During episodes of inflammation and hypoxia, vascular endothelial cells interact with polymorphonuclear leukocytes (PMN, neutrophils) in both direct and indirect ways. Under such conditions, PMN may significantly modulate endothelial function, including vascular barrier function. Our previous studies explored the possibility that activated PMN may liberate factors which promote endothelial barrier function, and as such, may serve as a counter-regulatory pathway for potentially detrimental factors liberated at such sites. These studies identified a number of previously unappreciated molecules important in endothelial permeability, including soluble mediators (e.g. 5'AMP, glutamate), membrane proteins (CD73, metabotropic glutamate receptors, alpha-v beta-3), and cytoskeletal crosslinking proteins [e.g. vasodilator-stimulated phosphoprotein (VASP)] in maintenance of junctional permeability during interactions with PMN. In this proposal, we will test the hypothesis that PMN-derived factors regulate endothelial permeability. As such, three specific aims are proposed to test this hypothesis. First, we will elucidate the molecular pathway(s) ecto-nucleotidase regulation of endothelial permeability. We will utilize extensive in vitro and in vivo models to answer basic questions regarding the cause and effect relationship between ecto-nucleotidase expression and vascular barrier function. Second, we will explore the molecular mechanisms of vascular barrier regulation by exogenous glutamate. As such, we will elucidate the cooperative contributions of endothelial glutamate receptors with VASP in vitro and in vivo. Third, we will determine the relationship between PMN-derived extracellular mediators with endothelial cell-cell junction coupling. In particular, we will concentrate efforts on understanding VASP interactions with endothelial tight junctions and the influence of glutamate and adenine nucleotides on junctional reorganization in real- time. The overall aim of this proposal is to elucidate the molecular pathways critical to vascular barrier regulation in hypoxia and during inflammation.
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