Hedgehog signaling controls fibroblast activation and tissue fibrosis in systemic sclerosis.

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 α-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.

Inhibition of activator protein 1 signaling abrogates transforming growth factor ß-mediated activation of fibroblasts and prevents experimental fibrosis.

OBJECTIVE: To investigate whether c-Jun and c-Fos contribute to the pathologic activation of fibroblasts in systemic sclerosis (SSc) and to evaluate the antifibrotic potential of selective activator protein 1 (AP-1) inhibition. METHODS: Expression of c-Jun and c-Fos was determined by real-time polymerase chain reaction (PCR) and immunohistochemical analysis. Fibroblasts were stimulated with transforming growth factor ß (TGFß) and incubated with T-5224, a small-molecule inhibitor of AP-1, or were transfected with small interfering RNA (siRNA) duplexes against c-Jun and c-Fos. Collagen synthesis was quantified by real-time PCR and hydroxyproline assay. Differentiation of resting fibroblasts into myofibroblasts was assessed by staining for α-smooth muscle actin and stress fibers. The antifibrotic potential of T-5224 was evaluated in mouse models of dermal fibrosis induced by bleomycin or by adenoviral overexpression of a constitutively active TGFß receptor type I. RESULTS: Up-regulation of c-Jun and c-Fos was detected in mouse models of SSc and in the skin and dermal fibroblasts of patients with SSc. Stimulation of healthy fibroblasts with TGFß induced the expression of c-Jun and c-Fos. Treatment with T-5224 or nucleofection with siRNA directed against c-Jun and c-Fos abrogated the profibrotic effects of TGFß. T-5224 decreased the release of collagen selectively in SSc fibroblasts. T-5224 was well tolerated and prevented dermal fibrosis induced by bleomycin or by adenoviral activation of TGFß signaling. CONCLUSION: AP-1 is up-regulated in a TGFß-dependent manner in SSc. The selective AP-1 inhibitor T-5224 reduced collagen synthesis selectively in SSc fibroblasts and efficiently prevented the development of experimental dermal fibrosis. Thus, AP-1 might be a promising new molecular target for the treatment of SSc.

Stimulation of soluble guanylate cyclase reduces experimental dermal fibrosis.

BACKGROUND: Fibrosis and vascular disease are cardinal features of systemic sclerosis (SSc). Stimulators of soluble guanylate cyclase (sGC) are vasoactive drugs that are currently being evaluated in phase III clinical trials for pulmonary arterial hypertension. OBJECTIVE: To study the antifibrotic potency of sGC stimulators. METHODS: The effect of the sGC stimulator BAY 41-2272 on the release of collagen from dermal fibroblasts was examined. The antifibrotic effects of BAY 41-2272 on prevention and regression of fibrosis in bleomycin-induced dermal fibrosis and in Tsk-1 mice were also studied. Telemetric blood pressure studies in conscious mice were used to study potential hypotensive effects of sGC stimulation. RESULTS: sGC stimulation with BAY 41-2272 dose-dependently inhibited collagen release in dermal fibroblasts from patients with SSc and healthy individuals. Furthermore, BAY 41-2272 stopped the development of bleomycin-induced dermal fibrosis and skin fibrosis in Tsk-1 mice, preventing dermal and hypodermal thickening, reducing the numbers of myofibroblasts and reducing the hydroxyproline content. In addition, BAY 41-2272 was highly effective in the treatment of established fibrosis in the modified models of bleomycin-induced skin fibrosis and Tsk-1 mice. Treatment with sGC stimulators was well tolerated. Relevant antifibrotic doses of BAY 41-2272 did not affect systemic blood pressure and heart rate in mice. CONCLUSIONS: These findings demonstrate potent antifibrotic effects and good tolerability of sGC stimulators in various experimental models of SSc. Given their potential vasoactive properties, sGC stimulators may be promising candidates for the dual treatment of fibrosis and vascular disease in SSc.

Inhibition of hedgehog signalling prevents experimental fibrosis and induces regression of established fibrosis.

OBJECTIVES: Tissue fibrosis is a leading cause of death in patients with systemic sclerosis (SSc). Effective antifibrotic treatments are not available. Here, the authors investigated inhibition of hedgehog signalling by targeting Smoothened (Smo) as a novel antifibrotic approach. METHODS: The activation status of the hedgehog pathway was assessed by immunohistochemistry for Gli transcription factors and by quantification of hedgehog target genes. Hedgehog signalling was inhibited by the selective inhibitor LDE223 and by small interfering RNA against Smo in the models of bleomycin-induced dermal fibrosis and in tight-skin-1 mice. RESULTS: Hedgehog signalling is activated in SSc and in murine models of SSc. Inhibition of Smo either by LDE223 or by small interfering RNA prevented dermal thickening, myofibroblast differentiation and accumulation of collagen upon challenge with bleomycin. Targeting Smo also exerted potent antifibrotic effects in tight-skin-1 mice and did prevent progression of fibrosis and induced regression of pre-established fibrosis. CONCLUSIONS: Inhibition of hedgehog signalling exerted potent antifibrotic effects in preclinical models of SSc in both preventive and therapeutic settings. These findings might have direct translational implications because inhibitors of Smo are already available and yielded promising results in initial clinical trials.

ß-catenin is a central mediator of pro-fibrotic Wnt signaling in systemic sclerosis.

OBJECTIVES: Pathologic fibroblast activation drives fibrosis of the skin and internal organs in patients with systemic sclerosis (SSc). ß-catenin is an integral part of adherens junctions and a central component of canonical Wnt signaling. Here, the authors addressed the role of ß-catenin in fibroblasts for the development of SSc dermal fibrosis. METHODS: Nuclear accumulation of ß-catenin in fibroblasts was assessed by triple staining for ß-catenin, prolyl-4-hydroxylase-ß and 4',6-diamidino-2-phenylindole (DAPI). The expression of Wnt proteins in the skin was analysed by real-time PCR and immunohistochemistry. Mice with fibroblast-specific stabilisation or fibroblast-specific depletion were used to evaluate the role of ß-catenin in fibrosis. RESULTS: The auhors found significantly increased nuclear levels of ß-catenin in fibroblasts in SSc skin compared to fibroblasts in the skin of healthy individuals. The accumulation of ß-catenin resulted from increased expression of Wnt-1 and Wnt-10b in SSc. The authors further showed that the nuclear accumulation of ß-catenin has direct implications for the development of fibrosis: Mice with fibroblast-specific stabilisation of ß-catenin rapidly developed fibrosis within 2 weeks with dermal thickening, accumulation of collagen and differentiation of resting fibroblasts into myofibroblasts. By contrast, fibroblast-specific deletion of ß-catenin significantly reduced bleomycin-induced dermal fibrosis. CONCLUSIONS: The present study findings identify ß-catenin as a key player of fibroblast activation and tissue fibrosis in SSc. Although further translational studies are necessary to test the efficacy and tolerability of ß-catenin/Wnt inhibition in SSc, the present findings may have clinical implications, because selective inhibitors of ß-catenin/Wnt signaling have recently entered clinical trials.

The 12/15-lipoxygenase pathway counteracts fibroblast activation and experimental fibrosis.

BACKGROUND: Idiopathic and inflammation-dependent fibrotic diseases such systemic sclerosis (SSc) impose a major burden on modern societies. Understanding endogenous mechanisms, which counteract fibrosis, may yield new therapeutic approaches. Lipoxins are highly potent lipid mediators, which have recently been found to be decreased in SSc. OBJECTIVES: To determine the potential role of 12/15-lipoxygenase (12/15-LO), the key enzyme for the synthesis of lipoxins, in fibrosis. METHODS: Two mouse models for experimental dermal fibrosis (bleomycin-induced dermal fibrosis and tight-skin 1 mouse model) together with bone marrow transfers were used in wildtype and 12/15-LO(-/-) mice to elucidate the role of this enzyme during dermal fibrosis. Primary dermal fibroblasts of wildtype and 12/15-LO(-/-) mice, and 12/15-LO-derived eicosanoids, were used to identify underlying molecular mechanisms RESULTS: In both models, 12/15-LO(-/-) mice exhibited a significant exacerbation of the fibrotic tissue response. Bone marrow transfer experiments disclosed a predominant role of mesenchymal cell-derived 12/15-LO in these antifibrotic effects. Indeed, 12/15-LO(-/-) fibroblasts showed an enhanced activation of the mitogen-activated protein-kinase pathway and an increased col 1a2 mRNA expression in response to stimulation with transforming growth factor ß (TGFß), whereas 12/15-LO-derived eicosanoids blocked these TGFß-induced effects. CONCLUSIONS: These data indicate that 12/15-LO and its metabolites have a prominent antifibrotic role during dermal fibrosis. This opens new opportunities for therapeutic approaches in the treatment of fibrotic diseases.

Synthetic cannabinoid ajulemic acid exerts potent antifibrotic effects in experimental models of systemic sclerosis.

BACKGROUND: Cannabinoids modulate fibrogenesis in scleroderma. Ajulemic acid (AjA) is a non-psychoactive synthetic analogue of tetrahydrocannabinol that can bind the peroxisome proliferator-activated receptor-γ (PPAR-γ). Recent evidence suggests a key role for PPAR-γ in fibrogenesis. OBJECTIVE: To determine whether AjA can modulate fibrogenesis in murine models of scleroderma. MATERIAL AND METHODS: Bleomycin-induced experimental fibrosis was used to assess the antifibrotic effects of AjA in vivo. In addition, the efficacy of AjA in pre-established fibrosis was analysed in a modified model of bleomycin-induced dermal fibrosis and in mice overexpressing a constitutively active transforming growth factor ß (TGFß) receptor I. Skin fibrosis was evaluated by quantification of skin thickness and hydroxyproline content. As a marker of fibroblast activation, α-smooth muscle actin was examined. To study the direct effect of AjA in collagen neosynthesis, skin fibroblasts from patients with scleroderma were treated with increasing concentrations of AjA. Protein expression of PPAR-γ, and its endogenous ligand 15d-PGJ2, and TGFß were assessed before and after AjA treatment. RESULTS: AjA significantly prevented experimental bleomycin-induced dermal fibrosis and modestly reduced its progression when started 3 weeks into the disease. AjA strongly reduced collagen neosynthesis by scleroderma fibroblasts in vitro, an action which was reversed completely by co-treatment with a selective PPAR-γ antagonist. CONCLUSIONS: AjA prevents progression of fibrosis in vivo and inhibits fibrogenesis in vitro by stimulating PPAR-γ signalling. Since therapeutic doses of AjA are well tolerated in humans, it is suggested that AjA as an interesting molecule targeting fibrosis in patients with scleroderma.

Jun N-terminal kinase as a potential molecular target for prevention and treatment of dermal fibrosis.

OBJECTIVES: The hallmark of systemic sclerosis (SSc) is the accumulation of extracellular matrix proteins by pathologically activated fibroblasts. This study analysed the antifibrotic effects of the selective c-Jun N-terminal kinase (JNK) inhibitor, CC-930, which recently entered first clinical trials as a novel antifibrotic approach. METHODS: Phosphorylated c-Jun was detected by western blot and immunohistochemistry. The model of bleomycin-induced dermal fibrosis and the tight skin 1 (TSK1) mouse model were used to investigate the effects of CC-930 on the prevention of experimental fibrosis. The potential of CC-930 to induce regression of fibrosis was assessed in a modified model of established fibrosis. RESULTS: Transforming growth factor beta (TGFß) and platelet-derived growth factor (PDGF) activate JNK and stimulate the phosphorylation of its downstream target c-Jun. Incubation with CC-930 prevented the phosphorylation of c-Jun and reduced the stimulatory levels of these cytokines on the release of collagen. Inhibition of JNK prevented dermal thickening, myofibroblast differentiation and the accumulation of collagen in a dose-dependent manner in mice challenged with bleomycin and in TSK1 mice. In addition to the prevention of fibrosis, treatment with pharmacologically relevant doses of CC-930 also induced regression of established experimental fibrosis. CONCLUSIONS: These data identify JNK as a downstream mediator of the pro-fibrotic effects of of TGFß and PDGF in SSc fibroblasts. Selective inhibition of JNK by CC-930 exerted potent antifibrotic effects in vitro and in different models in vivo. JNK might thus be a novel molecular target for the treatment of fibrosis in SSc.

Induction of apoptosis in circulating angiogenic cells by microparticles.

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune disease marked by aberrant activation and apoptosis of endothelial cells (ECs) and decreased numbers of circulating angiogenic cells (CACs). The aim of this study was to analyze whether microparticles might link pathologic activation and apoptosis of ECs with reduced numbers of CACs. METHODS: Apoptosis was quantified by staining for annexin V and measurement of caspase 3 activity. The uptake of microparticles by CACs was determined by fluorescence-activated cell sorting and by fluorescence microscopy. Tritiated arachidonic acid and phosphatidylinositol 3,5-bisphosphate were used to demonstrate the transfer of arachidonic acid and highlight the role of the acid sphingomyelinase in microparticle-induced apoptosis of endothelial progenitor cells. RESULTS: Microparticles derived from activated or apoptotic ECs, the expression of which is strongly increased in the blood of patients with SSc, induce apoptosis in CACs in a dose-dependent manner. Microparticles, which are rich in arachidonic acid, are phagocytosed by CACs. Inhibition of phagocytosis prevents the induction of apoptosis in CACs by microparticles. Microparticles can transport arachidonic acid from ECs to CACs, and purified arachidonic acid mimics the proapoptotic effects of microparticles. Arachidonic acid activates the acid sphingomyelinase, and inhibition of acid sphingomyelinase prevents microparticle-induced apoptosis of CACs. Thus, phagocytosis of microparticles might stimulate the activity of acid sphingomyelinase and activate the apoptotic machinery. CONCLUSION: The induction of apoptosis in CACs by microparticles derived from ECs provides a novel link between aberrant activation or apoptosis of ECs, decreased numbers of CACs, and impaired formation of new vessels in SSc.

Inhibition of Notch signaling prevents experimental fibrosis and induces regression of established fibrosis.

OBJECTIVE: Tissue fibrosis caused by pathologic activation of fibroblasts with increased synthesis of extracellular matrix components is a major hallmark of systemic sclerosis (SSc). Notch signaling regulates tissue differentiation, and abnormal activation of Notch signaling has been implicated in the pathogenesis of various malignancies. The present study was undertaken 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 levels of HES-1 messenger RNA. In the mouse model of bleomycin-induced dermal fibrosis and in tight skin 1 mice, Notch signaling was inhibited by the γ-secretase inhibitor DAPT and by overexpression of a Notch-1 antisense construct. RESULTS: Notch signaling was 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 antifibrotic effects were also obtained with DAPT treatment. In addition to prevention of fibrosis, targeting of Notch signaling resulted in almost complete regression of established experimental fibrosis. CONCLUSION: The present results demonstrate that pharmacologic as well as genetic inhibition of Notch signaling exerts potent antifibrotic 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 have yielded promising results in cancer trials.

Inactivation of the transcription factor STAT-4 prevents inflammation-driven fibrosis in animal models of systemic sclerosis.

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 α, interleukin-6 (IL-6), IL-2, and interferon-γ 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.

Microparticles stimulate angiogenesis by inducing ELR(+) CXC-chemokines in synovial fibroblasts.

Microparticles (MPs) are small membrane-vesicles that accumulate in the synovial fluids of patients with rheumatoid arthritis (RA). In the arthritic joints, MPs induce a pro-inflammatory and invasive phenotype in synovial fibroblasts (SFs). The present study investigated whether activation of SFs by MPs stimulates angiogenesis in the inflamed joints of patients with RA. MPs were isolated from Jurkat cells and U937 cells by differential centrifugation. SFs were co-cultured with increasing numbers of MPs. The effects of supernatants from co-cultures on endothelial cells were studied in vitro and in vivo using MTT assays, annexin V and propidium iodide staining, trans-well migration assays and modified matrigel pouch assays. MPs strongly induced the expression of the pro-angiogenic ELR⁺ chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in RASFs. Other vascular growth factors were not induced. Supernatants from co-cultures enhanced the migration of endothelial cells, which could be blocked by neutralizing antibodies against ELR⁺ chemokines. Consistent with the specific induction of ELR⁺ chemokines, proliferation and viability of endothelial cells were not affected by the supernatants. In the in vivo bio-chamber assay, supernatants from RASFs co-cultured with MPs stimulated angiogenesis with a significant increase of vessels infiltrating into the matrigel chamber. We demonstrated that MPs activate RASFs to release pro-angiogenic ELR⁺ chemokines. These pro-angiogenic mediators enhance migration of endothelial cells and stimulate the formation of new vessels. Our data suggest that MPs may contribute to the hypervascularization of inflamed joints in patients with rheumatoid arthritis.

Inhibition of glycogen synthase kinase 3ß induces dermal fibrosis by activation of the canonical Wnt pathway.

OBJECTIVE: Glycogen synthase kinase 3ß (GSK-3) regulates the phosphorylation and subsequent degradation of ß-catenin, thereby preventing aberrant activation of the canonical Wnt pathway. A study was undertaken to define the role of GSK-3 in fibroblast activation and in experimental models of systemic sclerosis (SSc). METHODS: siRNA and specific inhibitors were used to inhibit GSK-3 in cultured fibroblasts and in mice. Activation of the canonical Wnt signalling was analysed by determining the levels of nuclear ß-catenin and by measuring the mRNA levels of the Wnt target gene Axin2. The effects of GSK-3 on the release of collagen were evaluated in human dermal fibroblasts and in the mouse model of bleomycin-induced skin fibrosis in tight-skin-1 (tsk-1) mice. RESULTS: Targeting GSK-3 potently activated the canonical Wnt pathway in fibroblasts in vitro and in vivo. Inactivation of GSK-3 dose-dependently stimulated the release of collagen from cultured fibroblasts in a ß-catenin-dependent manner and further resulted in progressive accumulation of collagen and dermal thickening in mice. Inhibition of GSK-3 aggravated experimental fibrosis in bleomycin-challenged mice and in tsk-1 mice. CONCLUSION: Inhibition of GSK-3 activates the canonical Wnt pathway in fibroblasts, stimulates the release of collagen from fibroblasts, exacerbates experimental fibrosis and is sufficient to induce fibrosis. GSK-3 is therefore a key regulator of the canonical Wnt signalling in fibroblasts and inhibition of GSK-3 results in fibroblast activation and increased release of collagen.

The transcription factor JunD mediates transforming growth factor {beta}-induced fibroblast activation and fibrosis in systemic sclerosis.

OBJECTIVES: Transforming growth factor ß (TGFß) has been identified as a key player in fibrotic diseases. However, the molecular mechanisms by which TGFß activates fibroblasts are incompletely understood. Here, the role of JunD, a member of the activator protein 1 (AP-1) family of transcription factors, as a downstream mediator of TGFß signalling in systemic sclerosis (SSc), was investigated. METHODS: The expression of JunD was analysed by real-time PCR, immunofluorescence, western blotting and immunohistochemistry. The canonical Smad pathway was specifically targeted by small interfering (si)RNA. The expression of extracellular matrix proteins in JunD deficient (JunD(-/-)) fibroblasts was analysed by real-time PCR and hydroxyproline assays. The mouse model of bleomycin-induced dermal fibrosis was used to assess the role of JunD in experimental fibrosis. RESULTS: JunD was overexpressed in SSc skin and in cultured fibroblasts in a TGFß dependent manner. The expression of JunD colocalised with pSmad 3 in fibrotic skin and silencing of Smad 3 or Smad 4 by siRNA prevented the induction of JunD by TGFß. JunD(-/-) fibroblasts were less responsive to TGFß and released less collagen upon stimulation with TGFß. Moreover, JunD(-/-) mice were protected from bleomycin-induced fibrosis with reduced dermal thickening, decreased myofibroblast counts and lower collagen content of lesional skin. CONCLUSIONS: These data demonstrate that JunD is overexpressed in SSc and that JunD is a mediator of the profibrotic effects of TGFß. Considering that inhibitors of AP-1 signalling have recently been developed and are available for clinical trials in SSc, these findings may have translational implications.

Notch signalling regulates fibroblast activation and collagen release in systemic sclerosis.

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 α-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.

The cannabinoid WIN55, 212-2 abrogates dermal fibrosis in scleroderma bleomycin model.

OBJECTIVES: There is increasing evidence that the endocannabinoid system may be involved in pathological fibrosis, and that its modulation might limit fibrotic responses. The aim of this study was to examine the capacity of a synthetic cannabinoid receptor agonist to modify skin fibrosis in the bleomycin mouse model of scleroderma. METHODS: Skin fibrosis was induced by local injections of bleomycin in two groups of DBA/2J mice. One group was cotreated with the synthetic cannabinoid WIN55,212-2 at 1 mg/kg/day. Skin fibrosis was evaluated by histology and skin thickness and hydroxyproline content were quantified. Markers of fibroblast activation, including α smooth muscle actin and the profibrotic cytokines transforming growth factor (TGF)ß, connective tissue growth factor (CTGF) and platelet-derived growth factor (PDGF)-BB, were examined. Levels of PSMAD2/3, which are crucial in extracellular matrix overproduction, were analysed. RESULTS: Bleomycin treatment induced typical skin fibrosis. Upon WIN55,212-2 treatment dermal fibrosis was completely prevented. Subcutaneous inflammatory cell infiltration, dermal thickness and collagen content resulted similar to those of the control group. The synthetic cannabinoid prevented fibroblasts activation induced by bleomycin, paralleled by a strong inhibition of TGFß, CTGF and PDGF-BB expression. Phosphorylation of SMAD2/3 was significantly downregulated after WIN55,212-2 exposure. CONCLUSIONS: Taken together, the results indicate that the synthetic cannabinoid WIN55,212-2 is capable of preventing skin fibrosis in a mouse model of scleroderma.

Dysbalance of angiogenic and angiostatic mediators in patients with mixed connective tissue disease.

OBJECTIVE: Vascular disease is common in mixed connective tissue disease (MCTD). The aim of the present study was to investigate, whether dysbalance of angiogenic and angiostatic factors occurs in MCTD. METHODS: In all, 38 patients with MCTD, and 40 patients with systemic sclerosis (SSc) for comparison, were included. Four centres contributed to this cross-sectional analysis. A total of 66 healthy volunteers were used as controls. The serum levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endostatin were determined by ELISA. For comparisons between controls and patients with MCTD and detection of associations of serum levels with dichotomous clinical parameters in patients with MCTD the Mann-Whitney test was used. RESULTS: Serum levels of the angiogenic factor VEGF were significantly elevated in patients with MCTD and SSc. Significantly increased levels of the angiostatic factor endostatin were also detected in MCTD, but not in SSc. No differences were observed for bFGF. Levels of VEGF were higher in patients with MCTD with pulmonary arterial hypertension (PAH), acrosclerosis and myositis. In multivariate linear regression analysis, an additive model of PAH, myositis and lymphadenopathy accounted for 79% of the variability of the VEGF levels (r=0.889). CONCLUSIONS: Molecular factors modulating angiogenic responses are dysregulated in patients with MCTD and SSc with increases of VEGF in MCTD and SSc and selective upregulation of endostatin in MCTD. Furthermore, high serum levels of VEGF might characterise patients with MCTD with a more severe course of the disease with increased prevalence of PAH and myositis.

Inactivation of the cannabinoid receptor CB1 prevents leukocyte infiltration and experimental fibrosis.

OBJECTIVE: Cannabinoids are derivates of the marijuana component Δ(9) -tetrahydrocannabinol that exert their effects on mesenchymal cells and immune cells via CB1 and CB2 receptors. The aim of the present study was to evaluate the role of CB1 in systemic sclerosis. METHODS: CB1-deficient (CB1(-/-) ) mice and wild-type littermates (CB1(+/+) mice) were injected with bleomycin. CB1 signaling was activated in vivo with the selective agonist N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA). Bone marrow transplantation experiments were performed to investigate whether the phenotype of CB1(-/-) mice was mediated by leukocytes or mesenchymal cells. The role of CB1 was also investigated in the TSK-1 mouse model. RESULTS: CB1(-/-) mice were protected from bleomycin-induced dermal fibrosis, with reduced dermal thickening, hydroxyproline content, and myofibroblast counts. Inactivation of CB1 decreased the number of infiltrating T cells and macrophages in lesional skin. In contrast, activation of CB1 with ACEA increased leukocyte infiltration and enhanced the fibrotic response to bleomycin. The phenotype of CB1(-/-) mice was mimicked by transplantation of CB1(-/-) mouse bone marrow into CB1(+/+) mice, demonstrating that CB1 exerts its profibrotic effects indirectly by regulating leukocyte infiltration. Consistently, knockdown of CB1 did not prevent fibrosis in the inflammation-independent TSK-1 mouse model. CONCLUSION: We demonstrate that the cannabinoid receptor CB1 is crucial for leukocyte infiltration and secondary fibroblast activation and that inactivation of CB1 exerts potent antifibrotic effects in inflammation-driven models of fibrosis.

MicroRNA-29, a key regulator of collagen expression in systemic sclerosis.

OBJECTIVE: To investigate the role of microRNA (miRNA) as posttranscriptional regulators of profibrotic genes in systemic sclerosis (SSc). METHODS: MicroRNA, which target collagens, were identified by in silico analysis. Expression of miRNA-29 (miR-29) was determined by TaqMan real-time polymerase chain reaction analysis of skin biopsy and fibroblast samples from SSc patients and healthy controls as well as in the mouse model of bleomycin-induced skin fibrosis. Cells were transfected with precursor miRNA (pre-miRNA)/anti-miRNA of miR-29 using Lipofectamine. Collagen gene expression was also studied in luciferase reporter gene assays. For stimulation, recombinant transforming growth factor beta (TGFbeta), platelet-derived growth factor B (PDGF-B), or interleukin-4 (IL-4) was used. The effects of inhibiting PDGF-B and TGFbeta signaling on the levels of miR-29 were studied in vitro and in the bleomycin model. RESULTS: We found that miR-29a was strongly down-regulated in SSc fibroblasts and skin sections as compared with the healthy controls. Overexpression in SSc fibroblasts significantly decreased, and accordingly, knockdown in normal fibroblasts increased, the levels of messenger RNA and protein for type I and type III collagen. In the reporter gene assay, cotransfection with pre-miR-29a significantly decreased the relative luciferase activity, which suggests a direct regulation of collagen by miR-29a. TGFbeta, PDGF-B, or IL-4 reduced the levels of miR-29a in normal fibroblasts to those seen in SSc fibroblasts. Similar to human SSc, the expression of miR-29a was reduced in the bleomycin model of skin fibrosis. Inhibition of PDGF-B and TGFbeta pathways by treatment with imatinib restored the levels of miR-29a in vitro and in the bleomycin model in vivo. CONCLUSION: These data add the posttranscriptional regulation of collagens by miR-29a as a novel aspect to the fibrogenesis of SSc and suggest miR-29a as a potential therapeutic target.

Decreased lymphatic vessel counts in patients with systemic sclerosis: association with fingertip ulcers.

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease that is characterized by microvascular disease and tissue fibrosis. Progressive loss and irregular architecture of the small blood vessels are well characterized, but the potential involvement of the lymphatic vessel system has not been analyzed directly in SSc. This study was undertaken to assess whether the lymphatic vascular system is affected in SSc, and whether changes to the lymphatic vessels are associated with dystrophic changes and tissue damage in patients with SSc. METHODS: Lymphatic endothelial cells in skin biopsy samples from patients with SSc and age- and sex-matched healthy volunteers were identified by staining for podoplanin and prox-1, both of which are specifically expressed in lymphatic endothelial cells but not in blood vascular endothelial cells. CD31 was used as a pan-endothelial cell marker. Statistical analyses were performed using Kruskal-Wallis, Mann-Whitney U, and Spearman's rank correlation tests. RESULTS: The numbers of podoplanin- and prox-1-positive lymphatic vessels were significantly reduced in patients with SSc as compared with healthy individuals. The number of podoplanin-positive lymphatic precollector vessels was significantly lower in SSc patients with fingertip ulcers than in SSc patients without ulcers. Moreover, the number of lymphatic vessels correlated inversely with the number of fingertip ulcers at the time of biopsy and with the number of fingertip ulcers per year. The inverse correlation between lymphatic precollector vessel counts and fingertip ulcers remained significant after statistical adjustment for the blood vessel count, age, and modified Rodnan skin thickness score. CONCLUSION: These results demonstrate a severe reduction in the number of lymphatic capillaries and lymphatic precollector vessels in patients with SSc. Patients with decreased lymphatic vessel counts may be at particularly high risk of developing fingertip ulcers.