Oxidative Stress Induces Apoptosis in Cultured ARPE-19 Cells: Protective Effect of Erythropoietin

Abstract

Purpose:: Oxidative damage of the retinal pigment epithelium (RPE) might be involved in the pathogenesis of age related macular degeneration (ARMD) and apoptosis might play a role in dry ARMD. The following study investigates if free radicals are able to induce apoptotic cell death in cultured ARPE-19 cells. It has been shown recently that erythropoietin protects photoreceptor against degeneration in a rodent light damage model. Thus it is hypothesized in the present study that erythropoietin inhibits apoptotic cell death induced by oxidative stress.

Methods:: The experiments were carried out with confluent ARPE-19 cells. Apoptosis was determined by the Annexin V fluorescence assay (Clontech, CA, USA). Confirmative experiments were done with a fluorescence based TUNEL assay (Roche, Mannheim, Germany). The relative fluorescence in both cases is used as a measure of apoptosis development. Erythropoietin was obtained from Janssen-Cilag. Hydroxyl radicals (OH) were generated from 0.75mM H₂O₂ under catalysis of 10µM Fe³⁺ (Fenton reaction). This concentration of the Fenton reaction partners is taken as reference (1-fold concentration).

Results:: Exposure to the 1-fold OH-concentration does not change apoptosis rate, but starting at the 1.5-fold OH-concentration apoptosis rate determined by Annexin V labelling begins to rise by 4.3±0.2%, by 7.8±0.4% at the 2-fold OH-concentration and by 11.8±1.7% at the 3-fold concentration (P<0.05 and n=3 for each OH-concentration). At each OH-concentration the maximum apoptosis rate is reached 6h after radical exposure, although apoptosis rate keeps elevated even after 24h (between 1.3±0.4% to 6.4±2.6% depending on the OH-concentration). The TUNEL assay confirmed these findings (e.g. at 3-fold OH concentration an apoptosis rate of 12.2±1.0% was found; n=3; P<0.05 vs. control, n.s. vs. Annexin V results). Addition of 5 IU erythropoietin to the media reduces apoptosis rate (Annexin V) from 12.5±0.9% to 4.0±0.8% (P<0.001; n=3). The effect of erythropoietin is dose-dependent and reaches it maximum at 5 IU/ml.

Conclusions:: OH induces in concentration dependent manner apoptosis in cultured ARPE-19 cells. Erythropoietin inhibits apoptosis in these cells.

Keywords: retinal pigment epithelium • apoptosis/cell death • oxidation/oxidative or free radical damage