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N. Chan, S. He, S. J. Ryan, Jr., D. R. Hinton; Histone Acetylation is a Major Mediator of VEGF-Induced Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3499.
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Choroidal neovascularization (CNV) is a serious complication of age-related macular degeneration (AMD). An imbalance of pro- and anti-angiogenic factors leads to the formation of CNV. However, the mechanisms underlying the regulation of vascular endothelial growth factor (VEGF) expression in intraocular angiogenesis remain unclear. The current study was to investigate whether epigenetics, specifically the inhibition of histone deacetylation, is involved in the regulation of expression of VEGF and its signaling in human fetal retinal pigment epithelial (RPE) cells.
Cultured early passage human fetal RPE cells were used in the study. The viability of RPE cells treated with the histone deacetylase inhibitor, trichostatin A (TSA), was tested by the MTT assay. The expression of acetyl-histone H3 in RPE cells treated with TSA (0µM, 0.1µM, 0.5µM or 1µM) for 24 hours and in mouse CNV sections induced by laser was investigated by immunostaining and/or Western blot. The expression of VEGF, PEDF, Flk-1, HIF-1α, PPARγ, caspase 3, p-p38 and p-Akt in RPE cells was examined by Western blot after treatment with TSA (0µM, 0.05µM, 0.1µM, 0.3µM or 0.5µM) for 24 hours with or without 150µM of cobalt chloride for 6 hours.
TSA induced a strong acetylation of the histone H3 protein in RPE cells and mouse CNV lesion as revealed by Western blot and immunostaining. TSA also inhibited RPE cell proliferation and arrested the cell cycle at G1 and S phase. Most importantly, TSA significantly down-regulated the expression of VEGF and VEGF receptor 2, and up-regulated the anti-angiogenic and neuro-protective factor, PEDF, as demonstrated by Western blot. The decreased VEGF expression was consistent with the reduced production of HIF-1α, as the concentration of TSA reached 0.3µM. TSA also induced an increased phosphorylation of p38 and activation of caspase 3; however, the phosphorylation of Akt was dramatically reduced by the exposure of TSA. In addition, the anti-proliferative transcription factor, PPARγ, was up-regulated by TSA. The results implicated that epigenetic events are involved in the regulation of angiogenesis and histone acetylation is a major mediator of VEGF-induced angiogenesis in vitro in RPE cells.
TSA inhibits angiogenesis by down-regulating VEGF and its signaling and up-regulating the anti-angiogenic factor, PEDF. Epigenetics play a critical role in the regulation of angiogenesis and TSA can be a potential therapeutic candidate for the treatment of CNV.
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