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Piyush Kothary, Patrick Lee, Neil Parikh, Sara Abraham, Monte Del Monte; Role of Hypoxia-inducible Factor 1 Alpha in Hydrogen Peroxide-induced Human Retinal Pigment Epithelial Cell Death. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2010.
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Molecular biological investigations led the development of anti-vascular endothelial growth factor (VEGF) therapy for choroidal neovascularization (CNV). Since anti-VEGF therapy has not been effective in all patients, we investigated the mechanism of action of VEGF: whether there is a role for hypoxia-inducible Factor 1 Alpha in the VEGF pathway in our cultured human retinal pigment epithelial (hRPE) cell model.
Human RPE cells were cultured from normal eyes obtained from the Michigan Eye Bank. Cellular proliferation in the presence of increasing concentrations of FBS was measured by 3(H) thymidine incorporation (3H-thy) and cell viability by the trypan blue exclusion method. Intracellular synthesis of caspase 3, an apoptosis cell death marker, HIF 1 alpha, a regulatory subunit of hypoxia-inducible factor 1 and VEGF were quantitated and localized by immunoprecipitation and immunohistochemistry. Statistical analysis was done by Student t-test with p≤0.05 considered to be significant.
Fetal bovine serum stimulated hRPE cell proliferation and H2O2 exposure inhibited proliferation of viable hRPE cells also in a in a dose dependent manner. H2O2 (0.5 mM) exposure decreased hRPE cell number by 209.6% (5.70±0.73 vs 2.72±0.35, (Viable cells x 10,000±SEM, n=4, p≤0.05)) and increased 14C-caspase-3 and 14C-HIF1 alpha synthesis in a dose dependent manner. H2O2 (0.5 mM) exposure increased 14C-caspase synthesis by 209.4% (1022.44±432 vs. 2141.26± 666.31, n=4, p≤0.05)) and HIF1 alpha by 211.1% (197.62±43.44 vs. 417.20± 21.34, n=6, p≤0.05)). H2O2 (0.5 mM) exposure also increased 14C-VEGF synthesis by 130.0% (613.43.26± 143.49 vs.793.68 ±124.02, n=4, p≤0.05)). Phase contrast microscopy confirmed oxidative damage hRPE cell morphology following H2O2 exposure. Immunohistochemical analysis confirmed and localized increased expression of HIF1 alpha and VEGF expression in presence of H2O2 when compared with controls.
Our data suggest that H2O2 may stimulate angiogenesis in CNV by stimulating HIF1 alpha as well as VEGF, suggesting anti-HIF1 alpha targeted therapy may provide a novel approach to treating CNV.
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