ARPE–19 Cells Are Protected Against Hydrogen Peroxide–Induced Cell Death by Multiple Growth Factors

Abstract

Abstract: : Purpose: Human RPE cells are exposed to and are sensitive to oxidative stress in vivo. PEDF, an anti–angiogenic and neuroprotective protein secreted by the RPE, protects cells against oxidative stress. In this study, we compared the ability of PEDF to protect ARPE–19 cells against hydrogen peroxide (H₂O₂)–induced cell death with the actions of other neuroprotective factors. Methods: ARPE–19 cells were adapted into DMEM containing 1% FBS and 1mM Butathione Sulfoximine (BSO). Cells were pretreated with different doses of neuroprotective factors PEDF, brain–derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (FGF2), vascular endothelial growth factor (VEGF) (12.5 to 200 ng/ml), for 24 hours and then exposed in different concentrations of H₂O₂ (1:320000, 1:160000, 1:80000) for 24 hours. Inhibitors of signal transduction pathways were applied before growth factors and H₂O₂ treatment. Calcein fluorescence was used to detect the surviving cells. Results: Treatment with H₂O₂ for 24 hours induced significant death of ARPE–19 cells in a dose–dependent manner. Pretreatment with PEDF prior to the insult greatly attenuated H2O2–induced cytotoxicity and apoptosis, with the peak protective effect occurring at 100 ng/ml. Other factors showed equivalent or less protective effects. Combination of PEDF with BDNF, CNTF, FGF2, or VEGF significantly improved the protection. Addition of the mitogen–activated protein kinase (MAPK) inhibitor PD98059 did not block the protection given by CNTF. Conclusions: All factors tested gave significant protection from H₂O₂–inducd cell death in their physiologically active concentration range. In the presence of saturating concentrations of PEDF, the other factors were still protective suggesting they act, at least in part, through different transduction pathways. The effect of CNTF is probably mediated through STAT3 activation rather than mitogen–activated protein kinase (MAPK) activation. The data strongly suggest that PEDF is a useful protective agent and that a cocktail of growth factors might provide optimum rescue in conditions that promote RPE degeneration. Supported by the David Woods Kemper Memorial Foundation, the NIH, RPB Inc., and the Connecticut Lions.

Keywords: neuroprotection • oxidation/oxidative or free radical damage • retinal pigment epithelium