The receptor tyrosine kinase KDR/flk-1 is expressed uniquely in vascular endothelial cells. The ligand for KDR/flk-1 is the endothelial cell- specific mitogen and angiogenic peptide vascular endothelial growth factor (VEGF). KDR/flk-1 and VEGF function as the key regulators of physiologic as well as pathological angiogenesis. More importantly, KDR/flk-1 is one of the earliest markers for angioblasts, the precursors of vascular endothelial cells, and KDR/flk-1 is necessary for these precursors to develop into mature endothelial cells. We hypothesize that the restricted expression of KDR/flk-1 in mature vascular endothelial cells, and its expression in angioblasts, is regulated by the interaction of specific DNA sequences (cis-acting elements) and their cognate DNA- binding proteins (trans-acting factors). We have isolated the human KDR/flk-1 gene and characterized its 5'-flanking sequence. The goal of the proposed work is to isolate the cis-acting elements important for endothelial cell-specific expression of KDR/flk-1 and for its induction during angiogenesis in vitro. The cis-acting elements identified in vitro will be tested in vivo in transgenic mice to determine whether they are sufficient to confer developmental and endothelial cell-restricted expression of the reporter gene. With these cis-acting elements, it will be possible to isolate the genes coding for the cognate trans-acting factors and study their expression in the contexts of vasculogenesis and angiogenesis. These experiments will allow us to isolate nodal genes encoding the trans-acting factors that regulate cells of the endothelial lineage, thereby defining potential targets for the regulation of angiogenesis. In addition, the endothelial cell-specific cis-acting elements uncovered by this work may be useful tools in gene therapy aimed at restricting expression of transduced genes to the vessel wall.
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