The ability of normal tissues to mount an angiogenic response in a precise and regulated manner is fundamental to normal physiological processes such as wound healing. The process of angiogenesis is initiated by vascular endothelial cells and involves the orderly migration, proliferation and differentiation into new capillary channels. Cultured human umbilical vein endothelial cells (HUVEC) express many of the characteristics of the endothelium in vivo and are used as a model system to investigate the mechanisms of angiogenesis. For example, HUVEC proliferate in response to fibroblast growth factor (FGF)-l and are growth arrested by cytokines. The tumor promoter phorbol myristic acetate (PMA) serves as an example of a biochemical agent able to repress FGF-1-induced HUVEC growth in vitro. Prolonged treatment of HUVEC with PMA results in the formation of differentiated capillary-like tubular structures. To study the transcriptional events in angiogenesis, we have cloned several immediate-early (IE) genes induced by PMA in HUVEC. Analogous to growth factor-inducible IE genes, PMA-inducible IE genes may mediate key, regulatory events in endothelial cell growth inhibition and/or differentiation. A novel PMA-inducible IE gene, termed edg-1 was cloned and characterized. It encodes a novel G-protein-coupled receptor (GPR) polypeptide. The ligand for the edg-1 receptor as well as the signal transduction pathways regulated by edg-1 are unknown at present. Because the edg-1 mRNA is an IE gene induced by angiogenic stimuli, and because high-levels of edg-1 transcript are expressed in endothelial cells, it may be involved in the regulation of signal transduction events intrinsic to angiogenesis. This proposal will attempt to define the edg-l-regulated signal transduction mechanisms by using the chimeric receptor approach. The ability of edg-1 to modulate the in vitro angiogenic phenomenon of endothelial cells will be assessed as well. Furthermore, we will test whether known vasoactive and angiogenic response modifiers are ligands for edg-1. These experiments will systematically examine the ability of candidate ligands to activate the transfected edg-1 receptors. In addition, this proposal will attempt to characterize the in vitro expression and in vivo correlates of edg-1 polypeptide expression in angiogenesis that occurs during wound healing. The expression patterns of G-protein isotypes during migration, proliferation and differentiation of endothelial cells will be studied. Since dysregulated angiogenesis is a hallmark of many diseases including diabetic retinopathy, glomerulonephritis, impaired wound healing and solid tumor growth, the proposed characterization of edg-1 as a regulator of angiogenesis in vitro may be of value in the understanding the pathogenesis of many human diseases.
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