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

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THE ANAND-APTELABORATORYPOSTDOCTORAL FELLOWSQuteba Ebrahem, M.D.Philip Klenotic, Ph.D.Jian Hua Qi, Ph.D.Anand-Apte B., and B. Zetter (2000) Biological principles of angiogenesis. In:D’Amore, P., and E. Voest, eds. Tumor Angiogenesis and Microcirculation. New York:Marcel Dekker, pp. 59-72.Anand-Apte, B. and P. Fox (2001) Melanoma: Translating biologicals into effectivetherapies: In: Borden, E., ed. Tumor Angiogenesis. New York: Humana Press.Karasarides M, Anand-Apte B, Wolfman A. (2001) A direct interaction between oncogenicHa-Ras and phosphatidylinositol 3-kinase is not required for Ha-Ras-dependenttransformation of epithelial cells. J. Biol. Chem. 276:39755-39764.Qi, J.H., Ebrahem, Q., Yeow, K., Edwards, D.R., Fox, P.L., and B. Anand-Apte (2002)Expression of Sorsby’s fundus dystrophy mutations in human retinal pigment epithelialcells reduces matrix metalloproteinase inhibition and may promote angiogenesis. J.Biol. Chem. 277:13394-13400.Qi, J.H., Ebrahem, Q., Moore, N., Murphy, G., Claesson-Welsh, L., Bond, M., Baker,A., and B. Anand-Apte (2003) A novel function for tissue inhibitor of metalloproteinases-3(TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2.Nat. Med. 9:407-415.162Bela Anand-Apte, Ph.D.Choroidal Neovascularization Regulatedby Extracellular MatrixThe clinical significance of ocular angiogenesis is enormous, because in the West,retinal neovascularization resulting fromdiabetic retinopathy is the most common cause ofnew blindness in young patients, and choroidalneovascularization (CNV) resulting from agerelatedmacular degeneration (AMD) is the chiefcause of severe, irreversible loss of vision inelderly patients. Recently, much progresshas been made in angiogenesis research,fueled by the hypothesis that inhibition ofangiogenesis would be a useful strategy totreat cancers.Pathologic angiogenesis plays a rolein a number of other diseases. Retinalneovascularization involves the developmentof sprouts from retinal vessels,which usually penetrate the inner limitingmembrane and grow into the vitreous.Retinal neovascularization is observed inischemic retinopathies such as diabeticretinopathy, retinopathy of prematurity,central vein occlusion and branch retinalvein occlusion. CNV refers to theformation of new vessels in the subretinalor sub-retinal pigment epithelial (RPE)space, which arises from thechoriocapillaris. CNV is seen in oculardiseases such as AMD, presumed ocularhistoplasmosis, high myopia and angioidstreaks.Angiogenesis (formation of newblood vessels) is a multi-step processrequiring degradation of the basementmembrane of the parent vessel, endothelial cellmigration, capillary tube formation and endothelialcell proliferation. A precise spatial andtemporal regulation of extracellular proteolyticactivity appears to be important inneovascularization. The matrix metalloproteinases(MMPs), a major group of degradative enzymes,can break down most components of theextracellular matrix. Under physiologicalconditions, the matrix’s integrity is maintained byan orchestrated balance between MMPs and theirendogenous inhibitors, tissue inhibitors ofmetalloproteinases (TIMPs).TIMP-3 is a novel TIMP expressed by RPEcells and is unique in being a component of theextracellular matrix. It has been established thatSorsby’s fundus dystrophy (SFD), a dominantlyinherited form of blindness, is caused by Timp-3gene mutations. SFD is characterized bydevelopment of CNV, subretinal hemorrhages andchanges consistent with disciform degeneration.Although rare, SFD is of considerable interest, asit is the only genetic disease in which degenerationoccurs in most affected patients.We have shown that TIMP-3 is a potentinhibitor of angiogenesis. Since CNV is aprominent feature of SFD, we propose thatmutations in SFD may affect the angiogenesisinhibitory function of the wild-type protein andinduce the formation of new blood vessels. Weare investigating whether endothelial cells arenormally maintained in a quiescent state becauseof a regulated balance between angiogenesisinducers and inhibitors. Wild-type TIMP-3protein is secreted by RPE cells and deposited inBruch’s membrane, where it acts as an efficientangiogenesis inhibitor. During pathological retinalneovascularization, as seen in SFD, mutantTIMP-3 is either unable to inhibit angiogenesis byacting directly on the endothelial cells or is aninefficient inhibitor of MMPs, resulting in anincreased breakdown of matrix, release ofsequestrated angiogenic factors such as vascularendothelial growth factor, and increasedneovascularization. To this end, we havegenerated adenovirus-expressing wild-type TIMP-3 and mutated versions of the protein (TIMP-3156, and TIMP-3 181) to test in vivo in a mousemodel and in vitro in cell cultures. We are alsopurifying recombinant protein from Baculovirus totest in similar models.The goal of the laboratory is to gain anunderstanding of the mechanism(s) by whichalterations in matrix integrity may regulate retinalneovascularization. We have identified a novelADAM-TS (A Disintegrin-like AndMetalloprotease domain with ThromboSpondintype I modules)-like molecule that is expressed inthe retina and may play a role in angiogenesis. Weare attempting to identify other novel endogenousinducers and inhibitors of angiogenesis tounderstand the basic biology ofneovascularization and to design therapeuticapproaches to combat this process in diseasestates. Our ultimate goal is to prevent and/orreverse this process in an effort to control thedevastating consequences of CNV.
Oxidative Protein Modifications Situatedat Focal Point of Proteomic Studies onAge Related Macular DegenerationResearch in the Crabb laboratory seeks abetter understanding of the biochemistryof vision in two broad areas: proteomechanges associated with retinal degenerativediseases and the mammalian visual cycle.ProteomicsOur objective is to identify proteins andprotein chemical modifications associated withthe pathogenesis of age-related macular degeneration(AMD). AMD, the most common cause ofblindness in the U.S. forthose over age 55, is acomplex disease withboth genetic andenvironmental contributions.Significant riskfactors for AMDdevelopment areextracellular deposits ordebris termed drusen,which form beneath theretina on Bruch’smembrane. We arepursuing proteomestudies to test thehypothesis that proteinoxidative modificationscontribute to drusenformation and Bruch’smembrane thickening inAMD. Our proteomicsresearch involves avariety of methods.Microdissection methodsare used to isolate drusenand Bruch’s membrane.Proteins in tissue samples are fractionated by 1Dand 2D chromatographic and electrophoreticmethods. Mass spectrometry (MS) tools,including matrix-assisted laser desorption/ionization time-of-flight (MALDI-Tof) MS, triplequadrupole electrospray MS, capillary liquidchromatography quadrupole time-of-flight (QTof)MS and liquid chromatography MS/MS, are usedfor protein identification. Oxidative proteinmodifications are identified using bioinformatictools and a battery of antibodies. Immunocytochemistryprovides confirmation of drusen andBruch’s membrane localization. We anticipatethat this protein chemical approach will provideinsights into the etiology of AMD and newopportunities for finding cures for the disease.Visual Cycle StudiesThe rod visual cycle is the process bywhich all-trans-retinal, released from rhodopsinJohn W. Crabb, Ph.D.during bleaching, is enzymatically isomerized to11-cis-retinal in the retinal pigment epithelium(RPE), then shuttled back to the rod photoreceptorcells for rhodopsin regeneration. Themechanism for regeneration of bleached visualpigments in cone photoreceptors appears to bedifferent than in rods and may involve retinalMüller cells. Our research focuses on the cellularretinaldehyde-binding protein (CRALBP), whichendogenously carries 11-cis-retinol and/or 11-cisretinaland is expressed inboth the RPE and Müllercells. CRALBP functionsin the rod visual cycle asan 11-cis-retinol acceptorin the isomerization stepand as a substrate carrierfor 11-cis-retinoldehydrogenase. We areprobing CRALBPstructure-functionrelationships using acombination of proteinchemical, nuclearmagnetic resonance andmolecular biologicalapproaches. In additionto characterizing thestructure of CRALBPfunctional domains,CRALBP proteininteractions are sought.Using immunoaffinitypurification and MSmethods, we haveidentified an RPE visualcycle protein complex. Current efforts focusupon identifying additional CRALBP interactionsand the mechanisms controlling CRALBPexpression.THE CRABBLABORATORYPROJECT SCIENTISTBruce LevisonRESEARCH ASSOCIATESanjoy Bhattacharya, Ph.D.POSTDOCTORAL FELLOWSXiaorong GuJian SunLEAD RESEARCH TECHNOLOGISTKaren A. West, M.S.GRADUATE STUDENTSZhaoyan Jin 1Regganathan Kutralanathan 2Zhiping Wu 11Cleveland State Univ.,Cleveland, OH2Case Western Reserve Univ.,Cleveland, OHUNDERGRADUATE STUDENTJohn S. CrabbCase Western Reserve Univ.,Cleveland, OHCOLLABORATORSSuneel S.Apte, M.B.B.S., D.Phil. 1Charles L. Bevins, M.D., Ph.D. 2Joe G. Hollyfield, Ph.D. 3John C. Saari, Ph.D. 4Robert J. Salomon, Ph.D. 5Dennis J. Stuehr, Ph.D. 21Dept. of Biomed. Eng., CCF2Dept. of Immunology, CCF3Ctr. for Ophthalmic Res., CCF4Univ. of Washington, Seattle5Case Western Reserve Univ.,Cleveland, OHCrabb, J.W., O’Neil, J., Miyagi, M., West, K., and H.F. Hoff (2002) Hydroxynonenalinactivates cathepsin B by forming Michael adducts with active site residues.Protein Sci. 11:831-840.Miyagi. M., Sakaguchi, H., Darrow, R.M., Yan, L., West, K.A., Aulak, K.S., Stuehr,D.J., Hollyfield, J.G., Organisciak, D.T., and J.W. Crabb (2002) Evidence that lightmodulates protein nitration in rat retina. Mol. Cell. Proteomics 1:293-303.Sakaguchi, H., Miyagi, M., Darrow, R.M., Crabb, J.S., Hollyfield, J.G., Organisciak,D.T., and J.W. Crabb (2002) Intense light exposure changes the crystallin content inretina. Exp. Eye Res. 76:131-133.Crabb, J.W., Miyagi, M., Gu, X., Shadrach, K., West, K.A., Sakaguchi, H., Kamei,M., Hasan A., Yan, L., Rayborn, M.E., Salomon, R.G., and J.G. Hollyfield (2002)Drusen proteome analysis: an approach to the etiology of age-related macular degeneration.Proc. Natl. Acad. Sci. USA 99:14682-14687.Kennedy, B.N., Li, C., Ortego, J., Coca-Prados, M., Sarthy, V.P., and J.W. Crabb(2003) CRALBP transcriptional regulation in ciliary epithelial, retinal Müller and retinalpigment epithelial cells. Exp. Eye Res. 76:257-260.163
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