EDG-1 signaling in angiogenesis and vascular remodeling

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

The EDG-1 gene encodes a G-protein-coupled receptor for the platelet- derived bioactive lipid sphingosine I-phosphate (S1P). The applicant's laboratory has shown that S1P signaling via EDG-1 and -3 receptors induce angiogenesis and vascular maturation. Further, the Edg-1 gene was shown to be indispensable for pericyte ensheathment for developing vessels in the embryo. Thus, our central hypothesis is that signal transduction of the EDG-1 receptor in the endothelial cells regulates distinct phases of angiogenesis, namely, directed cell migration and cell- cell interactions between vascular endothelial cells and vascular smooth muscle/pericyte cells. Three specific aims are proposed in this project. First, we will critically explore the novel mechanism of protein kinase Akt phosphorylation of the EDG-1 GPCR. Specifically, we will identify the polypeptides that associate with the native as well as phosphorylated EDG-1. The role of the associated polypeptides on EDG-1 signaling will be determined. Since Akt is translocated specifically to the migrating front of cells, the role of polarized phosphorylation of EDG-1 will be explored. The relationship between Akt phosphorylation of EDG-1 on S1P-induced cell-cell adherens will be explored. The relationship between Akt phosphorylation of EDG-1 on S1p-induced cell-cell adherens junction assembly (mediated by VE-cadherin) and survival will be delineated. In the second specific aim, mechanisms involved in the S1P/EDG-1 regulate endothelial cell/vascular smooth muscle cell (heterotypic) interaction will be characterized. We have identified N- cadherin as a critical molecule that mediates heterotypic cell-cell interaction. The role of N-cadherin activation by S1P/EDG-1 in heterotypic EC/VSMC interactions will be assessed in co-culture systems. Signaling pathways from the EDG-1 receptor to the N-cadherin will be defined, with an emphasis on the role of Akt and EDG-1 phosphorylation. The role of N-cadherin in vascular maturation in vivo will be assessed in nude mouse models of angiogenesis. In the third aim, the physiological role of Akt-mediated phosphorylation of EDG-1 will be assessed in the transgenic rescue experiments in the Edg-1 null background. Wild-type and T236A mutants of EDG-1 will be expressed in transgenic mice using the endothelial cell-specific Tie II promoter. The ability of the wild-type and mutant receptors to rescue the embryonic lethality will be assessed. Developmental phenotypes will be characterized by morphological, histological and in situ gene expression analysis. These efforts are anticipated to yield novel mechanistic insights into the biology of S1P signaling in the formation and maintenance of the vascular system. Furthermore, the goals of this proposal are within the focused theme of the program project application, which is to investigate the interactive aspects of signaling pathways germane to vascular remodeling.