Characterizing the role of endogenous antiangiogenic factors in neovascular pathology and normal vascular development in the retina

Pigment epithelium-derived factor (PEDF) and tissue inhibitor of metalloproteinase-3 (TIMP-3) are thought to be involved in normal and aberrant ocular angiogenesis. The goal of this project is to examine the role that each plays in these processes. In human developing retinas, polyclonal antibody (pAb) anti-PEDF labeled the retinal pigment epithelium (RPE), cones, some neuroblasts and cells in the ganglion cell layer (GCL). In adult human retinas, pAb anti-PEDF labeled rods, cones, cells of the inner nuclear and GC layers, choroid, corneal epithelium and endothelium, and ciliary body. In situ hybridization revealed PEDF mRNA in the RPE, photoreceptors, INL and GCL. In developing and adult retinas, monoclonal antibody (mAb) anti-PEDF labeled the interphotoreceptor matrix (IPM). In the mouse retina, immunohistochemical and in situ hybridization showed PEDF expression in the ciliary body and choroid during mid-gestation. Near term, protein levels increased in the GCL and remained high through the first two weeks postnatal. Levels decreased after this point but persisted through adulthood. Expression levels in the inner retina were much higher at all timepoints than in the outer retina. Because PEDF protein levels have been found to be responsive to ambient oxygen tension, we also attempted to isolate the PEDF promoter and examine its activity under normoxic and hypoxic conditions. However, the portion of promoter isolated was weakly functional in luciferase assays, and further study was not pursued. We also demonstrated inhibition of ischemia-induced ocular neovascularization via adeno-assoicated viral-induced overexpression of PEDF, TIMP-3, and endostatin using fluorescein angiography and histologic techniques. The effects of mutations in TIMP-3 known to cause inherited choroidal neovascularization on angiogenesis in vivo were also examined. Mice injected subretinally with AAV.CMV.TIMP-3 172 2/5, AAV.CMV.TIMP-3 167 2/5, or AAV.CMV.TIMP-3 wild type 2/5 demonstrated more RPE changes and white spots when observed by indirect ophtalmoscopy than control virus-injected eye. Changes were most severe in eyes injected with AAV.CMV.TlMP-3 172 2/5. However, no choroidal neovascularization or retinal degeneration was seen histologically. Transduction efficiency of AAV.CMV.EGFP 2/1, 2/2, and 2/5 in human umbilical vein endothelial cells (HUVEC) as well as COS-7 cells was also determined using microscopy and flow cytometry

Characterizing the role of endogenous antiangiogenic factors in neovascular pathology and normal vascular development in the retina

Pigment epithelium-derived factor (PEDF) and tissue inhibitor of metalloproteinase-3 (TIMP-3) are thought to be involved in normal and aberrant ocular angiogenesis. The goal of this project is to examine the role that each plays in these processes. In human developing retinas, polyclonal antibody (pAb) anti-PEDF labeled the retinal pigment epithelium (RPE), cones, some neuroblasts and cells in the ganglion cell layer (GCL). In adult human retinas, pAb anti-PEDF labeled rods, cones, cells of the inner nuclear and GC layers, choroid, corneal epithelium and endothelium, and ciliary body. In situ hybridization revealed PEDF mRNA in the RPE, photoreceptors, INL and GCL. In developing and adult retinas, monoclonal antibody (mAb) anti-PEDF labeled the interphotoreceptor matrix (IPM). In the mouse retina, immunohistochemical and in situ hybridization showed PEDF expression in the ciliary body and choroid during mid-gestation. Near term, protein levels increased in the GCL and remained high through the first two weeks postnatal. Levels decreased after this point but persisted through adulthood. Expression levels in the inner retina were much higher at all timepoints than in the outer retina. Because PEDF protein levels have been found to be responsive to ambient oxygen tension, we also attempted to isolate the PEDF promoter and examine its activity under normoxic and hypoxic conditions. However, the portion of promoter isolated was weakly functional in luciferase assays, and further study was not pursued. We also demonstrated inhibition of ischemia-induced ocular neovascularization via adeno-assoicated viral-induced overexpression of PEDF, TIMP-3, and endostatin using fluorescein angiography and histologic techniques. The effects of mutations in TIMP-3 known to cause inherited choroidal neovascularization on angiogenesis in vivo were also examined. Mice injected subretinally with AAV.CMV.TIMP-3 172 2/5, AAV.CMV.TIMP-3 167 2/5, or AAV.CMV.TIMP-3 wild type 2/5 demonstrated more RPE changes and white spots when observed by indirect ophtalmoscopy than control virus-injected eye. Changes were most severe in eyes injected with AAV.CMV.TlMP-3 172 2/5. However, no choroidal neovascularization or retinal degeneration was seen histologically. Transduction efficiency of AAV.CMV.EGFP 2/1, 2/2, and 2/5 in human umbilical vein endothelial cells (HUVEC) as well as COS-7 cells was also determined using microscopy and flow cytometry