ROCK mediates thrombin's endothelial barrier dysfunction

Abstract

Thrombin-induced endothelial monolayer hyperpermeability is thought to result from increased F-actin stress fiber-related contractile tension, a process regulated by the small GTP-binding protein Rho. We tested whether this process was dependent on the Rho-associated protein kinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects of Y-27632 on thrombin-induced myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) and paxillin were measured by Western blotting. F-actin organization and content were analyzed by digital imaging, and endothelial monolayer permeability was measured in bovine pulmonary artery endothelial cell (EC) monolayers using a size-selective permeability assay. Y-27632 enhanced EC monolayer barrier function due to a decline in small-pore number that was associated with increased EC surface area, reduced F-actin content, and reorganization of F-actin to beta-catenin-containing cell-cell adherens junctions. Although Y-27632 prevented thrombin-induced MLCP, stress fiber formation, and the increased phosphotyrosine content of paxillin and p125(FAK), it attenuated but did not prevent the thrombin-induced formation of large paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to be partially ROCK dependent.

Inhibition of Protein Geranylgeranylation and RhoA/RhoA Kinase Pathway Induces Apoptosis in Human Endothelial Cells

“…Therefore, inhibition of cell spreading as well as adhesion may account for the reduction of cell survival produced by disrupting the RhoA/ RhoA kinase pathway. Consistent with this notion, Y-27632 has been reported to block formation of stress fibers and cell spreading in various cell types including EC (21). In addition, Y-27632 inhibited phosphorylation of focal adhesion kinase and the assembly of focal contacts (21,26,48).…”

“…Consistent with this notion, Y-27632 has been reported to block formation of stress fibers and cell spreading in various cell types including EC (21). In addition, Y-27632 inhibited phosphorylation of focal adhesion kinase and the assembly of focal contacts (21,26,48). Moreover, activation of RhoA by CNF-1 increased the expression of focal adhesion kinase and promoted cell spreading (49).…”

“…Recently, a new synthetic compound, Y-27632, has been widely used as a specific inhibitor of RhoA kinase to identify the roles of RhoA pathway in a variety of systems (21)(22)(23)(24). Here we used Y-27632 to determine the role of RhoA kinase in cell viability.…”

“…A variety of putative RhoA effectors have been identified (18,19). Among these effectors, the serine/threonine kinase, named RhoA kinase, has been found to mediate multiple RhoA effects, such as formation of actin stress fibers and focal adhesion (20), EC barrier dysfunction (21), vascular smooth muscle cell DNA synthesis and migration (22), and angiogenesis (23).…”

Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of VE–cadherin

“…4d). In fact, transwell assay suggested that Y-27632 treatment actually enhanced endothelial barrier function by about twofold presumably via recruitment of F-actin to a-or bcatenin-containing junctions 20 . Interestingly, TiO 2 -NM treatment to Y-27632 preconditioned samples still led to an increase in dextran-FITC flux, albeit to a much lower level compared with the untreated Y-27632 control (Fig.…”