVEC. Circulating EPC were slightly decreased in HHcy mice. In 

addition, Hcy inhibited proliferation and migration of HUVEC, and decreased adhesion of EPC 

and HUVEC to fibronectin. These data indicate that Hcy inhibits post-injury reendothelialization 

and leads to intimal hyperplasia. The capacity of Hcy to inhibit the proliferation and migration 

of EC, and adhesion of EC and EPC may be responsible for impaired reendothelialization and 

contribute to arteriosclerosis in HHcy. 

P238 

Expression of ABCA1 is Selectively Impaired in Macrophages and Kidneys 

in Diabetic Mice 

Chongren Tang, Timothy McMillen, Jenny E Kanter, Karin E Bornfeldt, Renee C LeBoeuf, 

John F Oram; Univ of Washington, Seattle, WA 

Abnormal high-density lipoprotein (HDL) metabolism may contribute to the increased atherosclerosis 

associated with diabetes. The ATP-binding cassette transporter ABCA1 is an 

atheroprotective membrane protein that mediates cholesterol transport from tissue macrophages 

to apolipoprotein A-I (apoA-I), the major protein component of HDL, raising the 

possibility that impaired ABCA1 function may contribute to the increased atherosclerosis 

associated with diabetes. This concept is supported by our findings that diabetes-associated 

factors, such as elevated fatty acids and precursors for advanced glycation end products, 

destabilize ABCA1 protein in vitro. We therefore examined the regulation of ABCA1 in two 

mouse models of diabetes. RT-PCR and western blot analysis of murine cells and tissues 

demonstrated that ABCA1 protein levels were reduced by 41.9% (p0.007) in freshly isolated 

peritoneal macrophages and by 44.4% (p0.004) in kidneys in type 1 diabetic NOD mice 

compared to non-diabetic animals, even though ABCA1 mRNA levels were not significantly 

different. A similar reduction (39.4%, p0.001) in ABCA1 protein was found in peritoneal 

macrophages from virus-induced type 1 diabetic mice compared to littermate controls. 

However, in liver and brain, neither ABCA1 protein nor ABCA1 mRNA levels were significantly 

changed in diabetic NOD mice compared with non-diabetic mice. These results indicate that 

diabetes impairs ABCA1 expression in a tissue-specific manner, and that this impairment might 

contribute to the increased atherosclerosis and renal disease associated with diabetes. 

Olmesartan and Pravastatin Additively Reduce Development of 


José W van der Hoorn, TNO Quality of life, Leiden, The Netherlands; J W Jukema, Leiden 

Univ Med Cntr, Leiden, The Netherlands; Louis M Havekes, Teake Kooistra, Robert 

Kleemann, Hans M Princen; TNO Quality of life, Leiden, The Netherlands 

P239 

Angiotensin II receptor blockers (ARBs) are recognized primarily for their use in hypertension, 

in heart failure, and after myocardial infarction. Emerging clinical data indicate that ARBs may 

modulate atherosclerosis as well. The present study was designed to evaluate the effects of 

ARB olmesartan (10 mg/kg body weight (bw)), cholesterol-lowering drug pravastatin (4 mg/kg 

bw) and the combination of both on the development of atherosclerosis in APOE*3Leiden 

transgenic mice, a well-established mouse model for hyperlipidemia and atherosclerosis. 

During the intervention period of 25 weeks, the control group had average plasma cholesterol 

and triglyceride levels of 18 and 1.5 mmol/L. Pravastatin and combination therapy decreased 

plasma cholesterol to 15 mmol/L (p0.05) and triglycerides to 0.9 mmol/L (p0.001). 

Olmesartan and combination therapy reduced systolic blood pressure (85 and 89 vs 101 

mmHg, p0.002). The atherosclerotic lesion area in the aortic root was significantly reduced 

by olmesartan (88 vs 164 m2 *1000, p0.05), by pravastatin (101 vs 164 m2 *1000, 

p0.05) and by combination therapy (15 vs 164 m2 *1000, p0.001). Combination therapy 

also resulted by guest in a strongly on June reduced 29, number 2013of 

lesion, lesion size, severity of lesions, number

E-94 Vol 25, No 5 May 2005 

of smooth muscle cells, number of macrophages and number of T cells per cross-section as 

compared to the control group, with the olmesartan and the pravastatin group in between. In 

all treatment groups, the general inflammation marker serum amyloid A was reduced to the 

same extent. Adhesion of monocytes to the vessel wall was significantly decreased in the 

groups receiving olmesartan as compared to the control and pravastatin group. Protein 

expression of IB, inhibitor of NF-B, was not up regulated by olmesartan in liver or aorta. 

Olmesartan and pravastatin did not significantly affect endothelial adhesion molecule (Eselectin, 

ICAM-1), aortic chemokine (MCP-1) or cytokine (MIF, IL-1, IL-6, CD40L) expression 

in the aorta. In conclusion, olmesartan reduced monocyte adhesion and combination therapy 

with olmesartan and pravastatin additively reduced number of macrophages and leukocytes, 

and development of atherosclerosis, reflecting their different anti-atherosclerotic modes of 

action. 

The Atherogenic Potential of Natural Killer T-Lymphocytes is Immune 

Context Dependent 

Paul A VanderLaan, Catherine A Reardon, Godfrey S Getz; Univ of Chicago, Chicago, IL 

P240 

Natural killer T (NKT) cells are a distinct subset of T-lymphocytes that respond to lipid antigens 

presented by the MHC class-I like CD1d molecule and have recently been implicated in the 

pathogenesis of atherosclerosis. Every study to date has demonstrated a pro-atherogenic 

phenotype for NKT cells, but it is unclear if macrophage foam cells are direct targets of NKT 

cell activity, or if this effect is mediated indirectly via interactions with other lymphocytes 

present in the plaque. To address this issue, NKT cells were isolated from the spleens of 

V14tg mice and then adoptively transferred into female RAG1 -/- LDLR -/- mice which completely 

lack functional T- and B-cells. Following the adoptive transfer, these mice were fed a Western 

type diet (WTD) for 12 weeks (n7–13 per group). Compared to PBS controls, the NKT cell 

recipients had a significant decrease in plasma total cholesterol (743 vs. 1174 mg/dL, 

p0.00001) and triglyceride (86 vs. 342 mg/dL, p0.0001) levels at 4 weeks, with a trend for 

lower plasma lipids at 8 and 12 weeks. This reduction was primarily localized to the VLDL and 

LDL fractions. The atherosclerosis in the aortic sinus of the NKT cell recipients was significantly 

less than the PBS controls (106,484 vs. 164,186m 2 ,p0.014). Similar reductions in lipids 

and atherosclerosis were observed in groups fed WTD for 8 weeks. Laser capture microdissection 

with subsequent RT-PCR analysis confirmed the presence of the invariant T-cell 

receptor (V14J18) in the atherosclerotic plaques of the NKT cell recipients but not the PBS 

controls. In a related set of experiments, total splenocytes from either V14tg or C57BL6 mice 

were adoptively transferred into female RAG1 -/- LDLR -/- mice and fed a WTD for 12 weeks. There 

was no difference in plasma lipids between groups and preliminary data indicate that V14tg 

recipients tend to have increased aortic sinus atherosclerosis compared to C57BL6 recipients. 

In summary, these studies indicate that the proatherogenic properties of NKT cells may require 

the presence of other T- or B-cells. Furthermore, in the absence of these lymphocyte subsets, 

NKT cells may even be atheroprotective: either indirectly by altering lipoprotein metabolism, or 

through direct interactions with other plaque cells. 

P241 

Characterization of Cells Contributing to Ectopic Vascular Calcification 

Kasey C Vickers, Douglas Brownfield, Joel D Morrisett; Baylor College of Medicine, Houston, 

TX 

Vascular mineralization is highly associated with cardiovascular risk, specifically atherosclerotic 

intimal calcification. The onset of osteogenesis in the vascular wall is correlated to additional 

atherosclerotic plaque burden. Ectopic calcification is highly regulated and several molecular 

mechanisms have been proposed. There is less known about the cellular contribution (in vivo) 

to vascular calcification and what mechanisms govern the regulated gene expression of the 

calcifying vascular cells (CVC). For our studies, we are using tissues resected by carotid 

endoarterectomy. Since almost half of these tissues contain calcified lesions, as determined by 

MRI, they are particularly appropriate for studying arterial calcification. The 2D MR images of 

calcified carotid arteries are being used to create 3D reconstructions, with appropriate 

landmarks, which are then used as a scaffold to map RNA message distribution within the 

calcified area. Specific cellular biomarkers and unique calcifying factors have been localized in 

frozen sections with immunohistochemistry. Laser capture microdissection (Veritas) has been 

used to identify selectively labeled fluorescent cells that contribute to mineralization and for 

capturing individual cells, localized to or nucleating the calcification. RT-PCR and microarray 

techniques are being used to observe changes in gene expression of CVCs and their precursors. 

Gene expression patterns mapped to the 3D reconstructed image of the vessel are being used 

to evaluate patterns of cellular contribution and message distribution. These data are being 

used to accurately predict the involvement of specific cells in ectopic vascular calcification. This 

approach is enabling the identification and mapping of factors and biomarkers involved in 

atherosclerotic calcification, with the goal of preventing, stabilizing, or even reducing 

osteogenesis in the vascular wall. 

P242 

Chlamydophila pneumoniae Alters the Metabolism of Sphingolipids and 

Triggers an Extensive Coalescence of Lipid-Raft in Human Monocytic Cells 

Silvana A Vielma, Ja-Ran Ku, Jacek Bielawski, Lina Obeid, Gabriel Virella, Med Univ of 

South Carolina, Charleston, SC; Maritza de Munoz, Universidad de Los Andes, Merida, 

Venezuela; Maria F Lopes-Virella; Med Univ of South Carolina, Charleston, SC 

Chlamydophila pneumoniae have been detected in human atheroma lesions providing direct 

evidence for an association between C. pneumoniae and human arteriosclerosis. Numerous in 

vitro studies investigating possible mechanisms by which C. pneumoniae could lead to the 

development of arteriosclerosis have supported this association. C. pneumoniae infects cells 

that play a main role in arteriosclerosis such as macrophages and endothelial cells leading to 

increased expression of adhesion molecules, chemokines Downloaded and FcRII from 

in endothelial cells as 



well as to the transformation of macrophages into foam cells and increased release of 

cytokines by these cells. Since C. pneumoniae requires sphingomyelin (SM) for its intracellular 

replication, we decided to measure sphingolipids content and to studied the patching behavior 

of raft-associated ganglioside GM1 proteins after C. pneumoniae infection using ESI/MS/MS 

and fluorescence analysis respectively. Our data showed that macrophages infected with C. 

pneumoniae have a decrease in SM and an increase in ceramides within 5 to 30 minutes of 

infection but at 4 and 24 hours a marked and significant increase in sphingosine-1-phosphate 

(S1P) and SM and a decrease in C16 ceramide is observed. We have also shown that in 

macrophages, C. pneumoniae triggers a rapid and extensive coalescence of lipid-raft 

ganglioside GM1 within 5 minutes of infection and becomes quite large 15 to 30 minutes 

post-infection. C. pneumoniae infection also induced co-localization of FcRII, CD36 and CD14 

within the lipid raft within the 15 min of infection. In conclusion by altering the metabolism of 

sphingolipids C. pneumoniae may contribute to arteriosclerosis. The formation of lipid rafts 

would lead to C. pneumoniae entry into the cell, play a role in its initial intracellular survival and 

by leading to the formation of large platforms that recruit several receptors important for the 

development of arteriosclerosis. Furthermore, by leading to a persistent increase in S1P the 

infection may become chronic and perpetuate the inflammatory process. 

P243 

Role of Iron in Coronary Atherosclerosis : Coronary Atherosclerotic Risk in 

South Indian Population (CARSI) 

Girish N Viswanathan, Regional Cardiac Cntr, Morriston Hosp, Swansea SA66LZ, UK, 

Swansea, United Kingdom; N Karunanithi; Madras Med College and Rsch Institute, Chennai 

600003, India, Chennai, India 

Background:Coronary artery disease prevalence has been rapidly increasing in developing 

countries including India. Low prevalence of conventional risk factors and ethinic predisposition 

has created interest in novel coronary risk factors.Iron has been strongly associated with 

oxidization of LDLc, pro inflammatory meditors and progression of atherosclerosis.This is the 

first study in this unique group of population with normal or near normal lipid levels.Methods:90 

consecutive patients who presented at our institute for diagnostic coronary angiography 

were included for the study after satisfying inclusion and exclusion criteriae.The biochemical 

analysis for lipids and iron status were carried out by blinded observers. Serum ferritin,the 

widely accepted marker for iron stores was chosen for the analysis. The coronary angiograms 

were reported by two blinded cardiologists. The patients were divided as having significant 

atherosclerosis in one or more arteries(cases)and angiographically normal coronary arteries- 

(controls).Results:The data of 66 cases with significant coronary artery disease and 16 controls 

with normal coronary arteries were analysed. The cases and controls matched well with their 

age, sex and social strata, dietary iron intake. Diabetes, smoking and raised waist hip ratio 

were strongly associated with coronary atherosclerosis. The levels of total cholesterol, LDLc, 

HDLc and triglyceride matched well between the cases and the controls(203 vs 196, 131 vs 

121, 45 vs 41 and 160 vs 167 mg/dl respectively).The mean serum ferritin levels were 241.68 

in cases and 155.62 in controls,ng/ml p 0.22. Among those with quartiles of ferritin more 

than 100 ng/ml, 72% had significant coronary artery disease against 50% of the controls. RR 

2.6 p 0.72.Conclusions:In our study we have observed a positive trend between iron stores 

and the burden of coronary atherosclerosis in normolipidemic individuals which highlight the 

role of oxidative stress in atherogenesis. We hypothesise that these results may carry clinical 

significance in the western population with progressive coronary artery disease who consume 

a diet rich in iron. However larger scale studies are warranted before making clinical 

recommendations. 

P244 

Biphasic Activation of Rho/Rho Kinase Pathway by Angiotensin II through 

Distinct G Protein in Vascular Smooth Muscle Cells 

Shu Wakino, Koichi Hayashi, Takeshi Kanda, Kazuhiro Hasegawa, Naoki Sugano, Satoru 

Tatematsu, Kyoko Yoshioka, Ichiro Takamatsu, Keio Univ, Tokyo, Japan; Yoshifumi 

Kawanabe, Kyoto Univ, Kyoto, Japan; Takao Saruta; Keio Univ, Tokyo, Japan 

[Backgrounds] A potent vasoconstrictor Angiotensin II (AngII) plays a key role in proliferation 

and migration of vascular smooth muscle cells (VSMC). Although Rho/Rho kinase pathway is 

an important intracellular signaling pathway elicited by Ang II, its precise mechanism has not 

been elucidated. [Aims] The activation mechanism of Rho/Rho kinase by AngII is characterized 

by using rat aortic smooth muscle cells (RASMC). [Methods] Serum-starved RASMC (passage 

4th to 8th) were stimulated with 100 nM of AngII and cells were harvested 10 min and 6 hours 

after the stimulation. Activation of Rho/Rho kinase pathway was measured by phosphorylation 

of myosin phosphatase target subunit (MYPT) and activation of guanidine nucleotide exchange 

factors (GEF) for Rho, Lbc, LARG, p115RhoGEF (p115), and Vav, were assessed by 

tyrosine-phosphorylation of each protein. GP-2A, the inhibitor for Gq protein, GP-2 for Gs and 

Gi proteins, AG 490 for Janus-activated kinase 2 (JAK2), and AG1478 for epidermal growth 

factor receptor (EGFR) were pretreated 30 min before Ang II-stimulation. Dominant-negative 

mutant for G12 or G13 was transfected before the stimulation by AngII to examine the 

contribution of G12 or G13 to the activation of Rho/Rho kinase pathway. [Results] AngII elicited 

biphasic activation of Rho/Rho kinase; a rapid phase which reached a peak within 10 minutes 

and the sustained phase lasting for 6 hours. The rapid activation was blocked by the 

transfection of dnG12 neither by that of dnG13, nor by the pretreatment with GP2A or AG490. 

The sustained activation was blocked by the pretreatment with GP2A and AG490, neither by the 

transfection of dnG12 nor dnG13. Among several RhoGEF, p115 was tyrosine-phosphorylated 

in the rapid phase and Vav in the sustained phase. Tyrosine-phosphorylation of Vav was 

blocked by AG490 and that of p115 was not. [Conclusion] AngII activates Rho/Rho kinase 

pathway by distinct two pathways; G12/p115 in the rapid phase, and Gq/EGFR/Vav, a 

transactivation pathway in the sustained phase. The former may contribute to Ca sensitizing 

effects onby theguest vasoconstriction on June and 29, the 2013 latter to the proliferation or migration of VSMC.

P245 

Serum Amyloid A Induces Macrophage Matrix Metalloproteinase-9 and -13 

Expression: Implications for Abdominal Aortic Aneurysm Formation 

Jassir Witta, Kathy Forrest, Christopher Calulot, Frederick C de Beer, Nancy R Webb; Univ 

of Kentucky, Lexington, KY 

Serum amyloid A (SAA) is a major acute phase reactant whose expression can increase 

1000-fold in response to inflammation. Clinical studies have suggested that serum levels of 

SAA may be associated with processes involved in the early phases of abdominal aortic 

aneurysm (AAA) formation. The most prominent characteristics of developing AAA are localized 

inflammation and enzymatic degradation of elastic lamellae and extracellular matrix proteins. 

Mounting evidence suggests that matrix metalloproteinases (MMPs) are the predominant 

proteinases in AAA. Given previous findings that SAA induces MMP secretion by a variety of cell 

types, we postulated that SAA plays a direct role in MMP activation and consequently, AAA 

formation. To test this hypothesis, we analyzed AAA induced in apoE -/- mice by 2 week 

infusion of angiotensin II (1000 ng/kg/min). Immunohistochemical analysis using anti-SAA 

antibody revealed intense SAA staining in aneurismal tissue. We have investigated the effects 

of SAA on the production of MMP-13 and MMP-9 in the murine macrophage-like cell line J774 

by real-time RT-PCR, Western Blot and zymography. MMP activity was stimulated by SAA in a 

dose-dependent manner. Lipoxin A4, a known endogenous ligand for the formyl peptide 

receptor-like 1/lipoxin A4 receptor (FPRL1/LXA4R), blocked SAA induction of MMPs. Pertussis 

toxin also significantly reduced SAA induction of MMPs in J774 cells. These results implicate 

SAA in the early phases of AAA formation through transcriptional upregulation of MMPs via a 

signaling pathway involving the G-protein-coupled FPRL1/LXA4R receptor. 

P246 

Cholesterol Enrichment of Endothelial Cells Activates Both an Inflammatory 

and the Endoplasmic Reticulum Stress Response 

Yi Xie, Thomas N Tulenko; Thomas Jefferson Univ, Philadelphia, PA 

Atherosclerosis is the leading cause of mortality in western cultures. It has been accepted that 

atherosclerosis results, primarily, from hypercholesterolemia and is mediated by chronic 

inflammatory activity in the cells of the arterial wall. Cholesterol enrichment in cardiovascular 

cells secondary to hypercholesterolemia results in the accumulation of this sterol in the plasma 

membrane. Incubation of endothelial cells (EC) with a cholesterol-rich liposome/cyclodextrin 

(CD) mixture resulted in a time- and concentration-dependent increase in EC membrane 

cholesterol content. This was associated with increased cell surface ICAM-1 expression and 

monocyte adhesion. In addition, cholesterol enrichment increased nitric oxide production, 

which was inhibited by the iNOS inhibitor i1400w. Furthermore, cholesterol enrichment resulted 

in a time-dependent increasing the endoplasmic reticulum (ER) chaoerone, BiP, mRNA 

expression, indicating induction of the ER stress response. In contrast, in the shared unfolded 

protein response (UPR) pathway, the activated transcription factor 4 (ATF4) was downregulated 

in a time-dependent manner upon cholesterol enrichment, consistent with activation 

of a negative feedback loop in the UPR. In addition, down-regulation of ATF4 was accompanied 

by a decrease in LPS-induced expression of ICAM, VCAM and E-Selectin mRNA and ICAM-1 

protein level in EC previously enriched with cholesterol. In conclusion, cholesterol enrichment 

of the EC membrane activates the ER stress response, as well as an EC inflammatory response. 

This result suggests the possibility of a novel molecular association between cellular 

cholesterol enrichment the ER stress response and the cellular inflammatory activity in EC. 

P247 

Inflammatory Molecular Expressions in Proteomic Study on a New Model 

of Atherosclerotic Rat 

Peng-Yuan Yang, Yao-Cheng Rui, Ling Lu, Zhen-Yu Huang, Mohamad Radwan Almofti; 

Second Military Med Univ, ShangHai, China 

A new atherosclerotic rat model was established in our laboratory induced by high cholesterol 

diets with injection of vitamin D3, which provide a suitable experimental model for advanced 

study on the pathological and pharmacological mechanisms of atherosclerosis. In this paper we 

used the techniques of proteomics to compare the protein expression profiles between control 

and atherosclerotic rat aorta. Using 2-DE and MALDI-TOF-MS, we identified 226 proteins with 

high abundance in rat aorta, and 78 proteins whose expressions were significantly altered in 

atherosclerotic lesions. Majority of these altered proteins are related with the inflammatory 

reactions of atherogenesis. Therefore, we further investigated these inflammatory molecule 

expressions, such as c-reactive protein (CRP), interleukin (IL)-1, tumor necrosis factor 

(TNF)-, adhesion molecule (ICAM)-1 and vascular endothelial growth factor (VEGF), by ELISA 

and Western Blot analysis. CRP was significantly increased in rat serum during atherosclerotic 

process. The IL-1 and TNF- levels were increased greatly, which showed protein content of 

IL-1 in aorta increased to be 2.3-fold of the control, TNF- levels increased to be 2.4-fold in 

atherosclerotic rat aorta. There was a massive increase for both ICAM-1 and VEGF in plaques, 

but ICAM-1 showed up-regulation much earlier than VEGF in time course in atherosclerotic rat. 

The comparative analysis of the proteome also provides some new proteins that play important 

roles in inflammation of atherogenesis. Our results suggested a new suitable rat model for 

these inflammatory marker molecule studies in pathologic and pharmacological researches, 

and proteomic study also provide possible discovery of novel diagnosis markers and 

therapeutic approaches. 





P248 

Macrophage Apolipoprotein E Reduces Atherosclerosis and Prevents 

Premature Death in Apolipoprotein E and Scavenger Receptor Class BI 

Double Knockout Mice 

Hong Yu, Patricia G Yancey, Wenwu Zhang, Youmin Zhang, Sergio Fazio, MacRae F Linton; 

Vanderbilt Univ, Nashville, TN 

Background Mice deficient in apolipoprotein E (apoE) and scavenger receptor class BI (SRBI) 

exhibit cardinal features of human atherosclerotic coronary heart disease: hypercholesterolemia, 

occlusive coronary atherosclerosis, myocardial infarction, and premature death. Reconstitution 

of macrophage apoE in apoE -/- mice by bone marrow transplantation (BMT) corrects 

the dyslipidemia and prevents atherosclerosis. In contrast, reconstitution of apoE -/- LDLR -/- mice 

with macrophage apoE reduces atherosclerosis but does not correct the dyslipidemia. The goal 

of the study was to examine the ability of macrophage apoE -/- to improve dyslipidemia, reduce 

atherosclerosis, and rescue the lethal phenotype of apoE -/- SRBI -/- mice. Methods and Results 

Probucol treatment, as described by Krieger and coworkers, was used to rescue the 

apoE -/- SRBI -/- mice during BMT as the mice were very sensitive to radiation and died 3–5 days 

after BMT. The apoE -/- SRBI -/- recipient mice were generated from mating pairs on 0.5% 

probucol in a normal chow diet. At 6–7 weeks of age, apoE -/- SRBI -/- recipient mice were lethally 

irradiated and transplanted with wild type (n11) or apoE -/- SRBI -/- (n10) bone marrow. Two 

weeks after BMT, probucol was removed from the diet. Six weeks after BMT, the mean serum 

cholesterol level (mg/dlSD) of control apoE -/- SRBI -/- 3apoE -/- SRBI -/- mice was 649.295.6, 

and these mice all died within 8 weeks after BMT. In the experimental group, apoE was 

detected in serum 2 weeks after BMT, and the dyslipidemia was significantly corrected after 

6 weeks (mean serum cholesterol levels 137.630.2 mg/dl). The lipoprotein profile and HDL 

subpopulations were very similar to those of SRBI deficient mice. Analysis of the proximal aorta 

7 to 8 weeks after BMT demonstrated an 85.2% decrease in mean atherosclerotic lesion area 

in apoE -/- SRBI -/- mice transplanted with wild type vs. apoE -/- SRBI -/- BM (94.227.4x10 3 vs. 

637.6218.6x10 3 m 2 ; meanSEM, P0.00015, n6 per group). In addition, the experimental 

mice were rescued from premature death with a lifespan of up to 37 weeks at present. 

Conclusions Transplantation of apoE -/- SRBI -/- mice with wild type BM corrects the dyslipidemia, 

reduces atherosclerotic lesion development and prevents premature death. 

P249 

Expression of Lp-PLA 2 in Peripheral Blood Mononuclear Cells is Associated 

with Augmented Inflammatory Responses in Patients with Atherosclerosis 

Ping Zhang, LiFeng Zhang, Anthony Carabasi, Paul J DiMuzio, Chris DiMatteo, Thomas 

Jefferson Univ, Philadelphia, PA; Andrew Zalewski, Colin Macphee, GlaxoSmithKline, King of 

Prussia, PA; Yi Shi; Thomas Jefferson Univ, Philadelphia, PA 

Epidemiologic studies suggest that circulating levels of lipoprotein-associated phospholipase A 2 

(Lp-PLA 2) are an independent predictor of cardiovascular events. The purpose of the study was 

to characterize the expression and the regulation of Lp-PLA 2 in human peripheral blood 

mononuclear cells (PBMC). Plasma samples and PBMC were obtained from patients undergoing 

carotid endarterectomy (CEA, n54) with healthy blood donors used as controls (n19). 

Plasma levels of Lp-PLA 2 were assessed by the activity assay, whereas the expression of 

Lp-PLA 2 and inflammatory genes were analyzed by real-time RT-PCR. Plasma Lp-PLA 2 activity 

(331.5 nmol/min/ml) was correlated with total cholesterol (r0.55, p0.001) and LDL 

(r0.46, p0.001), but not with hsCRP (r0.1, NS). PBMC from CEA patients exhibited 

augmented expression of Lp-PLA 2 and inflammatory genes compared with control subjects 

(Table). Additionally, close associations were noted between expression of Lp-PLA 2 and ICAM-1 

(r0.6, p0.001), TNF- (r0.45, p0.001) and gp91phox (r0.73, p0.01). Contrary to 

plasma Lp-PLA 2 activity, the expression of Lp-PLA 2 in PBMC was correlated with plasma level 

of hsCRP (r0.31, p0.05), but not with total cholesterol and LDL. Since PBMC-derived 

Lp-PLA 2 may influence circulating Lp-PLA 2 levels, an independent marker of cardiovascular 

events, we examined the regulation of Lp-PLA 2 in primary PBMC using various inflammatory 

stimuli (n3/condition). The expression of Lp-PLA 2 (3.70.5, control) was significantly 

augmented by LPS (6.40.9, p0.05), IL-1 (10.21.3, p0.01), G-CSF (6.80.7, p0.01), 

and TNF- (6.90.4, p0.01). LDL and ox-LDL, however, showed no significant effects. 

Conclusions: 1. Augmented expression of inflammatory mediators in PBMC may contribute to 

a low-grade inflammation in atherosclerosis. 2. Inflammatory mediators upregulate Lp-PLA 2 

expression in PBMC, which may explain dynamic changes in circulating Lp-PLA 2 in patients at 

risk. 

Target gene/cyclophilin 

Lp-PLA2 ICAM-1 TNF- gp91phox 

Control 6.20.8 0.70.1 0.150.03 2.50.3 

CEA patients 10.31.0* 2.00.3* 0.280.02** 3.20.2 

P250 

Endothelial Nitric Oxide Synthase Efficiency Enhanced Carotid Artery 

Ligation-Induced Vessel Remodeling by Promoting Vascular Inflammation 

Lening Zhang, Univ of Carlifornia, Davis, Davis, CA; Valdeci da Cunha, Baby Martin-McNulty, 

Berlex Biosciences, Richmond, CA; Dennis Wilson, Univ of Carlifornia, Davis, Davis, CA; 

Mark E Sullivan, Ronald Vergona, Berlex Biosciences, Richmond, CA; John C Rutledge, Univ 

of Carlifornia, Davis, Davis, CA; Yi-Xin Wang; Berlex Biosciences, Richmond, CA 

Objective: An animal model of carotid artery ligation (CAL), by creating a low-shear stress and 

turbulent blood flow, which represents restenosis in humans, can induce concentric remodeling 

in the vascular wall. Inflammation plays an important role in this process. The goal of this study 

was to test the hypothesis that endothelial derived nitric oxide (NO) protects such remodeling 

by inhibiting expression of pro-inflammatory mediators and vascular inflammatory responses. 

Methods and Results: The left common carotid artery was ligated in C57BL/6J wild type (WT), 

endothelial by NO guest synthase on June deficient 29, (eNOS-KO), 2013 or WT mice treated with a NOS inhibitor,

E-96 Vol 25, No 5 May 2005 

N G -nitro-L-arginine methyl ester (L-NAME). 1 or 4 weeks later, both the ligated and 

contralateral carotid arteries were pressure perfused and fixed for morphological and 

immunohistochemistry analysis, or incubated for 24 hours for measuring pro-inflammatory 

mediators by ELISA. In WT mice, CAL induced vascular inflammation, characterized by 

neutrophil and macrophage infiltration into the vessel wall at 1 week. Although the 

inflammation diminished at 4 weeks, the ligated carotid artery developed a prominent vascular 

remodeling, manifested by SMC-rich neointimal formation, medial thickening and adventitial 

proliferation with reduced luminal diameter. CAL also increased the expression of vascular cell 

adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1). VCAM-1 

peaked at 1 week, and then slightly declined at 4 weeks after CAL, while MCP-1 continued to 

increase. In eNOS deficient mice, the above changes were significantly exacerbated. Treatment 

of L-NAME can mimic the above changes in the eNOS-KO mice. Conclusion: Absence of eNOS 

exacerbated CAL-induced vascular remodeling, which was associated with aggravated 

vascular inflammation. Increased VCAM-1 may be associated with initiation of vascular 

inflammation, while increased MCP-1 may be an important molecular mechanism of SMC-rich 

neointimal formation in the CAL model. These data indicate that vasculoprotective effects of 

eNOS are mediated, at least in part, by inhibition of vascular inflammation and SMC migration 

and proliferation. 

Vascular Expression of Receptors for Prostaglandin D2 (PGD2) 

Lei Zhao, Suk-Hwan Baek, Garret A FitzGerald; Univ of Pennsylvania, Philadelphia, PA 

P251 

Background: The emergence of a cardiovascular hazard from selective inhibitors of COX-2 has 

fostered interest in therapeutic opportunities further downstream in the arachidonic cascade. 

PGD 2, the major COX-2 product of mast cells, has been implicated in asthma via activation of 

its DP1 receptor. A second receptor, DP2 (CRTH2), is expressed on basophils and Th2 

lymphocytes and is activated by the physiological concentrations of the PGD 2 metabolite, 15 - 

deoxy 12,14 PGJ 2. The expression and function of DPs in the cardiovascular system in vivo is 

essentially unknown. Objective: In the current study, we examined the expression of DP1 and 

DP2 in the vasculature of mouse models of atherosclerosis and abdominal aortic aneurysm. 

Methods and Results: DP expression was analyzed using realtime PCR in tissues of apoE -/mice 

(8 months, chow diet). The highest DP1 mRNA expression was found in lung, followed by 

spleen, aorta, muscle, brain and ileum. DP2 is highly expressed in lung, but is relatively poorly 

expressed in all other tissues examined. mRNA expression of both DPs was confirmed in 

atherosclerotic aorta in LDL-R -/- mice (high fat, high cholesterol diet for 6 weeks). Expression 

of DP1, but not DP2, markedly increased as atherosclerosis further evolved over the next 10 

weeks on the diet in LDL-R -/- mice. Immunofluorescence studies revealed that while the DP1 

protein was barely detectable in the intima, it was strongly expressed in the aortic media of 

LDL-R -/- mice. A similar pattern of expression was noted for DP2, which was considerably less 

abundant. Expression of DP1 protein was also detected in abdominal aortic aneurysmal 

dilatations induced in apoE -/- mice (3 months, chow diet) by infusion of angiotensin II (1.0 

g/min/kg, subcutaneously, for 4 weeks). Conclusion: Abundant expression of DP1 and less 

pronounced expression of DP2 in atherosclerotic and aneurysmal aorta suggest that PGD2 and 

its metabolite , 15 - deoxy 12,14 PGJ 2, may have important cardiovascular functions in vivo and 

that DPs may prove to be attractive therapeutic targets in modulating atherogenesis, 

hypertension and the response to vascular injury. 

P252 

Endothelial Progenitor Cells Recruitment to Endothelial Cells in Response 

to Shear Stress 

Chuhong Zhu, Dajun Ying, Jianhong Mi; Key Lab for Biomechanics in Chongqing, Third 

Military Med Univ, Chonqing, China 

Endothelial progenitor cells(EPCs) contribute not only to physiological and pathological new 

vessel formation, but also to maintenance of endothelial lining. Low shear stress can increase 

monocyte adhesion to endothelial cells in the pathogenesis of atherosclerosis. Here we 

investigate whether low shear stress can mobilize endothelial progenitor cells in vitro. EPCs 

migration and adhesion toward low shear -induced endothelial cells were measured in parallel 

flow chamber. This study shows Low shear (2 dynes/cm 2 ) for 5 h can express significantly 

higher level vascular endothelial growth factor (VEGF) and IL-8 than control group. Moreover, 

Low shear can increase the VCAM-1 expression on endothelial cells, which functioned to 

promote adhesion and homing of EPCs expressing 4 integrin. Low shear can increase circulated 

EPCs adhesion to endothelial cells. The adhesion force between endothelial progenitor cells and 

low shear -induced endothelial cells was higher than control. The results demonstrate that low 

shear flow can increase endothelial cells mobilization of endothelial progenitor cells, which may 

be helpful for EPCs maintenance of endothelial lining and playing a role in angiogenesis of 


P253 

Mechanisms of Hypochlorite-Induced Vascular Endothelial Dysfunction 

Jian Xu, Zhenglin Xie, Stacy Kirkpatrick, Ming-Hui Zou; Graduate Sch of Medicine, Knoxville, 

TN 

Increasing data suggest that in human, increased myeloperoxidase (MPO) activity is a 

prominent feature of developing atherosclerotic lesions and this enzyme is known to produce 

hypoclorite (HOCl) as its principal oxidant. Recent evidence indicates that HOCl participates in 

the pathogenesis of atherosclerosis. However, the targets and mechanisms by which HOCl 

modifies endothelial function are poorly understood. In present study, we investigated if HOCl 

oxidized the endothelial nitric oxide synthase (eNOS) and induced the enzyme uncoupling. Both 

cultured human umbilical vein endothelial cells (HUVEC) and recombinant eNOS were exposed 

to clinically relevant concentrations of HOCl and the eNOS dimer/monmers, eNOS activity, and 

eNOS-derived ROS were assayed as described previously (Zou et al., J. Clin. Invest. 

109:817– 826, 2002). Our results indicate that HOCl Downloaded dose-dependently from 

released zinc from the 

zinc-thiolate cluster of eNOS and disrupted the enzyme-active eNOS dimers into monomers 

under reducing conditions. The catalytic activities of eNOS were also sensitive to HOCl. Low 

concentrations of HOCl significantly decreased NO synthesis but increased superoxide anions 

(O 2 .- ) production by the enzyme. Exposure of HUVEC to HOCl (1–100 M) significantly 

attenuated the NO release, as assayed by the formation of 3 H-citrulline. In contrast, HOCl 

significantly increased ROS release, which was abolished by L-NAME, a NOS inhibitor, 

suggesting that eNOS was uncoupled and became the souce of oxidant in HUVEC after being 

exposed to HOCl. The co-factor of eNOS, tetrahydrobiopterin (BH 4), was not oxidized by HOCl 

(160.8 26.5 pmol/mg cell protein, control vs HOCl), suggesting that HOCl-induced eNOS 

uncoupling was independent of BH 4 oxidation. Furthermore, in the aortic atherosclerotic 

plaques obtained from 16 patients with hypertension, we found that eNOS predominantly 

existed as the eNOS monomers in parallel with an increased 3-nitrotyrosine staining and 

abnormal expression of adhesion molecules (ICAM-1 and VCAM-1). We conclude that oxidants 

such as hypochlorite uncouples eNOS by disrupting the zinc-thiolate center of the enzyme and 

the eNOS uncoupling might contribute to the development of vascular diseases such as 


P254 

Parental Longevity and Structure and Function of Large Arteries in Adult 

Offspring 

Mahmoud Zureik, INSERM, Lille, France; Sébastien Czernichow, INSERM, Paris, France; 

Pierre Ducimetière, INSERM, Villejuif, France; Serge Hercberg, INSERM, Paris, France; 

Michel E Safar, Hôtel Dieu Hosp, Paris, France; Pilar Galan; INSERM, Paris, France 

Background- Longevity in a family may protect against coronary heart disease in offspring but 

the underlying mechanisms are not known. In this report, we examined the associations of 

parental longevity with carotid intima-media thickness, carotid plaques and aortic arterial 

stiffness in adult offspring. Methods- A population of 1094 volunteers free of chronic diseases 

and apparently healthy who participated to the SUVIMAX Vascular Study (mean age 59.9 years, 

48.8% women, 33.7% hypertensives) were included. Carotid-femoral pulse-wave velocity 

(PWV) was used to assess aortic stiffness. Carotid B-mode ultrasound examination included 

measurements (at sites free of plaques) of intima-media thickness (IMT) at the common carotid 

arteries (CCA) and assessment of atherosclerotic plaques in the extracranial carotid arteries. 

Paternal and maternal longevity were analyzed separately. Results - The prevalence of carotid 

plaques in subjects whose father died before 65 years, in those whose father was alive by 65 

years but died by 80 year and in those whose father was alive by 80 years was respectively 

40.2 %, 29.6 and 29.6 % (p0.001). The multivariate-odds-ratios of carotid plaques in the 

three groups of paternal longevity adjusted for conventional cardiovascular risk factors were 

respectively 1, 0.68 [95 % confidence interval: 0.46–0.99], 0.66 [0.43–0.96] (p for trend 

0.007). Mean CCA-IMT was higher in subjects with paternal premature death in univariate (p 

for trend0.001) but not in multivariate analyses (p for trend0.34). Mean PWV decreased 

with increasing paternal longevity both in univariate and multivariate analysis. The multivariateadjusted 

means of PWV in the three groups of paternal longevity were respectively 11.80.13, 

11.60.11 and 11.10.12 m/s (p for trend0.001). In contrast, neither B-mode ultrasound 

measurements nor PWV measurements were associated with maternal longevity Conclusions- 

This study suggests that paternal premature death was associated with higher frequency of 

carotid plaques and increased aortic arterial stiffness. These results may indicate that there are 

modifications of structure and function of large arteries according to paternal longevity. 

P255 

Plasma Myeloperoxidase Levels do not Predict Risk in Patients with Stable 

Coronary Artery Disease 

Guijing Lu, Lukas Kubala, Lars Berglund, Jason P Eiserich; Univ of California Davis, 

Sacramento, CA 

Coronary artery disease (CAD) is a common cause of death in the Western world and is 

associated with increased inflammation and oxidative stress. Previous studies have demonstrated 

that serum levels of myeloperoxidase (MPO), a highly abundant redox-active hemoprotein 

expressed by leukocytes, are highly predictive of future myocardial infarction or death 

following acute coronary syndromes. However, it remains to be demonstrated whether MPO 

levels reflect disease risk in humans presenting with stable CAD. Herein, plasma samples of 

573 African American and Caucasian patients undergoing coronary angiography were analyzed 

for MPO protein level and CAD risk factors. The MPO level distribution was skewed with a 

median level of 125 g/l. For all statistical analyses, log transformation was used. While weak 

but significant associations existed between plasma levels of MPO and inflammatory markers 

associated with CAD risk [CRP (r0.13, p0.002); fibrinogen (r0.08, p0.007); and 

adiponectin (r0.12, p0.011)], MPO levels did not differ in patients with or without CAD (log 

MPO 2.080.14 vs 2.090.15 in CAD vs controls, respectively). Further, no difference in MPO 

levels was found between the four gender/ethnicity groups (African American or Caucasian men 

and women). Additionally, MPO antineutrophil cytoplasmic autoantibody (ANCA) titers, presumably 

a long-term reflection of circulating MPO also did not correlate with CAD incidence. These 

results demonstrate that the utility of plasma/serum MPO levels to predict risk of vascular 

complications or disease should be restricted to those individuals suffering from acute coronary 

syndromes, since no support for its use in predicting CAD risk was found. 

P256 

Determination of Serum Apolipoprotein B by Infrared Spectroscopy- A New 

Method for the Diagnosis and Screening of Cardiovascular Disease 

Kan-Zhi Liu, Angela Man, Institute for Biodiagnostics, Winnipeg, Canada; Thomas C 

Dembinski, Health Sciences Cntr, Winnipeg, Canada; Anthony R Shaw; Institute for 

Biodiagnostics, Winnipeg, Canada 

Objective: While the conventional approach to assess both the risk of coronary artery disease 

http://atvb.ahajournals.org/ (CAD) andby theguest adequacy on of June therapy 29, is LDL 2013 cholesterol testing, there is compelling evidence to 


suggest that apolipoprotein B (apo B) is superior to LDL cholesterol for both of these purposes. 

However, the measurement of apo B requires techniques that are expensive and difficult to 

standardize. The aim of this study is, therefore, to develop a new method, infrared (IR) 

spectroscopy, for the quantification of apo B in human serum. Methods: A total of 46 serum 

samples were obtained from patients with various disorders. Small volumes (2 l) of serum 

specimens were dried to films, and their IR absorption spectra (duplicated) measured. The 

reference apo B concentrations were determined separately using standard method, and the 

proposed IR method was then calibrated using partial least squares (PLS) regression analysis 

to quantitatively correlate the IR spectra with the reference results. Results: The apo B 

concentrations predicted from the IR spectra of serum were highly correlated and in excellent 

agreement with those determined by the reference method. The correlation coefficient (r) for 

apo B was 0.97, with the standard error 0.08 g/L between IR-predicted and reference values 

(Figure 1). Conclusion: In combination with earlier work demonstrating the accurate 

determination of LDL-C, HDL-C, total cholesterol, and triglycerides from a single infrared 

spectroscopic measurement, the addition of accurate apo B determination from the same 

spectrum makes the method very attractive for laboratory use in the routine evaluation of CAD 

risk. 

P257 

Platelet-Activating Factor-Acetylhydrolase Fraction may be a Useful New 

Marker for Coronary Restenosis as Well as Vasospasm 

Keisuke Okamura, Shin-ichirou Miura, Bo Zhang, Yoshinari Uehara, Hiroaki Nishikawa, 

Kazuyuki Shirai, Kunihiro Matsuo, Keijirou Saku; Fukuoka Univ Hosp, Dept of Cardiology, 

Fukuoka, Japan 

Background: Arterial inflammation and cholesterol plaque are associated with a risk of coronary 

stenosis. Recently, highly-sensitive C reactive protein (h-CRP) has been identified as a systemic 

marker of inflammation. We sought to identify new markers for ischemic heart disease (IHD) 

patients. Methods: In 138 patients undergoing coronary angiography, we measured plasma 

regulated upon activation, normal T cell-expressed and secreted (RANTES), monocyte 

chemotactic protein-1 (MCP-1), total platelet-activating factor-acetylhydrolase (T-PAF-AH), 

high-density lipoprotein (HDL)-PAF-AH, low-density lipoprotein (LDL)-PAF-AH, lysophosphatidylcholine 

(PC) and paraoxonase (PON)-1. Results: HDL, lyso-PC, HDL-PAF-AH in the 

IHD group (441 mg/dL, 1674 ìmol/L and 773 IU/L, respectively) were lower than those 

in the control group (512, 2058 and 1119, respectively) (p0.001). There were no 

significant differences among the number of significant stenosis vessels. T-PAF-AH and 

LDL-PAF-AH in the restenosis () group (49341 and 41137) tended to be higher than 

those in the restenosis (-) group (37546 and 30541) (p0.06). In addition, 61 patients 

were given the ergonovine test to diagnose coronary vasospasm. Triglyceride and lyso-PC in 

the ergonovine-positive group (14020 mg/dL and 22414 ìmol/L) were higher than those in 

the ergonovine-negative group (1036 mg/dL, 19310 ìmol/L) (p0.05 and p0.07). The 

ratio of LDL-PAF-AH/HDL-PAF-AH in the ergonovine-positive group was significantly higher 

than that in the negative group (4.7 vs. 3.6, p0.05). There were no significant differences in 

total cholesterol, LDL, the LDL/HDL ratio, h-CRP, MCP-1, RANTES or PON-1 between the control 

and IHD groups. Conclusion: Lyso-PC, T-PAF-AH and PAF-AH fraction might be useful as 

markers of coronary risk and coronary restenosis. Coronary vasospasm may also be associated 

with arterial inflammation and atherosclerotic change. 

P258 

Platelet-Activating Factor-Acetylhydrolase may be a Therapeutic Target for 

Preventing Atrial Fibrillation 

Keisuke Okamura, Shin-ichirou Miura, Yoshinari Uehara, Kunihiro Matsuo, Bo Zhang, 

Kouichirou Kumagai, Keijirou Saku; Fukuoka Univ Hosp, Dept of Cardiology, Fukuoka, Japan 

Background: Since higher levels of serum highly-sensitive C reactive protein (h-CRP) have been 

found in patients with atrial fibrillation (AF), we compared biochemical markers of inflammation 

between patients with AF and those with normal sinus rhythm (NSR). Methods: We investigated 

175 patients and classified them into an AF group (82 patients; 63 were paroxysmal (P) AF 

patients and 19 were chronic (C) AF patients) and an NSR group (93 patients). We analyzed 

plasma h-CRP, regulated upon activation, normal T cell-expressed and secreted (RANTES), 

monocyte chemotactic protein-1 (MCP-1), total Downloaded platelet-activating from 

factor-acetylhydrolase 



(T-PAF-AH), high-density lipoprotein (HDL)-PAF-AH, low-density lipoprotein (LDL)-PAF-AH, 

lyso-phosphatidylcholine (PC) and paraoxonase (PON). Results: LDL, T-PAF-AH and LDL- 

PAF-AH in the AF group (1193 mg/dl, 49521 IU/L and 40819 IU/L, respectively) were all 

significantly higher than those in the NSR group (1093, 42918 and 32817, respectively) 

(p0.05). The ratio of LDL-PAF-AH/HDL-PAF-AH was 4.9 in AF patients, and was significantly 

higher than that in NSR patients (3.9) (P0.005). RANTES in the AF group (5.50.8 ng/ml) was 

also significantly lower than that in the NSR group (10.71.5) (p0.005). MCP-1 in the CAF 

group (22422 pg/ml) was significantly higher than that in the PAF group (1829) (p0.05). 

There was a significant decrease in lyso-PC according to the AF burden (NSR: 2179 mmol/L, 

PAF: 1986 and CAF: 17710). These differences were not related to medication except for 

warfarin. Interestingly, RANTES in the warfarization () group (3.70.6 ng/ml) was significantly 

lower than that in the warfarization (-) group (6.90.8) (p0.05) among AF patients, 

while there was no difference in the RANTES level between CAF and PAF. Conclusion: AF is 

related to T-PAF-AH and the ratio of LDL-PAF-AH/HDL-PAF-AH, and may be a therapeutic target 

for preventing AF. Warfarization might have an anti-inflammatory effect associated with a 

reduction of plasma RANTES. 

Colesevelam is Safe and Effective in Patients with Statin Myotoxicity 

Paul S Phillips, Nancy L Gray, Frederick G McDonald, Margaret Blaszcak, Scripps Mercy, 

San Diego, CA; Tanya Wolfson, Univ California, San Diego, San Diego, CA; Michael J 

Sullivan; Scripps Mercy, San Diego, CA 

P259 

Background: Myotoxicity is an adverse effect that can limit the use of lipid lowering therapy. 

Evidence suggests myotoxicity is mediated by defects in fatty acid oxidation. It has been 

described with statins, fibrates, niacin and ezetimibe which lower triglycerides (TG). Limited 

information is available on the safety of bile acid sequestrants, which have neutral effects or 

raise TG, in patients with intolerance to lipid lowering therapies (ILLT). Methods: Seventeen 

patients with a history of ILLT and a fasting respiratory exchange ratio (RER) of 0.80 received 

6 weeks of colesevelam (3.75g/d). Lipids, creatine kinase (CK), grip strength, RER, anaerobic 

threshold (AT), maximal oxygen consumption (Vmax) and subjective status (SF36) were 

assessed at baseline, and following 6 weeks of therapy. Results: All patients had a history of 

statin myotoxicity and 9/11 (82%) also had myotoxicity when treated with ezetimibe. After 6 

weeks of colesevelam, only 2 patients experienced muscle complaints. Colesevelam was well 

tolerated, 15/17 (88%) had no muscle complaints or were improved. Mean LDL-C was reduced 

by 16% (P0.0009) and TG increased by 13% (PNS). There was no difference in QOL scores 

on therapy. RER was not increased on colesevelam suggesting no impairment in fat oxidation. 

There was no evidence of myotoxicity on colesevelam as assessed by functional testing or CK 

(Table). Conclusions: In this high risk group with ILLT, colesevelam was safe, causing no 

measurable increase in muscle complaints or decrease in functional capacity. Colesevelam was 

effective in reducing mean LDL-C by 16%. 

Grip 

(kg) RER 

Vmax 

(ml/kg/ 

min) 


AT 

(ml/kg/ 

min) 

CK 

(mg%) 

LDL 

(mg%) 

TG 

(mg %) 

OFF 40.810 0.85.06 239 143 283198 17645 16174 

ON 40.810 0.82.05 227 133 266272 14942 18297 

P-value NS NS NS NS NS p0.0009 NS 

P260 

Abnormal Fasting Respiratory Exchange Ratios Normalize over Time in 

Patients with Statin-Induced Rhabdomyolysis or Myositis 

Keith A Somma, Lyn M Puhek, Nancy L Gray, Frederich G McDonald, Scripps Mercy Hosp, 

San Diego, CA; Tanya Wolfson, Univ of California, San Diego, San Diego, CA; Paul S 

Phillips; Scripps Mercy Hosp, San Diego, CA 

Background: Muscle toxicity is a known complication of statin therapy. Recent research 

suggests a defect in fatty acid oxidation (FAO) in patients with statin-induced rhabdomyolysis 

and myositis (MYO). Exhaled gas analysis with fasting respiratory exchange ratio (RER), is a 

valuable tool in assessing substrate utilization including FAO. MYO, have been shown to have 

an elevated RER over the normal value of 0.75. It is unknown if statins cause the defect in FAO, 

or unmask an intrinsic abnormality in affected patients. In the absence of such a predisposing 

condition, we expect the RER to normalize over time from myotoxic event (T0). Methods: RER 

was measured in MYO by standard techniques at varying time intervals from T0. Data was 

plotted as RER vs. time from T0 and Spearman’s correlation determined. Results: RERs for 

32 MYO are shown in the graph. RER decreases towards normal with increasing time from 

T0. The p-value for this correlation is 0.058. Ten patients have two or more sequential tests. 

8 of these 10 patients trend toward normal with increasing time from T0. Three of those are 

displayed by best fit line. Conclusion: It is unknown if abnormal FAO reflected by an increased 

RER in patients with MYO preceds, or is a direct consequence of statin therapy. The trend 

toward normalization of RER with time, along with the clear trend toward normalization in 8 out 

of the 10 MYO with seqential testing, would suggest normal pre-statin FAO in MYO. Thus, the 

derangement in FAO in MYO may be solely related to statin therapy.

E-98 Vol 25, No 5 May 2005 

P261 

Lipoprotein Size, Cardiovascular Risks and Metabolic Syndrome in Children 

with Severe Obesity 

Daniel L Preud’Homme, Adrienne Stolfi; Wright State Univ, Dayton, OH 

Background: Obesity predisposes to early cardiovascular risk (CVR), especially if metabolic 

syndrome (MS) is present. Children with severe obesity may have abnormal levels of 

lipoprotein, which contributes to early CVR. Lipoprotein particle size and subclass also play a 

significant role in the assessment of early CVR. Having at least two of the following abnormal 

size particles- small LDL, low numbers of large high-density lipoprotein (HDL), and increased 

very low-density lipoprotein (VLDL) - is also associated with MS and increased CVR. Objective: 

To evaluate lipoprotein particles in severly obese children, and identify gender or race 

differences for high risk size/levels of lipoprotein particles. Methods: Lipoprotein particle size 

and subclass evaluations were performed on 160 severely obese children (BMI z-score 2.00) 

referred to the pediatric lipid clinic. Lipoprotein size was determined by nuclear magnetic 

resonance (NMR). Lipoprotein values were classified as high risk according to established 

criteria. Proportions with high risk values were compared for males vs. females and 

whites/others vs. blacks with chi-square tests. Mean lipoprotein values, age, and BMI were 

compared between groups with two sample t tests. Results: Of 160 children, 53% were male, 

and 70% were white/other. The mean (SD) age was 12.6 (3.4) years, and BMI was 36.0 (8.2) 

kg/m 2 . There were no differences between males and females for age, BMI, or lipoprotein 

variables. Whites/others did not differ from blacks in age or BMI, but differed significantly in 

most lipoprotein variables (table), with whites/others having higher risks. Conclusion: Severely 

obese children express abnormal lipoprotein particle numbers and subclasses. These represent 

increased modifiable CVR. Although no gender differences are identified, race differences are 

significant. Performing lipoprotein evaluation through NMR may identify the proportion of 

children at greater risk of early CVR and/or MS. 

Lipoprotein 

Variable Measure 

All Patients 

(n160) 

White/Other 

(n112) 

Black 

(n48) 

P 

Value 

LDL particle no. 

(nmol/L) 

Mean (SD) 1251 (316) 1267 (322) 1212 (301) 0.313 

LDL particle no. 

1300 nmol/L 

No. (%) 105 (66) 69 (62) 36 (75) 0.102 

LDL size (nm) Mean (SD) 20.7 (0.7) 20.6 (0.7) 20.9 (0.6) 0.013 

LDL pattern B No. (%) 59 (37) 50 (45) 9 (19) 0.002 

Large HDL 

(mg/dL) 

Mean (SD) 13.2 (7.4) 12.5 (7.4) 15.0 (7.2) 0.044 

Large HDL 11 

mg/dL 

No. (%) 66 (41) 52 (46) 14 (29) 0.042 

Large VLDL 

(mg/dL) 

Mean (SD) 52.2 (61.7) 61.4 (63.6) 30.7 (51.2) 0.002 

Large VLDL 27 

mg/dL 

No. (%) 84 (53) 69 (62) 15 (31) 0.000 

2 MS traits No. (%) 69 (43) 59 (53) 10 (21) 0.000 

P262 

Lipid-Lowering Therapy has Beneficial Effect on Large Artery Stiffness in 

Combined Hyperlipidaemia 

Jan Simek, Dan Wichterle, General Univesity Hosp, Prague, Czech Republic; Vojtech 

Melenovsky, Johns Hopkins Med Insts, Baltimore, MD; Jan Malik; General Univesity Hosp, 

Prague, Czech Republic 

Purpose: While the ability of statins to reduce arterial stiffness is well documented, data on the 

effect of fibrates are missing. Therefore, we conducted a study to compare the effects of 

atorvastatine and fenofibrate on large artery stiffness in patients with combined hyperlipidaemia. 

Methods: 29 otherwise healthy males (aged 47.4 7.8 years) with combined 

hyperlipidaemia (total cholesterol 7.55 1.20 mmol/l, triglycerides 5.41 4.54 mmol/l) were 

included into the randomized, single-blind, cross-over study to receive either 200 mg of 

micronised fenofibrate or 10mg of atorvastatin daily for a period of 10 weeks. Finger arterial 

pressure (FAP) waveforms (Finapres) were recorded at baseline, at cross-over, and at the end 

of the study. Recently introduced index of large artery stiffness (SI) was calculated as a ratio 

of subject height and time delay (dT) between the systolic and diastolic peaks of the FAP 

waveform (see the figure). Paired t-test was used for evaluation of effects of both drugs. 

Results: Both fenofibrate and atorvastatin treatment significantly reduced total cholesterol and 

triglycerides (not shown). Baseline SI of 5.82 0.45 m/s was reduced to 5.61 0.43 m/s 

(p 0.018) and 5.72 0.43 m/s (p 0.062) after fenofibrate and atorvastatine, respectively. 

The difference in the effects of both drugs on SI was not significant (p 0.135). Conclusions: 

Large artery stiffness was improved to a similar extent after both drugs. This finding is in 

agreement with other studies documenting beneficial effect of lipid-lowering therapy on arterial 

wall mechanical properties. 

P263 

Short and Long-Term Effects of Low-Density Lipoprotein Apheresis on the 

Oxidative Stress and Inflammatory Makers 

suppression or regression of coronary atherosclerosis. To investigate its mechanisms, we 

attempted to evaluate markers of oxidative stress and inflammation. [Methods and Results] 

Patients with hetero.FH and CAD, have undergone LDL apheresis using a dextran sulfate 

cellulose column once every 2 or 3 weeks, were enrolled into a short-term study ( 8 patients, 

5 male, 58 7 y.o.) and/or a long-term study (12 patients, 6 male, 56 12 y. o.). In the 

short-term study, blood and urine samples were measured before and after apheresis at 4 

times per patient. After a single apheresis treatment, the ratios of vitamin E to apolipoprotein 

B and LDL-cholesterol significantly increased by 55.3% and 36.4%, respectively (p0.0001). 

Plasma high sensitive C-reactive protein (hs-CRP) and ox-LDL significantly decreased by 77.2% 

and 66.0 %, but urine 8-isoprostane significantly increased by 28.6% (p0.0001). Plasma 

extracellular superoxide dismutase (EC-SOD) indicated no significant change. In the long-term 

study, hs-CRP and EC-SOD before apheresis were compared at every 3months for 63.1 23.6 

months. Hs-CRP significantly decreased in accordance with time (r-0.553, p0.0001) and 

EC-SOD kept staying at similar levels even after several years. [Conclusions] These findings 

indicate that inflammation has been decreased by single or repeated LDL apheresis and it 

might be related with changes of oxidative stress, such as reduction of ox-LDL and increase 

of vitamin E content. 

The CTP: Phosphocholine Cytidylyltransferase Gene Contains Multiple 

Silencer Elements within Intron 1 

Jiming Zhou, Alan Ryan, Rama Mallampalli; Univ of Iowa, Iowa City, IA 

P264 

Phosphatidylcholine (PC) is the major phospholipid of alveolar surfactant. PC synthesis is 

controlled by a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCT). There is 

limited information regarding transcriptional regulation of the CCT gene. We previously 

described a core promoter (-169/71) for CCT which exhibits robust activity, harbored a 

putative sterol-regulated enhancer (SRE) and an INF-activated site (GSA). In this study, we 

identified multiple silencer elements within intron 1 of the CCT gene between nucletide 

1075 and 1815, which abolished the promoter activities of both CMV and CCT core 

promoters in pGL3-based constructions. Further studies revealed that intron 1: i) harbored at 

least three independent silencer elements, ii) in combination, these silencers were both 

orientation and position dependent, showing significantly greater silencing effect in a reverse 

orientation, iii) one silencer was localized to a 20 bp region (1404/1423), with no 

identifiable binding partner, iv) a second silencer was localized to 44 bp region (1261/ 

1304), with binding sites for three consensus repressors, Barbie Box, Delta EF1, and OCT1. 

Progressive DNA deletional and site-directed mutagenesis assays however revealed that none 

of these sites were related to its repression of CMV promoter activity. These findings invite 

further investigation to identify novel repressors that might regulate expression of this key 

lipogenic gene. 

P265 

Effects of 8 Week Lifestyle Modification on Cardiovascular Structure and 

Function in Those at Risk for Cardiovascular Diseases 

Kunihiko Aizawa, Mauricio Marin, Isidro Torres Castro, Michele A Lawrence, Jeniffer A 

Manley, Leonard A Piche, Kevin J Shoemaker, Robert J Petrella; The Univ of Western 

Ontario, London, Canada 

The independent and combined effects of exercise and diet on blood pressure (BP) have been 

well-documented; however, their combined effects on overall cardiovascular structure and 

functioning in those who have preclinical risk factors for cardiovascular diseases are still 

unclear. The purpose of this study was to evaluate the effect of lifestyle (exercise and diet) 

modification on measures of left ventricular diastolic filling (LVDF), left ventricular mass (LVM), 

intima-media thickness (IMT), arterial distensibility (AD), clinic BP, and exercise capacity 

(VO 2max) in individuals with either high-normal BP (HNBP), impaired fasting glucose level (IFG), 

or impaired glucose tolerance (IGT). These preliminary findings are from a larger randomized 

trial of Staged Nutrition and Activity Counseling (SNAC) whose plan includes an individually 

customized Mediterranean diet with physical exercise vs usual care lifestyle counseling. Thirty 

eight subjects with either HNBP, IFG, or IGT (16 M and 22 F, 53.3 7.9 yrs) have participated 

in the preliminary analysis of the study cohort. Before and after 8 weeks of SNAC, measures 

of LVDF, LVM, IMT and AD in carotid (CA) and brachial (BA) arteries assessed using an 

ultrasound machine at rest, and VO 2max by treadmill test were determined. Clinic BP was also 

recorded. AD in CA improved significantly following SNAC (0.94 0.36 vs 1.09 0.33 

1/mmHgx10 -1 ,p.01). This improvement was accompanied by the reduction of body weight 

(BW) and improvement of VO 2max (92.5 18.0 vs 90.9 17.7 kg, 32.1 9.5 vs 35.3 10.1 

ml/min/kg, p.01, respectively). Clinic DBP tended to be lower after 8 weeks than baseline 

(84.3 11.2 vs 81.3 9.0 mmHg, p.11). No changes were observed in LVDF, LVM, IMT, 

AD in BA, and clinic SBP. These preliminary data suggest that our lifestyle modification 

significantly improved AD in CA with the reduction of BW and improvement of VO 2max after 8 

weeks. 

P266 

Effect of Chromium Picolinate and Biotin Combination on Coronary Risk 

Lipids and Lipoproteins in Subjects with non HDL -C (>130 mg/dL) in Type 

2 Diabetes Mellitus 

Cesar Albarracin, Alpha Therapy Cntr, Corpus Christi, TX; Burcham Fuqua, Mature Med 

Care, Corpus Christi, TX; Jeff Geohas, Radiant Rsch, Chicago, IL; Manley Finch, James R 

Komorowski; Nutrition 21, Purchase, NY 

Hiromi Tasaki, Kiyoshi Ozumi, Shun-ichi Nihei, Noriko Hirakawa, Kazuhiko Tamura, Kazuhito 

Yamashita, Yasuhide Nakashima; Univ. Occup. Environ. Health, Kitakyushu, Japan 

Cardiovascular disease (CVD) is the primary cause of mortality in type 2 DM (T2DM). Recent 

data suggests that non-HDL cholesterol (non HDL-C) might be a better predictive risk factor of 

[Background] Low-density lipoprotein (LDL) apheresis has been applied to patients with familial CVD than LDL-C. The Adult Treatment Panel (ATP-III) recommended using non-HDL-C in 

hypercholesterolemia (FH) with coronary arteryDownloaded disease (CAD) and from 

some http://atvb.ahajournals.org/ 

studies showed assessingby CVDguest risk inon patients June with 29, T2DM. 2013Recent 

studies suggest that chromium picolinate 


(CrPic) alone and in combination with biotin can reduce CVD risk factors. This 90-day, 

randomized, double blinded, placebo controlled multicenter study was conducted to evaluate 

the effects of the combination of CrPic and biotin on coronary risk lipids (total cholesterol (TC) 

and triglycerides (TG) and lipoproteins (LDL-C, HDL-C, non HDL-C, VLDL-C) in patients with 

T2DM. Subjects were randomized in a 2:1 ratio to active (600 mcg Cr as CrPic/biotin 2 mg/per 

day) or placebo for 12 weeks. Change in HbA1c (p0.01), total cholesterol (p0.02) and 

TG/HDL (p0.0001) were improved in actives compared to placebo. An analysis was 

conducted on a subset of the intent-to-treat (ITT; N368) population having a baseline non 

HDL-C 130 mg/dL (N234; Actives: 149, Placebo: 85). ITT was defined as any subject who 

took at least one dose and had one post baseline measurement. After 12 weeks, subjects in 

the active group had significant reduction in TG (225 vs. 278 mg/dL; p 0.05), TG/HDL-C (5.4 

vs. 6.6; p 0.02), TC/HDL-C (4.5 vs. 5.0; p 0.02), VLDL-C (37 vs. 49 mg/dL; p 0.0035), 

LDL-C/TG (0.79 vs. 0.66; p 0.04); change in TC/HDL-C ( -0.3 vs.-0.03; p 0.034), 

LDL-C/HDL-C ( -0.35 vs. -0.1; p 0.03), non HDL-C ( -14.0 vs. -4.0; p 0.05) and non 

adjusted HDL-C ( -14.0 vs. -4.0; p 0.05) levels compared to placebo. Change in Atherogenic 

Index (AI [TC-HDL] / HDL) was significantly reduced in actives compared to placebo ( -0.3 

vs. -0.03; p0.04). This study indicates that CrPic and biotin combination may significantly 

improve coronary risk lipid profiles in subjects with non HDL-C greater than 130 mg/dl. 

Carotid Intima-Media Thickness and Brachial Artery Flow Mediated 

Dilatation in Patients with and without Metabolic Syndrome 

Manish Bansal, Sweta Agrawal, Ravi R Kasliwal; Escorts Heart Institute & Rsch Cntr, New 

Delhi, India 

P267 

Metabolic syndrome (MS) is a constellation of cardiovascular (CV) risk factors (RFs) that is being 

increasingly recognized as a marker for increased risk of future CV events. However, the risk 

of CV events in patients with MS and its significance as compared to individual RFs is not 

exactly known. This is especially true for diabetes mellitus (DM), which has already been 

recognized as ‘CAD risk equivalent’. The purpose of the present study was to assess extent of 

subclinical atherosclerosis as determined by carotid intima-media thickness (CIMT) and 

brachial artery flow-mediated vasodilatation (BaFMD) in patients with MS and compare it with 

the same in individuals with CV RFs not having MS. Methods: 167 individuals visiting cardiac 

outpatient department for various indications were included in the study and were divided into 

4 groups- group 1: CV RFs present but not MS; group 2: MS without DM or coronary artery 

disease (CAD); group 3: MS with DM but without CAD; group 4: patients with documented CAD. 

MS was diagnosed on the basis of ATP III criteria. All patients underwent assessment of CIMT 

and BaFMD. For assessment of CIMT, common carotid IMT was measured on both sides and 

averaged. Results: Mean values of CIMT & BaFMD in the four groups and comparison between 

groups is presented in table 1. Both CIMT & BaFMD were similar in the groups 1&2but 

patients in group 3 had significantly higher CIMT & significantly lower BaFMD as compared to 

patients in group 1. There was no significant difference in the two parameters between groups 

3&4.Conclusions: Patients with MS had similar extent of subclinical atherosclerosis as the 

patients with individual CV RFs not having MS. However patients with MS with DM had 

significant endothelial dysfunction and evidence of subclinical atherosclerosis as compared to 

patients without MS. Absence of significant difference in CIMT & BaFMD between patients with 

MS with DM and patients with established CAD once again confirms the ‘CAD risk equivalence’ 

of DM. 

Grp 1 Grp 2 Grp 3 Grp 4 

n 71 21 27 48 

CIMT (mm) 0.698 0.706 0.774 0.789 

0.13 0.23 0.15 0.16 

BaFMD (%) 11.82 12.87 7.37 5.86 

5.83 7.04 6.12 3.85 

p-value 

(one way 

ANOVA) 

Group 

1vs2 

p value for comparison 

between groups 

Group 

1vs3 

Group 

2vs3 

Group 

3vs4 

0.008 0.828 0.014 0.223 0.681 

0.001 0.521 0.003 0.006 0.249 

Identification of Procyanidins as the Main Regulator of Endothelial 

Function in Red Wine 

Roger CORDER, William Harvey Rsch Institute, London, United Kingdom; William Mullen, 

Univ of Glasgow, Glasgow, United Kingdom; Noorafza Q Khan, Elizabeth G Wood, Martin J 

Carrier, William Harvey Rsch Institute, London, United Kingdom; Alan Crozier; Univ of 

Glasgow, Glasgow, United Kingdom 

P268 

Endothelial dysfunction is a precursor to atherosclerotic lesion formation; hence dietary agents 

that improve endothelial function are likely to contribute to vascular wellbeing. Regular 

consumption of red wine has been linked to a reduced incidence of coronary heart disease. 

Potent effects of wine polyphenols have been described in studies of endothelial cells, but the 

identity of the polyphenols involved are unclear. The goal of these studies was to identify the 

red wine polyphenols with greatest biological activity by using endothelial cells as a bioassay 

to monitor purification. Size-exclusion chromatography (Fractogel TSK HW-40), hydrophobic 

interaction chromatography (Sephadex LH-20), and reverse phase HPLC were used to purify 

polyphenols from an extract (2.5 g 2 L) of cabernet sauvignon wine. Fractions were screened 

for their ability to inhibit ET-1 release from cultured bovine aortic endothelial cells (BAEC); those 

causing the greatest inhibition were subjected to further purification. Purified samples were 

analyzed with a Thermo Finnigan LCQ DecaXP ion trap mass spectrometer. This identified all 

purified polyphenols as straight chain B-type procyanidins composed of three to five catechin 

units some with gallate groups attached. To confirm the contribution of procyanidins to the 

inhibition of ET-1 synthesis by red wine, representative wines were separated into low 

molecular weight polyphenols and oligomeric procyanidins using Sephadex LH-20. The 

procyanidin fraction was 5 fold more potent as an inhibitor of ET-1 synthesis. Red wines (n 

127) from several countries were analyzed by bioassay on BAEC to determine the concentration 

inhibiting ET-1 by 50%, and fractionated on Sephadex LH-20 to quantify chemically the 

procyanidin content. Biological activity (median IC50: Downloaded 1.86 ml/L; 10th, from 

90th percentiles: 0.86, 




4.97) and procyanidin content were closely correlated (R 0.85, P 0.001). In conclusion 

these laboratory investigations have identified oligomeric procyanidins as the main vasoactive 

component in red wine. These analyses show the potential of red wines to regulate ET-1 

synthesis varies markedly. Based on these results not all red wines should be considered 

equivalent in terms of their likely health benefit. 

P269 

Diabetes Induces Endothelial Dysfunction but does not Increase Neointimal 

Formation in High-Fat Diet Fed C57BL/6J Mice 

Judit Molnar, Mount Sinai Med Cntr, New York, NY; Shuiqing Yu, Nino Mzhavia, Clara Pau, 

Columbia Univ Med Cntr, New York, NY; Igor Chereshnev, Mount Sinai Med Cntr, New York, 

NY; Hayes M Dansky; Columbia Univ Med Cntr, New York, NY 

Studies of diabetic vascular disease have traditionally utilized murine models of islet cell 

destruction/ type 1 diabetes and genetic models of type 2 diabetes. Since the majority of 

patients with type 2 diabetes have diet induced obesity, we sought to study the effect of 

diabetes on arterial disease in a mouse model of diet induced obesity/diabetes. C57Bl/6 mice 

fed a high fat diet for 9 weeks developed type 2 diabetes characterized by elevated body 

weight, hyperglycemia, and hyperinsulinemia. Arteries from diabetic mice exhibited a marked 

decrease in endothelium-dependent vasodilation, a modest decrease in endothelium independent 

vasodilation, and an increase in sensitivity to adrenergic vasoconstricting agents. Insulin 

stimulated protein kinase B (akt) and endothelial nitric oxide synthase (eNOS) phosphorylation 

were preserved in the arteries of diabetic mice, but eNOS protein dimers were undetectable. 

The abnormal vasomotion was not an acute response to the high fat diet as short term (1 week) 

high fat diet feeding had no effect on endothelium dependent dilation. Bilateral femoral artery 

wire denudation injury of chow and high fat diet fed mice revealed a trend toward smaller 

neointimal lesions in high fat diet fed mice. In summary, disrupted eNOS dimer formation rather 

than impaired insulin mediated eNOS phosphorylation contributed to the endothelial dysfunction 

in diet induced obese/diabetic mice. The lack of an increase in neointimal formation 

indicates that additional diabetes associated parameters (such as hyperlipidemia and 

atherosclerotic vascular disease) may need to be present to increase neointimal formation in 

this model. 

P270 

Tetrahydrobiopterin Supplementation Augments Endothelium-Dependent 

Dilation in Sedentary but not Habitually Exercising Older Adults 

Iratxe Eskurza, Laura A Myerburg, Zachary D Kahn, Douglas R Seals; Univ of Colorado at 

Boulder, Boulder, CO 

Age is a major risk factor for the development of atherosclerotic diseases. Endotheliumdependent 

dilation (EDD) is impaired with aging in sedentary, but not regularly exercising 

adults. We recently demonstrated that oxidative stress mediates the impairment of brachial 

artery flow-mediated dilation (FMD) with sedentary human aging, and that the absence of 

oxidative stress explains the preservation of FMD with physically active aging. Oxidative stress 

reduces the bioavailability of tetrahydrobiopterin (BH 4), the essential cofactor for endothelial 

nitric oxide synthase-mediated NO synthesis. To test the hypothesis that differences in BH 4 

bioavailability is a key mechanism explaining the impairment in EDD with sedentary aging and 

the maintenance of EDD with aging in regularly exercising adults, we conducted a randomized 

double-blind crossover study in which brachial artery FMD was measured after acute oral 

placebo or BH 4 in young sedentary (YS) (n10; 221 years, meanSE) and older sedentary 

(OS) (n9; 622) and older aerobically exercising (OE) (n10; 651) healthy men. Results. 

At baseline, FMD (corrected for the hyperemic stimulus) was 60% lower in OS vs. YS 

(0.0540.009 vs. 0.1330.011 [mm/ (ml/min)]*10 -2 ,p0.01), but was preserved in OT 

(0.1260.013). BH 4 administration improved FMD by 45% in OS (to 0.0980.012, p0.001 

vs. baseline), but did not affect FMD in YS or OT. After BH 4 administration FMD was no longer 

significantly different among the 3 groups (p0.05). The improvement in FMD with BH 4 was 

positively related to measures of total and abdominal adiposity (r0.43–0.49, p0.05) and 

inversely related to maximal aerobic exercise capacity, measured by maximal oxygen 

consumption (r-0.64, p0.001). Endothelium-independent dilation neither differed between 

groups at baseline nor changed with BH 4 administration. Conclusions. These results 

demonstrate for the first time in humans that BH 4 bioavailability is a key mechanism in the 

impairment of conduit artery EDD with sedentary aging and the EDD-preserving effect of 

habitual exercise. Differences in total and abdominal adiposity may contribute to the differences 

in BH 4 bioavailability and EDD in sedentary and physically active older adults, perhaps by 

modulating oxidative stress. 

P271 

Effect of Exercise Induced Oxidative Stress and Vitamin E on Levels of LDL 

and HDL 

Mahdi O Garelnabi, Emir Veledar, Jerome Abramson, Drex Earle, Brandon Petro, Grant 

Anderson, Jill White-Welkley, William Weintraub, Emory Univ Sch of Medicine, Atlanta, GA; 

Sampath Parthasarathy; Louisiana State Univ Health Science Cntr, New Orleans, LA 

Background: Beneficial physiologic changes as a result of exercise have been observed in both 

men and women. Exercise results in favorable alteration in serum lipids. Objective: The study 

aim to investigate the effect of vitamin E supplementation and exercise induced oxidative stress 

on levels of LDL and HDL Methods: We measured plasma LDL and HDL, along with oxidized 

LDL (OX-LDL), Myeloperoxidase (MPO), sVCAM, MCP-1, CRP, and Autoantibodies to oxidized 

LDL (AALDL) as oxidative stress/inflammatory markers in 453 healthy men and women before 

and after two months of regular aerobic exercise in a randomized double blind clinical trial. 

Subjects were randomized on Vitamin E (VE), and Placebos (Pl). The base line parameters were 

assessed before they started to exercise and take the supplementation. Participants VO2 and 

duration time spent in treadmill before and after exercise were also measured. Spearman’s 

rank correlation by guest was on calculated June 29, to assess 2013the 

association between the LDL , and HDL and

E-100 Vol 25, No 5 May 2005 

oxidative stress/inflammatory markers and VO2. Linear regression models were run to examine 

the relationship between hormones levels and oxidative stress/inflammatory markers and VO2 

after adjustment for other factors. Results: The following table summarizes the levels of 

lipoproteins, oxidative stress and inflammatory markers in subjects before (1) and after two 

month (4) of exercise. The Spearman correlation coefficients of the lipoproteins verses the 

remaining measurements are also shown on the table. Conclusion: These finding indicates 

that levels of LDL and HDL are associated with oxidative stress/inflammatory markers; the 

association depends on the level of fitness and the status and type of oxidative stress/ 

inflammatory marker. Interestingly, Vitamin E failed to demonstrate a considerable role on 

reducing the level of oxidative stress and inflammation associated with exercise and improving 

lipoproteins status. 

Variable N Mean 

Pl LDL1(mg/dl) 

LDL4 VE 

LDL1(mg/dl) LDL4 

Pl HDL1 (mg/dl) 

HDL4 VE HDL1 

(mg/dl) HDL4 

Pl TC1 (mg/dl) TC4 

VE TC1 (mg/dl) 

TC4 

VO2–1(ml/kg/min) 

VO2–4 VE 

VO2–1(ml/kg/min) 

VO2–4 

DUT-1 Sec DUT-4 

VE DUT-1 Sec 

DUT-4 

Pl Isopro1(pg/ml) 

Isopro4 VE 

Isopro1(pg/ml) 

Isopro4 

Pl OXLDL-1(U/L) 

OXLDL-4 VE 

OXLDL-1(U/L) 

OXLDL-4 

Pl MPO1(ng/ml) 

MPO4 VE 

MPO1(ng/ml) 

MPO4 

Pl sVCAM 1(ng/ml) 

sVCAM-4 VE 

sVCAM1(ng/ml) 

sVCAM-4 

Pl MCP 

1–1m1(pg/ml) 

MCP-1–4 VE 

MCP-1 1(pg/ml) 

MCP-1–4 

Pl CRP1(ng/ml) 

CRP-4 VE 

CRP1(ng/ml) CRP-4 

Pl AALDL-1 (OD) 

AALDL-4 VE 

AALDL-1 (OD) 

AALDL-4 

222169 

225164 

225170 

227167 

226170 

227167 

227140 

225142 

224136 

225141 

217162 

220157 

216161 

216157 

221155 

219155 

214157 

217154 

207157 

204150 

203154 

203149 

209150 

208152 

112.55 

114.36 

108.76 

110.35 

53.34 

54.18 

54.48 

56.19 

185.78 

189.66 

183.91 

187.10 

30.75 

34.55 

29.54 

34.10 

468.67 

509.53 

458.21 

508.20 

15.16 

15.39 

15.63 

15.90 

26.23 

23.60 

23.28 

22.37 

3.43 

3.48 

3.72 

3.74 

426.83 

444.19 

422.84 

438.22 

118.80 

129.91 

145.03 

101.02 

4325 

3670 

4491 

3817 

0.26 

0.29 

0.28 

0.30 

R (Vs 

LDL) R(Vs HDL) 

P Value (Vs 

LDL) 

P Value (Vs 

HDL) 

– – – – 

– – – – 

– – – – 

0.203 

0.297 

0.215 

0.311 

0.001 

0.002 

0.021 

0.003 

0.534 

0.176 

0.672 

0.121 

0.124 

0.001 

0.001 

0.001 

0.001 

-0.945 

0.388 

0.962 

0.152 

0.993 

0.734 

0.291 

0.162 

0.189 

0.784 

0.138 

0.078 

0.679 

0.532 

0.268 

0.181 

0.811 

0.114 

0.329 

0.602 

0.203 0.207 

0.241 0.310 

0.025 0.032 

0.041 0.065 

0.128 0.017 

0.077 0.009 

0.914 0.805 

0.027 0.175 

0.721 0.998 

0.198 0.733 

0.001 0.001 

0.001 0.001 

0.342 0.154 

0.328 0.444 

0.532 0.217 

0.003 0.171 

0.896 0.067 

0.757 0.385 

-0.08(NS) 

-0.08(NS) 

-0.07(NS) 

-0.06(NS) 

0.125(NS) 

0.213(NS) 

0.136(NS) 

0.221(NS) 

0.042 

0.107(NS) 

0.028 

0.124(NS) 

0.319(NS) 

0.422(NS) 

0.339(NS) 

0.326(NS) 

0.004 

0.069(NS) 

-0.003 

0.116(NS) 

-0.0005 

-0.027 

0.072(NS) 

0.114(NS) 

0.092(NS) 

0.022 

0.104(NS) 

0.145(NS) 

0.029 0.050 

0.078(NS) 

0.110(NS) 

-0.016 

-0.129(NS) 

0.068(NS) 

0.043 

-0.09(NS) 

-0.08(NS) 

-0.07(NS) 

-0.06(NS) 

0.152(NS) 

0.251(NS) 

0.112(NS) 

0.231(NS) 

-0.103(NS) 

-0.186(NS) 

-0.119(NS) 

-0.207(NS) 

0.007(NS) 

0.019 

-0.150(NS) 

-0.108(NS) 

-0.024 0.0001 

0.087(NS) 

0.027 

-0.328(NS) 

-0.333(NS) 

-0.276(NS) 

-0.303(NS) 

-0.066(NS) 

-0.114(NS) 

0.068(NS) 

-0.062(NS) 

0.044 

0.099(NS) 

0.251(NS) 

0.112(NS) 

-0.009(NS) 

-0.149(NS) 

0.021 

0.071(NS) 

P272 

Effect of Vitamin E and Sex Hormones on Exercising Women Oxidative 

Stress Status: A Randomized Clinical Trial 

Mahdi O Garelnabi, Emir Veledar, Jerome Abramson, Abdelmoniem Younis, Drex Earle, 

Brandon Petro, Grant Anderson, Jill White-Welkley, William Weintraub, Emory Univ Sch of 

Medicine, Atlanta, GA; Sampath Parthasarathy; Louisiana State Univ Health Science Cntr, 

New Orleans, LA 

Background: Exercise reduces the risk of coronary heart disease in men and women but 

paradoxically, may promote free-radical formation, lipid peroxidation and vascular tissue injury. 

Objectives: In this study, we assessed the effect of vitamin E supplementation and sex 

hormones on oxidative stress and inflammatory markers in exercising women. Methods: We 

measured plasma estradiol (E2) and progesterone (PGN) hormones, and lipid profile levels (TC, 

HDL, LDL, and TG); along with oxidized LDL (OX-LDL), Myeloperoxidase (MPO), sVCAM, MCP-1, 

CRP, and Autoantibodies to oxidized LDL (AALDL) as oxidative stress/inflammatory markers in 

260 healthy women before and after two months of regular aerobic exercise in a randomized 

double blind clinical trial. Women were randomized on Vitamin E (VE), and Placebos (Pl). The 

base line parameters were assessed before they started to exercise and take the supplementation. 

Participants VO2 and duration time spent in treadmill before and after exercise was also 

measured. Spearman’s rank correlation was calculated to assess the association between the 

hormones and oxidative stress/inflammatory markers and VO2. Linear regression models were 

run to examine the relationship between hormones levels and oxidative stress/inflammatory 

markers and VO2 after adjustment for other factors. Results: The following table summarizes 

the levels of hormones, lipid profile, oxidative stress and inflammatory markers in women 

before (1) and after two weeks (2) of exercise. The correlation and P Value are shown on the 

table. Conclusion: These finding indicates that Downloaded exercise and from 

women sex hormone are 

associated with oxidative stress/inflammatory markers; the degree and line of association 

varies according to the level of fitness and type of biomarker. Interestingly, Vitamin E haven’t 

demonstrated considerable role on containing the level of oxidative stress and inflammation 

associated with exercise. 

Variable N Mean 

Pl TC1 (mg/dl) 

TC2 VE TC1 

(mg/dl) TC2 

Pl HDL1(mg/dl) 

HDL2 VE 

HDL1(mg/dl) 

HDL2 

Pl LDL1 (mg/dl) 

LDL2 VE LDL1 

(mg/dl) LDL2 

Pl TG1 (mg/dl) 

TG2 VE TG1 

(mg/dl) TG2 

Pl E2–1 (pg/ml) 

E2–2 VE E2–1 

(pg/ml) E2–2 

Pl PGN1 

(ng/ml) PGN2 

VE PGN1 

(ng/ml) PGN2 

Pl 


Isopro2 VE 


Isopro2 

Pl 

OXLDL-1(U/L) 

OXLDL-2 VE 

OXLDL-1(U/L) 

OXLDL-2 

Pl MPO1(ng/ml) 

MPO2 VE 

MPO1(ng/ml) 

MPO2 

Pl sVCAM 

1(ng/ml) 

sVCAM-2 VE 

sVCAM1(ng/ml) 

sVCAM-2 

Pl MCP 

1m1(pg/ml) 

MCP-1–2 VE 

MCP-1 1(pg/ml) 

MCP-1–2 

Pl CRP1(ng/ml) 

CRP-2 VE 

CRP1(ng/ml) 

CRP-2 

Pl AALDL-1 

(OD) AALDL-2 

VE AALDL-1 

(OD) AALDL-2 

128108 

130119 

128108 

130119 

126108 

129119 

124104 

127116 

119 95 

122110 

117 95 

116108 

121104 

126116 

125104 

126112 

125104 

126116 

121102 

124115 

112 96 

111101 

109 95 

112100 

126105 

130116 

184.42 

184.04 

183.37 

186.47 

59.27 

59.55 

59.53 

60.55 

107.94 

106.44 

103.84 

106.94 

88.37 

90.32 

89.16 

95.75 

66.25 

64.39 

61.32 

62.17 

3.36 

2.29 

3.42 

2.46 

13.52 

13.79 

13.00 

12.88 

23.39 

23.92 

24.6 

24.04 

3.26 

3.44 

3.68 

3.46 

314.36 

311.85 

308.3 

302.5 

109.43 

103.37 

122.24 

107.29 

4731 

4415 

6889 

6276 

0.257 

0.263 

0.269 

0.36 

R (Vs 

E2) R(Vs PGN) 

-0.242 

-0.633 

-0.847 

-0.119 

-0.565 

-0.405 

-0.292 

-0.823 

-0.366 

-0.978 

0.638 

-0.135 

-0.026 

-0.164 

-0.043 

-0.034 

-0.044 -0.001 

-0.124 -0.011 

-0.109 -0.010 

-0.042 0.293 

-0.507 -0.01 

-0.354 -.0068 

-0.069 -0.160 

-0.012 -0.811 

P Value 

(Vs E2) 

0.10(N.S) 

0.04 0.01 

0.14(N.S) 

0.05 

0.08(N.S) 

0.09(N.S) 

0.02 

0.08(N.S) 

0.002 0.04 

0.14(N.S) 

0.20(N.S) 

0.14(N.S) 

0.23(N.S) 

0.20(N.S) 

P Value (Vs 

PGN) 

0.184(N.S) 

0.324(N.S) 

0.143(N.S) 

0.242(N.S) 

0.18(N.S) 

0.10(N.S) 

0.14(N.S) 

0.24(N.S) 

0.06 (N.S) 

0.26 (N.S) 

0.08(N.S) 

0.06(N.S) 

0.16(N.S) 

0.14(N.S) 

0.01 0.02 

– – – 

– – – – 

0.650 

-0.606 

-0.330 

-0.962 

0.137 

0.674 

-0.503 

-0.922 

0.467 

0.588 

0.378 

0.089 

-0.400 

0.654 

-0.607 

-0.718 

0.818 

-0.197 

-0.471 

0.752 

-0.08 

-0.121 

-0.213 

-0.503 

0.188 

0.102 

-0.263 

-.551 

-0.957 0.885 

-0.303 0.327 

0.855 0.189 

0.122 0.600 

0.933 0.790 

0.904 0.803 

0.510 0.407 

-0.866 -0.760 

-0.985 -0.078 

-0.504 0.100 

-0.304 -0.020 

-0.149 -0.291 

0.382 0.021 

-0.148 -638 

0.04 0.05 

0.08(N.S) 

0.004 

0.13(N.S) 

0.04 

0.06(N.S) 

0.009 

0.06(N.S) 

0.05 

0.08(N.S) 

0.08(N.S) 

0.07(N.S) 

0.04 0.04 

0.03 

0.02 0.13 

0.07(N.S) 

0.03 

0.16 0.16 

0.12 0.06 

0.12(N.S) 

0.16(N.S) 

0.1(N.S) 

0.05 

Assessment of the Longer-Term Effects of a Dietary Portfolio of 

Cholesterol-Lowering Foods in Hypercholesterolemia 

Cyril Kendall; Univ of Toronto, Toronto, Canada 



0.005 0.01 

0.09(N.S) 

0.09(N.S) 

0.01 

0.13(N.S) 

0.12(N.S) 

0.05 

0.007 0.02 

0.01 0.02 

0.06(N.S) 

0.08(N.S) 

0.01 0.02 

0.001 

0.18(N.S) 

0.06(N.S) 

0.16(N.S) 

0.1(N.S) 

0.2(N.S) 

0.14(N.S) 

0.10(N.S) 

0.08(N.S) 

0.2(N.S) 

0.13(N.S) 

0.04 

P273 

Objective: To assess the effectiveness over a six-month period of dietary advice to follow a 

combination of cholesterol-lowering foods (dietary portfolio) ad libitum and to compare to 

LDL-cholesterol reductions achieved in shorter-term metabolic studies of a portfolio diet or 

after taking a statin. Methods: Thirty-five hyperlipidemic subjects took a diet high in plant 

sterols (1.0 g/1000 kcal), soy protein (21.4 g/1000 kcal), viscous fibers (9.8 g/1000 kcal) and 

almonds (14 g/1000 kcal) for six months. Their data were also compared with results on 26 

of these subjects who had taken the same diet for one month under metabolic conditions and 

had also taken a statin for a further month. Results: At 2-weeks and 24-weeks of the ad libitum 

diet the LDL-cholesterol reductions from baseline were similar at 13.42.0% (P0.001) and 

13.32.4% (P20% in 28.6% of subjects (mean 29.51.7%), 10–20% in 34.3% of subjects 

(mean15.41.0%) and 10% in 37.1% of subjects (mean 0.22.4%). Despite the 

differences in response on the ad libitum diet, subjects in the above tertiles responded similarly 

in their cholesterol reduction responses on the metabolically controlled portfolio diet (33.3 

2.8%, 27.52.1% and 29.42.1%) or after 20 mg lovastatin (39.4 3.7%, 33.52.7% and 

32.73.6%, respectively). No direct relation was found with compliance and LDL reduction 

although no subject with less than 50% compliance (n11) achieved 20% LDL-cholesterol 

reduction at 24-weeks. Conclusions: Dietary combinations may have approximately threequarters 

the potency of early statins, in just under one third of those prepared to take a dietary 

intervention. by guest on June 29, 2013

P274 

Stress-Induced Abdominal Obesity and Metabolic Syndrome: Neuronal 

Regulation of Adipogenesis? 

Lydia E Kuo, Edward W Lee, Joanna B Kitlinska, Zofia Zukowska; Georgetown Univ, 

Washington, DC 

Sympathetic nerve activation is commonly thought to induce weight-loss via -adrenoceptors. 

Some individuals, however, favor weight-gain during stress, thought to be due to glucocorticoids. 

Here we test the hypothesis that stress promotes obesity by releasing a cotransmitter, 

neuropeptide Y (NPY). Previously, we found that NPY is potently angiogenic and stimulates 

preadipocyte proliferation and differentiation into lipid-filled adipocytes (in insulin-primed cells) 

- all by the same Y2 receptors (Rs) which are up-regulated in the process. Subcutaneous 

administration of a NPY pellet increased while Y2R antagonist decreased in vivo abdominal fat 

deposit and its vascularization in ob/ob mice; the antagonist also improved glucose tolerance. 

We now used a new model of obesity, high fat diet (HF) and tested the effects of superimposed 

chronic stress of cold exposure (CS)(0.5cm 4 o C water/1hr/daily/14days) - previously proven to 

increase NPY release. Mice fed HF had 20% more total body fat, hyperglycemia, impaired 

glucose tolerance and hyperleptinemia, compared to non-stressed (NS) and standard chow fed 

(SC). These changes, except leptin levels, were augmented by CS, which increased abdominal 

obesity by 50%, without increasing food intake. CS actually decreased plasma leptin while 

increasing NPY levels (ELISA) - suggesting that in the periphery, like in the brain, the two 

antagonize each other. Y2R antagonist (slow-release pellet in abdominal area, s.c.) or Y2R 

knockout prevented or reversed hyperglycemia, hyperleptinemia and abdominal obesity due to 

CS and HF. CS, like NPY pellet previously, increased the number of small adipocytes in the 

subcutaneous abdominal fat pads - resembling NPY’s proliferative effect on preadipocytes in 

vitro. Like in ob/ob, CS in HF-fed mice, but not HF diet alone, increased Y2R expression 

(immunostaining and mRNA) in the subcutaneous abdominal fat, suggesting that these 

angiogenic and adipogenic Rs are activated by stress. Thus, this study suggests that stress 

promotes abdominal obesity and metabolic syndrome not only via previously known pathway 

of glucocorticoids, but by activating sympathetic release of NPY, which, via its endothelial and 

preadipocytes Y2Rs, stimulates both angiogenesis and adipogenesis. 

P275 

Suppression of Vegf and Weight Loss with Improved Glucose and Lipid 

Metabolism by Tegreen in a Dio Model 

Jihong Lu, Wei Chen, Pharmanex Beijing Pharmacology Cntr, Beijing, China; Jiashi Zhu; 

Pharmanex, Provo, UT 

Numerous studies demonstrated previously biological properties of green tea in promoting 

longevity in humans, antioxidant, anti-cancer, inhibiting angiogenesis, etc. We examined the 

effects of Tegreen, a green tea extract product containing 97% tea polyphenols or 65% 

catechins, in suppressing VEGF, reducing weight, and improving glucose & lipid metabolism in 

DIO rats. After several weeks on this diet, rats were given by gavage either placebo or Tegreen 

(25, 75, 250 mg/kg) for 8 weeks and examined for body weight, fasting blood glucose (FBG), 

insulin (Ins), triglycerides (TG), Glucagon (Glca), adiponectin (AN), and VEGF. Weight of 

abdominal fat pad around uterus and kidneys (AFI) and ratio of Ins/Glca (RIG) suggested a 

balance of fat deposition and burning. Reduction of body weight was found in the high dose 

group (-15%, p0.003) after Tegreen treatment, associated with a -29.8% reduction of VEGF 

(p0.001). Tegreen reduced FBG (-1622%, p0.004), TG (-3154%, p0.006) and AFI 

(-1222%, p0.011), increased FBG-Ins index (a measure of Ins sensitivity; 2026%, 

p0.002) and decreased ratio of Ins/Glca (-4669%, p0.037). Our findings suggest that 

Tegreen is capable of reducing VEGF and improving glucose-lipid metabolism, and reducing 

body weight at a high dose. 

P276 

Impact of Soybean Oils Modified in Fatty Acid Profile on Plasma Lipid and 

Lipoprotein Concentrations 

Alice H Lichtenstein, Nirupa R Matthan, Susan M Jalbert, Ernst J Schaefer, Lynne M 

Ausman; Jean Mayer USDA Human Nutrition Rsch Cntr on Aging at Tufts Univ, Boston, MA 

A major determinant of plasma lipoprotein concentrations is the fatty acid profile of the diet. 

One approach that is being used to alter this variable is to change the fatty acid profile of 

commonly used vegetable oils. To assess the efficacy of this strategy soybean oils developed 

to have modified fatty acid profiles were assessed in individuals 50 years (N27) with 

LDL-C concentrations 130 mg/dL at time of entry into the study. All diets contained 28% of 

energy as fat, 16% protein, 56% carbohydrate, 65 mg cholesterol and 16 g fiber/1000 kcal. 

Two-thirds of the fat in each diet was contributed by the experimental fat, native soybean oil 

(SO, 14%, 22%, 62%, 1%; SFA, MUFA, PUFA, trans, respectively), hydrogenated soybean oil 

(HydroSO; 19%, 33%, 36%, 12%), soybean oil low in saturated fatty acids (LoSFA; 7%, 23%, 

68%, 1%), soybean oil low in alpha-linolenic acid (LoALA18:3; 15%, 24%, 61%, 1%) and 

soybean oil high in oleic acid (HiOleic; 10%, 85%, 5%, 1%). Fasting total cholesterol 

concentrations at the end of each diet period were 223b , 233a , 216b , 225 a,b and 222 b mg/dL 

(SO, HydroSO, LoSFA, LoALA and HiOleic, respectively, values without common superscripts 

significantly different, P0.05); LDL-C concentrations were 142a,b , 149a , 136b , 143 a,b and 143 

a,b mg/dL (SO, HydroSO, LoSFA, LoALA and HiOleic, respectively); HDL-C concentrations were 

51 a,b ,52a,b ,51b ,52a,b and 53 a mg/dL (SO, HydroSO, LoSFA, LoALA and HiOleic, respectively) 

and triglyceride concentrations were 152, 156, 152, 158 and 149 mg/dL (SO, HydroSO, LoSFA, 

LoALA and HiOleic, respectively). Modifying the fatty acid profile of soybean oils, with the 

exception of hydrogenation, resulted in only small differences in plasma lipid and lipoprotein 

profiles. Within the context of the whole diet the magnitude of these changes were small, even 

when using modified oils as the major source of Downloaded dietary fat. from 




P277 

The Effect of Collagen Crosslink-Breaker ALT-711 on Carotid Pressure 

Augmentation in Older Subjects with Systolic Hypertension 

Vojtech Melenovsky, Lia Clattenburg, Patricia Fitzgerald, Ann Capriotti, David A Kass, Suzan 

Zieman; Johns Hopkins Hosp, Baltimore, MD 

Background: Amplification of central pressure increases LV afterload and is associated with 

higher CV risk. ALT-711 is a breaker of non-enzymatic collagen crosslinks that increases 

vascular compliance. The purpose of study was to determine whether short-term high-dose 

therapy with ALT-711 could decrease augmentation of pulse pressure. Methods: Older 

subjects with systolic hypertension on stable therapy (n13, age: 64y, m/f: 9/4, BMI 29 kg/m 2 ) 

received 2 weeks of run-in placebo, followed by 8 weeks of ALT-711 210 mg/bid. Carotid 

pressure waveforms and carotid-femoral pulse wave velocity (cfPWV) were measured using 

applanation tonometry before and after therapy. Results: Augmentation decreased by 37.3 % 

(from 0.310.15 to 0.2017, p 0.0007), augmented pressure declined from 16.410 to 

9.69 mmHg (p 0.0008) (Figure 1). Heart rate did not changed (from 63 to 63 bpm, p 

0.92). Brachial systolic and diastolic blood pressure did not change (from 146/82 mmHg to 

142/82 mmHg, p 0.28 and 0.90) and also carotid mean, systolic and diastolic pressures 

remained unchanged (from 103/132/82 mmHg to 102/130/82 mmHg, p 0.55, 0.58 and 

0.91). As cfPWV did not change (from 1482837 cm/s to 1250488, p0.30), lower pressure 

augmentation cannot be explained by slowing of wave travel, but rather by decreased 

reflectivity of more compliant peripheral arteries. Conclusion: Short-term therapy with 

ALT-711 markedly decreases carotid pressure augmentation. This effect may favorably modify 

cardiovascular risk in older hypertensive subjects, independently from lowering of blood 

pressure. 

New Mechanisms of Anti-Atherosclerotic Action of Bioflavonoids from 

Chokeberry (Aronia Melanocarpa) Fruit 

Marek Naruszewicz, Danuta Zapolska-Downar, Mariusz Kaczmarczyk; Pomeranian Med 

Univ., Sczecin, Poland 

P278 

In the previous clinical studies we have proven that the extract of chokeberry fruit containing 

anthocyanins (27%) and catechins (55%) administered for 6 weeks (3 x 100 mg/dl) to 

post-myocardial infarction patients significantly lowered the level of inflammatory marker 

C-reactive protein (CRP)s and oxidized LDL (ox-LDL). Apart from the drop in the levels of hs CRP 

by 31%, (p 0.001), and ox-LDL by 27% (p0,001) also a significant reduction in the systolic 

and diastolic pressure in comparison with the placebo group was found. The above effect was 

independent from statins and ACE inhibitors used in these patients. In this study we verified the 

hypothesis that in addition to their antioxidative properties, chokeberry bioflavonoids may exert 

a direct protective action on endothelial cells through stimulation of expression of the nitric 

oxide synthase gene and through down-regulation of the lectin-like oxidized LDL receptor-1 

(LOX-1) endothelin-1 gene. The study was performed on non-stimulated HUVEC cells incubated 

for 3 and 24 hours with chokeberry extract at increasing concentrations (from 0,1g to 

5g/ml/). The content of mRNA for eNOS and LOX-1 ET-1 was determined with the use of 

RT-PCR. It was found that chokeberry extract in dose dependent manner increased 2–53 times 

eNOS expression in theendothelial cells without an effect on ET-1 expression with simultaneous 

down-regulation of LOX-1.. The properties of chokeberry bioflavonoids indicate their additional 

pleiotropic action on vascular endothelium, which may prove useful for the prevention and 

treatment of ischaemic heart disease. 

Diet-Induced Obesity Increases the Severity of Angiotensin II-Induced 

Abdominal Aortic Aneurysms in C57BL/6 Mice 

Sara B Police, Alan Daugherty, Lisa A Cassis; Univ of Kentucky, Lexington, KY 

P279 

Objective: Infusion of angiotensin II (AngII) to LDL receptor -/- mice fed a fat enriched (21% 

wt/wt) diet results in the formation of abdominal aortic aneurysms (AAA). In contrast, 

normolipidemic C57BL/6 mice exhibit a low incidence (10%) of AngII-induced AAA formation. 

The purpose of this study was to determine whether obesity induced by high fat feeding of 

C57BL/6 mice by guest would increase on June AAA29, susceptibility. 2013 Methods: Male C57BL/6 mice were fed either

E-102 Vol 25, No 5 May 2005 

a low fat (LF, 10% kcal as fat), medium fat (MF, 45% kcal as fat), or a high fat (HF, 60% kcal 

as fat) diet for 19 weeks. The extent of fat enrichment in the diet was directly related to the 

degree of obesity. Systolic blood pressure was not altered by diet-induced obesity. At week 15, 

mice in each diet group were randomized to receive either saline (n 3) or AngII (1,000 

ng/kg/min; n 7) by Alzet mini-pump for 28 days. AngII-induced elevations in systolic blood 

pressure were similar in all groups. Cholesterol concentrations were increased in HF mice 

infused with AngII, compared to LF and MF groups (LF: 152.9 10.7; MF: 214.0 23.1; HF: 

242.8 8.3 mg/dl, P0.05). Lipoprotein distribution revealed a significant increase in 

cholesterol carried in apoB containing particles in the HF-fed animals. Despite the modest 

hypercholesterolemia, atherosclerotic lesions were not detected in the aortic arch and thoracic 

aorta from mice in any group. The incidence of AAAs was greatest in the HF group (LF: 43%, 

MF: 43%, HF: 71%; P0.05). Measurement of aortic weight as an index of AAA severity 

demonstrated an increase in AAA severity in the HF group (HF 54.8 9.6 vs. MF 31.6 3.0 

vs. LF 35.2 2.6 mg; P0.05). Conclusion: Diet-induced obesity elevated the incidence and 

severity of AngII-induced AAA in C57BL/6 mice. Future studies will establish the specific 

component of dietary fat that augments AAA formation. 

Microarray Analysis of the Actions of Cranberry and Red Wine 

Procyanidins on Human Aortic Endothelial Cells 

Mark R Pothecary, Noorafza Q Khan, Elizabeth G Wood, Delphine M Lees, Roger Corder; 

William Harvey Rsch Institute, London, United Kingdom 

P280 

Diet is a key influence on the incidence of coronary heart disease. The protective role of high 

flavonoid consumption is recognised but not yet fully understood. Experimental studies indicate 

that the vascular endothelium is an important site of action for dietary flavonoids. To discover 

how procyanidins, an abundant type of flavonoid, modify endothelial function we investigated 

by microarray analysis the changes in gene expression induced by cranberry juice (CJ) (A type) 

and red wine (RW) (B type) procyanidins. In separate experiments human aortic endothelial 

cells (HAEC) from 3 donors were grown to confluence in cell culture. HAEC were incubated for 

6h with control medium alone or containing CJ or RW procyanidins (prepared using Sephadex 

LH-20). At the end of the treatment period conditioned media were collected and total RNA was 

extracted. Prior to microarray analysis the response to treatment was verified by immunoassay 

of ET-1 release, and by qRT-PCR for KLF2 and eNOS mRNA levels. Microarray analysis was 

performed with Affymetrix U133 Plus 2.0 Arrays for mRNA transcript detection. Significant 

changes in mRNA transcript signal were determined by comparison analysis using Affymetrix 

software. ET-1 release was reduced by CJ and RW (45 6%, 67 8%, p 0.05). Levels of 

KLF2 and eNOS mRNA were increased by both treatments (KLF2 CJ 59 9%, RW 124 19 

%, P0.01; eNOS: CJ 25 4%, RW 43 12 %, P0.05). Microarray analysis revealed 

significant changes in a wide spectrum of genes (increased: CJ 124, RW 178, with 84 in 

common; decreased: CJ 237, RW 237, with 159 in common). Significant decreases in 

microarray signal for IL6, IL8 and BMP4 were confirmed by immunoassay of the corresponding 

medium samples. Heme oxygenase 1 was significantly increased by CJ and RW (microarray: 

6.7, 4.5 fold). This increase was confirmed by qRT-PCR (13.2 1.7, 7.5 1.2 fold, p 0.01). 

Microarray analysis also revealed a co-ordinated profile of changes in anti-thrombotic and 

thrombolytic genes. In conclusion, these experiments suggest that dietary procyanidins may 

induce an atheroprotective phenotype in the endothelium. These results may help define a 

group of biomarkers to monitor for assessing the vascular response to dietary flavonoid 

consumption. 

WITHDRAWN 

P281 

P282 

Effects of Dietary Fat Content, Glycemic Index, and Caloric Restriction on 

Heart Disease Risk Factors in Obesity 

Ernst J Schaefer, Joi L Gleason, Paul Fuss, Judith R McNamara, Helen Rasmussen, Alice H 

Lichtenstein, Julie Richards, Susan B Roberts; Tufts Univ, Boston, MA 

Introduction Obesity is a growing problem in the US and is contributing to CHD risk. There is 

controversy about the optimal diet with regard to dietary fat content as well as glycemic index 

of carbohydrate. Objectives Our goals in this study were to determine the effects of calorie 

restriction, dietary fat content and glycemic index on weight loss and CHD risk factors in 

overweight and obese subjects under controlled conditions. Methods We enrolled 80 male and 

female subjects, with a BMI of 28 –38, mean 33 kg/m2, mean age 55 years into a 22 week 

feeding trial in which all subjects received an average American diet under isoweight conditions 

for 5 weeks, and then were randomly assigned to one of 4 diets, all of which contained about 

5% of calories as saturated fat, 15% protein, 60 mg of cholesterol and 16 grams of fiber/1000 

calories. The diets differed in fat content (15% versus 30%) and glycemic index (low at 55 

versus high at 95). They were on these diets for 12 weeks with 1/3 caloric restriction (ad 

libitum-so they could get more unit snack foods if they requested them), and then for 5 weeks 

under isoweight conditions. All food and drink was provided. Blood analysis for a variety of lipid 

and other parameters were carried in the fasting state, 4 hours after an evening meal (PP), and 

2 hours after a standard 75 gram glucose challenge. Results All four diets promoted significant 

(p0.05) weight loss (range 6.5– 8.2%), fasting LDL cholesterol reduction (8.5–13.1%), PP 

triglyceride reduction (25.5–33.0%), and fasting and PP cholesterol/HDL cholesterol ratio 

reductions (7.3–19.8%). Low GI diets lowered fasting insulin (23.1%)and PP insulin (39.9%) 

more than high GI diets (14.3%, 2.7%). These finding were true for HOMA as well. In addition 

fasting insulin and HOMA was were lowered less by the low fat diets (14.7%, 16.8%) than by 

the moderate fat diets (22.8%, 25.3%). All diets promoted CRP reduction (range 12.5–33.9%). 

Conclusion The data are consistent with the concept that calorie restriction is paramount in 

weight loss, and that dietary composition plays a limited role in affecting this process. However 

the glycemic index and fat content of the diet appears to play a role in insulin resistance, such 

that low glycemic index, moderate fat diets appear Downloaded to have a more from 

favorable effect. 



Skin Microvascular Flow is not Associated with Circulating Levels of 

Leptin or Adiponectin 

Xenia T Tigno, Shi Ying Ding, Barbara C Hansen; Obesity and Diabetes Rsch Cntr, Univ of 

Maryland at Baltimore, MD 

P283 

Leptin and adiponectin are adipose tissue hormones which have differential actions on energy 

balance and food intake. Adiponectin levels are decreased while, leptin levels are elevated in 

obese humans and monkeys. Adiponectin increases insulin-sensitivity, suppresses hepatic 

gluconeogenesis, and may possess anti-inflammatory and anti-atherogenic properties. Leptin 

suppresses food intake, enhances muscle glucose uptake and metabolism, increases energy 

expenditure through shivering, and may be involved in obesity- related hypertension via 

sympathetic excitation. Moreover, leptin has been suggested to stimulate nitric oxide 

production and ischemia- induced retinal neovascularization. We have previously reported that 

correction of dyslipidemia in prediabetic monkeys using a lipid-lowering agent resulted in 

improvement of both metabolic and microvascular functions, concomitant with elevation of 

both leptin and adiponectin. In this study we examine further the relationship between hormone 

levels and microvascular flow in a cross-section of diabetic and non-diabetic rhesus monkeys. 

Microcirculatory function was evaluated using the heat-induced hyperemic response of the skin 

(% increase over baseline, %PU change), % increase per degree rise in temperature (PU/ o C), 

baseline perfusion, peak perfusion, and increase in flow/sec, as measured by laser Doppler 

fluximetry. Correlation analysis was used to test the significance of associations. As expected, 

adiponectin was significantly related to insulin sensitivity, whereas leptin showed a significant 

negative correlation. The increase in flow over baseline (%PU change), PU/ o C , and PU/sec 

were found to be negatively associated with insulin sensitivity. Neither plasma adiponectin nor 

leptin levels were associated with %PU change or PU/ o C. Although adiponectin levels tended 

to be associated with both basal perfusion and peak perfusion during hyperemia, the 

relationships did not attain statistical significance (p’s 0.07). Leptin levels correlated 

significantly with the rate of rise of flow (PU/sec), but not with basal or peak flows. We conclude 

that cutaneous hyperemia due to heat provocation is not directly influenced by circulating levels 

of either leptin or adiponectin. 

The Clinical Impact of Fiber Supplementation on Cardio-Vascular Risk 

Parameters in Type 2 Diabetes 

Peter J Verdegem, Unicity International, Orem, UT; Steven H Freed, David J Joffe; 

DiabetesInControl.com, Deerfield, IL 

P284 

Introduction. Fiber supplementation, in particular of the soluble kind, has known beneficial 

effects in lowering the cardio-vascular risk profile by lowering serum cholesterol. It is thought 

that bile-acid sequestration of cholesterol in the digestive system is the main mechanism for 

cholesterol reduction. This study investigates the efficacy of BiosLife 2, a fiber supplement 

combining soluble and insoluble fiber, that has been specifically designed for cholesterol 

lowering. Methods. This study included 78 type 2 diabetes patients with an average age of 59. 

At baseline, total cholesterol, triglycerides, LDL, and HDL were assessed. The subjects then 

added 10 - 15 gram of the fiber supplement to their diet for 90 days. At the final visit, the 

parameters were re-assessed. The fiber supplement is taken as a drink, and consists of guar 

gum, gum arabic, locust bean gum, pectin, and oat fiber dispersed in calcium carbonate. In 

addition this product contained chromium, and B-vitamins. Five grams were taken 2-3times 

daily 5 -10 minutes prior to eating. Results. The compliance with the fiber supplementation 

was excellent. The supplementation with fiber resulted in beneficial changes to the assessed 

parameters. The changes are listed in the table. Conclusion. Supplementing the diet with this 

fiber drink to a level as recommended by the American Heart Association has clear beneficial 

effects on the lipid profile of type 2 diabetics. This specially designed fiber supplement has 

promising effects as an alternative treatment to pharmaceutical intervention for hyperlipidemia. 

RESULTS 

Parameter Baseline average 90-day average % change 

Tot. Chol. 215 mg/dL 184 -14.4 

Triglycerides 299 mg/dL 257 -14.0 

LDL 129 mg/dL 92 -28.7 

HDL 43 mg/dL 55 21.8 

A Nutritional Supplement Program Halts the Progression of Plaque 

Formation in Carotid Artery Disease 

Peter J Verdegem, Unicity International, Orem, UT; Stewart Lonky, Private practice, Pacific 

Palisades, CA; William Curley; Private Practice, Las Vegas, NV 

P285 

Introduction Carotid artery disease or carotid artery stenosis is a major risk factor for stroke. 

Narrowed carotid arteries may block the blood flow to the brain. Common medical treatment 

includes carotid endarterectomy, or surgical removal of the plaque in the carotid. Previous work 

suggests that a nutritional supplement program may reduce plaque sizes in arteriosclerosis. 

This work investigates if that program can also reduce carotid plaques in carotid artery disease. 

Methods Twenty four carotid artery disease patients were followed for a period of 12 months. 

Fourteen patients used Cellular Essentials, a dietary supplement that contains all major 

vitamins and minerals in adequate levels, a variety of amino acids, and other phytochemicals. 

Ten patients were used as a control group and did not use any supplementation during the trial 

period. The patients were evaluated at baseline and after 12 months using carotid 

ultrasonography. Statistical analysis was performed on the difference in changes of stenosis 

between the two groups after twelve months using Student’s t-tests. Results The compliance 

with the supplement program was excellent. The mean difference in calcification area after 12 

months in the supplement user group was -18.2% vs. 5.5% in the control group. The 

difference in changes in calcification area between the two groups was statistically significant 

(p0.05). by Conclusion guest on ThisJune pilot trial 29, supports 2013 the theory that plaque formation in carotid artery

stenosis can be reduced by taking a nutritional supplement program involving vitamins, 

minerals, amino acids and phytochemicals. Early intervention with such a program may reverse 

the need for surgical procedures. 

Decreased Coronary Flow Reserve in an Obese Cohort Determined by 

Positron Emission Tomography Myocardial Perfusion Imaging 

P286 

James A Vitarius, Irini M Youssef, Arlene R Travis, Samprit Chatterjee, Josef Machac; Mount 

Sinai Sch of Medicine, New York, NY 

Obesity is a major predisposing risk factor for the development of coronary heart disease. 

Coronary flow reserve (CFR) calculations from positron emission tomography (PET) imaging can 

be used to noninvasively evaluate coronary endothelial function. To see if obesity affects 

PET-determined CFR, a cohort of individuals with body mass indices (BMI) 30 were randomly 

selected from a population that underwent pharmacological cardiac stress testing with 

Rubidium-82 PET myocardial perfusion imaging. Those with a prior positive stress test or 

history of coronary artery disease were excluded. The global CFR was calculated. The CFR of 

the obese group (N18) was significantly lower compared with healthy controls (N20) 

(2.179 0.81 vs 2.913 0.59, p0.0028). (Normal CFR is 2.0.) Univariate ANOVA 

revealed BMI, age, diabetes, hypertension, but not gender or hyperlipidemia as significant 

predictors of lower CFR (p0.05). Multivariate stepwise logistic regression analysis showed 

that BMI was a significant independent predictor of CFR (p0.003), while age, gender, 

diabetes, hypertension, and hyperlipidemia were not. These data suggest that in a retrospective 

analysis, BMI has the strongest independent effect on CFR determined by PET myocardial 

perfusion imaging. 

P287 

Exercise Reduce Adipocyte Hypertrophy and Up-Regulation Expression of 

Peroxisome Proliferator Activated Receptor in Rats with Metabolic 

Syndrome 

Zhiming Zhu, Lili Zhang, Liquan Wang; Dept of Hypertension and Endocrinology, Cntr for 

Hypertension and Meatbolic Disease, Third Military Med Univ, Daping Hosp, Chongqing, 

China 

Obesity is characterized by hypertrophy and hyperplasia of adipocyte. Peroxisome proliferator 

activated receptor(PPAR) plays an important role in modulation of adipocyte differentiation 

and lipids metabolism. Many studies showed that administration of PPARagonists can 

improve insulin resistance and prevent visceral obesity. This study aims to investigate whether 

exercise can prevent adipocyte hypertrophy and change of PPAR in visceral adipocyte tissues. 

Methods: high fat induced metabolic syndrome (MS) in Wistar rat was according to our previous 

report (Shen CY, et al. Am J Hypertens, 2004, 17(5) part2: 220A). Wistar rats were divided into 

4 groups: rats on normal diet, rats on high fat diet, swimming rats on normal die and swimming 

rats on high fat diet. PPARexpression in mesenteric adipocyte tissue was detected by western 

blot. The morphologic change of adipocyte of mesenteric fat was observed using HE staining 

and measured by image analysis system. Compared with rats on normal diet, body weight 

(55757gvs44226 g ) and visceral fat weight (245.5gvs5011g) were significantly 

higher in MS rats (p0.01), but exercise can prevent high fat induced visceral obesity 

(21.475.68) and MS. Compared with rats on normal diet, adipocyte size was significantly 

larger in rats with MS (0.1070.003m 2 vs 0.0480.002m 2, p0.01). In contrast, 

adipocyte size was no difference between swimming rats on normal diet and swimming rats 

on high fat diet rats (0.0850.0037m 2 vs 0.0530.0026m 2 ,p0.05). Compared with rats 

on normal diet, rats on high fat diet reduced 20 % of PPAR expression in mesenteric fat, 

however swimming rats on high fat diet increased two fold of PPAR expression in mesenteric 

fat compared with rats on high fat diet. It concluded that exercise can significantly prevent 

adipocyte hypertrophy, which may be related to increase endogenous PPAR expression ( 

Supported by NSFC 30470830). 

P288 

Comprehensive Intervention of Multiple Cardiovascular Risk Factors in 

Patients with Metabolic Syndrome 

Qian Li, Zhiming Zhu, Jin Chen, Zhigang Zhao; Dept of Hypertension and Endocrinology, 

Cntr for Hypertension and Metabolic Disease, Third Military Med Univ, Daping Hosp, 

Chongqing, China 

Metabolic syndrome (MS) is characterized by a clustering of multiple of cardiovascular (CV) risk 

factors. Control these risk factors are very important to reduce CV events. This follow-up study 

evaluates the outcome in patients with MS. From 2000 to 2003, subjects were admitted to 

hospital by initial diagnoses of type 2 diabetes mellitus (T2DM), hypertension and T2DM 

combined with hypertension. Among them, 246 patients with metabolic syndrome were 

included in this study. MS was diagnosed using modified NCEP-ATPIII definition: waist 

circumference 90 cm for male and 80 cm for female, triglycerides150mg/dl; HDLcholesterol40 

mg/dl, blood pressure 130/85 mmHg; and fasting blood glucose 110mg/ 

dl. Period of follow up is over 6 month to 2 year (mean follow up period 9 months). Lipid profile, 

fasting blood glucose (FBG), blood pressure, waist circumference, and adherence to medications 

were measured. Compared with before follow up, the control rates of MS components 

were significantly higher (p0.05): plasma TG ( 46% vs 57% ), plasma HDL-C ( 65% vs 73% 

), SBP ( 43% vs 55% ), DBP ( 61% vs 83%), and FBG ( 39% vs 48%). But, waist circumference 

was not significantly difference before and after follow-up ( 35% vs 34% , p0.05). Analysis 

of MS components showed that control rate of components: 24% for one components, 18% for 

two, 34.3% for three, 13.4% for four, and only 4.9% for five. There were significantly different 

between among groups ( P0.05 ). Compliance for medication was significantly higher ( 

p0.05): 38% vs 58% for antihypertensive agents, antidiabetic agents (including insulin ): 46% 

vs 65%, but administration rate of lowering lipidDownloaded drugs was still lower from 

before and after follow 

up ( 17% vs 20%). This study indicated that through the regular follow up, most MS 

components were obviously improved in patients with MS, but control rate of abdominal obesity 

was not optimal. Comprehensive intervention should be paid more intensive attention in MS. 

Plasminogen Activator Inhibitor-1 has a Circadian Rhythm in Blind 

Individuals 

John A Schoenhard, Johns Hopkins Univ, Baltimore, MD; James A Muldowney, III, 

Vanderbilt Univ Med Cntr, Nashville, TN; Jonathan S Emens, Alfred J Lewy, Oregon Health 

Sciences Univ, Portland, OR; Douglas E Vaughan; Vanderbilt Univ Med Cntr, Nashville, TN 

P289 

Circadian variation in plasminogen activator inhibitor-1 (PAI-1) production likely contributes to 

increased risk of myocardial infarction and decreased efficacy of thrombolytic therapy in the 

morning. We have recently shown that PAI-1 is transcriptionally regulated by molecular 

components of the body’s endogenous circadian clock. As this clock is most potently regulated 

by daily light-dark cycles, we employed blind individuals to study the relative contribution of 

light perception to PAI-1 rhythmicity. We studied 4 subjects rendered totally blind by prior 

bilateral enucleation. Plasma PAI-1 levels (expressed in ng/ml) were measured hourly for 24h 

during 4–6 overnight admissions per subject, and evaluated by fitting the curve y M A 

sin (2/24h t ), where M is the rhythm-adjusted mean, A is the amplitude, and is the 

acrophase. Corresponding plasma melatonin levels allowed for assessment of the period and 

phase of each subject’s intrinsic clock. Despite total blindness, 2 subjects were entrained at 

a relatively normal phase, with melatonin onset (MO) occurring between 8 and 10 pm on each 

admission. In contrast, 2 other subjects possessed free-running central clocks, with period 

lengths of 23.9 and 24.9 h and MO alternating from night to day between clinic visits. Although 

significant circadian variation in PAI-1 was observed in all subjects (p 0.05), the amplitude 

of this rhythm was two-fold greater in entrained subjects than in free-running subjects (M 34.4, 

A 20.3 vs. M 29.5, A 9.5). In addition, PAI-1 levels peaked 3 hours earlier in entrained subjects 

than in free-running subjects (8:17 vs. 11:20 am), at a time more in line with previous studies 

of sighted individuals. In general, goodness-of-fit was better for sine curves describing PAI-1 

variability on individual clinic visits of entrained vs. free-running subjects (average R 2 0.47 vs. 

0.32). These results indicate that circadian variation in PAI-1 expression is not wholly 

dependent on light perception, although the magnitude and timing of PAI-1’s oscillation may 

be influenced by centrally-located intrinsic circadian pacemakers. These results also suggest 

that peripheral factors may play a role in PAI-1 rhythmicity, which may in turn impact the 

pathogenesis, timing, and treatment of acute MI. 

WITHDRAWN 

P290 

P291 

Cholesterol Enrichment Enhances the Release of Tissue Factor-Positive 

Microparticles from THP-1 Monocytes 

Ming-Lin Liu, Michael P Reilly, Steven E McKenzie, Kevin J Williams; Thomas Jefferson 

Univ, Philadelphia, PA 

Cholesterol loading of cultured cells has been reported to stimulate the expression of tissue 

factor (TF), a potent pro-coagulant. In addition to subendothelial tissue sources, TF has been 

detected on circulating monocyte-derived microparticles (P). Moreover, the total number of 

circulating monocyte-derived P is significantly increased in hyperlipidemic patients compared 

to normolipidemic controls, and we recently observed increased immune complex-stimulated 

thrombosis in hypercholesterolemic mice. Direct effects of cholesterol on P release, however, 

have not been examined. In the current study, we enriched THP-1 cells, a human monocytic 

cell line, with cholesterol by incubating them in the presence of 10g cholesterol/ml 

complexed to methyl--cyclodextrin (1:6 molar ratio) for 4hat37°C. The total number of P 

in the culture supernatant was quantified by flow cytometry, based on forward (size) and side 

(granularity) scatter and staining with annexin-V. TF-positive P were quantified using a 

FITC-labeled anti-TF monoclonal antibody. Results are reported as P per 1000 cells. In the 

absence of cholesterol enrichment, the total P count was 1223, of which 332 were TF 

positive. In contrast, with cholesterol loading, we observed 6281, of which 1045 were 

TF-positive, an increase of several fold. Large increases were also observed at other time 

points and cholesterol doses. Next, we examined the effects of a known activator, 

lipopolysaccharide (LPS). Addition of 10g LPS/ml increased the total P count to 5915 at 4h, 

with 1014 being TF-positive. Incubation of THP-1 cells with LPS and cholesterol/methyl-cyclodextrin 

together showed an additive effect on P generation, reaching a total of 12842 

at 4h, of which 1470 were TF-positive. In addition to flow cytometry, we confirmed an increase 

in TF-positive P in cell culture supernatant by quantitative ELISA after cholesterol enrichment 

or LPS stimulation and an additive effect after incubation with LPS and cholesterol/methyl-cyclodextrin 

combined. Taken together, our results indicate that acute cholesterol enrichment 

of human monocytic cells enhances TF release in a biologically relevant form. Increased levels 

of this potent procoagulant factor may contribute to atherothrombosis in vivo. 

Polyphosphates - A Novel Modulator of Coagulation 




Stephanie A Smith, Nicola J Mutch, Roberto Docampo, James H Morrissey; Univ of Illinois 

at Urbana-Champaign, Urbana, IL 

P292 

Polyphosphate (polyP), a ubiquitous polymer of inorganic phosphate residues, is found in 

acidocalcisomes of many prokaryotic and unicellular eukaryotic cells. Recently, platelet dense 

granules were discovered to resemble acidocalcisomes, including the presence of mM levels 

of polyP (chain length, 70–75; polyP75). Platelet polyP is released following thrombin activation, 

suggesting by aguest role in on coagulation. June 29, We2013 found that polyP75 prolonged tissue factor-mediated

E-104 Vol 25, No 5 May 2005 

clotting (prothrombin time, PT), especially with dilute PT reagents (longer base clot times) 

suggesting that polyP might block a coagulation inhibitor. PolyP had no impact on antithrombin 

but profoundly antagonized tissue factor pathway inhibitor (TFPI) activity: TFPI added to plasma 

markedly prolonged clot times (closed triangles), an effect which was totally abrogated by 

polyP 75 (open circles). The anti-TFPI effect of polyP was concentration-dependent, with an 

optimal range of 67 nM to 1 M. PolyP chains 25 to 75 residues long had potent anti-TFPI 

activity, but short polymers did not. Hydrolysis of polyP by alkaline phosphatase destroyed its 

anti-TFPI effect. These results show that polyP enhances clot formation by preventing TFPI 

inhibition. PolyP is labile in plasma and it also inhibits fibrinolysis. The burst of release of polyP 

from activated platelets may therefore provide a “timed” switch, which could enhance fibrin 

formation and stability, allowing sufficient time for clot formation in response to injury. 

P293 

Rosuvastatin Reduces Platelet Activation in Heart Failure: Role of Nitric 

Oxide Bioavailability 

Andreas Schäfer, Daniela Fraccarollo, Martin Eigenthaler, Stefan Frantz, Ulrich Walter, Georg 

Ertl, Johann Bauersachs; Univ of Würzburg, Würzburg, Germany 

Objectives: Endothelial dysfunction and platelet activation are part of the cardiovascular 

phenotype in congestive heart failure (CHF). We investigated whether HMG-CoA reductase 

inhibition would beneficially modulate vascular nitric oxide (NO) bioavailability and platelet 

activation in experimental CHF. Methods and Results: Chronic myocardial infarction was 

induced by coronary ligation in male Wistar rats. Animals were either treated with placebo or 

the HMG-CoA reductase inhibitor rosuvastatin. After 10 weeks, hemodynamic assessment was 

performed and endothelial function was determined in organ bath studies. NO bioavailability 

was assessed by in vivo platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. 

Markers of platelet degranulation (surface expression of P-selectin and glycoprotein 53) were 

determined as well as the amount of circulating platelet-leukocyte aggregates. Endotheliumdependent, 

acetylcholine-induced vasorelaxation was significantly impaired in aortic rings from 

CHF rats and improved by rosuvastatin. In parallel, in vivo VASP phosphorylation reflecting NO 

bioavailability was significantly attenuated in platelets from CHF rats and normalized by 

rosuvastatin. Platelet activation, which was increased in CHF, was reduced by treatment with 

rosuvastatin. Conclusion: HMG-CoA reductase inhibition improved endothelial function, 

increased systemic NO bioavailability and inhibited exaggerated platelet activation in CHF rats. 

These mechanisms may contribute to the beneficial effects of statin treatment in CHF. 

P294 

Inhibition of Platelet Activation in Diabetic Rats by Chronic Treatment with 

the Guanylyl Cyclase Activator HMR1766 

Andreas Schäfer, Ulrike Flierl, Christian Vogt, Melinda Hemberger, Martin Eigenthaler, Ulrich 

Walter, Georg Ertl, Johann Bauersachs; Univ of Würzburg, Würzburg, Germany 

Background: Diabetes is associated with increased platelet activation contributing to the 

enhanced risk for atherothrombosis and thromboembolism. Under physiological conditions, 

endothelium-derived nitric oxide (NO) inhibits platelet activation by stimulating platelet guanylyl 

cyclase and cGMP. NO bioavailability and platelet sensitivity towards NO is significantly 

decreased in diabetes. We investigated whether chronic treatment of diabetic rats with the 

guanylyl cyclase activator HMR1766 would inhibit in vivo platelet activation. Methods and 

results: Diabetes was induced in male Wistar rats by intravenous injection of streptozotocin. 

Treatment with HMR1766 or placebo was started at day 14 and continued for another 2 weeks. 

Thereafter, whole blood was taken from the inferior vena cava for FACSS analysis during 

terminal anesthesia. Compared to healthy controls, platelets from diabetic rats displayed 

increased fibrinogen binding on activated glycoprotein IIb/IIIa and enhanced surface expression 

of P-selectin indicating in vivo platelet activation. Chronic treatment with HMR1766 reversed 

both changes during diabetes. Furthermore, in vitro stimulation of diabetic platelets with ADP 

resulted in stronger expression of P-selectin than on platelets from control rats. This was 

suppressed by chronic treatment with HMR1766. Chronic treatment of diabetic rats with the 

guanylyl cyclase activator resulted in enhanced phosphorylation of platelet vasodilatorstimulated 

phosphoprotein, an indicator of platelet NO bioavailability and inhibitor of platelet 

activation. Conclusion: Chronic activation of guanylyl cyclase by HMR1766 prevents enhanced 

platelet activation in diabetes and might be a promising option for future pharmaceutical 

treatment/ prevention of cardiovascular complications Downloaded in diabetes. from 

Lysophophatidic Acid Receptors in Phenotypic Modulation of Smooth 

Muscle Cells 

Christopher Vallanat, Zehra Pamuklar, Christopher End, Trevor Dundon, Andrew Morris, 

Jerold Chun, Susan Smyth; Carolina Cardiovascular Biology Cntr, Chapel Hill, NC 

P295 

Lysophosphatidic acid (LPA), the simplest glycerophospholipid, exerts growth-factor like 

activities on many mammalian cells by acting through G-protein coupled receptors (GPCR). To 

date, four GPCR for LPA have been identified and named LPA1–4. Previous studies indicated 

that LPA is a major component in serum responsible for dedifferentiation and proliferation of 

vascular smooth muscle cells (SMC) in culture and that plasma LPA may contribute to 

atherothrombotic vascular disease progression. To identify the roles for specific LPA receptors 

in these responses, we used cultured SMCs from mouse aorta (MASMC). LPA induces ERK1/2, 

p38 MAPK, and Rho activation in MASMCs. In addition, LPA stimulates focal adhesion formation 

and tissue factor activity. By real time PCR analysis, MASMCs express LPA123 and 4. 

Preliminary analysis of MASMCs from LPA 1 and 2-deficient mice reveals reduced early ERK1/2 

and p38MAPK activity in response to LPA in LPA1-null cells. Our results indicate that MASMCs 

will prove to be a useful model system to define LPA effects on vascular cells and suggest that 

LPA1 may partially mediate SMC phenotypic modulation by LPA. 

P296 

Upregulation of Arterial Intima-Enriched (AIE) Genes in Vascular Smooth 

Muscle Cells in Response to Shear and Cyclic Stress–Possible Role in 

Vascular Adaptation to Biomechanical Stress 

Amy Pyle, Bin Li, AmyLynn Teleron, Paul Chang, Raul Guzman, Pampee Young; Vanderbilt 

Univ, Nashville, TN 

The underlying molecular variations which govern the physiological differences between adult 

arteries and veins are not known. To understand these differences, we previously used 

microarray analysis to compare the gene expression profile of murine aorta and vena cava. We 

identified a group of genes designated as the AIE genes which were expressed highly in large 

arteries, but in low or undetectable amounts in large veins. Furthermore, by immunohistochemistry 

and western blot analysis we have confirmed artery-enriched expression of many of 

these genes in human tissue. Most of these genes were previously unknown to be expressed 

in vascular tissue but are well characterized members of the cornified cell envelope, an 

insoluble physical barrier just inside the plasma membrane of terminally differentiated epithelial 

cells, such as skin, where it contributes to the mechanical and barrier properties of the tissue. 

We therefore hypothesized that AIE genes may be important in vascular adaptation to 

biomechanical stress. In order to test this hypothesis, we utilized primary and immortalized 

human vascular smooth muscle cells (SMCs) in culture to determine if shear (5 and 10 dynes) 

and/or cyclic (20% elongation) strain could upregulate AIE gene expression. After exposing 

cells to stress, RNA was harvested for quantitative RT-PCR to assay for changes in gene 

expression levels. Preliminary data using several different aortic primary and immortalized 

SMCs showed a subset of AIE gene transcripts, most notably sciellin and periplakin, increased 

by as much as 8-fold over static controls after exposure to both shear and cyclic strain. The 

transcripts appeared at 12hrs and peaked between 24–48hrs. Furthermore, protein expression 

of sciellin and periplakin in the SMCs was confirmed by immunofluorescent staining. 

Additionally, by immunohistochemistry we detected sciellin and periplakin protein expression 

in the intima of arterialized, but not normal saphenous veins, further implying that they are 

important in vascular response to changes in stress. Our data suggest that sciellin and 

periplakin are positively regulated by biomechanical stress both in vitro and in vivo and thus 

may be novel mediators of the response of vascular tissues to biomechanical stress. 

P297 

Hypoxia and Adenosine Differentially Express Angiogenic Factors in Human 

Microvascular Endothelial Cells 

Sergey Ryzhov, Anna Goldstein, Italo Biaggioni, Igor Feoktistov; Vanderbilt Univ, Nashville, 

TN 

Hypoxia increases extracellular adenosine concentrations and stimulates expression of 

angiogenic factors. Here we tested the hypothesis that adenosine contributes to the effects of 

hypoxia by expressing additional angiogenic factors. We studied the effect of hypoxia on the 

expression of VEGF and IL-8 in human microvascular endothelial cells (HMEC-1). The effects 

of endogenous adenosine were prevented by adding adenosine deaminase (1 U/ml). Hypoxia 

upregulated only VEGF but not IL-8. Incubation of HMEC-1 in 5% O 2 for 1 hour increased VEGF 

mRNA by 4-fold, but decreased IL-8 mRNA by 41%. VEGF concentrations in the media of 

HMEC-1 incubated for 12 hours increased from 47012 pg/ml during normoxia, to 50817 

pg/ml in 10% O 2, 86179 pg/ml in 5% O 2, 128758 pg/ml in 2%O 2 and 1603122 pg/ml 

in anoxia, whereas IL-8 concentrations did not change significantly. The stable adenosine 

analog NECA (10 -4 M) increased IL-8 concentration in media, from 802 pg/ml to 35649 

pg/ml in HMEC-1 incubated in normoxia and from 604 pg/ml to 31146 pg/ml in 5% O 2.We 

conclude that adenosine contributes to the production of angiogenic factors (e.g., IL-8) not 

induced by hypoxia alone in HMEC-1. 

Inhibition of Experimental Abdominal Aortic Aneurysm Progression by 

Nifedipine 

Naruya Tomita, Keita Yamasaki, Yasuo Kunugiza, Keiko Izawa, Mariana K Osako, Hiromi 

Koike, Toshio Ogihara, Ryuichi Morishita; Osaka Univ Graduate Sch of Medicine, Suita, 

Japan 

To follow up the abdominal aortic aneurysm (AAA) we must control blood pressure, first of all. 

http://atvb.ahajournals.org/ Recently, by twoguest phenomena, on June inflammation 29, 2013 and matrix degradation are thought to contribute to 


P298

the progression of AAA. Nifedipine, one of the calcium channel blocker, is most often used for 

the control of blood pressure. However, several effects beyond blood pressure lowering effect 

of calcium channel blocker attract a lot of interests, recently. In this study we examined how 

Nifedipine contribute to the inhibition of AAA progression. AAA was induced in rats by transient 

aortic perfusion with elastase. Then, Nifedipine (10 mg/kg/day) and placebo were given to rats 

by osmotic mini-pump. At 7 and 14 days after starting the treatments with drugs the size of 

AAA was assessed by ultrasound, and both blood pressure and heart rate were also measured 

at day 7 and 14. Then, we examined the mechanism of this inhibitory effect of Nifedipine on 

AAA using human vascular smooth muscle cells (VSMCs). Especially, we focused on one of the 

transcription factors, NF-kB and matrix metalloproteinase-2 (MMP-2). To test effects of 

Nifedipine on NF-kB activity we used luciferase reporter assay, and on MMP-2 activity we used 

zymography. Treatment with Nifedipine resulted in a significant inhibition of the progression of 

AAA such as aneurysmal dilation at 7 and 14 days compared to the control treated with placebo 

(Day 7; Placebo: 2.98 0.71 mm, Nifedipine: 2.37 0.64 mm, p0.05 and Day 14; Placebo: 

3.28 0.98 mm, Nifedipine: 2.41 0.17 mm, p0.05). Both Nifedipine and placebo did not 

change blood pressure and heart rate significantly. Nifedipine (0.1 and 1 mM) significantly 

suppressed luciferase activity in VSMCs that was induced by angiotensin II (10 -6M) (p0.01). 

Moreover, this inhibitory effect was dose-dependent. Futhermore, Nifedipine (1 mM) inhibited 

MMP-2 activity as assessed by zymography. Taken together, Nifedipine can inhibit the 

progression of experimental AAA possibly through suppression of NFkB activity and MMP-2 

activity. Calcium channel blocker, Nifedipine, may have protective effects to AAA beyond blood 

pressure lowering effects. 

P299 

Transient High-Flow Stimulation Programs Endothelial Cell Proliferation 

Misa Yamauchi, Masato Takahashi, AKITA Univ Sch of Medicine, Akita, Japan; Hiroshi 

Nanjo, AKITA Univ Hosp, Akita, Japan; Mikio Kobayashi, Kouichi Kawamura, AKITA Univ Sch 

of Medicine, Akita, Japan; Eiketsu Sho, Stanford Univ Sch of Medicine, Stanford, CA; 

Hirotake Masuda; AKITA Univ Sch of Medicine, Akita, Japan 

[Objective] Endothelial cells (ECs) are activated in response to high-flow. Our previous studies 

using arterio-venous fistula (AVF) have demonstrated that high-flow induces an early and rapid 

proliferation of ECs. We reported ECs, which had been stimulated by high-flow loading, could 

proliferate in a situation without the influence of high-flow in vivo and ex vivo experiments. That 

is to say that, they are programmed to proliferate when they are once stimulated by high-flow 

loading. However, these experiments lacked individual cell follow-up. To ensure the presence 

of these programmed ECs, we tried in vitro study. [Methods] First, we induced high-flow in the 

rabbit common carotid artery (CCA) by using AVF for 1.5 days. Then, the segments of the left 

CCAs were resected after bromodeoxyuridine (BrdU) administration. Cells were isolated after 

the vessels were treated with collagenase. Isolated cells obtained by this procedure were in the 

range of 5000 - 15000 cells in each resected CCA. Some cells were clustered in various sizes 

consisting of up to about 60 cells. The isolated cells were cultured at 37 °C in an atmosphere 

of5%CO 2. The cultures were maintained for 1.5 days (for observation of BrdU-labeled ECs) 

and 3.5 days (for evaluation of cell growth). The attached cells were observed with 

phase-contrast microscopy. BrdU-labeled ECs were detected immunohistochemically. Samples 

from animals without an AVF operation were used as control. [Results] Immunohistochemistry 

revealed that almost all the cultured cells were ECs expressing CD31. Number of cells in 

clusters increased from 0.5-day to 2.0-day of culture nearly to 14-fold as much as those of 

cells at 0.5-day of culture. After 2.5-day of culture, number of cells in clusters remained almost 

unchanged. At 1.5-day of culture, BrdU-labeled ECs appeared in 16.7 9.6 %. Isolated cells 

from normal-flow animals were in the range of 2000 - 3000 cells. CD31 positive cultured ECs 

did not increase at 1.5-day of culture and disappeared at 2.5-day. [Conclusions] From transient 

high-flow stimulated CCAs, actively and automatically proliferated ECs could be cultured. They 

are considered to correspond with ECs, which are known to proliferate in vivo in the high-flow 

stimulated arteries. 


predominant myosin II heavy chain from SM1 and SM2 (in the contractile phenotype) to 

nonmuscle myosin (in the proliferative phenotype). Previously, we showed that NM-A heavy 

chain knockdown using siRNA inhibited focal adhesion formation and disturbed stress filament 

formation in cultured rat aortic smooth muscle cells (RASMC). In this study, we examined the 

function of NM-A in proliferation, migration and adhesion of RASMC. METHODS AND RESULTS: 

Transfection of RASMC with small interfering RNA (siRNA) duplexes directed against NM-A 

heavy chain markedly inhibited NM-A protein expression. siRNA transfection disturbed NM-A 

thick filament formation resulting in a disorganized microfilament network with extensive 

clumping and this was associated with a decreased number of focal adhesions (as measured 

by vinculin staining) when cells were treated with thrombin or maintained in 10% FBS. NM-A 

siRNA reduced RASMC adhesion to non-coated tissue culture plates by 27% but had no effect 

on apoptosis as measured by TUNEL staining. siRNA to NM-A inhibited random migration and 

also reduced directed migration to PDGF-BB by 69%. NM-A heavy chain siRNA partially 

inhibited SMC proliferative responses to serum. Cell number increased by 205% in nontransfected 

RASMC exposed to 10% FBS for 4 days. In contrast, under similar conditions cell 

number increased by 144% in RASMC transfected with NM-A heavy chain siRNA. CONCLU- 

SIONS: NM-A heavy chain knockdown inhibited RASMC proliferation, migration and adhesion, 

but had no effect on apoptosis. 

P301 

G Protein-Coupled Receptor Kinase 5 Diminishes Injury-Induced Vascular 

Neointima Formation 

Rui-Hai Zhou, Cntr for Translational Medicine, Thomas Jefferson Univeristy, Philadelphia, 

PA; Jihee Kim, Dept of Medicine, Duke Univ Med Cntr, Durham, NC; Matthew Kuhn, Cntr for 

Translational Medicine, Thomas Jefferson Univeristy, Philadelphia, PA; Walter J Koch, Cntr 

for Translational Medicine,Thomas Jefferson Univ, Philadelphia, PA; Andrea D Eckhart; Cntr 

for Translational Medicine, Thomas Jefferson Univeristy, Philadelphia, PA 

Alterations in signaling through G protein-coupled receptors (GPCRs) are involved in cardiovascular 

diseases such as heart failure, hypertension, and postintervention restenosis (PIRS). 

GPCRs are regulated by a family of GPCR kinases (GRKs) through phosphorylation-dependent 

and -independent mechanisms. GRK5 is present in vascular smooth muscle (VSMC) and 

elevated levels have been associated with heart failure and hypertension. However, it is not 

known whether GRK5 is involved in PIRS, an event resulting from aberrant proliferation and 

migration of VSMCs leading to neointima formation. Therefore in this study we investigated the 

role of GRK5 in the VSMC activation in response to vascular injury using transgenic (Tg) mice 

with VSMC-specific overexpression of GRK5 compared with non-transgenic littermate control 

(NLC). Our results revealed that in VSMC GRK5 overexpression functionally uncoupled the 

receptors of thrombin and lysophosphatidic acid (LPA), mitogens known to contribute to 

neointima formation, as well as that of 1-adrenergic receptors (ARs). Consistently, the 

proliferation of GRK5-Tg VSMCs in response to LPA and thrombin and the migration to LPA 

were decreased when compared with those of NLC VSMCs. In accordance, the molecular 

signaling pathways responsible for these biological processes were affected by GRK5. ERK1/2 

and Akt signaling activation in response to thrombin and/or LPA were diminished in GRK5-Tg 

VSMCs as compared to those in NLC VSMCs. To test our hypothesis that GRK5 is inhibitory to 

post-injury neointima formation we performed in vivo studies using mouse left carotid artery 

(LCA) injury model and delivered GRK5 to the vascular wall by infecting vessels with adenoviral 

GRK5 (adeno-GRK5) and adenoviral LacZ (adeno-LacZ) as control at the time of injury. Four 

weeks after injury, neointima formation of the LCA was reduced dramatically as measured by 

the area of luminal stenosis of 963% (n8) in adeno-LacZ mice versus 4212% (n8) in 

adeno-GRK5 mice (P0.002). Thus, these data strongly suggest that GRK5 plays a critical role 

in curbing post-injury neointima formation and may become an effective anti-restenosis 

approach to the prevention and treatment of PIRS. 

Post-Transcriptional Regulation of AP-1 Family Expression in Human 

Endothelial Cells Stimulated with Thrombin: Variations on a Theme 

Douglas I Schmid, Huimiao Jiang, Andrew S Weyrich, Guy A Zimmerman, Larry W Kraiss; 

Univ of Utah, Salt Lake City, UT 

Non-Muscle Myosin-A Functions in Cell Proliferation, Migration, and 

Adhesion, but not Apoptosis in Cultured RASMC 

P300 

Introduction JunB, an AP-1 transcription factor, is upregulated within minutes in thrombinstimulated 

HUVEC, primarily via enhanced translation of pre-existing JunB mRNA (ATVB 2002; 

22:878). We asked whether other AP-1 members were subject to translational regulation. 

Methods The expression of c-Jun, c-Fos and FosB (and JunB) in HUVEC was studied over a 

four-hour period after thrombin stimulation using immunoblotting, PCR, polysome profiling and 

EMSA. Results FosB protein, undetectable at baseline, was readily apparent at one hour; FosB 

mRNA was upregulated 20-fold by 15 minutes. Polysome profiles revealed redistribution of 

FosB mRNA from the inefficiently translated fraction to the efficiently translated fraction, 

suggestive of added translational control. Protein levels of c-Jun and c-Fos were unchanged 

following thrombin stimulation as were c-Jun mRNA levels. c-Fos mRNA was 16-fold higher at 

15 and 30 minutes before returning to baseline. Polysome profiles showed no change in the 

distribution of c-Fos and c-Jun between the monosome and polysome fractions; the overall 

amount of c-Fos mRNA present in the ribosomal fraction did not increase despite the increase 

in total c-Fos mRNA. Consistent with our previous results, JunB protein was upregulated within 

15–30 minutes with little change in mRNA level. JunB mRNA was redistributed from the 

monosome to polysome fraction. EMSA revealed increased JunB and FosB in DNA-bound AP-1 

complexes after thrombin stimulation but no change in c-Jun or c-Fos. Conclusion Several 

post-transcriptional mechanisms regulate expression of AP-1 transcription factors in thrombintreated 

HUVEC. JunB is primarily under translational control. FosB induction occurs as a result 

Renyi Zhao, Alok Pathak, George Stouffer; Univ of North Carolina at Chapel Hill, Chapel Hill, of both transcriptional activation and enhanced translation of its mRNA. An undefined 

NC 

mechanism appears to block translation of newly transcribed c-Fos mRNA preventing a change 

in c-Fos protein. Neither c-Jun mRNA nor protein is affected by thrombin, consistent with data 

BACKGROUND: Smooth muscle cells undergo phenotypic modulation during development and that nuclear c-Jun activity is regulated by phosphorylation. These data illustrate the regulatory 

at sites of vascular injury. Phenotypic modulation Downloaded is accompanied from 

byhttp://atvb.ahajournals.org/ a change in the complexity by and guest versatility on June of AP-129, transcription 2013 factors in EC. 


P302

E-106 Vol 25, No 5 May 2005 

P303 

Human Monocyte-Derived Macrophages Synthesize Collagen Type VI to 

Support Cell-Cell and Cell-Matrix Interactions 

Michael Schnoor, Paul Cullen, Katrin Stolle, Jürgen Rauterberg, Stefan Lorkowski; 

Leibniz-Institute for Arteriosclerosis Rsch, Münster, Germany 

Several collagens are expressed in every section of the arterial wall, mainly by smooth muscle 

cells. Due to the production of several proteases, macrophages are mainly considered as being 

of proteolytic nature. We recently showed that macrophages produce collagen type VIII in vitro 

and in the atherosclerotic plaque, indicating that macrophages may contribute to atherosclerotic 

plaque stability. Following from this result, we screened human monocyte-derived 

macrophages in vitro for the expression of other collagens using RT-PCR with mRNA-specific 

primers. Surprisingly, we found that both monocytes and macrophages as well as the 

monocytic leukemia cell line THP-1 express mRNAs for a wide variety of collagens. We selected 

collagen type VI for further studies because this type of collagen is strongly expressed by 

macrophages and has been shown to anchor cells and extracellular matrix components, 

suggesting that it may help to stabilize the atherosclerotic plaque. Western blot analysis 

showed that secretion of collagen type VI by macrophages occurs in a time-dependent manner. 

While in the medium of freshly isolated monocytes no protein could be detected, the amount 

of secreted collagen type VI increased during differentiation to mature macrophages. The 

secretion occurs via a classical pathway and is upregulated by TGF- and PDGF. Adhesion 

assays revealed that the function of collagen type VI for the macrophage in vitro may be 

1-integrin-mediated cell adhesion. In addition, confocal microscopic images showed that 

macrophages are able to bind collagen type VI on their cell surface probably to mediate cell-cell 

contacts of macrophages. The results presented here support a new role for macrophages in 

the arterial wall. The classical hypothesis that the macrophage has only a degradative function 

in the plaque must be reconsidered due to the ability of the macrophage to synthesize at least 

two different types of collagens. We conclude that depending on the physiological state, 

macrophages may also help to stabilize atherosclerotic plaques. 

P304 

Micro vs Macrovascular Extracellular Matrix Gene Expression in Type 2 

Diabetes: Role of Endothelin-1 

Weiwei Song, Alex K Harris, Kamakshi Sachidanandam, Jim R Hutchinson, Vera 

Portik-Dobos, Adviye Ergul; Univ of Georgia, Augusta, GA 

Introduction: Hyperglycemia-induced change in vascular wall structure contribute to the 

pathogenesis of diabetic microvascular and macrovascular complications. Matrix metalloproteinases 

(MMP), a family of proteolytic enzymes that degrade extracellular matrix (ECM) 

proteins, are essential for vascular remodeling. Endothelin-1 (ET-1), a vasoactive peptide that 

is chronically elevated in diabetes, promotes VSMC growth and collagen deposition. However, 

ET-1 regulation of vascular structure and MMP expression in Type 2 diabetes remained 

unknown. In this study, we investigated the effect of diabetes and/or ET-1 on relative gene 

expression in macro and micro circulation. Methods: Aorta and mesenteric artery samples 

were isolated from control wistar, Type 2 diabetic Goto-Kakizaki (GK) rats and GK rats treated 

with ET A antagonist ABT-627 for 4 weeks. All oligonucleotide primer sets (MMP2, MMP9, 

MT1-MMP, fibronectin, procollagen, c-fos and c-jun.) were designed to yield amplicon lengths 

100bp-200bp. The real-time PCR was performed in a SmartCycler II by using SYBR® Green 

PCR Master Mix. All reactions were performed in triplicate. Glyceraldehyde-3-phosphate 

dehydrogenase (GAPDH) primers were used to normalize samples. Results: Table Conclusions: 

These results suggest that the expression of both matrix protein and matrix degrading 

MMP genes are altered in macro- and microvascular beds in Type 2 diabetes. ET A antagonism 

restores the changes in gene expression in the mesenteric bed but not aorta suggesting that 

ET-1 differentially regulates microvascular gene expression in Type 2 diabetes. 

Aorta Mesenteric artery 

GK vs GK 

ABT627 C vs GK 

Fold Change; Fold Change; 

P Value 

P Value 

GK vs GK 

ABT627 

Fold Change; 

P Value 

MMP-2 

CvsGK 

Fold Change; 

P Value 

110 P0.0001 No change 111 P0.001 23 P0.05 

MMP-9 13 P0.05 No change 110 P0.05 22 P0.05 

MT1-MMP 16 P0.001 No change 17 P0.001 27 P0.0001 

COL1A1 127 P0.001 No change 17 P0.05 28 P0.0001 

Fibronectin 11.23 P0.05 21.25 P0.05 19 P0.05 23 P0.05 

c-Fos 23 P0.001 12 P0.05 23 P0.0001 12 P0.05 

c-Jun 13 P0.05 No change 13 P0.05 25 P0.0001 

P305 

A Role for Endothelial A 2B Adenosine Receptors in Inflammation Associated 

with Type 2 Diabetes 

Suseela Srinivasan, Univ of virginia, Charlottesville, VA; Robert Figler, Adenosine 

Therapeutics LLC, Charlottesville, VA; Nicole Ferger, Shankar Srinivasan, David Bolick, Joel 

Linden, Catherine C Hedrick; Univ of virginia, Charlottesville, VA 

There is a growing body of evidence to suggest that inflammation underlies the pathogenesis 

of Type 2 diabetes. The B6.Cg-m /Leprdb /J mouse (db/db) is an established genetic mouse 

model of Type 2 diabetes. We found a 190-fold elevation of A2B adenosine receptor (A2BAR) mRNA in aortic endothelial cells (EC) isolated from diabetic db/db mice as compared to 

non-diabetic, control C57BL/6 mice. The expression of mRNA for the pro-inflammatory 

chemokines, IL-6 and KC, were also increased by several-fold in db/db EC. Given the increased 

level of transcription of the A2BAR gene in the db/db mouse we sought to identify a possible role 

for adenosine in mediating inflammatory processes in Type 2 diabetes. Endothelial cells were 

freshly isolated from murine db/db and B6 aortae. Downloaded Mouse aortic EC (MAEC) from 

were treated for 36h 



with 1nM-10M N-ethylcarboxamidoadenosine (NECA), a non-selective pharmacological 

agonist of the 4 four adenosine receptor subtypes. Media and cell lysates were collected and 

assayed for IL-6 and KC protein and mRNA. quantification. IL-6 and KC secretion into media 

in response to NECA was dose-dependent with a half-maximal response occurring at a dose 

of 100 30nM NECA for IL-6 and 72 nM NECA for KC. IL-6 mRNA levels significantly increased 

as early as within 5 minutes of after of adding 100nM NECA, peaked at 2 hr (35-fold) and then 

decreased rapidly. KC mRNA levels significantly increased within 30 minutes after adding 

100nM NECA, and also peaked at 2 hr (2-fold). IL-6 and KC proteins were both detectable in 

cell supernatants at 2 hours and secretion of both chemokines increased continuously through 

36 hours. Treatment of aortic EC with an array of adenosine receptor-specific agonists 

indicates that NECA-induced IL-6 and KC secretion from endothelial cells is mediated, at least 

partially, by the A 2BAR. These new findings implicate the A 2BAR as a pro-inflammatory receptor 

in vascular endothelium that is substantially induced in diabetes. . 

Macrophage - Vascular Smooth Muscle Cell Cooperation Leads to 

Amplification of the MT1-MMP - Pro-MMP-2 Proteolytic Cascade 

Philipp Stawowy, Heike Meyborg, Dietger Stibenz, Núbia Borges Pereira Stawowy, Mattias 

Roser, Usan Thanabalasingam, Deutsches Herzzentrum Berlin, Berlin, Germany; John P 

Veinot, Michel Chrétien, Ottawa Health Rsch Institute, Ottawa, Canada; Nabil G Seidah, 

Clinical Rsch Institute Montréal, Montréal, Canada; Eckart Fleck, Kristof Graf; Deutsches 

Herzzentrum Berlin, Berlin, Germany 

P306 

Expression of MMPs by macrophages is a key determinant of plaque stability. Macrophages 

express soluble MMPs and membrane-bound MT-MMPs. Whereas soluble MMPs are released 

as zymogens and are than activated by other proteases, MT-MMPs are activated intracellular 

by furin-like proprotein convertases (PCs) and than anchored to the cell surface as active 

enzymes. Activation of pro-MMP-2 depends on the formation of an MT-MMP/TIMP-2/MMP-2 

complex. Macrophages express mostly MMP-9, but little MMP-2. MMP-2 is constitutively 

synthesized by VSMCs and potentially stored in the ECM. The aim of this study was to 

investigate the activation of MMPs in macrophages, as well as the role of PCs (namely furin and 

PC5) in macrophages. Macrophages maturation of monocytic THP-1 cells (PMA 100 nM; 48h) 

was accompanied by increased MT1-MMP, as well as its activating enzymes furin and PC5. 

Inhibition of furin/PC5 with the pharmacological furin-like PC inhibitor dec-CMK inhibited 

MT1-MMP activation, but did not affect its sorting/routing to the cell surface. Monocytes 

synthesized little MMP-2 and MMP-9 and stimulation with TNF or LPS enhanced only MMP-9. 

In contrast, macrophages abundantly expressed MMP-9 and some pro-MMP-2 activation was 

found in these MT1-MMP competent cells. However, this could not be further enhanced by 

stimulation with the inflammatory mediators. Culturing of macrophages in medium derived 

from serum-starved VSMCs resulted in an enhanced pro-MMP-2 activation, whereas no 

activation was found when monocytes were used. This demonstrates, that pro-MMP-2 

activation from VSMCs dependents on macrophage MT1-MMP. Activation of VSMC pro-MMP-2 

by macrophage MT1-MMP was inhibited by dec-CMK or a furin-silencing siRNA, as well as the 

MMP inhibitors GM6001 or an excess of TIMP-2. Immunohistochemistry demonstrated the 

colocalization of furin and PC5 with MT1-MMP in CD68 positive macrophages in human 

endarterectomy lesions and FACS analysis revealed the presence of furin, PC5 and MT1-MMP 

on human peripheral blood mononuclear cells obtained from healthy volunteers. Conclusion: 

The present study demonstrates, that furin/PC5 are central regulators of an MT1-MMP - 

MMP-2 proteolytic amplification cascade, which requires interaction of macrophages and 

VSMCs. 

PJ34 Enhanced Spinal Cord Tissue Viability and Differential Gene 

Expression in a Murine Model of Thoracic Aortic Reperfusion Injury 

P307 

David H Stone, Mark F Conrad, Hassan Al-Badawi, Fateh Entabi, Michael C Stoner, Richard 

P Cambria, Michael T Watkins; Massachusetts General Hosp, Boston, MA 

Introduction: The inflammatory response associated with thoracic aortic ischemia reperfusion 

(TAR) has been implicated as a likely source of morbidity and mortality. Spinal cord ischemia 

(SCI), remains among the most dreaded complications of complex aortic reconstruction. These 

experiments were designed to determine whether PJ34, a novel Poly-ADP Ribose Polymerase 

Inhibitor, could ameliorate the sequelae of TAR by evaluating spinal cord tissue viability using 

a murine model of thoracic aortic reperfusion injury. In addition, experiments were performed 

to determine transcriptional differences in PJ34 treated and untreated spinal cord tissue 

exposed to TAR to identify potential mediators of injury versus tissue protection. Methods: 

Twenty-seven 129S1/SvImj mice were subjected to TAR followed by 48 hours of normothermic 

reperfusion. The thoracic aorta was clamped at the level of the left subclavian artery for 11 

minutes. Animals were treated with either normal saline Control (UC), (n11) 0.5 ml NS IP) or 

PJ34 (PJ) (n11) 10 mg/kg IP both 1 hour before and after TAR. Sham (SH) mice (n5) 

underwent median sternotomy without TAR. After 48 hours, mice were euthanized and spinal 

cord viability index (MTT assay) was measured. Expression differences among sham, untreated 

control, and PJ34 treated mice were determined using microarray technology. COX-2 

expression differences were measured using Real time RT-PCR. Statistical analysis was 

performed using unpaired t-test. Results: PJ34 improved spinal cord tissue viability following 

TAR (UC: 53.1 6.3, P: 73.5 4.1 * * , p0.01). Transcriptional interrogation revealed 

numerous genes, which were differentially expressed following TAR. COX-2 was induced by 

TAR but was unaffected by PJ34 administration (UC: 1.72 0.29, PJ: 1.66 0.13 pNS). 

Data is depicted as fold change versus sham. Conclusions: PJ34 administration enhances 

spinal cord mitochondrial activity following TAR. COX-2 expression is induced by TAR, however 

was not ameliorated by PJ34 administration. These findings suggest that PJ34 enhances spinal 

cord viability via COX-2 independent mechanisms. This data also identifies COX-2 inhibition as 

a potential by alternative guest on novel June therapeutic 29, 2013 adjunct for thoracoabdominal aortic reconstruction.

Diverse Effects of T-type Voltage-Dependent Calcium Channels on 

Rho/rho-Kinase Pathway in Vascular Smooth Muscle Cells 

P308 

Naoki Sugano, Koichi Hayashi, Shu Wakino, Takeshi Kanda, Ichiro Takamatsu, Satoru 

Tatematsu, Koichiro Homma, Kyoko Yoshioka, Kazuhiro Hasegawa, Keio Univ, Tokyo, Japan; 

Goro Tokudome, Tatsuo Hosoya, Jikei Univ Sch of Medicine, Tokyo, Japan; Takao Saruta; 

Keio Univ, Tokyo, Japan 

(Backgrounds and Objectives) We have recently demonstrated that T-type calcium channels 

prevail not only in the renal afferent arteriole but also in the efferent arteriole, and that T-type 

calcium channel blockers (T-CCBs) protect glomeruli from systemic hypertension by regulating 

vascular tone of renal arterioles. On the other hand, Rho/Rho-kinase pathway also regulates 

intraglomerular pressure, which suggested the link between the two regulatory systems. We 

therefore examined whether T-CCBs affected Rho/Rho-kinase pathway. (Methods) Vascular 

smooth muscle cells (VSMCs) derived from Sprague-Dawley rats’ aorta were used. After 

30-min pretreatment with T-CCBs mibefradil and efonidipine, VSMCs were stimulated by 

angiotensin (Ang) II or platelet-derived growth factor BB (PDGF-BB) Six hours after the 

stimulation, cells were collected and Rho-kinase activity was assessed by immunoblotting 

using antibody against phospho-MYPT, a substrate of Rho-kinase. Activation of Rho/Rho-kinase 

pathway was also assessed by pull-down assay which detected GTP-bound active Rho. 

(Results) Both mibefradil and efonidipine suppressed Rho-kinase activity stimulated by Ang II, 

and mibefradil, a more specific T-CCB, blocked Rho kinase activity more potently than 

efonidipine. Both drugs also blocked GTP-GDP exchange reaction of Rho. On the other hand, 

neither drug inhibited Rho-kinase activity stimulated by PDGF-BB.(Conclusion) This study 

demonstrates that T-CCBs suppress Rho/Rho-kinase pathway, suggesting a more important 

role of T-type than L-type channels in this regulation. Although both Ang II and PDGF activate 

Rho-kinase, Ang II requires T-type Ca channels in its activation. In contrast, PDGF could 

enhance Rho-kinase activity without participation of these channels. This study suggests a 

novel function of T-type channels to block Rho/Rho kinase activation, probably through its 

effects on G proteins. 

P309 

Enhanced Susceptibility to Doxorubicin-Induced Heart Failure in 

Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1) Deficient Mice 

Suman Tandon, Kerry S Russell, Joseph A Madri; Yale Univ Sch of Medicine, North Haven, 

CT 

Endothelial cell-cardiomyocyte interactions are crucial for preservation of normal cardiac 

function. Loss of cardiac endothelium has been shown to have negative effects on myocyte 

contractility. PECAM-1 (CD31) is expressed on endothelial cells and is involved in the 

maintenance of endothelial cell function and survival. We investigated whether the absence of 

CD31 in the vasculature is associated with enhanced susceptibility to cardiac dysfunction in 

response to Doxorubicin. C57BL/6 wild type (WT) and PECAM-1 deficient (KO) mice were 

injected with Doxorubicin (10mg/Kg). Cardiac function was assessed by 2D M-mode 

echocardiography at baseline and at 1 and 2 weeks. Histopathology, immunohistochemistry 

and confocal microscopy were performed at 2 weeks. In vivo permeability studies were done 

1, 6, 24 and 48 hours after Doxorubicin injection. Immortalized CD31 KO and CD31 

reconstituted mouse lung endothelial cells were used for evaluating cell death. At baseline, the 

average fractional shortening (FS) was 52% for both the WT and KO mice. Following 

Doxorubicin injection, FS declined to 41% at 1 week (p0.004) and there was a further decline 

to 31% at 2 weeks (p0.0001) in the KO mice, whereas, it was preserved in the WT mice. 

Significant differences in FS were noted between the WT mice and KO mice at both 1 week 

(p0.003) and 2 weeks (p0.00005). Histology was consistent with Doxorubicin toxicity, 

characterized by cytoplasmic vacuolization and no significant inflammation or fibrosis was seen 

in both groups. There were no differences in TUNEL labeling between the WT and the KO 

cardiomyocytes. Similar amounts of Doxorubicin cumulated in the hearts of KO and WT mice. 

No differences in organ permeability to Evans Blue were noted. Light microscopy revealed a 

Doxorubicin dose-dependent increase in the number of dead cells in the KO cultures. 

Annexin-FACS showed a significant increase in apoptosis in CD31 KO cells on treatment with 

500ng/ml Doxorubicin for 48 hours. Since PECAM-1 signaling may impact anti-apoptotic 

pathways as well as responsiveness to oxidant stress, it is possible that dysregulation of 

PECAM-1 mediated endothelial cell function in the KO mice leads to impaired cardiac 

contractility in response to Doxorubicin. 

P310 

Inactivation of Src Family Tyrosine Kinases by Reactive Oxygen Species in 

Vivo 

Hua Tang, Qin Hao, Brad Low; Univ of Texas Health Cntr at Tyler, Tyler, TX 

Reactive oxygen species (ROS) are related to the development of cardiovascular diseases 

including atherosclerosis. Failure of clinical trials of antioxidants prompts us to question 

whether ROS are deleterious, protective or both. Here, we report a novel finding that ROS 

inactivate Src family tyrosine kinases in primary human endothelial cells (ECs) and protect the 

cells from expression of adhesion molecule by cytokines. We found that H2O2 markedly 

inactivated temporally and spatially Src family kinases, the central kinases in cell signaling. 

However, H2O2 did not affect Src kinases activities in vitro. Furthermore, we found that 

oxidation and inactivation of receptor protein tyrosine phosphatases such as RPTP and the 

subsequent phosphorylation of Src at Tyr-527 were involved in the inactivation of Src family 

kinases by H2O2. Finally, pretreatment of ECs with H2O2 abolished the cell responses to growth 

factors and markedly suppressed the expression of vascular cell adhesion molecule-1 by 

thrombin and tumor necrosis factor. Thus, we showed for the first time that ROS inactivated 

Src family kinases through the oxidation/inhibition of RPTPs, downregulated cell signaling, and 

protected ECs from expression of adhesion molecule by cytokines. Thus, ROS can be protective 

as an inhibitor of endothelial cell inflammatory activation. Downloaded from 

WITHDRAWN 

P311 

P312 

Cyclic Strain-Induced Mmp-9 Activity in Bovine Aortic Endothelial Cells is 

Mediated via a Notch/cbf-1 Dependent Pathway 

Nicholas von Offenberg Sweeney, JP Cullen, Univ of Rochester Med Cntr, Rochester, NY; PA 

Cahill, Vascular Health Rsch Cntr, Dcu, Ireland; EM Redmond; Univ of Rochester Med Cntr, 

Rochester, NY 

Mechanical forces associated with blood flow play an important role in dictating vascular cell 

functions and phenotype. Notch proteins are important signaling receptors that regulate 

intercellular communication and direct cell fate decisions. As they are robustly expressed in the 

vasculature and are upregulated in response to vascular injury, they are believed to play a role 

in angiogenesis and vascular remodeling. Matrix metalloproteinases (MMPs), a family of 

extracellular matrix-degrading endopeptidases, are produced by vascular cells and have also 

been implicated in vascular remodeling. The aim of our study was (i) to examine the regulation 

of Notch receptors and MMPs in endothelial cells in response to cyclic strain and (ii) to 

investigate the regulation of MMPs by the Notch signaling pathway. Methods: The Flexercell 

system was used to expose bovine aortic endothelial cells (BAEC) to physiological levels of 

cyclic strain; (0–10% strain, 60 cycles/min, 0–24h). MMP-2 and MMP-9 activity in conditioned 

media was determined by gelatin zymography. BAEC were transfected using Lipofectamine 

with the intracellular fragment of Notch-1 (N1-IC), Notch-3 (N3-IC), Notch-4 (N4-IC), or with 

inhibitory constructs (RPMS-1 and R218H) for the CBF-1 transcription factor. Results: Following 

exposure of BAEC to 5% or 10% cyclic strain MMP-9 activity was increased 1.60.08 fold and 

2.10.5 fold, respectively, with no observable effect on MMP-2 activity. In parallel 

experiments, 5% strain increased the expression of Notch-1 and Notch-4, 1.80.16 fold and 

1.740.26 fold, respectively, while 10% strain increased expression of Notch-4 only 

(2.30.3fold). Cyclic strain had no effect on Notch-3. Transfection of BAEC with N1-IC, N3-IC 

or N4-IC had no significant effect on MMP-2/-9 activity under static conditions. While 

overexpression of either RPMS-1 or R218H had no effect on MMP-2/-9 activity under static 

conditions, both attenuated the strain-induced increase in MMP-9 activity. Conclusions: This 

study demonstrates that cyclic strain selectively stimulates MMPs and modulates the 

expression of specific Notch receptors in BAEC. Moreover, cyclic strain-induced MMP-9 activity 

is mediated, at least in part, via a Notch/CBF-1 dependent pathway. 

Hedgehog Signaling in the Retinal Vasculature 




Tony Walshe, Vascular Health Rsch Cntr, Dublin, Ireland; Lorna Cryan, Colm O’Brien, 

Institute of Ophthalmology,, Dublin, Ireland; Paul Cahill; Vascular Health Rsch Cntr, Dublin, 

Ireland 

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Abnormalities in retinal blood flow are a precursor of remodeling of the retinal vasculature. The 

Hedgehog (hh) signaling pathway is a highly conserved mechanism of cellular signaling 

involved in cell fate decisions. Using a perfused transcapillary coculture of bovine retinal 

endothelial cells (BRECs) and bovine retinal pericytes (BRPs), we examined the role of hh in 

controlling the proliferative and apoptotic profile of each cell type in vitro and determined the 

expression of hh components in normal and glaucomatous human eyes. Cocultures of BRECs 

with BRPs were exposed to low and high pulsatile flow rates for 72 hours (low: 0.3 mls/min; 

high: 24 mls/min) before cell proliferation and apoptosis was determined by FACs analysis, real 

time PCR and immunoblotting. Human eyes were examined by immunohistological staining and 

RT-PCR analysis. FACs analysis of BRP exposed to high pulsatile flow revealed a significant 

increase in apoptosis and a decrease in proliferation compared to cells exposed to low flow. 

The expression levels of pro-apoptotic bax were significantly increased in cells exposed to high 

flow with a concomitant decrease in the anti-apoptotic marker, Bcl-2. High flow reduced BRP 

Ptc and Shh protein expression and Ptc, Shh, and Gli2 mRNA levels, whereas Ihh protein 

expression remained unchanged. In contrast, BREC apoptosis but not proliferation was reduced 

under high flow conditions. High flow also decreased BREC bax mRNA and protein expression, 

whereas bcl-2 and bcl-xl expression was increased in these cells concomitant with an increase 

in the expression of Ptc, Ihh, Shh, Smo and Gli2 mRNA and protein levels. Moreover, the 

anti-apoptotic effect of high flow was significantly reduced in BRECs following hh inhibition with 

cyclopamine. Immunohistological staining for Shh and Ptc was negative in both normal & 

glaucomatous eyes, while Ihh was present in all normals, but not in glaucomas. Ihh mRNA 

levels were significantly increased in normal eyes when compared to glaucomas. Shh and Ptc 

mRNA levels were present in these eyes, as determined by RT-PCR, with no difference between 

groups. These results demonstrate the potential importance of hh signalling within the retinal 

microvasculature and the differential modulation by changes in pulsatile flow in vitro. 

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Conditional Deletion of Cyclooxygenase -2 in Endothelial Cells in Vivo 

Dairong Wang, yan cheng, yiqun Hui, Hannah NewBorn, Wenliang Song, Garret A FitzGerald; 

Univ of Pennsylvania, Philadelphia, PA 

Background: Selective inhibitors of cyclooxygenase-2 (COX-2) have increased the incidence of 

myocardial infarction and stroke in placebo controlled trials. These drugs depress prostacyclin 

(PGI2) biosynthesis in humans and deletion of the PGI2 receptor ( the IP) augments the platelet 

activation and vascular proliferation evoked by vascular injury in vivo. Deletion of COX-2 results 

in multiple reproductive defects in mice and those that survive gestation exhibit renal and 

cardiac anomalies. We adopted a cre/lox strategy to generate COX-2 conditional knockout mice 

to study the by role guest of the on enzyme June 29, in a 2013 time and site-specific fashion. Objectives: To achieve

E-108 Vol 25, No 5 May 2005 

conditional deletion of COX-2 and to initiate studies of its role in vascular function in vivo. 

Methods and Results: We flanked exons 6, 7, 8 of COX-2 by two directly repeated loxp sites 

in the corresponding introns to generate mice floxed for COX-2. Macrophages from floxed mice 

have unaltered expression of COX-2 and production of its major product PGE 2. Universal 

deletion of the floxed COX-2 gene was first achieved by crossing them into a suitable cre line, 

suggesting that it is possible to generate conditional knockout models via cre/lox strategy. We 

have now generated endothelium specific COX-2 knockout mice by crossing the floxed-COX-2 

mice with Tie-2 cre mice. Renal architecture is preserved in contrast to a disordered phenotype 

resultant from universal deletion of the floxed-COX-2 gene. Inhibition of COX-2 with a selective 




inhibitor reduced the time to carotid artery occlusion following induction of thrombosis 

secondary to free radical dependent vascular injury. Deletion of the PGI 2 receptor - the IP- also 

augmented the response to the thrombotic stimulus in a gene dose dependent manner. The 

time to vascular occlusion was also significantly decreased ( p 0.05 ), from 5329 to 

3216 minutes compared to littermate controls, in mice lacking one copy of the COX-2 gene 

in endothelium. Summary: We have successfully generated mice conditionally lacking COX-2. 

These data are consistent with the hypothesis that selective inhibition of COX-2 augments the 

response to thrombotic stimuli in vivo by depressing endothelial COX-2 dependent activation of 

the IP.

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

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