Human RPE Cell Injury Mediated by Oxidative Stress and Alternative Complement Cascade

Abstract

Purpose: : Complement activation has been increasingly implicated in the pathogenesis of AMD. RPE cells are continually exposed to oxidative stress. Herein, we investigate the effect of repetitive and single exposure to hydroquinone (HQ) and hydrogen peroxide (H₂O₂) on alternative complement pathway (AP)-mediated injury in human RPE (hRPE) cells.

Methods: : Cultured hRPE cells from 3 adult donors (CFH_YY402, CFH_YH402, and CFH_HH402) were stimulated with various concentrations of HQ and H₂O₂ for 1 week (four hits) or for 90 minutes (one hit) and then primed with a complement-fixing antibody for 30 minutes followed by incubation with C1q-depleted human serum for various times. Tetrazolium salt WST-1, lactate dehydrogenase release and cell count assays were used to assess cell permeability, cell viability and cell number, respectively. DNA fragmentation was determined using ELISA and TUNEL assays; cells transfected with Bcl-xL splice-switching oligonucleotide (SSO) to induce apoptosis were used as a positive control.

Results: : AP attack significantly increased RPE cell permeability, and decreased cell viability and cell number in a dose-dependent manner in 3 donor RPE cells (P<0.05). Repetitive hits with low dose HQ significantly increased cell viability (P<0.01), and AP attack significantly reduced this HQ-induced effect (P<0.001). Repetitive and single hits with either HQ or H₂O₂ significantly enhanced AP-driven cell death in 3 donor RPE cells as compared to cells treated with HQ, H₂O₂ and AP alone (P<0.05). Nuclear shrinkage was observed by DAPI stain in cells treated with HQ+AP. Cells treated with H₂O₂+AP appeared bloated. DNA fragmentation was modestly but significantly increased in cells treated with HQ+AP as compared to cells treated with HQ (P=0.02) and AP alone (P=0.02). TUNEL positive cells were detected in SSO transfected cells, but not in cells treated with HQ+AP or H₂O₂+AP.

Conclusions: : For both the repetitive hit and single hit model, oxidative stress renders RPE cells more susceptible to AP-induced cell death. The repetitive hit model which simulates chronic oxidant exposure will be useful in future investigations to explore mechanisms of observed differences in HQ- and H₂O₂-enhanced AP-driven RPE cell death.

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