Biography
Biography: Alexander D Verin
Abstract
It is established that edema genic agonists (thrombin, LPS)-induced endothelial cell (EC) barrier dysfunction is tightly linked to myosin light chain (MLC)-dependent EC contraction and cytoskeletal reorganization. In this study, we examined the role of MAP kinases (p38 and p42/44) as potentially important enzymes in MLC-independent, agonist-mediated EC contractile responses and permeability. We demonstrated that thrombin significantly increased MAPK activities. Specific inhibition of p38 and p42/44 MAPK significantly attenuated thrombin-induced increases in F-actin stress fibers and permeability reflecting the involvement of MAPK in thrombin-mediated EC barrier compromise. Next, we examined potential cytoskeletal targets of thrombin-induced MAPK activation. MAPK inhibition did not alter basal and thrombin-induced EC MLC phosphorylation but significantly increased phosphorylation of caldesmon (Cad), an actin- and myosin-binding regulatory protein. Similar to smooth muscle, phosphorylation of Cad can potentially facilitate agonist-induced contraction and lead to EC barrier dysfunction. Under basal conditions Cad co-immunoprecipitated with actin and myosin suggesting a functional complex. Thrombin decreased the amount of myosin, but not actin in non-denaturing Cad immunoprecipitates suggesting decreased Cad/myosin interaction. Immunoblotting with anti-phospho Cad antibody to MAPK phosphorylation sites on Cad demonstrated that thrombin-mediated EC activation leads to direct phosphorylation of Cad by MAPK. Inhibition of MAPK significantly attenuated thrombin-induced Cad phosphorylation. These data strongly suggest the direct link between edema genic agonists-mediated EC contractile response and permeability, activation of MAPK cascades and Cad phosphorylation