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C. Zhang, N. Strunnikova, K. Csaky; Hsp70 Over Expression Protects Human Retinal Pigment Epithelium (HRPE) Against Oxidant Induced Cell Death . Invest. Ophthalmol. Vis. Sci. 2005;46(13):180.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Retinal pigmented epithelium are continually exposed to oxidative stress. RPE appears to tolerate low level of the stress and may only undergo cell death either when exposed to high levels of oxidative damage or in the context of retinal disease such in atrophic age–related–macular degeneration (ARMD). Our previous study indicated that prolonged sublethal oxidative stress induced the expression of Hsp70, a prominent cytoprotective factor. The following study examined further the role Hsp70 expression in protecting RPE from oxidative stress–induced cell death. Methods: Exogenous expression of Hsp70/GFP was achieved by retroviral transduction of ARPE–19 cells. Cell viability exposed to the oxidant agent menadione was determined by XTT assay. Immuno–localization of proapoptotic factors was performed with organelle fractions and in intact AREP–19 cells expressing Hsp70 /GFP (RPE–70GFP), GFP (RPE–GFP) or sham transduced (RPE–S). Mitochondrial potential was monitored by mitotracker fluorescence. Results: Hsp70/GFP preferentially expressed in the cytoplasm where the estimated molar ratio to endogenous Hsp70 was 2.1. While the dose responsiveness of RPE–GFP and RPE–S was comparable, over expression Hsp70/GFP increased significantly the LD50 concentration from 25 µM to 85 µM menadione. After exposure of a lethal dose of menadione (50 µM), RPE–GFP cells demonstrated nuclear shrinkage and 50 kbp DNA fragmentation which was not seen in RPE–70/GFP cells. In contrast to RPE–GFP cells, which demonstrated a marked m depolarization, RPE–70/GFP cells maintained a membrane potential (m). Redistribution of AIF to nuclei, which was observed in RPE–GFP cells, could not be detected in RPE–70/GFP cells. Additionally, RPE–70 /GFP cells exposed to 50 µM menadione showed minimal change in nuclear AIF redistribution which was present in RPE–GFP cells. Caspase–3 activation was not observed under any condition. Conclusions: Exogenous expression HSP70 promotes hRPE survival. The pro–survival effect appears upstream of mitochondria permeability transition.
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