Microtubules in lung endothelial cell barrier regulation

DESCRIPTION (Applicant's Abstract): Disturbances in endothelial cell (EC) barrier regulation is a hallmark of lung inflammation, angiogenesis and cancer, and is critically dependent upon rearrangements in the cytoskeleton (microfilaments and microtubules). We have previously shown the involvement of microfilaments in actomyosin-driven contraction in specific models of lung EC permeability. For example, edemagenic agents such as thrombin increase the level of phosphorylation of myosin "regulatory" light chain (MLC20), which is tightly linked to stress fiber formation, gap formation and barrier dysfunction. In contrast, cAMP elevation has a barrier protective effect and significantly attenuates thrombin-induced MLC phosphorylation and barrier dysfunction. Although information is limited as how to changes in microtubule network regulate EC contractility, careful temporal analysis of the MLC phosphorylation status in cultured EC reveals that disruption of the cellular microtubule structure with the clinically relevant agents such as vinblastine significantly increases MLC phosphorylation, decreases electrical resistance of EC monolayer and increases transendothelial neutrophil migration reflecting EC barrier dysfunction. Stabilization of microtubule structure by paclitaxel completely abolishes these effects and significantly attenuates thrombin-induced EC stress fiber formation. In addition, microtubule disruption activates MAP kinase (p38 and ERKs) pathway in endothelium, which also potentially can be involved in MLC-independent EC contractility via phosphorylation of key cytoskeletal proteins, like caldesmon and HSP-27. In this proposal, we will explore the role of MLC-dependent and MLC-independent mechanisms in the regulation of EC contraction and barrier dysfunction, induced by microtubule disruption in both macro- and microvascular endothelium. In SA 1 we will identify mechanisms leading of microtubule-mediated increases in EC MLC phosphorylation by examining how MLC-specific kinase and phosphatase activities are affected by microtubule alterations. In SA 2 we will identify MLC-independent pathways involved in microtubule-mediated EC contractility and barrier dysfunction and examine the effect of MAPK activities on cytoskeletal protein phosphorylation, cytoskeletal rearrangement and barrier function. In SA 3 we will examine the ole of cAMP-mediated barrier protection on EC barrier failure, induced by microtubule disruption. These studies will provide an understanding of novel signaling pathways involved in lung EC barrier regulation and promise new directions and targets for treatment of lung disorders.