Hypoxia-Induced COX-2 and VEGF Gene Expression in Retinal Cells: Repression by CGP43182

Abstract

Abstract: : Purpose: Up-regulation of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) gene expression are associated with the initiation of neovascularization (NV) in hypoxia triggered retinal cells. This study examined transcription factor (TF) AP-1-, HIF-1- and NF-ΚB-DNA binding in relation to COX-2 and VEGF RNA and protein levels in monkey choroidal endothelial (RF/6A) cells exposed to short periods of hypoxia. We also examined the effects of the carboxamide CGP43182, a secretory phopholipase A₂ and NF-ΚB inhibitor, on TF-DNA binding, COX-2 and VEGF activation, prostaglandin E₂ (PGE₂) release and de novo tube formation. Methods: RF/6A cells were subjected to hypoxia (90% N₂/5% CO₂/5%O₂) for 0, 1 and 3 hours at which times RNA and nucleoproteins were isolated. AP-1-, HIF-1- and NF-ΚB-DNA binding was assayed using gel-shift assay; RNA, cellular protein and PGE₂ levels were quantitated using RT-PCR, Western and enzyme-immunoassay, respectively. Results: HIF-1 and NF-ΚB activation and HIF-1- and NF-ΚB-DNA binding immediately preceded up-regulation of COX-2 and VEGF gene expression during hypoxia. COX-2 RNA was elevated after 1 and 3 hours of hypoxia 4- and 5-fold, respectively. VEGF RNA and protein abundance consistently lagged behind COX-2 induction, but each were increased 2- to 3-fold 3 hours post-hypoxia. CGP43182 was found to inhibit NF-ΚB- (but not HIF-1-)-DNA binding, COX-2 and VEGF gene expression, PGE₂ release and hypoxia-induced tubular morphogenesis. Conclusions: Increased HIF-1- and NF-ΚB-DNA-binding just before COX-2 expression suggests that these TFs are important precursors to both COX-2 and VEGF induction in hypoxic RF/6A cells. CGP43182 appears to be an effective inhibitor of NF-ΚB-mediated signaling pathways culminating in NV. VEGF expression may be regulated through dual-interdependent mechanisms involving HIF-1 directly and indirectly via NF-ΚB-mediated COX-2 expression and PGE₂ production. These data underscore the importance of HIF-1 and NF-ΚB activation in orchestrating genetic programs which culminate in de novo tube formation and retinal NV.

Keywords: neovascularization • transcription factors • hypoxia