A Dual-Hit Hypothesis in Retinal Pigmented Epithelial Cell Dysfunction: Oxidative Stress Renders Cells Susceptible to Complement-Mediated Injury

Abstract

Purpose: : Genetic variations in complement factor H (fH), an inhibitor of the alternative complement pathway (AP), and oxidative stress are associated with all forms of age-related macular degeneration (AMD). Here we hypothesized whether oxidative stress renders the RPE more susceptible to complement activation.

Methods: : Human embryonic RPE or ARPE-19 were grown as monolayers on transwell plates. Effects on RPE tight junctions were determined by transepithelial electrical resistance (TER) measurements and immunohistochemistry. Endogenous complement inhibitors were analyzed by flow-cytometry and immunohistochemistry, and VEGF secretion by ELISA. Analysis of long-term effects on RPE cell physiology included analysis of energy metabolism, ROS uptake and cell death assays.

Results: : (1) At low H₂O₂ levels or when exposed to serum alone, RPE monolayers have a stable TER. TER deteriorated rapidly in H₂O₂-exposed monolayers upon adding serum as a source of complement, which was concomitant with a disruption of junctional proteins. The effect was dependent upon sublytic activation of the membrane attack complex. (2) H₂O₂ reduced levels of CD55 and CD59 cell-surface complement inhibitors and impaired fH activity. (3) Only H₂O₂ + serum led to C3-deposition and apical VEGF secretion. (4) TER could be stabilized and VEGF secretion blunted in H₂O₂ + serum treated cells by inhibiting the AP or VEGF-receptor-2. (5) Long-term H₂O₂ + serum impaired RPE physiology and ATP production, but did not result in apoptosis.

Conclusions: : While RPE cells are resistant to low level oxidative stress, oxidative stress sensitizes RPE cells to effects of a misregulated complement system, resulting in VEGF secretion and disruption of RPE barrier integrity. Long-term oxidative stress and complement exposure impairs RPE physiology and energy metabolism. In summary, we have developed a model whereby oxidative stress in RPE can be linked to the molecular events involved in AMD, including activation of the AP and local production of VEGF.

Keywords: age-related macular degeneration • oxidation/oxidative or free radical damage • immunomodulation/immunoregulation