HYPOXIC REGULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR

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The primary objective of this project is to delineate the regulation of rat vascular endothelial growth factor gene expression by hypoxia in vitro and ischemia in vivo. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and potent endothelial cell specific mitogen. Preliminary data have shown that the steady state mRNA for VEGF is markedly upregulated by hypoxia in vitro. The specific aims of the studies described herein are as follows: 1) to define the contribution of new transcription versus mRNA stabilization to the increase in the steady state VEGF mRNA levels following a hypoxic stimulus; 2) to define the cis- acting regulatory sequences responsible for mediating VEGF transcriptional activation and/or mRNA stabilization by hypoxia; 3) to identify specific nucleic acid binding proteins that bind to these cis regulatory sequences; 4) to utilize reporter gene constructs to allow for a rational pharmacological approach to manipulate-endogenous VEGF gene expression. Methods to be employed include functional assays (nuclear run-off transcription assays,transient expression assays, in vitro RNA degradation analyses) and structural analyses (DNA and RNA mobility shift assays, in vitro DNAase l footprint analysis, protein purification and characterization). Acquiring a better understanding of the molecular mechanisms governing the regulation of VEGF, and hence angiogenesis, by hypoxia may permit the development of novel new strategies to treat human conditions resulting from vascular insufficiency such as coronary artery disease and peripheral vascular disease.