Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

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THE CHISOLMLABORATORYRESEARCH ASSOCIATESara G. Carlson, Ph.D.POSTDOCTORAL FELLOWSudesh Agrawal, Ph.D.TECHNOLOGISTSRichard A. Cole, B.S.Charles A. Kaul, B.S.STUDENTSShu-Ling Liang, M.S.Cleveland State Univ., Cleveland,OHTina ChoudhriJohn Carroll Univ., UniversityHts., OHCOLLABORATORSMunna L. Agarwal, Ph.D. 1Alex Almasan, Ph.D. 2Martha K. Cathcart, Ph.D. 3Paul E. DiCorleto, Ph.D. 3Donna M. Driscoll, Ph.D. 3Stanley L. Hazen, M.D., Ph.D. 3,4Roger M. Macklis, M.D. 5Marc S. Penn, M.D., Ph.D. 3,4George R. Stark, Ph.D. 1Yan Xu, Ph.D. 21Dept. of Molecular Biology, CCF2Dept of Cancer Biology, CCF3Dept. of Cell Biology, CCF4Dept of Cardiovascular Medicine,CCF5Dept of Radiation Oncology,CCFLow-density lipoprotein (LDL) is theprincipal cholesterol-carrying molecularcomplex in normal human plasma, andLDL levels correlate strongly with risk ofatherosclerosis. For twodecades, our laboratory hasbeen evolving and testing atheory that the oxidativemodification of lipoproteinspromotes the development ofatherosclerotic lesions.Alterations in cellfunction induced by oxidizedLDL in vitro mimic events inlesion development observedin vivo. Our research focuses oncellular changes brought aboutby oxidized LDL that aredistinct from those resultingfrom exposure of cells tounaltered LDL. We haveidentified constituents ofoxidized LDL that arebioactive and probedmechanisms by which theseconstituents change particularcell functions.Oxidized Lipoproteins and Cell InjuryWe have shown that oxidized LDL injurescells in culture, that the cytotoxicity is independentof the mode of LDL oxidation, that cellsare significantly more susceptible during the S-phase of the cell cycle, and that the delivery ofthe toxin does not require lipoprotein receptors.Lipoproteins oxidized in vivo, such as thoseisolated from diabetic rats or human lesions, arealso cytotoxic to cells in culture. Infusion ofoxidized LDL injures vascular endothelial cells invivo and impairs their function.The Department of Cell BiologyLipoprotein Oxidation, LipoproteinRegulation of Vascular Cell Function:Roles in Apoptosis, Thrombosis,Inflammation and AtherosclerosisGuy M. Chisolm, III, Ph.D.We have identified multiple cytotoxinsborne by oxidized LDL that accumulate invascular lesions and we have studied cell deathmechanisms. For example, a hydroperoxide ofcholesterol kills cells byperoxidation of cell lipids; itsderivative oxysterols kill byapoptosis. With Dr. DonnaDriscoll, we are studying theregulation of antioxidantselenoenzymes that can reducetoxic lipid peroxides and protectcells from apoptosis. With Drs.Munna Agarwal, Martha Cathcartand George Stark, we arestudying Stat1-dependent cellsignaling pathways of apoptosisinduced by oxysterols. These arebeing explored in cell culture andin vivo in genetically altered mice.Prothrombotic Actions ofOxidized LDLWith Drs. Marc Penn andPaul DiCorleto, we have shownthat LDL and oxidized LDL, aswell as various of their lipids,can induce the gene or enhancethe activity of the clotting cascade initiator,tissue factor, on the surfaces of vascular smoothmuscle cells (SMCs). These effects may beimportant contributors to lesion development,since tissue factor induction could lead toincreased local production of thrombin, andthrombin not only enhances coagulation, but isalso mitogenic for SMCs. We are studying theregulation by specific oxidized LDL constituentsof tissue factor gene expression, proteinproduction and cell surface tissue factor activity.Penn, M.S., Cui, M.Z., Winokur, A.L., Bethea, J., Hamilton, T.A., DiCorleto, P.E., and G.M. Chisolm (2000) Smooth muscle cell surface tissuefactor pathway activation by oxidized low-density lipoprotein requires cellular lipid peroxidation. Blood 96:3056-3063.Chisolm, G.M., and D. Steinberg (2000) The oxidative modification hypothesis of atherogenesis: an overview. Free Radical Biol. Med. 28:1815-1826.Agrawal, S., Agarwal, M.L., Chatterjee-Kishore, M., Stark, G.R., and G.M. Chisolm (2002) Stat1-dependent, p53-independent expression ofp21(waf1) modulates oxysterol-induced apoptosis. Mol. Cell. Biol. 22:1981-1992.Hazen, S.L., and G.M. Chisolm (2002) Oxidized phosphatidylcholines: pattern recognition ligands for multiple pathways of the innate immuneresponse. Proc. Natl. Acad. Sci. USA 99:12515-12517.Chen, Q., Chai, Y.C., Mazumder, S., Jiang, C., Macklis, R.M., Chisolm, G.M., and A. Almasan (2003) The late increase in free radical oxygenspecies during apoptosis is associated with cytochrome c release, caspase activation and mitochondrial dysfunction. Cell Death Differ. 10:323-334.Cui, M.Z., Zhao, G., Winokur, A.L., Laag, E., Bydash, J.R., Penn, M.S., Chisolm, G.M., and X. Xu (2003) Lysophosphatidic acid induction oftissue factor expression in aortic smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 23:224-230.70
The central objective of the research in thislaboratory is to understand the role of geneexpression by vascular endothelial cells(ECs) in the physiological maintenance of vesselwall homeostasis and in the pathogenesis ofvascular diseases. The working hypothesis is thataberrant expression of embryonic or “pathological”genes in the endothelium of adults leads toatherosclerotic plaque development and otherdiseases of large blood vessels. Two specificresearch areas are under investigation. The firstfocuses on the regulation of expression ofleukocyte adhesion molecules, including E-selectinand VCAM-1, by the endothelium in response tothrombin, TNF-alpha and cytokines. The secondarea is the regulation of expression of the plateletderivedgrowth factor (PDGF) A and B chain genesby ECs. In both pursuits, we are defining thecascade of intracellular signaling events that lead tothe expression of these genes, the transcriptionalfactors and DNA elements responsible for theirinduction, and post-transcriptional events thatcontribute to the expression of active proteins.Endothelial Cell Expression of LeukocyteAdhesion MoleculesMonocyte binding to the endothelium is arequisite first step in the emigration of thisleukocyte from the circulation to tissue duringinflammation and wound healing. The overallobjective of this project is to understand themonocyte/endothelium interaction and to definethe intracellular signaling pathways and agonistinducedgenes that are involved in the regulationof monocyte binding to ECs. We have beenstudying thrombin- and TNF-induced signalingpathways and transcription factors that areresponsible for induction of the leukocyteadhesion molecules. In addition, we are studyingthe relative importance of the two TNF-alphareceptors in the induction of leukocyte adhesionin vitro and in vivo using mice that are null for oneor both of the receptor genes. We have recentlyobserved that TNFR2 appears to perform a criticalrole in TNF-induced firm adhesion to postcapillary venules in a mouse cremaster musclemodel.The Department of Cell BiologyEndothelial Cell Gene Expression Linkedto Vascular DiseaseThrombin-induced PDGF Production by theEndotheliumOur major objective in this project is toidentify and characterize regulatory pathwaysinvolved in the expression of the polypeptidemitogen PDGF by ECs in response to the proteasethrombin. We have recently identified a novel modeof transcription factor activation using this modelsystem. We first identified a thrombin responseelement in the PDGF B chain promoter and a novelthrombin-dependent trans-acting factor that boundto this element to cause increased transcription. Wepurified this factor, and amino acid sequenceinformation identified it as a member of the Y-boxfamily of transcription factors. Y-box-bindingproteins are known to act constitutively to positivelyor negatively regulate the expression of severalcellular and viral genes; however, activation of thisclass of transcription factors in response to cellstimulation had not previously been reported. Wehave shown that the Y-box protein is bound tomRNA in the cytosol under basal conditions, but inresponse to thrombin stimulation, it is cleaved andtranslocated to the nucleus, where it induces PDGFexpression. The cleaved protein has a DNA-bindingsequence distinct from the full-length Y-box protein,which is present in the nucleus constitutively.We have used micro-array gene-screeningtechnique to identify seven novel thrombinresponsivegenes that were verifiable by Northernblot analysis in human umbilical vein ECs. Amongthem CL-100, a.k.a. MAP kinase phosphatase-1, a dualspecificityphosphatase, was found to be inducedstrongly, but transiently by thrombin-- mRNApeaked at 1 h with a t 1/2of 45 min. CL-100induction by thrombin was protease-activatedreceptor-1 (PAR-1)-mediated, protein synthesisindependent and at the transcriptional level. Srckinase and p42/p44 ERK activity are critical for theinduction of CL-100 by thrombin, whereas PKCactivation is not required. CL-100 inhibition by aspecific anti-sense oligonucleotide inhibitedthrombin-induced PDGF-A and -B genes, whileVCAM-1 and E-selectin expression levels were upregulatedfurther. These results suggest an importantregulatory role of CL-100 in thrombin-inducedsignaling and gene expression in EC.THE DICORLETOLABORATORYRESEARCH ASSOCIATESSmarajit Bandyopadhyay, Ph.D.Unni Chandrasekharan, Ph.D.POSTDOCTORAL FELLOWSZahid Ashraf, Ph.D.Lin Yang, M.D.TECHNICIANLori Mavrakis, B.A.STUDENTSCorttrell KinneyPam DaherCOLLABORATORSGuy Chisolm, Ph.D. 1Stephen Ellis, M.D. 2Stanley Hazen, M.D., Ph.D. 1Marc Penn, M.D., Ph.D. 1Maria Siemionow, M.D., Ph.D. 31Dept. of Cell Biology, CCF2Dept. of CardiovascularMedicine, CCF3Dept. of Plastic and ReconstructiveSurgery, CCFPaul E. DiCorleto, Ph.D.Patel, C., Sharangpani, R. Bandyopadhyay, S., and P.E. DiCorleto (1999) Endothelial cells express a novel, TNF-alpha-regulated variant of HOXA9. J.Biol. Chem. 274: 1415-1422.Stenina, O.I., Poptic, E.J., and P.E. DiCorleto (2000) Thrombin activates a Y box-binding protein (DNA-binding protein B) in endothelial cells. J. Clin.Invest. 106:579-587.Mao, C.D., Hoang, P., and P.E. DiCorleto (2001) Lithium inhibits cell cycle progression and induces stabilization of p53 in bovine aortic endothelialcells. J. Biol. Chem. 276:26180-8. [Stenina, O.I., Shaneyfelt, K. , and P.E. DiCorleto (2001) Thrombin induces the release of the Y-box protein dpbB from mRNA: a new mode oftranscription factor activation. Proc. Natl. Acad. Sci. USA 98:7277-82.Byzova, T.V., Goldman, C.K., Jankau, J., Chen, J., Cabrera, G., Achen, M.G., Stacker, S.A., Carnevale, K.A., Siemionow, M., Deitcher, S.R.,and P.E. DiCorleto, (2002) Adenovirus encoding vascular endothelial growth factor-D induces tissue-specific vascular patterns in vivo. Blood99:4434-42.71
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