Leukocyte migration across the endothelium is a critical event in inflammation. The goals of this project are to understand the signals initiated by leukocyte adhesion to endothelial cells that promote passage of leukocytes across the endothelium. We will focus on two aspects of this process: the generation of leukocyte-induced cup-like structures that form on the surfaces of endothelial cells, and on leukocyte passage through endothelial cell-cell junctions. In the first aim, we will test the hypothesis that leukocyte adhesion to endothelial cells activates specific Rho GTPases and that these contribute both to formation of cups and to the disassembly of endothelial cell-cell junctions. We will explore the pathways by which leukocyte adhesion regulates these GTPases, using techniques to identify relevant guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Particular attention will be paid to SGEF, which co-localizes with ICAM-1 in cups. SGEF activates RhoG, a Rho protein which induces dorsal membrane ruffles. We will also investigate the pathways downstream from RhoA and Rac1 that promote junctional disassembly. In the second aim, the hypothesis that endothelial junctions are regulated by Rap1 activity in response to leukocyte adhesion will be examined. We will use mouse models of inflammation to investigate the roles of Rap isoforms in endothelial cells in mice that are null for Rapla or Raplb. In preliminary work, we have shown that leukocyte adhesion stimulates the tyrosine phosphorylation of endothelial junctional components. In the third aim, we will investigate the pathway by which this occurs, whether it is in response to the activation of Rac1 and generation of reactive oxygen species. We will look for the tyrosine kinases and phosphatases involved and determine whether the tyrosine phosphorylation of VE-cadherin leads to its removal from junctions by endocytosis. Several receptor tyrosine phosphatases reside in endothelial junctions. We will test the hypothesis that these may interact with and be inhibited by extravasating leukocytes so as to elevate levels of phosphotyrosine in junctions. Because leukocyte migration across the endothelial barrier lining blood vessels is a critical step in inflammation, the elucidation of signaling pathways that regulate this process may reveal novel targets for the development of therapies to control inflammation and inflammatory diseases.
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