Effect of Pigment Epithelium-Derived Factor on Hydrogen Peroxide-Exposed Human Retinal Pigment Epithelial Cells

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • age-related macular degeneration