The transforming growth factor-β (TGF-β) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-β and the canonical Wnt pathway. TGF-β stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases.

Blocking 5-HT2B receptor provides a therapeutic target for fibrotic diseases caused by activated platelet release of serotonin during vascular damage.

Objective. Hedgehog signaling not only plays crucial roles during human development but also has been implicated in the pathogenesis of several diseases in adults. The aim of the present study was to investigate the role of the hedgehog pathway in fibroblast activation in systemic sclerosis (SSc).Methods. Activation of the hedgehog pathway was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). The effects of sonic hedgehog (SHH) on collagen synthesis were analyzed by reporter assays, real-time PCR, and Sircol assays. Myofibroblast differentiation was assessed by quantification of ␣-smooth muscle actin and stress fiber staining. The role of hedgehog signaling in vivo was analyzed by adenoviral overexpression of SHH and using mice lacking 1 allele of the gene for inhibitory receptor Patched homolog 1 (Ptch ؉/؊ mice).Results. SHH was overexpressed and resulted in activation of hedgehog signaling in patients with SSc, with accumulation of the transcription factors Gli-1 and Gli-2 and increased transcription of hedgehog target genes. Activation of hedgehog signaling induced an activated phenotype in cultured fibroblasts, with differentiation of resting fibroblasts into myofibroblasts and increased release of collagen. Adenoviral overexpression of SHH in the skin of mice was sufficient to induce skin fibrosis. Moreover, Ptch ؉/؊ mice with increased hedgehog signaling were more sensitive to bleomycin-induced dermal fibrosis.Conclusion. We demonstrated that the hedgehog pathway is activated in patients with SSc. Hedgehog signaling potently stimulates the release of collagen and myofibroblast differentiation in vitro and is sufficient to induce fibrosis in vivo. These findings identify the hedgehog cascade as a profibrotic pathway in SSc.

Purpose Tissue fibrosis caused by pathological activation of fibroblasts with increased synthesis of extracellular matrix components is a major hallmark of systemic sclerosis (SSc). Notch signalling regulates tissue differentiation and pathologic activation of Notch signaling has been implicated in the pathogenesis of various malignancies. The aim of the present study was to investigate the role of Notch signaling in SSc and to evaluate the therapeutic potential of Notch inhibition for the treatment of fibrosis. Methods Activation of the Notch pathways was analyzed by staining for the Notch intracellular domain (NICD) and quantification of the mRNA levels hes-1. Notch signaling was inhibited in the mouse model of bleomycin induced dermal fibrosis and in tight-skin-1 mice by the γ-secretase inhibitor DAPT and by overexpression of a Notch-1 antisense construct. Results Notch signaling is activated in SSc in vivo with accumulation of the NICD and increased transcription of the target gene hes-1. Overexpression of a Notch antisense construct prevented bleomycin-induced fibrosis and hypodermal thickening in tight-skin 1 mice. Potent anti-fibrotic effects were also obtained by treatment with DAPT. In addition to prevention of fibrosis, targeting Notch signaling resulted in almost complete regression of pre-established experimental fibrosis. Conclusion We demonstrate that pharmacologic as well as genetic inhibition of Notch signaling exerts potent anti-fibrotic effects in different murine models of SSc. These findings might have direct translational implications because different inhibitors of the γ-secretase complex are available and yielded promising results in cancer trials.

Objective. To investigate whether JAK-2 contributes to the pathologic activation of fibroblasts in patients with systemic sclerosis (SSc) and to evaluate the antifibrotic potential of JAK-2 inhibition for the treatment of SSc.Methods. Activation of JAK-2 in human skin and in experimental fibrosis was determined by immunohistochemical analysis. JAK-2 signaling was inhibited by the selective JAK-2 inhibitor TG101209 or by small interfering RNA. Bleomycin-induced dermal fibrosis in mice and TSK-1 mice were used to evaluate the antifibrotic potential of specific JAK-2 inhibition in vivo.Results. Increased activation of JAK-2 was detected in the skin of patients with SSc, particularly in fibroblasts. The activation of JAK-2 was dependent on transforming growth factor ␤ (TGF␤) and persisted in cultured SSc fibroblasts. Inhibition of JAK-2 reduced basal collagen synthesis selectively in SSc fibroblasts but not in resting healthy dermal fibroblasts. Moreover, inhibition of JAK-2 prevented the stimulatory effects of TGF␤ on fibroblasts. Treatment with TG101209 not only prevented bleomycin-induced fibrosis but also effectively reduced skin fibrosis in TSK-1 mice.Conclusion. We demonstrated that JAK-2 is activated in a TGF␤-dependent manner in SSc. Considering the potent antifibrotic effects of JAK-2 inhibition, our study might have direct translational implications, because inhibitors of JAK-2 are currently being evaluated in clinical trials for myeloproliferative disorders and would also be available for evaluation in patients with SSc.

BACKGROUND: Dermal fibroblasts from patients with systemic sclerosis (SSc) release excessive amounts of collagen resulting in tissue fibrosis. The molecular mechanisms underlying this pathological activation are incompletely understood. OBJECTIVE: To investigate whether Notch signalling contributes to the uncontrolled activation of fibroblasts in SSc. METHODS: Activation of the Notch pathway was assessed by immunohistochemistry or Western blot for the Notch intracellular domain and the Notch ligand Jagged-1 (Jag-1) and real-time PCR for the target gene hes-1. Differentiation of resting dermal fibroblasts into myofibroblasts was assessed by staining for -smooth muscle actin. The synthesis of collagen was quantified by real-time PCR and Sircol assays. RESULTS: Notch signalling was activated in lesional skin of patients with SSc. The activation persisted in cultured dermal SSc fibroblasts. Stimulation of healthy dermal fibroblasts with recombinant human Jag-1-Fc chimera resulted in an SSc-like phenotype with increased release of collagen and differentiation of resting fibroblasts into myofibroblasts. Consistent with the selective activation of the Notch pathway in dermal SSc fibroblasts, DAPT or siRNA against Notch strongly reduced the basal collagen expression in SSc fibroblasts, but not in fibroblasts from healthy volunteers. CONCLUSION: It was shown that Notch signalling is activated in SSc and plays an important role in fibroblast activation and collagen release. Inhibition of Notch signalling might be an effective strategy to selectively prevent the aberrant activation of SSc fibroblasts. Conclusion:We demonstrated that Notch signaling is activated in SSc and plays an important role in fibroblast activation and collagen release. Inhibition of Notch signaling might be an effective strategy to selectively prevent the aberrant activation of SSc fibroblasts.

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Objective. The transcription factor STAT-4 has recently been identified as a genetic susceptibility factor in systemic sclerosis (SSc) and other autoimmune diseases. The aim of this study was to investigate the contribution of STAT-4 in the development of a fibrotic phenotype in 2 different mouse models of experimental dermal fibrosis. Methods. STAT-4-deficient (stat4؊/؊ ) mice and their wild-type littermates (stat4 ؉/؉ ) were injected with bleomycin or NaCl. Infiltrating leukocytes, T cells, B cells, and monocytes were quantified in the lesional skin of stat4 ؊/؊ and stat4 ؉/؉ mice. Inflammatory and profibrotic cytokines were measured in sera and lesional skin samples from stat4 ؊/؊ and stat4 ؉/؉ mice. The outcome of mice lacking STAT-4 was also investigated in the tight skin 1 (TSK-1) mouse model.Results. Stat4 ؊/؊ mice were protected against bleomycin-induced dermal fibrosis, with a reduction in dermal thickening (mean ؎ SEM 65 ؎ 3% decrease; P ‫؍‬ 0.03), hydroxyproline content (68 ؎ 5% decrease; P ‫؍‬ 0.02), and myofibroblast counts (71 ؎ 6% decrease; P ‫؍‬ 0.005). Moreover, the number of infiltrating leukocytes, especially T cells, was significantly decreased in the lesional skin of stat4 ؊/؊ mice (mean ؎ SEM 63 ؎ 5% reduction in T cell count; P ‫؍‬ 0.02). Stat4؊/؊ mice also displayed decreased levels of inflammatory cytokines such as tumor necrosis factor ␣, interleukin-6 (IL-6), IL-2, and interferon-␥ in lesional skin. Consistent with a primary role of STAT-4 in inflammation, STAT-4 deficiency did not ameliorate fibrosis in TSK-1 mice.Conclusion. The results of this study demonstrate that the transcription factor STAT-4 exerts potent profibrotic effects by controlling T cell activation and proliferation and cytokine release. These findings confirm the results of genetics studies on the role of STAT-4 in the development of SSc.