The Effect of Combined Oxidant Exposure and AP Complement Attack on Oxidant Production and Gene Expression in Human RPE Cells

Abstract

Purpose: : Human RPE (hRPE) cells are chronically exposed to blood-borne complement proteins and oxidative stress, a combination that is thought to contribute to diseases such as AMD. At physiological levels, oxidants such as hydrogen peroxide (H₂0₂) and hydroquinone (HQ) alter RPE cell function, as does complement attack through alternative pathway (AP) activation. It is crucial to understand how oxidants interact with AP activation to influence RPE cell survival and gene expression. In the current study, we determined the effect of H₂0₂ or HQ combined with AP activation on hRPE cell survival factor, cytokine, and antioxidant enzyme gene expression.

Methods: : Cultured confluent hRPE cells were treated with varying H₂0₂ or HQ doses for 90 min. The cells were then primed with a complement-fixing Ab for 30 minutes, and incubated in C1Q-depleted serum (C1Qd). To examine intracellular reactive oxidative species (ROS), cells were loaded with fluorophore dye and fluorescence was quantified with a plate reader following 30 minute incubation in C1Qd. Cells were counted following fluorescence measurement to calculate fluorescence per cell for each treatment. For gene expression analysis, cells were incubated in C1Qd for 1 to 4 hrs. RNA was harvested, and mRNA expression was determined by qRT-PCR.

Results: : Following AP activation, intracellular ROS increased, and further enhancement occurred relative to oxidants alone and AP alone when AP activation was coupled with H₂0₂ or HQ. Anti-survival factor Bax was expressed more highly than Bcl-xL when AP activation was combined with H₂0₂ or HQ. MCP-1 was up-regulated following AP activation, and this up-regulation was attenuated when AP was combined with H₂0₂ or HQ. HO-1 and GCLC were unaffected by AP activation, but were up-regulated by H₂0₂ and HQ. This oxidant- mediated up-regulation was attenuated when AP was combined with H₂0₂ or HQ.

Conclusions: : When AP activation is combined with oxidative stress, the balance between pro- and anti- survival factors is shifted toward greater expression of anti-survival factors. The combination of oxidative stress and AP attenuated the effect of AP or oxidant mediated up-regulation of cytokine and antioxidant enzyme genes, providing a possible mechanism of hRPE dysfunction.

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