Expression of angiopoietin-1 in hypoxic pericytes: Regulation by hypoxia-inducible factor-2α and participation in endothelial cell migration and tube formation

We previously reported that hypoxia increases angiopoietin-1 (Ang1), but not Ang2, mRNA expression in bovine retinal pericytes (BRP). However, the mechanism underlying Ang1 expression is unknown. Here, we report that Ang1 protein expression increased in hypoxic BRP in a dose- and time-dependent manner. This increase was accompanied by an increase in hypoxia-inducible factor-2 alpha (HIF2 alpha) expression. Transfection with an antisense oligonucleotide for HIF2 alpha partially inhibited the hypoxia-induced increase in Ang1 expression. HIF2 alpha overexpression further potentiated hypoxia-stimulated Ang1 expression, suggesting that HIF2 alpha plays an important role in Ang1 regulation in BRP. When fused the Ang1 promoter (-3040 to +199) with the luciferase reporter gene, we found that hypoxia significantly increased promoter activity by 4.02 +/- 1.68 fold. However, progressive 5'-deletions from 3040 to 1799, which deleted two putative hypoxia response elements (HRE), abolished the hypoxia-induced increase in promoter activity. An electrophoretic mobility shift assay revealed that HIF2 alpha was predominantly bound to a HRE site, located specifically at nucleotides -2715 to -2712. Finally, treatment with conditioned medium obtained from hypoxic pericytes stimulated endothelial cell migration and tube formation, which was completely blocked by co-treatment with anti-Ang1 antibody. This study is the first to demonstrate that hypoxia upregulates Ang1 expression via HIF2 alpha-mediated transcriptional activation in pericytes, which plays a key role in angiogenesis. (C) 2015 Elsevier Inc. All rights reserved.