The Rho Kinase Inhibitor, Fasudil, Inhibits Wound Contraction

Abstract

Introduction: Scar contracture affects approximately 40 million people worldwide. There are currently no effective preventative treatments. Scar contracture is caused by fibroblast and myofibroblast contractility. Cellular contractility is stimulated by calcium independent activation of the small GTP-ases, Rho A and Rho C, which phosphorylate and activate the downstream kinase Rho-associated coiled coil-containing protein kinase (ROCK). ROCK has recently been found to cause renal, pulmonary, and cardiovascular fibrosis in vivo. This study evaluates the expression profile of RhoA and ROCK in human scar tissue and determines the effects of ROCK inhibition on rodent wound contraction. Methods: Human scar tissue and surrounding normal skin in the same specimen was immunostained for RhoA and ROCK. Full-thickness wounds (2×2cm) were created on the backs of age-matched Wistar-han rats (n=10). Fasudil (30mg/kg/d) or vehicle alone were continuously delivered systemically by subcutaneous implanted osmotic pumps for 21d. Wound contraction was measured by gravitational planimetry and rodents were assessed for signs of toxicity by measuring weight changes and observing behavior. On day 21, rodents were euthanized and wounds were harvested for Mason's trichrome, hematoxylin and eosin (H&E), and picosirius red staining, and immunohistochemistry performed for α-SMA (myofibroblasts), Ki-67 (proliferation) and CD31 (angiogenesis). Results: Human scar tissue showed significantly increased expression of RhoA and ROCK in dermal fibroblasts compared to surrounding normal tissue. Fasudil significantly inhibited wound contraction as compared to controls. Fasudil was non-toxic. H&E, Masson's, and Sirius Red staining was similar between groups. Fasudil did not alter angiogenesis nor proliferation (p>0.05). Conclusion: The increased expression of RhoA and ROCK in human scar tissue suggests an association between protein presence and scar contracture. Fasudil significantly inhibited rodent wound contraction without adversely affecting healing. Fasudil may be a potential drug to prevent scar contractures. Future work will involve a dose response curve of fasudil in rodents and application of Fasudil to the red duroc pig scar model.