Dermal scarring affects more than 40 million patients annually, ranging from fine lines to severely disfiguring scars and pathologic contractures. Pathologic contractures are the most severe form of scarring. Scars can limit joint motion and there is a need for better preventative therapies. By providing new insights into the molecular mechanisms into signaling pathways of fibroblast activity, more focused treatments can be developed It is possible that the increased expression or activity of signaling kinases could play a role in contracture. One such signaling kinase is ROCK, which is activated by the upstream GTPase, RhoA. Immunohistochemical staining of human scar tissue for RhoA, ROCK I and II showed an increase in expression as compared to normal surrounding tissue. This increase in staining supports the hypothesis that ROCK is likely active in remodeling scars. The mechanism for increased ROCK expression is unknown. Following injury RhoA/ROCK expression could be stimulated due to increased inflammatory response and infection. RhoA/ROCK expression is increased by prostaglandins. 23
and endotoxin both of which are expressed in wound healing and contribute to scarring.24–26
The rodent excisional wound model is the most frequently used animal model to study and test anti-scar contracture therapies.27–30
In this model, a 2×2cm full thickness wound is created on the dorsum of the animal between the scapula. The animal is treated with the therapy and the wound is allowed to heal by second intention. The primary endpoint that is most relevant to scar contracture is the rate of wound contraction, determined by gravitational planimetry. Secondary healing parameters include: (i) epithelialization, (ii) granulation tissue formation, (iii) inflammation, (iv) collagen deposition quantity and pattern, and (v) angiogenesis. In our wound healing studies, fasudil inhibited wound contraction as measured by gravitational planimetry and was non toxic as demonstrated by the presence of grossly normal appearing wound healing, normal behaving animals which consistently gained weight, and similar microscopic appearing wounds. H&E staining, proliferation (Ki-67) and angiogenesis (CD-31) revealed ROCK inhibition did not alter epithelialization, angiogenesis or tissue architecture. Despite reports that fasudil decreases collagen production and deposition, and increases collagenase activity 31, 32
, we found that 21d of fasudil treatment did not alter the normal collagen architecture in healing wounds as determined by Masson’s trichrome staining. Therefore fasudil likely inhibits wound contraction by different mechanisms.
Our data shows that ROCK inhibition by fasudil prevents FPCL contraction. This suggests that the mechanism by which fasudil prevented wound healing in our in vivo
model is due to inhibition of fibroblast and myofibroblast contractility. Fibrotic disease is characterized by the failure to halt normal tissue repair and the persistence of myofibroblasts which are highly contractile.2
Myofibroblast contractility is promoted by several extracellular agonists, such as platelet derived growth factor, TGF-β, sphingosine-1-phosphate, lysophosphatidic acid, through activation of the small GTPase RhoA and its downstream effector, ROCK.33–40
Inhibition of ROCK prevents granulation tissue contraction.38–46
ROCK promotes contractile force generation through sustained phosphorylation of myosin regulatory light chain, and myosin regulatory light chain phosphatase.47
This prolonged phosphorylation of myosin regulatory light chain, and myosin regulatory light chain phosphatase can incremental scar contractures to form at approximately 1cm per month.48
Fasudil blocks ROCK-dependent tension generation, preventing dermal fibroblasts from responding to extracellular agonists that normally cause contractility. In addition to fasudil blocking ROCK dependent tension, as an alternative mechanism for the attenuation of wound contraction by fasudil, we evaluated the effect of fasudil on myofibroblast formation. In vitro
we found that fasudil decreased αSMA expression; however, these finding were not corroborated with our analysis of the in vivo
tissue. Fasudil did not significantly effect αSMA expression on d21. This discrepancy may reflect limited analyses of time points. We conclude that fasudil inhibition of wound contraction is by blockade of cell contractility and may possibly involve altered myofibroblast formation.
Fasudil has been shown to decreases pulmonary 49
, cardiac 50, 51
, ocular 19
, and renal fibrosis.10
In a rat model of renal fibrosis, fasudil decreased the fibrotic area, histological injury and improved renal function by 30%–50% based on microscopic and macroscopic parameters.10, 17, 18, 52
Fasudil has not been evaluated for its effect on dermal fibrosis. Our dermal healing study showed that fasudil did not inhibit collagen deposition, cell proliferation or angiogenesis. However, fasudil did decrease contraction in vivo
and in vitro
. Since scar contracture is a significantly debilitating component of fibrosis, the data supports the hypothesis that fasudil has anti-fibrotic effects.
The RhoA/Rho-kinase pathway contributes to inflammatory 53
proliferative 7, 54
, and remodeling 48
in wound healing. Because of these multiple roles, RhoA and ROCK have emerged as critical regulators of the fibrotic response, including scar contracture formation.55, 56
Our results provide evidence that ROCK inhibition may be a therapeutic target for preventing scar contractures. It remains to be determined whether ROCK is a critical target for reducing scar contracture. A greater understanding how ROCK is activated and regulated during repair is necessary to develop future therapeutics. Future studies will need to determine dose response curves, dosing routes and the prolonged effects of fasudil on scarring. Beyond the rodent dermal wound healing model, the red-duroc pig and rabbit ear animal models will serve as higher-order assays to validate the efficacy of fasudil in reducing scarring. As drug discovery efforts continue to develop more potent and specific ROCK inhibitors, these drugs could be the first-in-man therapies to treat scarring.