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R. J. Grierson, M. J. Berna, B. Meyer-Rüsenberg, A. Wiermann, G. Richard, K. G. Csaky, M. Thill; A Role for Angiogenic Growth Factors in Hypermethylation in Endothelial Cells and Neovascular Disease. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2938.
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© ARVO (1962-2015); The Authors (2016-present)
Hypermethylation of epigenetically silenced genes is observed in tumor cells and tissue and implicated in cancerogenesis. This study set out to identify hypermethylation and the underlying mechanisms in endothelial cells of different origin particularly late outgrowth endothelial progenitor cells (OECs) from patients with neovascular AMD.
Peripheral blood mononuclear cells were collected from patients and cultured under defined conditions until outgrowth of OECs was observed. OECs, HUVEC and MVEC cells grown in EGM-2MV media were screened for hypermethylation on 84 different gene promoters using a methylation array. Expression of hypermethylated genes was investigated at the DNA and protein level. To achieve demethylation, cells were grown in EBM-2MV media containing 2 % fetal calf serum in the absence of growth factors or complete EGM2-MV media containing 5-aza-2’deoxycytidine. VEGF and bFGF were reintroduced to media to determine if promoters were remethylated by the addition of growth factors. Promoter methylation status was assessed by methylation-specific PCR and protein expression was detected by Western blotting. The global methylation pattern of enucleated eyes with ocular neovascularisation was studied using immunohistochemistry.
Hypermethylation was observed in the promoter regions of the HOXA2, IL4, VHL and NME2 genes. Growth of cells in EBM-2MV media triggered demethylation of the hypermethylated promoter regions after 48 h. 1 µM 5-aza-2’deoxycytidine demethylated promoters after 24 h. Reintroduction of bFGF and VEGF following demethylation resulted in promoter hypermethylation after 48 h. Levels of global methylation in ocular neovascularisation were significantly higher compared with normal eyes.
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