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Dec 12, 2011

Targeting Mechanical Stress Responses Reduces Fibrosis and Inflammation

  • Scientists say targeting specific factors that respond to the mechanical stresses resulting from tissue injury may help limit inflammation and fibrosis that occur in skin wounds and in diseases such as pulmonary fibrosis or rheumatoid arthritis. A team at Stanford University’s department of surgery, and Oregon Health and Science University’s department of surgery, have found that chemically inhibiting or genetically deleting focal adhesion kinase FAK in mouse fibroblasts reduces inflammatory and fibrotic responses following cutaneous injuries.

    Reporting in Nature Medicine, the team’s in vitro and in vivo studies showed that  FAK acts through extracellular-related kinase (ERK) to trigger secretion of the chemokine monocyte chemoattractant protein-1 (MCP-1, also known as CCL2). As with the FAK-knockout or FAK-inhibited mice, animals with global MCP-1 deletions also demonstrated reduced scar formation. The researchers suggest that targeting this FAK-ERKL-MCP-1 pathway might represent a therapeutic strategy to remove the additional effects that mechanical forces have on scar formation. Stanford’s Geoffrey C. Gurtner, M.D., and colleagues, describe their findings in a paper titled “Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signalling.”

    It has long been suspected that physical cues, such as mechanical forces, will influence the level of fibrosis in response to cutaneous injury, and a number of studies have suggested that reducing wound tension decreases scar formation. Research by the Stanford and Oregon team has in addition demonstrated that mechanical force induces hypertrophic scare (HTS)-like fibrosis in mouse models of cutaneous scarring, and that mechanical offloading of incisions can decrease HTS formation in human patients. These studies have all reinforced the notion that the mechanical environment plays a key role in cutaneous pathology, the authors write.

    To look at pathways that might be involved in early scar formation, they carried out genome-wide microarray analysis of scars in wild-type mice that had been subjected to human-like amounts of skin tension, between the fourth and fourteenth days after injury. The transcriptome networks that were constructed around the mechanically regulated genes highlighted a key role for FAK as a transducer of inflammatory and physical signals. Studies in mice confirmed that FAK was activated as a result of cutaneous injury, and that mechanical force increased this effect substantially “leading us to hypothesize that FAK is a key mediator of both inflammation and fibrosis,” the team writes.

    They next looked at the effects of deleting FAK specifically in dermal fibroblasts, as previous work had suggested that knocking out keratinocyte-specific FAK had no effect on scar formation or wound healing. Interestingly, when the team applied their previous HTS model to the dermal fibroblast FAK-knockout animals, normal healing of the incisions wasn’t affected, but compared with wild-type mice, the engineered animals exhibited less scar formation and reduced matrix density. These effects were associated with impaired proliferation, although not with changes in apoptosis, “which is consistent with our recent report suggesting that activation of survival pathways is not a primary factor in scar formation,” they state.

    The microarray analyses had suggested that FAK modulates cytokine/chemokine signaling, and further studies confirmed that transforming growth factor-β1 (TGF-β1) expression levels and protein levels were much lower in FAK knockout scars compared to wild-type scars. Concentrations of the chemokine MCP-1, which is associated with inflammatory cell recruitment and fibrotic skin diseases, were also lower in FAK knockouts, and the scars in these animals had lower numbers of α smooth muscle actin (α-SMA)-positive myofibroblasts and  F4/80+ macrophages. “In addition, overall wound expression of Ccr2 (the surface receptor for MCP-1) was less in FAK-deficient wounds compared to wild-type scars, indicating a defect in MCP-1–mediated cell trafficking,” the investigators remark. In fact, administering recombinant mouse MCP-1 intradermally to the FAK-knockout animals resulted in the development of scarring patterns that were similar to those in injured wild-type mice.

    Importantly, global MCP-1 knockout mice developed 70% less scar formation than wild-type mice, and scars in the engineered animals demonstrated significantly lower recruitment of macrophages, which act as key regulators of matrix remodeling: this indicated that MCP-1 inflammatory pathways also play a major role in scar mechanotransduction.

    In order to try and identify intracellular signaling pathways connecting FAK expression with MCP-1 secretion, the team looked at downstream mediators of FAK. They found that ERK specifically was activated by mechanical stimuli and differentially regulated by FAK, which again tied in with previous research linking ERK as a key target of FAK mechanotransduction.

    The findings in mice were substantiated in human cells in vitro. Applying strain to human fibroblasts led to the activation of FAK within five minutes, which was sustained during the static strain period. This was accompanied by the FAK-dependent phosphorylation of ERK within 10 minutes. Applying either mechanical or inflammatory stimuli also induced MCP-1 secre­tion, and when both stimuli were applied together there was a potentiating effect on MCP-1 secretion.

    In contrast, treating fibroblasts with a small molecule inhibitor of FAK (PF573228) reduced the formation of focal adhesions, impaired their spread morphology, and reduced cell motility, contraction, and α-SMA expression. Small molecule inhibition of FAK blocked both strain and inflammatory stimuli from activating MCP-1, while inhibiting ERK also blocked strain-induced MCP-1 release, “highlighting the key role of the FAK-ERK-MCP-1 axis in mechanotransduction and inflammation in human fibroblasts,” the authors state.

    Finally, in vivo studies demonstrated that administration of PF573228 daily to cutaneous wounds in the mouse HTS model resulted in reduced scar formation. At 10 days after injury, the gross scar area was 170% lower in the inhibitor-treated animals than in control mice, scar matrix density was comparatively reduced, and epithelial thick­ness, epithelial proliferation and dermal proliferation were 35%, 57%, and 28% lower, respectively, in the scars of mice treated using PF573228.

    Notably, the researchers add, in vitro inhibition of either FAK or ERK blocked collagen production in human fibroblasts in vitro, and in vivo there were lower ratios of collagen I (thicker) to collagen III (thinner) expression in FAK-knockout scars and FAK-inhibitor-treated scars.

    “Based on these studies, we propose a model for load-induced fibrosis whereby mechanical force activates both MCP-1 secretion and collagen production through FAK to perpetuate a ‘vicious cycle’ of fibroproliferation after injury,” the authors conclude. The admit that other mechanoresponsive cell types and cytokines will be involved in scar mechanotransduction, but nevertheless suggest their findings will provide a framework for further understanding how mechanical stimuli trigger local and systemic responses that mediate scar hypertrophy. “More broadly, these results suggest that targeted strategies to uncouple mechanical force from inflammation and fibrosis may prove clinically successful across diverse organ systems.”


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