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Originally published In Press as doi:10.1167/iovs.09-3381 on May 14, 2009
 (Investigative Ophthalmology and Visual Science. 2009;50:4957-4966.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.09-3381
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Enhanced HtrA2/Omi Expression in Oxidative Injury to Retinal Pigment Epithelial Cells and Murine Models of Neurodegeneration

Xiaoyan Ding,1,2 Mrinali Patel,1,3 Defen Shen,1 Alexandra A. Herzlich,1 Xiaoguang Cao,1,4 Rafael Villasmil,5 Kristina Klupsch,6 Jingsheng Tuo,1 Julian Downward,6 and Chi-Chao Chan1

1From the Section of Immunopathology, Laboratory of Immunology, and the 5Flow Cytometry Core, National Eye Institute, National Institutes of Health, Bethesda, Maryland; the 2Zhongshan Ophthalmic Center, Sun Yet-sen University, Guangzhou, China; the 3Howard Hughes Medical Institute, Chevy Chase, Maryland; the 4Department of Ophthalmology, People’s Hospital, Peking University, Beijing, China; and the 6Signal Transduction Laboratory, Cancer Research UK London Research Institute, London, United Kingdom.

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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