[Early recognition and prevention of systemic sclerosis].

Systemic sclerosis is a disease characterized by skin and internal organ fibrosis, lacking specific therapeutic drugs and having a poor prognosis. Early diagnosis and intervention of the disease is of significant value in improving patient prognosis. This article provides a systematic review of the early diagnosis and treatment of systemic sclerosis, including early symptom recognition, laboratory testing, and drug intervention. It will provide a reference for the prevention of this disease.

2-Aminoethyl diphenylborinate inhibits bleomycin-induced skin and pulmonary fibrosis via interrupting intracellular Ca2+ regulation.

BACKGROUND: Systemic sclerosis (SSc) causes progressive fibrosis of multiple organs with the low efficacy of immunosuppressive therapies. Our previous study indicated the SSc pathological pathways are closely correlated with Ca2+ signals, and blockage of the intracellular Ca2+ elevation facilitates inhibition of SSc pathogenesis. OBJECTIVE: Transforming growth factor ß (TGF-ß)-modulated SMAD signaling is crucial in regulating SSc pathogenesis. Whether Ca2+ signals are involved in TGF-ß1/SMAD signaling-induced fibrotic process has been further investigated. METHODS: We utilized TGF-ß1-induced myofibroblasts as a model to detect how Ca2+ signals affected SSc pathogenesis, and investigated the combination of treatment with store-operated Ca2+ entry (SOCE) associated inhibitors, 2-aminoethyl diphenylborinate (2-APB) and SKF96365 to restrain the increased Ca2+ signaling in myofibroblasts. In addition, the SSc bleomycin mouse model was used to detect the effect of 2-APB on SSc pathogenesis in vivo. RESULTS: Our findings revealed increased levels of TGF-ß1 production in SSc was associated with intracellular Ca2+ activity, and inhibition of intracellular Ca2+ regulation by 2-APB resulted in the dedifferentiation of TGF-ß1-induced myofibroblasts. This was due to the fact that 2-APB restrained the expression fibrotic markers, α-SMA, fibronectin and vimentin through inhibiting TGF-ß1/SMAD3 signaling. Thus, subcutaneous injection of 2-APB improved bleomycin-induced skin and pulmonary fibrosis. CONCLUSION: 2-APB is a potential candidate for treating fibrosis, by disrupting intracellular Ca2+ regulation in SSc to induce the dedifferentiation of myofibroblasts and meliorates fibrosis pathogenesis via inhibiting TGF-ß1/SMAD3 signaling.

Secukinumab and metformin ameliorate dermal fibrosis by decreasing tissue interleukin-17 levels in bleomycin-induced dermal fibrosis.

Although the pathogenesis of systemic sclerosis is not exactly known, it is thought that immune activation has prominent roles in pathogenesis. Secukinumab is a monoclonal antibody against interleukin (IL)-17A. Metformin, a widely used antidiabetic medication, has anti-proliferative, immunomodulating and anti-fibrotic activities. The purpose of our study is to determine the therapeutic efficacy of secukinumab and metformin on bleomycin (BLM) induced dermal fibrosis. Fifty Balb/c female mice were divided into 5 groups: (group 1 control, 2 sham, 3 secukinumab, 4 metformin and 5 secukinumab + metformin). The mice in the control group received 100 µL phosphate-buffered saline (PBS), while the mice in other groups received 100 µL (100 µg) BLM in PBS subcutaneously (sc) every day for 4 weeks. In addition, mice in groups 3 and 5 received secukinumab at a dose of 10 mg/kg/wk sc, and mice in the groups 4 and 5 received oral metformin 50 mg/kg/d for 28 days. All groups of mice were sacrificed at the end of the 4th week and tissue samples were taken for analysis. In addition to histopathological analysis, skin tissue messenger RNA (mRNA) expressions of IL-17 and collagen 3A were measured by real-time polymerase chain reaction. Repeated BLM injections had caused dermal fibrosis. In addition, the mRNA expressions of IL-17 and collagen 3A were increased in the BLM group. Secukinumab and metformin ameliorated dermal fibrosis. They decreased dermal thickness and tissue IL-17A and collagen 3A mRNA levels. Secukinumab and metformin exhibit anti-fibrotic effects in the BLM-induced dermal fibrosis.

Echinochrome A Treatment Alleviates Fibrosis and Inflammation in Bleomycin-Induced Scleroderma.

Scleroderma is an autoimmune disease caused by the abnormal regulation of extracellular matrix synthesis and is activated by non-regulated inflammatory cells and cytokines. Echinochrome A (EchA), a natural pigment isolated from sea urchins, has been demonstrated to have antioxidant activities and beneficial effects in various disease models. The present study demonstrates for the first time that EchA treatment alleviates bleomycin-induced scleroderma by normalizing dermal thickness and suppressing collagen deposition in vivo. EchA treatment reduces the number of activated myofibroblasts expressing α-SMA, vimentin, and phosphorylated Smad3 in bleomycin-induced scleroderma. In addition, it decreased the number of macrophages, including M1 and M2 types in the affected skin, suggesting the induction of an anti-inflammatory effect. Furthermore, EchA treatment markedly attenuated serum levels of inflammatory cytokines, such as tumor necrosis factor-α and interferon-γ, in a murine scleroderma model. Taken together, these results suggest that EchA is highly useful for the treatment of scleroderma, exerting anti-fibrosis and anti-inflammatory effects.

Targeting of canonical WNT signaling ameliorates experimental sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGVHD remain poorly understood, and targeted therapies for clinical use are not well established. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGVHD (sclGVHD). WNT signaling was activated in human sclGVHD with increased nuclear accumulation of the transcription factor ß-catenin and a WNT-biased gene expression signature in lesional skin. Treatment with the highly selective tankryase inhibitor G007-LK, the CK1α agonist pyrvinium, or the LRP6 inhibitor salinomycin abrogated the activation of WNT signaling and protected against experimental cGVHD, without a significant impact on graft-versus-leukemia effect (GVL). Treatment with G007-LK, pyrvinium, or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d) → BALB/c (H-2d) and LP/J (H-2b) → C57BL/6 (H-2b) models of sclGVHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect inflammation-dependent effects in sclGVHD. Our findings may have direct translational potential, because pyrvinium is in clinical use, and tankyrase inhibitors are in clinical trials for other indications.

DZ2002 ameliorates fibrosis, inflammation, and vasculopathy in experimental systemic sclerosis models.

BACKGROUND: Systemic sclerosis is a multisystem inflammatory and vascular lesion leading to extensive tissue fibrosis. A reversible S-adenosyl-l-homocysteine hydrolase (SAHH) inhibitor, DZ2002, modulates the pathologic processes of various inflammatory diseases and autoimmune diseases. This study is designed to investigate the therapeutic potentiality of DZ2002 for experimental systemic sclerosis models. METHODS: The anti-inflammatory and anti-fibrotic features of DZ2002 and its mechanisms were investigated in a bleomycin (BLM)-induced dermal fibrosis mice model. The effects of DZ2002 on expression of extracellular matrix components and TGF-ß signaling in human dermal fibroblasts were analyzed. Simultaneously, the effects of DZ2002 on macrophage activation and endothelial cell adhesion molecule expression were also evaluated. RESULTS: DZ2002 significantly attenuated dermal fibrosis in BLM-induced mice. Consistently, DZ2002 inhibited the expression of various molecules associated with dermal fibrosis, including transforming growth factor ß1, connective tissue growth factor, tumor necrosis factor-α, interferon-γ, IL-1ß, IL-4, IL-6, IL-10, IL-12p40, IL-17A, and monocyte chemotactic protein 1 in the lesional skin of BLM-induced mice. Furthermore, DZ2002 decreased the proportion of macrophages, neutrophils, and T cells (especially T helper cells) in the skin tissue of BLM-induced mice. In addition, DZ2002 attenuated both M1 macrophage and M2 macrophage differentiation in vivo and in vitro. Importantly, DZ2002 directly reversed the profibrotic phenotype of transforming growth factor-ß1-treated dermal fibroblasts and suppressed ICAM-1, VCAM-1, VEGF, bFGF, and ET-1 expression in endothelial cells. Finally, our investigations showed that DZ2002 relieved systemic sclerosis by regulating fibrosis TGF-ß/Smad signaling pathway. CONCLUSIONS: DZ2002 prevents the development of experimental dermal fibrosis by reversing the profibrotic phenotype of various cell types and would be a potential drug for the treatment of systemic sclerosis.

Sirtuin1 Protects against Systemic Sclerosis-related Pulmonary Fibrosis by Decreasing Proinflammatory and Profibrotic Processes.

Pulmonary fibrosis is the leading cause of death in systemic sclerosis (SSc). Sirtuin1 (SIRT1) is a deacetylase with known antiinflammatory and antifibrotic activity in the liver, kidney, and skin. The role of SIRT1 in SSc-related pulmonary fibrosis is unknown. In the present work, we determined that the expression of SIRT1 in peripheral blood mononuclear cells of patients with SSc with pulmonary fibrosis is lower than that in patients with SSc without pulmonary fibrosis. In in vivo studies of bleomycin-induced lung fibrosis in mice, SIRT1 activation with resveratrol reduced collagen production when it was administered either prophylactically during the inflammatory stage or after the development of fibrosis. Furthermore, SIRT1 activation or overexpression inhibited tumor necrosis factor-α-induced inflammatory responses in vitro in human fetal lung fibroblasts, depletion of SIRT1 in fibroblasts enhanced inflammation, and these effects were related to changes in the acetylation of NF-κB. In addition, SIRT1 activation or exogenous overexpression inhibited collagen production in vitro, and these manipulations also inhibited fibrosis via inactivation of transforming growth factor-ß/mothers against decapentaplegic homolog and mammalian target of rapamycin signaling. Taken together, our results show that a loss of SIRT1 may participate in the pathogenesis of SSc-related pulmonary fibrosis, and that SIRT1 activation is an effective treatment for both the early (inflammatory) and late (fibrotic) stages of pulmonary fibrosis. Thus, SIRT1 may be a promising therapeutic target in the management of SSc-related pulmonary fibrosis.

Anti-connective tissue growth factor (CTGF/CCN2) monoclonal antibody attenuates skin fibrosis in mice models of systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. Although the involvement of connective tissue growth factor (CTGF/CCN2) has been well-documented in SSc fibrosis, the therapeutic potential of targeting CTGF in SSc has not been fully investigated. Our aim was to examine the therapeutic potential of CTGF blockade in a preclinical model of SSc using two approaches: smooth muscle cell fibroblast-specific deletion of CTGF (CTGF knockout (KO)) or a human anti-CTGF monoclonal antibody, FG-3019. METHODS: Angiotensin II (Ang II) was administered for 14 days by subcutaneous osmotic pump to CTGF KO or C57BL/6 J mice. FG-3019 was administered intraperitoneally three times per week for 2 weeks. Skin fibrosis was evaluated by histology and hydroxyproline assay. Immunohistochemistry staining was used for alpha smooth muscle actin (αSMA), platelet-derived growth factor receptor ß (PDGFRß), pSmad2, CD45, von Willebrand factor (vWF), and immunofluorescence staining was utilized for procollagen and Fsp1. RESULTS: Ang II-induced skin fibrosis was mitigated in both CTGF KO and FG-3019-treated mice. The blockade of CTGF reduced the number of cells expressing PDGFRß, procollagen, αSMA, pSmad2, CD45, and Fsp1 in the dermis. In addition, inhibition of CTGF attenuated vascular injury as measured by the presence of vWF-positive cells. CONCLUSIONS: Our data indicate that inhibition of CTGF signaling presents an attractive therapeutic approach in SSc.

Pharmacological inhibition of porcupine induces regression of experimental skin fibrosis by targeting Wnt signalling.

OBJECTIVES: Wnt signalling has been implicated in activating a fibrogenic programme in fibroblasts in systemic sclerosis (SSc). Porcupine is an O-acyltransferase required for secretion of Wnt proteins in mammals. Here, we aimed to evaluate the antifibrotic effects of pharmacological inhibition of porcupine in preclinical models of SSc. METHODS: The porcupine inhibitor GNF6231 was evaluated in the mouse models of bleomycin-induced skin fibrosis, in tight-skin-1 mice, in murine sclerodermatous chronic-graft-versus-host disease (cGvHD) and in fibrosis induced by a constitutively active transforming growth factor-ß-receptor I. RESULTS: Treatment with pharmacologically relevant and well-tolerated doses of GNF6231 inhibited the activation of Wnt signalling in fibrotic murine skin. GNF6231 ameliorated skin fibrosis in all four models. Treatment with GNF6231 also reduced pulmonary fibrosis associated with murine cGvHD. Most importantly, GNF6231 prevented progression of fibrosis and showed evidence of reversal of established fibrosis. CONCLUSIONS: These data suggest that targeting the Wnt pathway through inhibition of porcupine provides a potential therapeutic approach to fibrosis in SSc. This is of particular interest, as a close analogue of GNF6231 has already demonstrated robust pathway inhibition in humans and could be available for clinical trials.

The Protective Effects of Bevacizumab in Bleomycin-Induced Experimental Scleroderma.

BACKGROUND: The capillary networks are less dense and have irregular structures in scleroderma. These abnormalities result in lower capillary blood flow causing severe tissue hypoxia, which is a major stimulus for angiogenesis. However, current knowledge about compensatory angiogenesis is ambiguous in scleroderma. Bevacizumab is an inhibitor of vascular endothelial growth factor (VEGF). OBJECTIVES: The aim of the present study is to evaluate the protective effects of bevacizumab in bleomycin (BLM)- -induced dermal fibrosis. MATERIAL AND METHODS: This study involved 4 groups of Balb/c mice (n = 10 per group). Mice in the control group received 100 µL/day of phosphate-buffered saline (PBS) subcutaneously, while the other 3 groups were given 100 µg/day of BLM (dissolved in 100 µL PBS) subcutaneously, for 4 weeks. Mice in BLM-treated 3rd and 4th groups also received bevacizumab (1 or 5 mg/kg twice a week, intraperitoneally). At the end of the fourth week, all mice were sacrificed and blood and tissue samples were obtained. RESULTS: The BLM applications increased the dermal thicknesses, tissue hydroxyproline contents, and α-smooth muscle actin-positive (α-SMA+) cell counts, and led to histopathologically prominent dermal fibrosis. The bevacizumab treatments decreased the tissue hydroxyproline contents and dermal thicknesses, and these improvements were more prominent at doses by which α-SMA+ cell counts were markedly decreased, in the BLM-injected mice. CONCLUSIONS: In our study, inhibition of VEGF with bevacizumab treatments prevented the BLM-induced dermal fibrosis suggesting that VEGF expression contributes to the pathogenesis of scleroderma.

Azacytidine mitigates experimental sclerodermic chronic graft-versus-host disease.

BACKGROUND: Previous studies have demonstrated that regulatory T cells (Tregs) play a protective role in the pathogenesis of chronic graft-versus-host disease (cGVHD). Tregs constitutively express the gene of the transcription factor Foxp3 whose CNS2 region is heavily methylated in conventional CD4(+) T cells (CD4(+)Tconvs) but demethylated in Tregs. METHODS: Here, we assessed the impact of azacytidine (AZA) on cGVHD in a well-established murine model of sclerodermic cGVHD (B10.D2 (H-2d) → BALB/cJ (H-2d)). RESULTS: The administration of AZA every 48 h from day +10 to day +30 at the dose of 0.5 mg/kg or 2 mg/kg mitigated chronic GVHD. Further, AZA-treated mice exhibited higher blood and thymic Treg frequencies on day +35, as well as higher demethylation levels of the Foxp3 enhancer and the IL-2 promoter in splenocytes at day +52. Interestingly, Tregs from AZA-treated mice expressed more frequently the activation marker CD103 on day +52. AZA-treated mice had also lower counts of CD4(+)Tconvs and CD8(+) T cells from day +21 to day +35 after transplantation, as well as a lower proportion of CD4(+)Tconvs expressing the Ki67 antigen on day +21 demonstrating an anti-proliferating effect of the drug on T cells. CONCLUSIONS: Our results indicate that AZA prevented sclerodermic cGVHD in a well-established murine model of cGVHD. These data might serve as the basis for a pilot study of AZA administration for cGVHD prevention in patients at high risk for cGVHD.

Spleen tyrosine kinase (Syk) inhibitor fostamatinib limits tissue damage and fibrosis in a bleomycin-induced scleroderma mouse model.

OBJECTIVES: The pathogenesis of fibrosis in scleroderma (SSc) is unknown. TGF-ß and platelet-derived growth factor are important in the development of fibrosis and tyrosine kinases are involved in these pathways. The possible antifibrotic effects of various kinase inhibitors in SSc have been studied before. Spleen tyro-sine kinase (Syk) is a protein tyrosine kinase which activates intracellular signal transduction pathways; and has been claimed to be involved in the pathogenesis of systemic autoimmune diseases. Inhibition of Syk suppresses IgE- and IgG-associated FcR signal activation in various cell types; and suppresses experimental arthritis and skin and kidney disease in lupus-prone mice. We investigated the ability of a small drug, the Syk inhibitor, fostamatinib, to protect mice from bleomycin-induced SSc. METHODS: Four study groups of BALB/c mice were included into this study: control, bleomycin (administered subcutaneously to BALB/c mice for 21 days), bleomycin and fostamatinib (mice fed with chow containing a Syk inhibitor for 21 days), and fostamatinib alone groups. Skin and lung tissue specimens were obtained and evaluated histologically. RESULTS: Treatment with fostamatinib significantly reduced skin thickness and fibrosis. Mice treated with fostamatinib also displayed less fibrosis and inflammation in the lung tissue. Following fostamatinib treatment, Syk, phospho-Syk, and TGF-ß expression decreased in both skin and lung tissues. CONCLUSIONS: The Syk inhibitor fostamatinib prevented bleomycin-induced fibrosis and inflammation in the skin and in the lung. The anti-fibrotic effect of fostamatinib is linked to reduced Syk phosphorylation and TGF-ß expression. The Syk pathway appears as a potential molecular target for therapeutic intervention in SSc.

Pantethine Prevents Murine Systemic Sclerosis Through the Inhibition of Microparticle Shedding.

OBJECTIVE: Endothelial cell (EC) damage in systemic sclerosis (SSc) is reflected by the shedding of microparticles (MPs). The aim of this study was to show that inhibiting MP release using pantethine or by inactivating ATP-binding cassette transporter A1 (ABCA1) ameliorates murine SSc. METHODS: First, the effects of pantethine on MP shedding and on basal oxidative and nitrosative stresses in ECs and fibroblasts were determined in vitro. The effects of pantethine were then tested in vivo. SSc was induced in BALB/c mice by daily intradermal injection of HOCl. Mice were simultaneously treated daily with pantethine by oral gavage. RESULTS: In vitro, pantethine inhibited MP shedding from tumor necrosis factor-stimulated ECs and abrogated MP-induced oxidative and nitrosative stresses in ECs and fibroblasts. Ex vivo, pantethine also restored redox homeostasis in fibroblasts from mice with SSc. In vivo, mice with SSc displayed skin and lung fibrosis associated with increased levels of circulating MPs and markers of oxidative and endothelial stress, which were normalized by administration of pantethine or inactivation of ABCA1. CONCLUSION: Pantethine is a well-tolerated molecule that represents a potential treatment of human SSc.

Amelioration of tissue fibrosis by toll-like receptor 4 knockout in murine models of systemic sclerosis.

OBJECTIVE: Bleomycin-induced fibrosis and the tight skin (TSK/+) mouse are well-established experimental murine models of human systemic sclerosis (SSc). Growing evidence has demonstrated the pivotal role of Toll-like receptors (TLRs) in several autoimmune inflammatory diseases, including SSc. This study was undertaken to determine the role of TLR-4 in the fibrotic processes in these murine models. METHODS: We generated a murine model of bleomycin-induced SSc using TLR-4(-/-) mice and TLR-4(-/-) ;TSK/+ mice. The mechanisms by which TLR-4 contributes to pathologic tissue fibrosis were investigated in these 2 models by histologic examination, hydroxyproline assay, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and flow cytometry. RESULTS: Dermal and lung fibrosis was attenuated in bleomycin-treated TLR-4(-/-) mice compared with their wild-type counterparts. Inflammatory cell infiltration, expression of various inflammatory cytokines, and pathologic angiogenesis induced by bleomycin treatment were suppressed with TLR-4 deletion. Furthermore, the increased expression of interleukin-6 (IL-6) in fibroblasts, endothelial cells, and immune cells in response to bleomycin in vivo and to lipopolysaccharide in vitro was notably abrogated in the absence of TLR-4. Moreover, TLR-4 deletion was associated with alleviated B cell activation and skew toward a Th2/Th17 response against bleomycin treatment. Importantly, in TSK/+ mice, another SSc murine model, TLR-4 abrogation attenuated hypodermal fibrosis. CONCLUSION: These results indicate the pivotal contribution of TLR-4 to the pathologic tissue fibrosis of SSc murine models. Our results indicate the critical role of TLR-4 signaling in the development of tissue fibrosis, suggesting that biomolecular TLR-4 targeting might be a potential therapeutic approach to SSc.

Attenuation of fibrosis with selective inhibition of c-Abl by siRNA in systemic sclerosis dermal fibroblasts.

Cellular abelson (c-Abl), a non-receptor tyrosine kinase, is an important molecule in the pathogenesis of systemic sclerosis. There have been reports of beneficial effects of pharmacological tyrosine kinase inhibitors, such as imatinib mesylate, on fibrosis. However, these inhibitors affect multiple tyrosine kinases including c-Abl, c-kit, and platelet-derived growth factor receptor. The effects of selective inhibition of c-Abl using small interfering RNA (siRNA) on dermal fibrosis have not yet been explored. The aim of this study is to evaluate whether specific inhibition of c-Abl by siRNA can influence the transforming growth factor-ß1 (TGF-ß1)-induced fibrotic responses. Dermal fibroblasts from systemic sclerosis patients and healthy controls were transfected with c-Abl siRNA. The expression levels of collagen type I, fibronectin, connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) were measured at both the mRNA and protein levels in the absence or presence of TGF-ß1 pro-fibrotic cytokine. In healthy dermal fibroblasts, the expression of collagen type 1, fibronectin, α-SMA, and CTGF mRNAs and proteins that were upregulated after stimulation with TGF-ß1 was markedly decreased by c-Abl siRNA. Silencing of c-Abl via siRNA efficiently reduced the basal synthesis of collagen type I, fibronectin, α-SMA, and CTGF mRNAs and proteins in systemic sclerosis fibroblasts, but it had no effect on the baseline expression of these genes and proteins in healthy dermal fibroblasts. In conclusion, specific c-Abl gene silencing using siRNA effectively reduced fibrosis-related gene expression. Inhibition of c-Abl by siRNA may be a potential therapeutic approach for fibrotic diseases such as systemic sclerosis.

Heat shock protein 90 (Hsp90) inhibition targets canonical TGF-ß signalling to prevent fibrosis.

OBJECTIVES: Targeted therapies for systemic sclerosis (SSc) and other fibrotic diseases are not yet available. We evaluated the efficacy of heat shock protein 90 (Hsp90) inhibition as a novel approach to inhibition of aberrant transforming growth factor (TGF)-ß signalling and for the treatment of fibrosis in preclinical models of SSc. METHODS: Expression of Hsp90 was quantified by quantitative PCR, western blot and immunohistochemistry. The effects of Hsp90 inhibition were analysed in cultured fibroblasts, in bleomycin-induced dermal fibrosis, in tight-skin (Tsk-1) mice and in mice overexpressing a constitutively active TGF-ß receptor I (TßRI). RESULTS: Expression of Hsp90ß was increased in SSc skin and in murine models of SSc in a TGF-ß-dependent manner. Inhibition of Hsp90 by 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) inhibited canonical TGF-ß signalling and completely prevented the stimulatory effects of TGF-ß on collagen synthesis and myofibroblast differentiation. Treatment with 17-DMAG decreased the activation of canonical TGF-ß signalling in murine models of SSc and exerted potent antifibrotic effects in bleomycin-induced dermal fibrosis, in Tsk-1 mice and in mice overexpressing a constitutively active TßRI. Dermal thickness, number of myofibroblasts and hydroxyproline content were all significantly reduced on treatment with 17-DMAG. No toxic effects were observed with 17-DMAG at antifibrotic doses. CONCLUSIONS: Hsp90 is upregulated in SSc and is critical for TGF-ß signalling. Pharmacological inhibition of Hsp90 effectively blocks the profibrotic effects of TGF-ß in cultured fibroblasts and in different preclinical models of SSc. These results have translational implications, as several Hsp90 inhibitors are in clinical trials for other indications.

Inactivation of evenness interrupted (EVI) reduces experimental fibrosis by combined inhibition of canonical and non-canonical Wnt signalling.

OBJECTIVES: Canonical as well as non-canonical Wnt signalling pathways have emerged as core pathways of fibrosis. Their profibrotic effects are mediated via distinct intracellular cascades independently of each other. Thus, inhibition of both pathways may have additive antifibrotic effects. Here, we knocked down evenness interrupted (EVI) to simultaneously target for the first time canonical and non-canonical Wnt signalling in experimental fibrosis. METHODS: The antifibrotic effects of siRNA-mediated knockdown of EVI were evaluated in the mouse models of bleomycin-induced skin fibrosis and in fibrosis induced by adenoviral overexpression of a constitutively active TGF-ß receptor I (AdTBRI). RESULTS: Knockdown of EVI decreased the release of canonical and non-canonical Wnt ligands by fibroblasts and reduced the activation of canonical and non-canonical Wnt cascades in experimental fibrosis with decreased accumulation of ß-catenin and phosphorylated JNK and cJun. Inactivation of EVI exerted potent antifibrotic effects and reduced dermal thickening, myofibroblast differentiation and accumulation of collagen in the mouse models of bleomycin-induced and AdTBR-induced fibrosis. CONCLUSIONS: Inhibition of Wnt secretion by knockdown of EVI inhibits canonical and non-canonical Wnt signalling and effectively reduces experimental fibrosis in different preclinical models. Inhibition of Wnt secretion may thus be an interesting approach for the treatment of fibrosis.

The natural organosulfur compound dipropyltetrasulfide prevents HOCl-induced systemic sclerosis in the mouse.

INTRODUCTION: The aim of this study was to test the naturally occurring organosulfur compound dipropyltetrasulfide (DPTTS), found in plants, which has antibiotic and anticancer properties, as a treatment for HOCl-induced systemic sclerosis in the mouse. METHODS: The prooxidative, antiproliferative, and cytotoxic effects of DPTTS were evaluated ex vivo on fibroblasts from normal and HOCl mice. In vivo, the antifibrotic and immunomodulating properties of DPTTS were evaluated in the skin and lungs of HOCl mice. RESULTS: H2O2 production was higher in fibroblasts derived from HOCl mice than in normal fibroblasts (P < 0.05). DPTTS did not increase H2O2 production in normal fibroblasts, but DPTTS dose-dependently increased H2O2 production in HOCl fibroblasts (P < 0.001 with 40 µM DPTTS). Because H2O2 reached a lethal threshold in cells from HOCl mice, the antiproliferative, cytotoxic, and proapoptotic effects of DPTTS were significantly higher in HOCl fibroblasts than for normal fibroblasts. In vivo, DPTTS decreased dermal thickness (P < 0.001), collagen content in skin (P < 0.01) and lungs (P < 0.05), αSMA (P < 0.01) and pSMAD2/3 (P < 0.01) expression in skin, formation of advanced oxidation protein products and anti-DNA topoisomerase-1 antibodies in serum (P < 0.05) versus untreated HOCl mice. Moreover, in HOCl mice, DPTTS reduced splenic B-cell counts (P < 0.01), the proliferative rates of B-splenocytes stimulated by lipopolysaccharide (P < 0.05), and T-splenocytes stimulated by anti-CD3/CD28 mAb (P < 0.001). Ex vivo, it also reduced the production of IL-4 and IL-13 by activated T cells (P < 0.05 in both cases). CONCLUSIONS: The natural organosulfur compound DPTTS prevents skin and lung fibrosis in the mouse through the selective killing of diseased fibroblasts and its immunomodulating properties. DPTTS may be a potential treatment for systemic sclerosis.

Integrin-modulating therapy prevents fibrosis and autoimmunity in mouse models of scleroderma.

In systemic sclerosis (SSc), a common and aetiologically mysterious form of scleroderma (defined as pathological fibrosis of the skin), previously healthy adults acquire fibrosis of the skin and viscera in association with autoantibodies. Familial recurrence is extremely rare and causal genes have not been identified. Although the onset of fibrosis in SSc typically correlates with the production of autoantibodies, whether they contribute to disease pathogenesis or simply serve as a marker of disease remains controversial and the mechanism for their induction is largely unknown. The study of SSc is hindered by a lack of animal models that recapitulate the aetiology of this complex disease. To gain a foothold in the pathogenesis of pathological skin fibrosis, we studied stiff skin syndrome (SSS), a rare but tractable Mendelian disorder leading to childhood onset of diffuse skin fibrosis with autosomal dominant inheritance and complete penetrance. We showed previously that SSS is caused by heterozygous missense mutations in the gene (FBN1) encoding fibrillin-1, the main constituent of extracellular microfibrils. SSS mutations all localize to the only domain in fibrillin-1 that harbours an Arg-Gly-Asp (RGD) motif needed to mediate cell-matrix interactions by binding to cell-surface integrins. Here we show that mouse lines harbouring analogous amino acid substitutions in fibrillin-1 recapitulate aggressive skin fibrosis that is prevented by integrin-modulating therapies and reversed by antagonism of the pro-fibrotic cytokine transforming growth factor ß (TGF-ß). Mutant mice show skin infiltration of pro-inflammatory immune cells including plasmacytoid dendritic cells, T helper cells and plasma cells, and also autoantibody production; these findings are normalized by integrin-modulating therapies or TGF-ß antagonism. These results show that alterations in cell-matrix interactions are sufficient to initiate and sustain inflammatory and pro-fibrotic programmes and highlight new therapeutic strategies.