Oxidative Stress Renders Retinal Pigment Epithelial Cells Susceptible to Complement-mediated Injury

Uncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H₂O₂ (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H₂O₂ reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H₂O₂ and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD.Age-related macular degeneration (AMD)⁶ is the leading cause of blindness in the elderly (1). Clinically, AMD is categorized as “dry” or “wet.” In the dry form of the disease, deposits (drusen) develop between the retinal pigment epithelium (RPE) and the underlying basement membrane (Bruch''s membrane). The loss of photoreceptor function and vision observed in patients is attributed to atrophic changes in the RPE (1, 2). Wet AMD is characterized by choroidal neovascularization extending through Bruch''s membrane and the RPE into the subretinal space. Subsequent leakage of exudative fluid and blood is thought to contribute to the eventual development of fibrosis characteristic of wet AMD. AMD is hypothesized to be a progressive disease, with the dry and wet forms likely representing different points on a spectrum of disease severity. Approximately 10–15% of patients with the less severe dry AMD go on to develop wet AMD (1).Several observations suggest that uncontrolled activation of the complement cascade contributes to the development and progression of AMD. Polymorphisms in complement factor H, a circulating inhibitor of the alternative pathway of complement, are strongly associated with the development of AMD (3–6). Drusen-like lesions also develop in patients with dense deposit disease, a form of glomerulonephritis caused by dysregulation of the alternative pathway (7, 8). Analysis of the composition of drusen demonstrates that they contain important complement proteins, including C3, C5, membrane attack complex (MAC), and endogenous complement regulatory proteins (7, 8). Mice with a genetic deletion of factor H (cfh^?/? mice) accumulate C3 throughout the RPE and the outer segment layer of the neuroretina, and lose visual function faster during aging than their wild type littermates (9). Furthermore, in a murine model of laser-induced choroidal neovascularization, blockade of signaling by C3a and C5a reduced the production of VEGF in the eye and reduced neovascularization (10). Taken together, these studies suggest that in AMD, inadequate control of the alternative pathway 1) contributes to the structural changes observed in RPE and Bruch''s membrane, including drusen formation; and 2) is upstream of VEGF-mediated mechanisms.The alternative pathway of complement is continually activated in the fluid phase, and inadequate inhibition of this pathway on tissue surfaces may permit spontaneous complement activation with rapid amplification and generation of pro-inflammatory activation fragments (11). In late-onset diseases such as AMD, local regulation of the alternative pathway may gradually be overwhelmed by cellular injury or the accumulation of debris (12, 13). Several environmental factors contribute to a high level of oxidative stress at the RPE layer, and oxidative injury of the RPE cells may be an important cause of AMD (14). Therefore, we hypothesized that oxidative stress may impair the ability of the RPE to regulate complement on its surface. In the intact adult human eye, only one cell surface complement inhibitor, membrane cofactor protein (MCP; CD46), has been identified on RPE cells (15). In the current study, we investigated whether ARPE-19 cells express the three cell surface complement inhibitors, CD46, decay accelerating factor (DAF; CD55), and CD59; and whether oxidative stress of RPE cells in culture alters surface expression of the complement inhibitory proteins or reduces inhibition of the alternative pathway on the surface of the cells by factor H. Second, we tested the hypothesis that rather than causing cell lysis, sublytic activation of complement on RPE cells induces VEGF release by these cells, which is known to compromise barrier function. The goal of these studies was to construct a model whereby oxidative stress in the eye could be linked to the inflammatory events that cause AMD, including uncontrolled activation of complement.