title: Inhibition of rho-kinase impairs fibroblast stress fiber formation, confluence, and contractility in vitro.
abstract: 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 microM 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.
authors: Department of Surgery, , The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7228, USA.
authors: Douglas H
authors: DH
authors: Gates
authors: Jin Sun
authors: JS
authors: Lee
authors: C Scott
authors: CS
authors: Hultman
authors: Bruce A
authors: BA
authors: Cairns
mesh_terms: D000199:Actins / Q000378:metabolism*; D000577:Amides; D000959:Antihypertensive Agents; D002056:Burns / Q000150:complications; D017439:Cicatrix, Hypertrophic / Q000503:physiopathology*; D003094:Collagen; D003286:Contracture / Q000503:physiopathology*; D005347:Fibroblasts / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors*; D008962:Models, Theoretical; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011725:Pyridines; D022502:Stress Fibers; D014945:Wound Healing; D054460:rho-Associated Kinases
chemical_list: D000199:Actins; D000577:Amides; D000959:Antihypertensive Agents; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases