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E.G. Rojo-Niersbach, A. Ottlecz, H. Buehler-Normi, G.N. Lambrou; Molecular Analysis of VEGF and IL-8 Release in Cultured Monkey Choroidal-Retinal Endothelial Cells Exposed to Hypoxia . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2920.
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Purpose: The role of oxygen-sensitive transcription factors (TFs) in activating genes involved in retinal neovascularization (NV) was examined. Methods: RF/6A rhesus monkey choroid-retinal endothelial cells were incubated under hypoxic conditions for 8 hours and treated with the phosphoinositol-3 (PI-3) kinase inhibitor LY294002 (Calbiochem), the MAP kinase (MEK) inhibitor U0126 (Promega), or vehicle. Cells and culture media were harvested at 0, 24, 48, and 72 hours after hypoxic challenge. Nuclear extracts were used in 1) electrophoretic mobility shift assays (EMSA) with hypoxia-inducible-factor-1 (HIF1) and NF-ΚB elements in the vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) promoters and 2) Western Blots for relevant TF detection (HIF1, NF-ΚB, and p53). VEGF and IL-8 release was determined by enzyme-linked immunoabsorbent assay (ELISA). Results: In addition to HIF1, a larger protein species interacted with the HIF1 binding site of the VEGF promoter during 24-72 hours post-hypoxia. LY294002 (10 µM) inhibited the binding of this species but left HIF1 binding unaffected. At later post-hypoxic timepoints, VEGF release in LY294002-treated cells was abolished, while IL-8 secretion was up to sevenfold higher. In contrast, VEGF levels were similar in cells treated with 1 µM U0126, while IL-8 release was 25-30% lower than untreated post-hypoxic controls. NF-ΚB binding on the IL-8 promoter was observed at 0-48 hours post-hypoxia but was abrogated in LY294002-treated cells. HIF1/NF-ΚB expression was observed at 48-72 hours post-hypoxia, while p53 expression was seen up to 48 hours post-hypoxia. Conclusions: Our results suggest that HIF1 activation and DNA binding may exert a stronger inducing effect on VEGF expression than NF-ΚB-mediated activation of IL-8 release in the ocular NV phenotype. It needs to be clarified whether the IL-8 stimulation is related to PI-3 kinase inhibition or if it is an independent effect of LY294002. VEGF activation in ocular endothelial cells may be due to the HIF1-mediated recruitment of a larger protein species to the VEGF promoter.
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