Disruption of the endothelial cell (EC) barrier is a prominent feature of acute lung injury (ALI). EC permeability is regulated by a balance between contractile and tethering forces and is dependent on the functional coordination of interrelated elements of the cytoskeleton, namely microfilaments (MF) and microtubules (MT). Our novel data indicate that MT remodeling is directly involved in thrombin-induced EC barrier compromise. Similar to thrombin, lipopolysaccharide (LPS) induces partial dissolution of the MT network, which correlates with an increase in EC permeability in vitro. Our novel preliminary data demonstrated that single intravenous dose of MT stabilizer, taxol, produced significant reductions in multiple indices of LPS-induced inflammatory lung injury suggesting the involvement of MT remodeling in sepsis-induced ALI in vivo. Thrombin-induced signaling involves activation of heterotrimeric G-proteins, G12 and G13. We have demonstrated that depletion of subunits of these proteins attenuates thrombin-induced EC permeability, whereas their overexpression led to MT disassembly and massive stress fiber formation, indicative of contraction. Inhibition of Rho and p38 MARK pathways attenuates the effect of thrombin on MT structure suggesting the involvement of these pathways in MT remodeling. Our data indicate that significant pools of Rho kinase and p38 MARK are tightly associated with MT. Thrombin induces phosphorylation of several MT- and MF-associated regulatory proteins, including caldesmon (CaD), HSP-27 and tau, which are potentially responsible for thrombin-induced changes in MT and MF structure. We hypothesize that thrombin-induced activation of G12 and G13 leads to activation of Rho and p38 MARK signaling, phosphorylation of cytoskeletal regulatory proteins, coordinated MT and MF remodeling and finally to barrier compromise.
Specific Aim 1 will define the role of G12 and G13 in thrombin-induced MT disassembly and EC permeability.
Specific Aim 2 will examine the role of MT- and MF-binding regulatory proteins, tau, CaD and HSP-27, in thrombin-induced MT remodeling and barrier failure.
Specific Aim 3 will examine the role of MT remodeling in sepsis-induced lung injury using a murine model of ALI. These studies will examine basic molecular mechanisms by which MT are involved in EC barrier regulation and promise new directions and targets for treatment of lung disorders. ? ? ?
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