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Walter J. Lukiw, Anna Ottlecz, George Lambrou, Monika Grueninger, Joelle Finley, Hilary W. Thompson, Nicolas G. Bazan; Coordinate Activation of HIF-1 and NF-κB DNA Binding and COX-2 and VEGF Expression in Retinal Cells by Hypoxia. Invest. Ophthalmol. Vis. Sci. 2003;44(10):4163-4170. doi: 10.1167/iovs.02-0655.
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© ARVO (1962-2015); The Authors (2016-present)
purpose. Proinflammatory signaling mechanisms are implicated in the induction of retinal neovascularization (NV) during ischemic retinopathies. This study examined transcription factor (TF) AP-1, HIF-1, and NF-κB DNA-binding in relation to cyclooxygenase (COX)-2 and VEGF RNA and protein levels in hypoxia-triggered monkey choroidal retinal (RF/6A) endothelial cells. Effects of the carboxamide CGP43182 were tested on COX-2 and VEGF activation and prostaglandin (PG)E2 release.
methods. RF/6A cells were subjected to hypoxia for 1 and 3 hours, at which times RNA and proteins were isolated. Potential AP-1, hypoxia-inducible factor (HIF)-1 and NF-κB DNA-binding sites were identified using DNA sequence search algorithms and were analyzed using gel-shift assay. COX-2 and VEGF RNA, protein, and PGE2 levels were quantified by RT-PCR, Western analysis, and enzyme immunoassay, respectively. Tubular morphogenesis was analyzed with phase-contrast imaging microscopy.
results. Nuclear AP-1, HIF-1 and NF-κB promoter DNA binding increased 1.5-, 4-, and 3-fold, respectively, after 1 hour of hypoxia. COX-2 RNA was elevated five- and fourfold after 1 and 3 hours of hypoxia, respectively. VEGF RNA and protein abundance lagged behind COX-2 induction but were each increased two- to threefold 3 hours after hypoxia. CGP43182 was found to inhibit NF-κB DNA binding, COX-2 and VEGF gene expression, PGE2 release, and hypoxia-induced tubular morphogenesis.
conclusions. Maximum HIF-1 and NF-κB DNA binding immediately before COX-2 expression suggests that these TFs are important regulators of COX-2 induction in hypoxic RF/6A cells. IL-1β emulated AP-1, HIF-1, and NF-κB DNA binding during hypoxia and may be a novel cytokine trigger for NV. CGP43182 appears to be an effective inhibitor of NV. VEGF expression appears to be regulated through dual interdependent mechanisms involving HIF-1 directly and indirectly through NF-κB-mediated COX-2 expression and PGE2 production.
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