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

Abstract

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 H 2 O 2 (to induce oxidative stress) and complementsufficient 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 H 2 O 2 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 H 2 O 2 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.

Sublytic Membrane-Attack-Complex (MAC) Activation Alters Regulated Rather than Constitutive Vascular Endothelial Growth Factor (VEGF) Secretion in Retinal Pigment Epithelium Monolayers

“…It has previously been reported that doses of up to 1 mM are not cytotoxic, and do not lead to disruption of barrier function in these cells (19). After treatment with H 2 O 2 , monolayers were immediately exposed to 25% NHS as a source of complement proteins (18).…”

“…FBS was removed completely for the final 5-7 days (2-3 media changes) prior to measurements, which we have previously shown does not alter survival or monolayer formation in these cells (29). TER of the cell monolayer on the Transwell filters was determined by measuring the resistance across the monolayer with an EVOM volt-ohmmeter (World Precision Instruments) (18). The value for cell monolayers was determined by subtracting the TER for filters without cells and then multiplying by the surface area of the filters.…”

“…Microplates were coated with the anti-human VEGF polyclonal capture antibody (Antigenix America, Inc.; Huntington Station, NY) (18), and 100 l of supernatant was added. The captured proteins were detected with the same VEGF-specific antibody conjugated to horseradish peroxidase (HRP), followed by development with the chromogenic substrate, OPD (Sigma Aldrich).…”

“…Activation-Sublytic complement activation, which is known to result in Ca 2ϩ influx through the pores generated by the MAC, is associated with an increase in VEGF secretion (18). Here we examined whether the known kinase pathways involved in VEGF secretion, are activated by sublytic MAC activation.…”

“…To further examine this concept, we investigated RPE cells (ARPE-19 cells) grown as stable monolayers (18). These monolayers exhibit stable transepithelial resistance of ϳ40 -45 ohm/ cm 2 ; are polarized; and stain for markers of tight and adherence junctions (19).…”