This is a competitive renewal of a grant in its eleventh year. The overall objective of the proposed studies is to address critical signaling mechanisms regulating the loss of endothelial barrier function induced by thrombin. The investigators have shown that thrombin increases the permeability of the pulmonary microvascular barrier by activation of discrete signaling pathways which involve the release of intracellular calcium and activation of protein kinase C isoforms leading to phosphorylation of myosin light-chain. In addition, they have shown that adherens junctional permeability may also be controlled by the cadherin-catenin complex, which contributes to the tethering force in endothelial cells. In the proposed studies, they will determine (1) the basis by which linkage of PAR-1 (endothelial cell surface receptor mediating thrombin-induced increased endothelial permeability) to specific G-proteins mediates the permeability response; (2) the relationship between heterotrimeric and monomeric G-proteins in the mechanism of increased endothelial permeability; (3) the role of oxidant generation in endothelial cells in signaling increased permeability; and (4) the role of cadherin-catenin complex in regulating the loss of endothelial barrier function. Studies will utilize molecular approaches to dissect the signaling pathways as well as physiologic assessment of endothelial permeability in the endothelial monolayer and pulmonary microvessels. It is hoped that that at the completion of these studies, they will define the mechanisms by which cell surface signaling involving the binding of the agonist to receptor and its linkage to specific G-proteins activates the signaling pathways that promote cell retraction and loss of cell tethering and thereby increasing endothelial permeability. These studies will be important in defining the basis of inflammatory states, such as the adult respiratory syndrome associated with increased permeability of the endothelial barrier.
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