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N.V. Strunnikova, J. Baffi, C. Zhang, D. Teichberg, K. Becker, S.W. Cousins, K.G. Csaky; Cellular and Molecular Responses to Sublethal Oxidative Injury in Retinal Pigment Epithelial Cells (RPE) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3145.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: A number of studies have indicated that oxidative injury could play a role in the pathogenesis of age-related macular degeneration (AMD). During the lifetime retinal pigment epithelium cells (RPE) exposed to a high degree of oxidative stress because of their location and function. RPE response to oxidative injury may help explain the development of certain anatomic changes of early AMD including absence of RPE cell death and sub-RPE deposit formation. The aim of the present study was to examine the different cellular and molecular responses of the RPE to sublethal injury and to examine these responses in relationship to the development of AMD. Methods: Human differentiated ARPE-19 cells were exposed to different concentrations of the oxidative stimulus hydroquinone. Viability of the cells was measured by XTT conversion, 3H leucine incorporation, trypan blue exclusion methods and localization of apoptotic proteins including apoptosis-induced factor (AIF).AIF localization, changes in mitochondrial potential, phosphorylation of SAPK and p38, actin reorganization were detected by immunocytochemistry and Western blot analysis. Cell membrane blebbing was detected using GFP-membrane labeled RPE cells (GFP-c’ras-ARPE). Changes in gene expression were evaluated by microarray analysis. Results: Absence of cell death at sublethal doses of hydroquinone was verified by XTT conversion, trypan blue exclusion and confirmed by the absence of translocation of AIF or changes in mitochondrial potential. At sublethal oxidative doses, an increase in 3H leucine incorporation and morphological changes which included actin rearrangement, activation of p38 and SAPK stress factors and cell membrane blebbing were observed. Microarray analysis revealed alteration in genes involved in the protection and recovery of injured cells. Specifically an up-regulation of genes involved in protection from oxidative stress (MGST1, TNX1 ets) and a down-regulation of cell death related genes (CASP9, RB1 ets) were observed. Full recovery of the RPE 48 hours after the injury was confirmed by the normalization of the gene expression profile back to baseline levels. Conclusion: These results indicate that RPE cells have an elaborate death avoidance system when exposed to lower doses of oxidative stress. However, sublethal injury does elicit specific sets of both cellular and molecular response, which may be involved in AMD development.
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