PEDF Gene-modified Pigment Epithelial Cells In Vitro Effect Of The Choroidal Neovascularization

PURPOSE: To determine the effect of pigment epithelium-derived factor (PEDF) modified iris pigment epithelium and retinal pigment epithelium in an in vitro model of choroidal neovascularization.METHODS: IPE and RPE were cultured. RNA was prepared from freshly isolated bovine IPE and retinal pigment epithelium (RPE) cells and reverse transcribed. The expression of mRNA for cellular retinaldehyde binding protein (CRALBP), 11-cis-dehydrogenase (11cisRDH), retinol binding protein (RBP) and Cat D (cathepsin D), was determined by RT-PCR using specific primers. The isolation of human choroidal endothelial cells (CECs) used micro dissection followed by the use of superparamagnetic beads (Dynabeads) coated with the CD31, which selectively binds to the endothelial cell surface. PEDF gene was transfected into IPE and RPE cells by lipofactamine 2000. The PEDF expressing IPE and RPE were cocultured with CECs. The mitogenic and motogeneic effects of VEGF on cultured choroidal capillary endothelial cells were examined in the presence or absence of PEDF or the epithelium cells' using cell counts and migration assays. The effects on CNV were tested using forming capillary structure analysis.RESULTS: Bovine IPE and RPE cells have similar biological characters. They expressed mRNA for CRALBP, 11cis- RDH, and RPE63. The mRNA expression for CRALBP, RBP, 1 lcis- RDH and Cat D was almost equal in both cell types. Cells bound to beads are isolated using a magnetic particle concentrator. Isolated cells grew to confluence in a monolayer with a cobble stone morphology and were shown to be endothelial cells by their greater than 95% immunoreactivity to von Willebrand factor. Isolated cells grew as tubes in three-dimensional cultures. PEDF and PEDF expressing cells suppressed VEGF-induced retinal microvascular endothelial cell proliferation and VEGF-induced migration. The CNV models in vitro were also be...