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S.A. Rosenzweig, M.G. Slomiany, B.A. Slomiany; Regulation of VEGF Expression in Human Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):479.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To examine the effect of insulin-like growth factor 1 (IGF-1), growth hormone (GH), and hypoxia on the expression of hypoxia inducible factor 1 alpha (HIF-1α) and the secretion of vascular endothelial growth factor (VEGF) and IGF binding protein-3 (IGFBP-3) in human retinal pigment epithelial cell lines ARPE and D407. Methods: RPE cell lines ARPE and D407 cultured in polystyrene dishes or transwell® inserts were treated with cobalt chloride, hypoxia, or varying doses of IGF-1 or GH. Whole cell lysates were assayed by immunoblot for HIF-1αexpression, whereas conditioned medium was TCA precipitated and assayed by immunoblot for VEGF and ligand blot for IGFBP-3. Cells grown on coverslips were similarly treated and probed with antibodies to HIF-1α, VEGF, and IGFBP-3 and visualized using epifluorescence microscopy. Electrophoretic mobility shift assays of D407 nuclear extracts were conducted using a HIF-1–specific consensus sequence from the VEGF promoter. Results: IGF-1 and hypoxia stimulated dose-dependent increases in the secretion of three distinct isoforms of VEGF, as well as glycosylated and unglycosylated IGFBP-3. Cells grown on transwell® inserts exhibited predominantly apical release of VEGF and exclusively apical secretion of IGFBP-3. In preliminary experiments, growth hormone (GH; 500 ng/ml) stimulated HIF-1α expression, as well as VEGF and IGFBP-3 secretion. When combined with IGF-1 and hypoxia, these effects were additive. Epifluorescence microscopy confirmed that HIF-1α accumulation was localized to the nucleus whereas preliminary electrophoretic mobility shift assays indicated that IGF-1 stimulates HIF-1 DNA binding activity. Conclusions: IGF-1, GH, and hypoxia stimulate increased HIF-1α expression as well as VEGF and IGFBP-3 secretion. D407 cells exclusively secrete IGFBP-3 from their apical pole while VEGF release was not polarized. We are currently examining the contribution of VEGF transcytosis from the apical to the basolateral surface. These studies will aid in defining the roles of hypoxia, IGF-1, and GH in the progression of CNV.
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