The goal of this K08 application is to provide the PI with essential skills in which to be a successful academician and achieve independent scientific investigator status. The PI will focus upon the endothelium, a functionally dynamic cellular monolayer which performs critical roles in angiogenesis and as a semi-selective barrier between the vasculature and surrounding tissues. Despite its importance in acute lung injury syndromes, the cellular regulation of endothelial barrier function, especially barrier restoration and enhancement, is poorly understood. Recent work in endothelial cells (EC) demonstrates that phosphorylation of myosin light chains (MLC) by the enzyme myosin light chain kinase (MLCK) is critical to induction of vascular permeability in some models. Studies in our laboratory have revealed that EC express a novel, high molecular weight MLCK isoform that when tyrosine phosphorylated is found in stable complex with the actin-binding protein, cortactin, an actin-binding protein implicated in multiple aspects of cytoskeletal organization and rearrangement including Arp2/3-mediated actin polymerization. We hypothesize that cortactin plays an active and critical role in endothelial cell cytoskeletal rearrangement during barrier enhancement and migration. SA#1 will examine the role of cortactin in cultured pulmonary EC barrier function and migration using molecular biology techniques. SA#2 will explore the role of cortactin phosphorylation in pulmonary EC cytoskeletal regulation. SA#3 will characterize the role of cortactin-EC MLCK interaction in pulmonary endothelial cytoskeletal rearrangement through biochemical, immunohistochemical, and molecular biology techniques. Finally, SA#4 will investigate the effect of biophysical shear stress and cyclical stretch on pulmonary endothelial cortactin biology and regulation. Through these complementary techniques and coursework, the PI will develop new skills and generate novel data regarding EC barrier restoration/enhancement, and angiogenesis which may potentially lead to new therapies for the devastating consequences of pulmonary edema.
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