Rho kinases are major regulators of actin cytoskeletal organization and cell motility. Depending on the model system, inhibitors of Rho kinases (ROCK) have been reported to increase or decrease endothelial cell migration. In the present study we investigated the effect of Rho kinase inhibitors on microvascular endothelial cell migration with a special focus on the isoform ROCK2. Migration of microvascular endothelial cells was analyzed in a wound-healing, a spheroid-on-collagen migration assay and in cells embedded in collagen-1 gels. The non-selective Rho kinase inhibitor H1152 was compared to the selective ROCK2 inhibitor SLX2119 and to siRNA knock down. Non-selective inhibition of Rho kinases decreased cell-spanning F-actin fibers, loosened cell-cell contacts visualized by VE cadherin staining, and reduced cell-matrix interactions as shown by reduced Hic-5 expression in focal contacts. Rho kinase inhibitors facilitated directed migration of endothelial cells away from spheroids on fibronectin-coated plates and in collagen-1 gels. By contrast, migration of firmly attached endothelial cells, resembling intact vessels, was not promoted by Rho kinase inhibition. Selective inhibition of ROCK2 mimicked the cytoskeletal effects of H1152 and also increased cell motility, although to a lesser extent. In summary, Rho kinase inhibition enhanced the migration and cytoskeletal restructuring preferentially in freshly attached endothelial cells. ROCK2 may be a potential target to manipulate endothelial cell migration after vessel injury.
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