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

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.