Regulation of vascular endothelial growth factor gene expression by hypoxia

Abstract

Vascular endothelial growth factor (VEGF), a specific endothelial cell mitogen and regulator of microvascular permeability plays a central role in angiogenesis. Hypoxia stimulates VEGF gene expression in a variety of cultured cell types. The ovary is one of the few sites where angiogenesis normally occurs. Granulosa cells are probably exposed to hypoxic conditions during follicular development; this environment likely leads to enhanced VEGF secretion. Increased VEGF expression may play a role in the processes of ovulation and corpus luteum development. This dissertation investigates the hypoxic induction of VEGF expression in granulosa cells and characterizes the hypoxia response element in the VEGF gene.Using reverse transcription-polymerase chain reaction (RT-PCR), we have shown that rat ovarian granulosa cells exposed to hypoxia respond by increasing steady-state levels of VEGF mRNA. This increase is apparent within 3 h and is maximal by 12 h. Reducing oxygen from 20% to just 15 or 10% induces an increase in VEGF message. Maximal induction is seen at 5, 2 or 1% O{dollar}\sb2{dollar}.;Apart from the female reproductive system, neovascularization in adults is essentially limited to disease states. Tumor growth, characterized by the development of hypoxic regions, is angiogenesis-dependent. VEGF is secreted at high levels by tumors of various origins. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric protein known to be a transcriptional activator of the human erythropoietin gene in hypoxic cells. We show the involvement of HIF-1 in activating VEGF transcription in Hep3B cells. VEGF sequences mediated transcriptional activation of luciferase (luc) reporter gene expression in response to hypoxia in Hep3B cells. A 47-base pair (bp) sequence, located 985 to 939 bp upstream of the VEGF transcription initiation site, mediated hypoxia-inducible gene expression. Reporters containing VEGF sequences, in the context of either the native VEGF or the heterologous SV40 promoter, were co-transfected with expression vectors encoding HIF-1{dollar}\alpha{dollar} and HIF-1{dollar}\beta{dollar}. Luc expression was significantly increased in both hypoxic and non-hypoxic cells relative to cells transfected with reporters alone. A HIF-1 binding site was identified in the 47-bp hypoxia response element. A 3-bp substitution eliminated HIF-1 mediated transcriptional activation in response to hypoxia and/or recombinant HIF-1. Co-transfection of a luc reporter containing the 47-bp element with a vector expressing a dominant negative form of HIF-1{dollar}\alpha{dollar} inhibited transcriptional activation in hypoxic cells in a dose-dependent manner. These data indicate that VEGF expression responds rapidly to a reduction in oxygen and that this response is partly due to an increase in transcription mediated by HIF-1.