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Jacob E. Berchuck, Ping Yang, Brett A. Toimil, Peter Baciu, Glenn J. Jaffe; All-trans-Retinal Sensitizes Human RPE Cells to Alternative Complement Pathway-Induced Cell Death. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1649.
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RPE cells are exposed to numerous oxidants, including all-trans-retinal (ATR), a chromophore released in photoreceptors after photon absorption. ATR is the precursor of A2E, a fluorophore believed to cause RPE cell death, and ATR itself is reportedly toxic to RPE cells, yet its direct role in the pathogenesis of AMD is unclear. It is thought that chronic oxidative stress injures RPE cells, impairing their ability to regulate surface complement deposition leading to uncontrolled alternative complement pathway (AP) activation. In this study, we used an in vitro model to determine whether pre-treatment with ATR sensitizes primary human RPE (hRPE) cells to AP-induced cell death. Further, we explored the signal transduction pathways by which ATR and AP exert their cytotoxic effects.
hRPE cells were treated with a non-lethal dose of ATR for 90 minutes, followed by 30-minute incubation with a complement-fixing antibody. Cells were then treated with C1q-depleted human serum (C1qD) to activate AP. After 90 minutes, the effect of ATR and AP activation on hRPE cell viability was assessed by tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) release assays. Following the same treatment conditions, changes in ERK and P38 phosphorylation were examined by Western blot.
Treatment with ATR alone caused a dose-dependent decrease in hRPE cell viability, as determined by LDH release and WST-1 assays. AP activation alone also caused hRPE cell death. In both LDH (p<0.01) and WST-1 (p<0.005) assays, AP activation in hRPE cells pre-treated with a non-lethal dose of ATR decreased viability more than the additive effect of both independent treatments. Following the same treatment conditions, when measured 5 minutes after C1qD addition, P38 phosphorylation increased, while ERK phosphorylation decreased.
We have shown that ATR causes dose-dependent cell death in hRPE cells. More importantly, at a non-lethal dose, ATR sensitizes hRPE cells to the cytotoxic effects of AP activation and when combined, the two synergistically cause hRPE cell death. Although their role in ATR and AP-induced hRPE cell death is not yet defined, P38 and ERK may be important mediators and represent potential targets to inhibit the oxidative stress and complement-induced injury that contributes to AMD.
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