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Xiaoyan Ding, Mrinali Patel, Defen Shen, Alexandra A. Herzlich, Xiaoguang Cao, Rafael Villasmil, Kristina Klupsch, Jingsheng Tuo, Julian Downward, Chi-Chao Chan; Enhanced HtrA2/Omi Expression in Oxidative Injury to Retinal Pigment Epithelial Cells and Murine Models of Neurodegeneration. Invest. Ophthalmol. Vis. Sci. 2009;50(10):4957-4966. doi: 10.1167/iovs.09-3381.
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purpose. To investigate the role of HtrA2/Omi, a nuclear-encoded mitochondrial serine protease with a proapoptosis function, under H2O2-induced oxidative stress in human RPE, in the Ccl2 − / − Cx3cr1 − / − double-knockout (DKO) mouse retina, and the HtrA2/Omi-deficient mice.
methods. Oxidative stress was induced in ARPE-19 cells by 1 mM H2O2 for 2 hours. HtrA2/Omi and caspase-3 expression was evaluated using RQ-PCR, immunohistochemistry, or Western blot. Cell viability was detected by MTT assay. HtrA2/Omi expression in the subcellular components and activated caspase-3 were measured. These processes were also evaluated in cells treated with UCF-101, an HtrA2/Omi inhibitor or in cells subjected to RNAi against HtrA2/Omi. Oxidative stress was assayed and compared in retinas of DKO and wild-type (WT) mice by determining serum NADPH oxidase subunits and nitrite levels. Transmission electron microscopy was used to view the retinal ultrastructure of the HtrA2/Omi-deficient mice.
results. H2O2-induced oxidative damage resulted in HtrA2/Omi translocation from mitochondria to cytosol, leading to RPE cell apoptosis via a caspase-mediated pathway. Treatment of RPE cells with UCF-101 reduced the cytosolic translocation of HtrA2/Omi, attenuated caspase-3 activation, and decreased apoptosis. After specific HtrA2 downregulation, increased cell viability was measured in H2O2-treated ARPE-19 cells. Retina of DKO mice exhibit increased oxidative stress and upregulation of HtrA2/Omi. Fewer and abnormal mitochondria were found in HtrA2/Omi − / − photoreceptors and RPE.
conclusions. These findings suggest that HtrA2/Omi is related to RPE apoptosis due to oxidative stress, which may play an important role in the integrity of mitochondria and the pathogenesis of AMD.
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