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Ping Yang, Christine Shieh, Jessica K. Huang, Glenn J. Jaffe; Complement-mediated Injury in Cultured Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):894.
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Complement activation has been implicated increasingly in the pathogenesis of age-related macular degeneration (AMD). We have previously shown that complement attack causes RPE cell injury when membrane complement regulatory proteins (mCRPs; CD46, CD55, CD59) are functionally blocked. In non-ocular cells, programmed necrotic cell death contributes to complement-mediated cell death. Herein, we investigate the mechanisms involved in complement-mediated RPE cell injury.
Density-arrested, non-proliferating cultured human RPE (hRPE) cells were incubated with function-blocking antibody cocktail (mCRP Abs; anti-CD46 Ab, anti-CD55 Ab and anti-CD59 Ab) for 1h and then cells were exposed to 10% normal human serum (NHS) and 10% heat inactivated NHS (HiNHS) in the presence or absence of 1h pretreatment with z-VAD-fmk (pan caspase inhibitor) or 10% C7-depleted serum (C7D) with or without reconstituted C7 for various times. Apoptosis and necrosis were determined with "Cell Death Detection ELISA" kit. Subcellular morphologic change induced by complement attack was evaluated by electron microscopy. Western blot was performed to detect ERK, Akt, and P38 protein phosphorylation. Cell viability was assessed with tetrazolium salt (WST-1) assay.
When RPE cell mCRP function was blocked, complement attack induced necrotic cell death 24h after complement incubation. There were vacuoles, organelle loss and mitochondria disorganization in cells treated with mCRP Abs+NHS and mCRP Abs+C7D+C7 when compared to those treated with mCRP Abs+HiNHS and mCRP Abs+C7D 1h following complement treatment. RPE cell ERK phosphorylation was decreased 30 min after complement attack induced by mCRP Abs+C7D+C7 when compared to that in cells treated with mCRP Abs+C7D, without affecting p-Akt and p-P38. RPE cell viability was modestly but significantly increased in cells treated with mCRP Abs+zVAD+NHS when compared to that in cells treated with mCRP Abs+DMSO+NHS.
Our data suggest that complement primarily induces necrotic RPE cell death. ERK signaling and caspases may be involved in complement-mediated RPE injury.
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