Alterations in vascular permeability are a defining feature of diverse processes including atherosclerosis, inflammation, ischemia/reperfusion injury and angiogenesis. Mechanisms which govern increases in vascular permeability are under intense investigation. However, little is known about processes which determine barrier protection or restoration. Platelets and platelet-derived products are essential to maintaining the integrity of the endothelial cell barrier and are intimately involved in vascular homeostasis and pathobiology. We have shown that sphingosine 1-phosphate (Sph 1-P) is a phosphorylated lipid angiogenic factor released from activated platelets which ligates specific endothelial differentiation gene (Edg) receptors to stimulate endothelial cell chemotaxis and angiogenic responses. Sph 1-P accounts for the majority of the strong endothelial cell chemotactic activity of blood serum, and was strikingly effective in enhancing the ability of fibroblast growth factor to induce angiogenesis in the avascular mouse cornea. Importantly, Sph 1-P produced rapid, sustained, and dose-dependent increases in the barrier integrity of human pulmonary artery and lung microvascular endothelial cells. Furthermore, Sph 1-P potently reversed barrier dysfunction elicited by the edemagenic agent, thrombin. The exact mechanisms by which Sph 1-P enhances barrier function are unknown, however, our data strongly implicate an essential role of endothelial cell cytoskeletal dynamics in this response. Sph 1-P-mediated barrier enhancement was dependent upon actin filament rearrangement and Rac GTPase-dependent recruitment to the cortical actin cytoskeleton of known cytoskeletal regulatory proteins such as cortactin, p21-associated kinase (PAK), LIM kinase and cofilin. In this proposal we will examine the molecular basis of Sph 1-P- induced barrier enhancement and have targeted cortical cytoskeletal interactions with cellular adhesive proteins which promote vascular integrity. SA number 1 will characterize the Sph 1-P-mediated rearrangement of the cortical cytoskeleton following shear stress and assess cortactin, PAK, and cofilin involvement. SA number 2 will investigate Sph 1-P-induced alterations in zona adherens (beta/gamma catenin complex) interaction with the actin cytoskeleton via platelet-endothelial cell adhesion molecule (PECAM) and tyrosine phosphorylation. SA number 3 will define the role of activated p125 focal adhesion kinase (FAK) and Rho/Rac GTPases in Sph 1-P-mediated regulation of focal adhesion structure/function. Finally, SA number 4 will define the role of the barrier-protective PKC delta isotype in Sph 1-P-mediated cytoskeletal rearrangement using antisense strategies, PKC over-expression constructs, pharmacologic and myristoylated peptide inhibitors, and immortalized stable PKC over-expressing cell lines. Given the profound physiologic derangements which accompany the vascular leak seen in multiple vascular pathobiologies, Sph 1-P infusion may provide a novel therapeutic intervention for consideration in these devastating disorders.
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