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S. A. Medearis, C. Shieh, P. Yang, G. J. Jaffe; Effect of Complement and Oxidants on Human Retinal Pigment Epithelial (RPE) Cell Survival in Age related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2009;50(13):708.
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RPE cells, a critical target in AMD, are exposed to repetitive oxidative damage. Complement-mediated damage may also play a role, and the Y402H variant of complement factor H is a risk factor for advanced AMD. The combined effects of repetitive oxidative stress and complement-mediated damage on RPE cell survival have yet to be elucidated. Herein we determined the effect of oxidants and complement on RPE cell viability and survival protein expression.
Cultured human donor WT or Y402H homozygote variant RPE cells were exposed to repetitive sub-lethal doses of hydrogen peroxide (100 uM) or hydroquinone (100 uM), with or without normal human serum (1:10 dilution). qRT-PCR was performed on cDNA synthesized from harvested RNA. Cell viability was assessed with a WST-1 assay, and cell lysis was determined by a calcein-release assay. The functional effects of complement regulatory proteins (CD46, CD55, and CD59) were studied via antibody blocking and PIPLC removal.
Under the combination of oxidative stress and complement attack, there was density-dependent decreased RPE cell viability and a decrease in RPE pro-apoptotic factor mRNA expression as compared to anti-apoptotic factor. When complement regulatory protein function was blocked, RPE cells had significantly more cell death. There were no significant differences in WT or Y402H variant RPE cell viability, resistance to lysis, or survival factor up-regulation response to oxidative stress and/or complement.
Complement and sublethal oxidants together mediate RPE survival in a density-dependant manner. The membrane-bound complement regulatory proteins likely play a key role in RPE survival against complement attack, but a role for complement factor H has yet to be determined.
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