Endothelial cells which line blood vessels are a critical element in barrier function. They regulate the passage between blood and tissues of macromolecules, nutrients and waste products. During inflammation, endothelial barrier function is reduced, leading to accumulation of leukocytes and leakage of inflammatory molecules into target tissue. While inflammation is a necessary response to pathogens and tissue damage, when it becomes chronic or dysregulated it is associated with tissue injury and serious diseases such as atherosclerosis, asthma, diabetes, septic shock and autoimmune diseases. Control of inflammation is therefore central to many clinical interventions, and reversing the loss of endothelial barrier function during inflammation has significant therapeutic value. Several mediators have been identified that augment endothelial barrier function. For example, signals to receptors which couple to Gs trigger an increase in the second messenger cAMP. This typically results in improved barrier function in vitro and in vivo, largely through regulation of endothelial junctions. In endothelium, the major effector for cAMP was thought to be cAMP dependent protein kinase (PKA). Recently, activation of the small GTPase Rap1 has been shown to dramatically improve the endothelial barrier, causing polymerization of actin at the junctions. Junctional actin is an essential component of adherence and tight junctions. This proposal will focus on proteins that promote actin filament nucleation at endothelial junctions. Preliminary studies indicate that WAVE2, a member of the WASP-WAVE family of actin nucleation promoting factors, may cooperate with the Arp2/3 complex at endothelial junctions to cause actin polymerization. Based on these observations it is hypothesized that WAVE2 lies downstream of cAMP signaling, and plays a critical role in regulating endothelial barrier function by nucleating actin polymerization at the junctions. This hypothesis will be tested in the following specific aims: 1. Identify and localize components of the WAVE2 signaling pathway in endothelial cells;2. Measure the functional effect of WAVE2 on monolayer permeability and the structural effect on junctional composition using a variety of strategies to interfere with its signaling. These studies will test the role for WAVE2 in polymerization of junctional actin and barrier function in endothelial cells in response to barrier strengthening and weakening stimuli. Identification of the molecular mechanisms that regulate endothelial barrier function will lead to better understanding of the inflammatory process, and is a necessary step towards design of more specific therapies to combat it dysregulation. PUBLIC HEALTH RELEVANCE: Inflammation is a necessary response to pathogens and tissue damage, but when it becomes chronic or dysregulated it is associated with tissue injury and serious diseases such as atherosclerosis, asthma, diabetes, septic shock and autoimmune diseases. During inflammation, endothelial barrier function is reduced, leading to accumulation of leukocytes and leakage of inflammatory molecules into target tissue. This proposal will identify one of the molecular mechanisms that regulate endothelial barrier function which will lead to better understanding of the inflammatory process, and is a necessary step towards design of more specific therapies to combat its dysregulation.