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The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin-null and wild-type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, showing that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild-type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle α-actin were expressed in smooth muscle α-actin-null myofibroblasts, as shown by immunostaining, real-time polymerase chain reaction, and mass spectrometry. These results show that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts., (© 2012 by the Wound Healing Society.)

The ability to regulate wound contraction is critical for wound healing as well as for pathological contractures. Matrix metalloproteinases (MMPs) have been demonstrated to be obligatory for normal wound healing. This study examined the effect that the broad-spectrum MMP inhibitor BB-94 has when applied topically to full-thickness skin excisional wounds in rats and its ability to inhibit the promotion of myofibroblast formation and function by the latent transforming-growth factor-beta1 (TGF-beta1). BB-94 delayed wound contraction, as well as all other associated aspects of wound healing examined, including myofibroblast formation, stromal cell proliferation, blood vessel formation, and epithelial wound coverage. Interestingly, BB-94 dramatically increased the level of latent and active MMP-9. The increased levels of active MMP-9 may eventually overcome the ability of BB-94 to inhibit this MMP and may explain why wound contraction and other associated events of wound healing were only delayed and not completely inhibited. BB-94 was also found to inhibit the ability of latent TGF-beta1 to promote the formation and function of myofibroblasts. These results suggest that BB-94 could delay wound closure through a twofold mechanism; by blocking keratinocyte migration and thereby blocking the necessary keratinocyte-fibroblast interactions needed for myofibroblast formation and by inhibiting the activation of latent TGF-beta1.

During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating myofibroblasts. We examined whether regulation of myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of myofibroblasts necessary for wound closure.