Dasatinib inhibits actin fiber reorganization and promotes endothelial cell permeability through RhoA-ROCK pathway

Treatment with dasatinib, a tyrosine kinase inhibitor, is associated with edema, pleural effusion, and pulmonary edema. We investigated the effect of dasatinib on the barrier function of human microvascular endothelial cells-1 (HMEC-1) in vitro and in vivo. The permeability of HMEC-1 to fluorescein isothiocyante (FITC)-dextran increased in Transwell chambers within 5 min following the addition of therapeutic concentrations of dasatinib. The change in permeability was associated with increased activation of RhoA GTPase and its effector Rho-associated coiled-coil kinase 1(ROCK1). RhoA inhibitor C3 transferase almost completely inhibited dasatinib-induced increase in permeability. Under similar conditions, imatinib had no effect on permeability or activation of RhoA. Since integrin-induced cell spreading suppresses RhoA activation, we examined the effect of dasatinib on cell spreading on fibronectin substrate. Dasatinib impaired endothelial cell spreading in a concentration-dependent manner and induced disorganization of actin fibers. Tyrosine kinases play an essential role in transmitting signals from integrins to RhoA and we examined tyrosine phosphorylation of several cytoskeletal proteins. Dasatinib markedly inhibited tyrosine phosphorylation of p130 Crk-associated substrate (p130cas), paxillin and vinculin. These results suggest that the inhibition of tyrosine phosphorylation of the focal adhesion plaque components by dasatinib may alter the assembly of actin fibers resulting in the activation of RhoA/ROCK pathway. Consistent with these findings, dasatinib-induced increase in the permeability was blocked by ROCK inhibitor y27632. In vivo administration of y27632, significantly inhibited the dasatinib-induced extravasation of Evans blue in mice and dasatinib-induced increase in microvascular permeability was attenuated in ROCK1-deficient mice. These findings suggest that ROCK inhibitors could serve as therapeutic modalities to ameliorate the dasatinib-induced pulmonary changes.