Inhibition of rho-kinase impairs fibroblast stress fiber formation, confluence, and contractility in vitro

Rho kinase is an enzyme that has been implicated in pathological wound healing. In our study, we aimed to characterize the dosages of Y-27632 (a competitive inhibitor of rho kinase) at which fibroblast stress fiber formation is inhibited, to determine the effect of Y-27632 on fibroblast confluence, and to determine the effect of varying concentrations of Y-27632 on fibroblast-mediated gel contraction. First, 3T3 fibroblasts were exposed to various concentrations of Y-27632, and stress fiber dissolution was assessed with immunofluorescence. Next, 10 μM Y-27632 was applied to 3T3 fibroblast monolayer cultures immediately after plating and at 8 hours after plating to determine the effect on fibroblast confluence. Finally, a three-dimensional model was used to assess the effect of Y-27632 on gel contraction. Increasing doses of Y-27632 resulted in a dose-dependent increase in stress fiber dissolution as well as a dose-dependent inhibition of gel contraction. Median number of days to confluence were 3, 5, and 6 after no, immediate, and delayed incubation with Y-27632, respectively (P < .05). Our results further characterize the dosages at which Y-27632 may be most effective in the modulation of wound contraction in burn patients. In addition, our confluence study suggests that patients need not necessarily be pretreated with Y-27632.