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S. Jiang, M.L. Orr, P. Sternberg, Jr., D.P. Jones; Fas Activation Mediates Mitochondria Changes in Oxidant-induced Apoptosis in Cultured Human Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1625.
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Purpose:Oxidant-induced apoptosis of retinal pigment epithelium (RPE) may serve an important mechanism in the pathogenesis of age-related macular degeneration. We have previously shown that mitochondrial permeability transition and Fas activation are involved in oxidant-induced apoptosis of cultured human RPE (hRPE). Fas activation can cause mitochondria changes in apoptotic cells through activation of caspase 8 and cleavage of Bid, a proapoptotic member of Bcl-2 family. The purpose of this study was to determine whether oxidant-induced apoptosis in hRPE cells occurs as a result of direct damage to mitochondria or whether mitochondria changes are secondary to activation of Fas-mediated pathway. Methods: The signal pathway upstream to Bid cleavage were blocked by pretreating cultured hRPE cells with antibodies that interfere Fas and FasL interaction and agents that inhibit caspase 8 activation. Oxidant tert-butylhydroperoxide (tBH) was then added to induce apoptosis in hRPE cells. Mitochondrial potential was measured by flow cytometry using rhodamine 123 and tetramethylrhodamine, methyl ester. Release of cytochrome c from mitochondria and activation of caspase 3 were determined by Western blot. Apoptosis was assessed by measuring phosphatidylserine exposure with Annexin-V staining. Cell death was determined by the cleavage of a tertrazolium salt WST-1. Results: Caspase inhibitors z-VAD-FMK, IETD-CHO, and IETD-FMK, and antagonistic anti-Fas antibody ZB4 partially inhibited tBH-induced loss of mitochondrial membrane potential. Inhibiting the signal pathway upstream of Bid cleavage also blocked cytochrome c releases from mitochondria and activation of caspase 3. In the present of these reagents, the down-stream apoptotic changes, phosphatidylserine exposure and cell death, were also inhibited. Conclusions: The results are consistent with the involvement of Fas pathway in apoptosis triggered by tBH. It also indicates that mitocondrial changes seen in tBH-induced apoptosis are partially mediated by the cleavage of Bid upon Fas activation.
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