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Yao Wang, Piyush Kothary, Monte Del Monte; Effect of Pigment Epithelium-Derived Factor on Hydrogen Peroxide-Exposed Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4274.
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Oxidative injury results in human retinal pigment epithelial (hRPE) cell damage, which has been postulated to be involved in the pathogenesis of age-related macular degeneration (AMD). We hypothesize that pigment epithelium-derived factor (PEDF) can serve as an anti-angiogenic factor and a neuroprotective factor in hRPE cells. This study examines the effects of PEDF on hRPE cell proliferation and morphology under oxidative stress induced by hydrogen peroxide (H2O2). We also investigated the effects of H2O2 on endogenous PEDF production.
Human RPE specimens were cultured from postmortem non-pathological eyes. Cellular proliferation and viability in the presence of increasing concentrations of H2O2 and PEDF were measured by [3H]thymidine incorporation and by the trypan blue exclusion method, respectively. After exposure to varying concentrations of H2O2, synthesis of PEDF was measured quantitatively by utilizing [14C]methionine immunoprecipitation with anti-PEDF and qualitatively via immunocytochemical methods.
Fetal bovine serum (FBS) stimulated hRPE cell proliferation in a dose-dependent manner. In contrast, H2O2 (0.1mM-0.5mM) decreased hRPE cell proliferation and viability, again in a dose-dependent manner as measured by [3H]thymidine incorporation and trypan blue exclusion method, respectively. The addition of PEDF (10ng/mL) to H2O2-exposed cells resulted in cell rescue, indicated by increased hRPE cell proliferation (358.43±57.03, n=12 vs. 1,087.22±235.58, n=6, CPM±SEM, p<0.05) and increased cell viability (60,000±10,700 vs. 164,500±20,100, n=28, cells±SEM, p<0.05). H2O2 also stimulated endogenous [14C]methionine-PEDF synthesis in hRPE cells in a dose dependent manner. Immunocytochemical studies confirmed endogenous production of PEDF in H2O2-exposed hRPE cells as well as nuclear rescue after the addition of PEDF to H2O2-induced hRPE cells.
These studies suggest that endogenous synthesis of PEDF is stimulated by H2O2. PEDF rescues H2O2-exposed hRPE cells, potentially by stimulating DNA synthesis and reversing nuclear damage induced by H2O2. Therefore, PEDF may have therapeutic value in treating AMD.
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