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S. Kase, S. He, Z. Wang, S. J. Ryan, Sr., D. R. Hinton, Sr.; Expression of Vascular Endothelial Growth Factor and Pigment Epithelium-Derived Factor Is Modulated by 5-aza-2’-Deoxycytidine (5-aza), in RPE. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4430.
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Choroidal neovascularization (CNV) is a serious complication of age-related macular degeneration. Retinal pigment epithelial cells (RPE) and their secreted growth factors play an important role in the pathogenesis of CNV. However, more information is needed about the factors that regulate expression of VEGF and PEDF in RPE. This study examined the role of DNA methylation in modulating VEGF and PEDF expression in RPE and cell proliferation in choroidal endothelial cells (CEC).
Cultured human fetal RPE and bovine CEC were used in this study. 1x105 RPE were seeded in 6-well plates and were adapted to serum-free conditions or kept in 10% FBS containing medium. 5-AZA, a methylation inhibitor, (0.1, 1, 2, 6 µmol) was administrated to the cells for 72 hr with or without the addition of TNF-α (10 ng/ml) for 24 hr before the cells were harvested. In a separate experiment, RPE were treated with 6 µmol 5-AZA for 24, 48, or 72 hr. The supernatant and cell lysates for all experiments were collected for examination of VEGF and PEDF expression by Western blot and ELISA. CEC proliferation was analyzed by MTT assay in 96-well plates in the presence of 5-AZA and VEGF (10 ng/ml) for 48-72 hr.
Pretreatment of RPE with 5-AZA (6 µmol) decreased the expression of VEGF induced by TNF-α. Western blot analysis of supernatant and cell lysates demonstrated a time-dependent inhibitory effect on VEGF expression that was maximal at 72 hr after 5-AZA exposure. Expression of PEDF in cell lysates and supernatant was low and gradually reduced in serum-free conditions without 5-AZA treatment. However, PEDF was significantly increased 48 hour after 5-AZA addition and sustained until 72 hr. ELISA assay confirmed Western blot data. These changes resulted in a decreased VEGF/PEDF ratio in 48 hr 5-AZA-treated RPE cultures compared to untreated controls. 5-AZA did not affect the cell proliferation of untreated CEC cultures; however, CEC proliferation stimulated by VEGF was significantly inhibited by 5-AZA.
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