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S.G. Barreiro, V.L. Marcheselli, N.G. Bazan; Human Retinal Pigment Epithelial Cells Protected by NPD–1 After A2E–epoxide Induction . Invest. Ophthalmol. Vis. Sci. 2005;46(13):250.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: N–retinylidene–N–retinylethanolamine (A2E) is the major hydrophobic fluorophore that is formed during the visual cycle and becomes part of the retinal pigment epithelial cell lipofuscins. Photo–oxidation promotes the accumulation of A2E and its subsequent conversion to A2E oxiranes (epoxides). These metabolites promote RPE cell damage and death, and in turn, photoreceptor death. A2E/A2E–oxirane accumulation has been implicated in retinal degenerations such as Stargardt’s disease. The present study assessed the ability of neuroprotectin–D1 (NPD1), a stereospecific docosahexaenoic acid–derived messenger, to counteract A2E/A2E–epoxide RPE cytotoxicity. Methods: ARPE–19 cells grown in 6–well plates were exposed to blue light for 15 minutes after adding 100 µM A2E. NPD1 (50 nM) was added 15 minutes after light exposure and then cells were further incubated for 15 hours. Hoechst staining was used to monitor apoptosis using a fluorescence confocal microscope. Another set of ARPE–19 cells was transfected by Fugene with NF–ΚB promoter sequence linked to a luciferase reporter gene and with IΚB–EGFP and 4 hours after transfection, cells were washed and further incubated 8 to 10 hours before adding inducers. The cells were serum–starved 4 hours before the addition of A2E, TNF–α / H2O2, and NPD1, and further incubated for 15 hours before they were harvested. The luciferase activity was measured using luciferin as substrate. The IΚB–EGFP probe was measured by fluorescence confocal microscopy. Results: Accumulation of A2E–epoxides under these conditions was detected by LC–PDA–ESI–MS–MS. A2E/blue light exposure triggered apoptosis in ARPE–19 cells. NF–ΚB is up–regulated after induction by A2E and oxidative stress (TNF–α /H2O2). Neuroprotectin D1 prevented apoptosis when it was added 15 minutes after A2E/blue–light exposure, and decreased the expression of NF–ΚB and IΚB Conclusions: Blue light accelerates the formation of oxiranes and photo–oxidation produces apoptosis in ARPE–19 cells. NF–ΚB and IΚB expressions are enhanced by A2E–epoxides and oxidative stress; however after the addition of 50 nM NPD1, cell damage is inhibited, and NF–ΚB and IΚB are inactivated. Moreover, LC–MS–MS shows high levels of oxiranes after A2E/blue–light exposure, but low levels in dark controls. The NF–ΚB pro–inflammatory pathway and photo–oxidation by A2E–epoxides are potential drug targets for preventing cell damage in retinal pigment epithelial cells and photoreceptors. NPD1 produces significant neuroprotection. NIH EY05121.
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