Coordinate activation of HIF-1 and NF-kappaB DNA binding and COX-2 and VEGF expression in retinal cells by hypoxia.

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-kappaB 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)E(2) 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-kappaB DNA-binding sites were identified using DNA sequence search algorithms and were analyzed using gel-shift assay. COX-2 and VEGF RNA, protein, and PGE(2) 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-kappaB 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-kappaB DNA binding, COX-2 and VEGF gene expression, PGE(2) release, and hypoxia-induced tubular morphogenesis. CONCLUSIONS: Maximum HIF-1 and NF-kappaB DNA binding immediately before COX-2 expression suggests that these TFs are important regulators of COX-2 induction in hypoxic RF/6A cells. IL-1beta emulated AP-1, HIF-1, and NF-kappaB 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-kappaB-mediated COX-2 expression and PGE(2) production.

Hypoxia activates matrix metalloproteinase expression and the VEGF system in monkey choroid-retinal endothelial cells: Involvement of cytosolic phospholipase A2 activity.

PURPOSE: To determine whether the gene expression of matrix metalloproteinases (MMPs) as well as that of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF) and its receptors change in response to hypoxic exposure in a primate choroid-retinal endothelial cell line, and furthermore, whether cytosolic phospholipase A2 (cPLA2) plays a role in this process. METHODS: Rhesus macaque choroid-retinal endothelial (RF/6A) cells were incubated under hypoxic conditions for 1, 2, 4, or 8 h prior to RNA extraction. In some experiments cells were pretreated with the cPLA2 inhibitor AACOCF3 (10 microM) for 30 min prior to hypoxia. Changes in gene expression were determined by RT-PCR and quantified by real-time PCR for urokinase plasminogen activator (uPA), collagenase-1 (MMP-1), membrane type-1 metalloproteinase (MT1-MMP), gelatinases A and B (MMP-2, MMP-9), tissue inhibitor-2 (TIMP-2), VEGF and its receptors, Flt-1 (VEGFR-1), KDR (VEGFR-2), and neuropilin-1 (NP-1). MMP-2 secreted by the cells was evaluated by zymography. VEGF release was measured by ELISA. In tube-formation studies, endothelial cells (EC) were seeded into collagen gel, exposed to hypoxia for 4 h, then incubated under normoxic conditions for 72 h. RESULTS: Hypoxia triggered a three fold increase in the gene expression of MT1-MMP, MMP-2, and TIMP-2, and a ten fold increase in MMP-2 levels. Moreover it also induced tube formation in EC. Expression of uPA, MMP-1, and MMP-9 mRNA was not detected. Pretreatment with AACOCF3 abolished hypoxia-induced tube formation and MT1-MMP, MMP-2, and TIMP-2 transcription. Furthermore, hypoxia produced a significant, sustained increase in the gene expression and release of VEGF-165, the only VEGF-A isoform detected in these cells. AACOCF3 reduced the hypoxia-induced VEGF release at 8 h of hypoxia. VEGF receptors KDR and NP-1 were constitutively expressed in EC and up-regulated under hypoxic conditions. CONCLUSIONS: In monkey choroid-retinal EC, hypoxia selectively induces MMP-2 activity. This induction is preceded by MT1-MMP, MMP-2, and TIMP-2 mRNA expression, as well as that of the VEGF-165 isoform and its receptors KDR and NP1. These increases possibly result from hypoxia-induced activation of cPLA2 and subsequent release of arachidonic acid and its conversion to prostaglandins. These molecular changes in EC could, in part, contribute to the angiogenic response that occurs in the development of ischemic retinopathies and choroidal neovascularization.