Pan-Lysyl Oxidase Inhibitor PXS-5505 Ameliorates Multiple-Organ Fibrosis by Inhibiting Collagen Crosslinks in Rodent Models of Systemic Sclerosis.

Systemic sclerosis (SSc) is characterised by progressive multiple organ fibrosis leading to morbidity and mortality. Lysyl oxidases play a vital role in the cross-linking of collagens and subsequent build-up of fibrosis in the extracellular matrix. As such, their inhibition provides a novel treatment paradigm for SSc. A novel small molecule pan-lysyl oxidase inhibitor, PXS-5505, currently in clinical development for myelofibrosis treatment was evaluated using in vivo rodent models resembling the fibrotic conditions in SSc. Both lysyl oxidase and lysyl oxidase-like 2 (LOXL2) expression were elevated in the skin and lung of SSc patients. The oral application of PXS-5505 inhibited lysyl oxidase activity in the skin and LOXL2 activity in the lung. PXS-5505 exhibited anti-fibrotic effects in the SSc skin mouse model, reducing dermal thickness and α-smooth muscle actin. Similarly, in the bleomycin-induced mouse lung model, PXS-5505 reduced pulmonary fibrosis toward normal levels, mediated by its ability to normalise collagen/elastin crosslink formation. PXS-5505 also reduced fibrotic extent in models of the ischaemia-reperfusion heart, the unilateral ureteral obstruction kidney, and the CCl4-induced fibrotic liver. PXS-5505 consistently demonstrates potent anti-fibrotic efficacy in multiple models of organ fibrosis relevant to the pathogenesis of SSc, suggesting that it may be efficacious as a novel approach for treating SSc.

Lysyl oxidase directly contributes to extracellular matrix production and fibrosis in systemic sclerosis.

Pulmonary fibrosis is one of the important causes of morbidity and mortality in fibroproliferative disorders such as systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Lysyl oxidase (LOX) is a copper-dependent amine oxidase whose primary function is the covalent crosslinking of collagens in the extracellular matrix (ECM). We investigated the role of LOX in the pathophysiology of SSc. LOX mRNA and protein levels were increased in lung fibroblasts of SSc patients compared with healthy controls and IPF patients. In vivo, bleomycin induced LOX mRNA expression in lung tissues, and LOX activity increased in the circulation of mice with pulmonary fibrosis, suggesting that circulating LOX parallels levels in lung tissues. Circulating levels of LOX were reduced upon amelioration of fibrosis with an antifibrotic peptide. LOX induced ECM production at the transcriptional level in lung fibroblasts, human lungs, and human skin maintained in organ culture. In vivo, LOX synergistically exacerbated fibrosis in bleomycin-treated mice. Further, LOX increased the production of interleukin (IL)-6, and the increase was mediated by LOX-induced c-Fos expression, the nuclear localization of c-Fos, and its engagement with the IL-6 promoter region. Our findings demonstrate that LOX expression and activity correlate with fibrosis in vitro, ex vivo, and in vivo. LOX induced ECM production via upregulation of IL-6 and nuclear localization of c-Fos. Thus, LOX has a direct pathogenic role in SSc-associated fibrosis that is independent of its crosslinking function. Our findings also suggest that measuring circulating LOX levels and activity can be used for monitoring response to antifibrotic therapy.

Cytokine-mediated induction of human xylosyltransferase-I in systemic sclerosis skin fibroblasts.

Systemic sclerosis (SSc) is an inflammatory fibrotic disease characterized by an excessive extracellular matrix deposition in the skin and internal organs. One fibrotic key event remains the fibroblast-to-myofibroblast differentiation that is controlled by a combination of mechanical and soluble factors, such as transforming growth factor-ß1 (TGF-ß1) and interleukin-1ß (IL-1ß). One important myofibroblast biomarker is human xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan biosynthesis. Increased serum XT activity was quantified in SSc, but the underlying cellular mechanisms remain elusive. This study aims to determine the cellular basis of XT-I induction in SSc by using a myofibroblast cell culture model with SSc fibroblasts (SScF) and healthy control fibroblasts. We found that SScF exhibit a higher extracellular XT-I activity compared to control fibroblasts. This increased XT-I activity in SScF was demonstrated to be mediated by an enhanced autocrine TGF-ß signaling. Upon IL-1ß treatment, SScF showed an increased mRNA expression level of XT-I and TGF-ß receptor II (TGFBR2), while healthy control fibroblasts did not, pointing towards an involvement of IL-1ß in the cytokine-mediated XT-I induction. Performing microRNA (miRNA) inhibition experiments in the presence of TGF-ß1, we showed that the pro-fibrotic effect of IL-1ß may be mediated by a miRNA-21/TGF-ß receptor II axis, enhancing the autocrine TGF-ß signaling in SScF. Taken together, this study improves the mechanistic understanding of fibrotic XT-I induction in SSc by identifying a hitherto unknown IL-1ß-mediated miRNA-21/TGFBR2 regulation contributing to the enhanced XYLT1 expression and XT-I activity in SScF.

Overexpression of ubiquitin-specific peptidase 15 in systemic sclerosis fibroblasts increases response to transforming growth factor ß.

OBJECTIVE: Ubiquitination of proteins leads to their degradation by the proteasome, and is regulated by ubiquitin ligases and substrate-specific ubiquitin-specific peptidases (USPs). The ubiquitination process also plays important roles in the regulation of cell metabolism and cell cycle. Here, we found that the expression of several USPs is increased in SSc tenosynovial and skin biopsies, and we demonstrated that USP inhibition decreases TGF-ß signalling in primary fibroblast cell lines. METHODS: High-density transcriptomic studies were performed using total RNA obtained from SSc tenosynovial samples. Confirmatory immunostaining experiments were performed on tenosynovial and skin samples. In vitro experiments were conducted in order to study the influence of USP modulation on responses to TGF-ß stimulation. RESULTS: Tenosynovial biopsies from SSc patients overexpressed known disease-associated gene pathways: fibrosis, cytokines and chemokines, and Wnt/TGF-ß signalling, but also several USPs. Immunohistochemistry experiments confirmed the detection of USPs in the same samples, and in SSc skin biopsies. Exposure of primary fibroblast cell lines to TGF-ß induced USP gene expression. The use of a pan-USP inhibitor decreased SMAD3 phosphorylation, and expression of COL1A1, COL3A1 and fibronectin gene expression in TGF-ß-stimulated fibroblasts. The effect of the USP inhibitor resulted in increased SMAD3 ubiquitination, and was blocked by a proteasome inhibitor, thereby confirming the specificity of its action. CONCLUSION: Overexpression of several USPs, including USP15, amplifies fibrotic responses induced by TGF-ß, and is a potential therapeutic target in SSc.

Lysyl oxidase-a possible role in systemic sclerosis-associated pulmonary hypertension: a multicentre study.

OBJECTIVE: Lysyl oxidase (LOX) is an extracellular enzyme that cross-links collagen fibrils. LOX was found to be increased in serum of SSc patients and was suggested to be related to skin fibrosis, yet a vascular source of LOX has been demonstrated in idiopathic pulmonary arterial hypertension (iPAH). We aimed to validate elevated LOX serum levels in SSc and to study its correlation with clinical characteristics and investigate its main source at the tissue level. METHODS: A total of 86 established SSc patients were compared with 86 patients with very early diagnosis of systemic sclerosis (VEDOSS), 110 patients with primary RP (PRP) and 80 healthy controls. LOX serum levels were determined by ELISA. Five lung and 12 skin biopsies from SSc patients were stained for LOX and compared with controls. RESULTS: Serum levels of LOX in SSc were significantly higher than in VEDOSS, PRP and healthy controls (P < 0.001). LOX inversely correlated with the diffusing capacity of the lung for carbon monoxide diffusing capacity (DLCO) in diffuse SSc (r = -0.376, P = 0.02). Patients with moderate to severe estimated systolic PAH had higher LOX levels (P < 0.01). Lung biopsies demonstrated intense LOX staining in SSc patients with PAH that was predominantly located in the endothelium of the remodelled pulmonary vessels. CONCLUSION: Serum LOX levels are increased in established SSc and inversely correlate with the DLCO. LOX is elevated in patients with moderate to severe PAH and is located in the proliferating endothelium in lung arterioles, suggesting a possible role for LOX in SSc-associated PAH.

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.

The histone demethylase Jumonji domain-containing protein 3 (JMJD3) regulates fibroblast activation in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis. METHODS: JMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP. RESULTS: The expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFß)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFß. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2. CONCLUSION: We present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.

Poly(ADP-ribose) polymerase-1 regulates fibroblast activation in systemic sclerosis.

OBJECTIVES: The enzyme poly(ADP-ribose) polymerase-1 (PARP-1) transfers negatively charged ADP-ribose units to target proteins. This modification can have pronounced regulatory effects on target proteins. Recent studies showed that PARP-1 can poly(ADP-ribosyl)ate (PARylate) Smad proteins. However, the role of PARP-1 in the pathogenesis of systemic sclerosis (SSc) has not been investigated. METHODS: The expression of PARP-1 was determined by quantitative PCR and immunohistochemistry. DNA methylation was analysed by methylated DNA immunoprecipitation assays. Transforming growth factor-ß (TGFß) signalling was assessed using reporter assays, chromatin immunoprecipitation assays and target gene analysis. The effect of PARP-1 inactivation was investigated in bleomycin-induced and topoisomerase-induced fibrosis as well as in tight-skin-1 (Tsk-1) mice. RESULTS: The expression of PARP-1 was decreased in patients with SSc, particularly in fibroblasts. The promoter of PARP-1 was hypermethylated in SSc fibroblasts and in TGFß-stimulated normal fibroblasts. Inhibition of DNA methyltransferases (DNMTs) reduced the promoter methylation and reactivated the expression of PARP-1. Inactivation of PARP-1 promoted accumulation of phosphorylated Smad3, enhanced Smad-dependent transcription and upregulated the expression of TGFß/Smad target genes. Inhibition of PARP-1 enhanced the effect of TGFß on collagen release and myofibroblast differentiation in vitro and exacerbated experimental fibrosis in vivo. PARP-1 deficiency induced a more severe fibrotic response to bleomycin with increased dermal thickening, hydroxyproline content and myofibroblast counts. Inhibition of PARylation also exacerbated fibrosis in Tsk-1 mice and in mice with topoisomerase-induced fibrosis. CONCLUSION: PARP-1 negatively regulates canonical TGFß signalling in experimental skin fibrosis. The downregulation of PARP-1 in SSc fibroblasts may thus directly contribute to hyperactive TGFß signalling and to persistent fibroblast activation in SSc.

Molecular recognition patterns of anti-topoisomerase I-antibodies in patients with systemic sclerosis before and after autologous stem cell transplantation.

OBJECTIVES: To evaluate the effect of autologous stem cell transplantation (aSCT) on antibody (ab) reactivity towards linear epitopes of topoisomerase-I (topo-I/Scl70) in patients with systemic sclerosis (SSc) and to correlate antibody reactivities with clinical outcome after aSCT. METHODS: Fourteen anti-topo-I/Scl70-positive SSc-patients were analysed before and after non-myeloablative aSCT. Five patients showed ongoing good response (group 1), 9 had primarily responded but later relapsed or did not respond (group 2). Patients' sera were tested by ELISA against full length (fl) topo-I and 45 overlapping 25-mer peptides. Furthermore, for comparison sera from patients with anti-topo-I-negative SSc (n=12), other collagen disorders (n=6), and from 21 healthy controls (HC) were analysed. RESULTS: Anti-topo-I-positive SSc-sera showed significantly higher IgG-reactivity as compared to HC towards 34 of the 45 peptides. Especially peptide 39 (aa647-671) emerged as a immunodominant epitope being recognised predominantly by anti-topo-I-positive SSc-sera. Reactivity towards 17 of the 45 peptides decreased after aSCT in group 1- and 2-patients. Before aSCT, group 1-patients had lower antibody reactivity towards peptide 39 than group 2-patients. There was no change in peptide-specificity after aSCT. CONCLUSIONS: Reactivity towards topo-I-epitopes is heterogeneous in SSc, but peptide 39 (aa647-671) may be another immunodominant epitope besides the published epitope aa489-573. Antibody reactivity to this peptide 39 was higher in group 2- than in group 1-patients. Peptide recognition pattern did not change after aSCT.

Targeting of NADPH oxidase in vitro and in vivo suppresses fibroblast activation and experimental skin fibrosis.

Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan-Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α-smooth muscle actin (α-SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22phox and Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF-ß1 had a dose- and time-dependent upregulating effect on NADH activity and Nox4 gene expression in HDFs. Genetic silencing of Nox4 as demonstrated by siRNA in HDFs as well as in murine fibroblasts established from Nox4 knockout mice confirmed that TGF-ß1 -mediated collagen type I gene, α-SMA and fibronectin 1 gene expressions were Nox4-dependent. This TGF-ß1 effect was mediated by Smad3 as shown by in silico promoter analysis, pharmacological inhibition and gene silencing of Smad3. The relevance of these findings is highlighted in the bleomycin-induced scleroderma mouse model. DPI treatment attenuated skin fibrosis and myofibroblast activation. Moreover, Nox4 knockdown by siRNA reduced skin collagen synthesis, α-SMA and fibronectin 1 expression in vivo. Finally, analyses of HDFs from patients with systemic sclerosis confirmed the expression of Nox4 and its adaptors, whereas Nox1 and Nox2 were not detectable. Our findings indicate that Nox4 targeting is a promising future treatment for fibrotic skin diseases.

Protein Tyrosine Phosphatases in Systemic Sclerosis: Potential Pathogenic Players and Therapeutic Targets.

PURPOSE OF REVIEW: The pathogenesis of systemic sclerosis depends on a complex interplay between autoimmunity, vasculopathy, and fibrosis. Reversible phosphorylation on tyrosine residues, in response to growth factors and other stimuli, critically regulates each one of these three key pathogenic processes. Protein tyrosine kinases, the enzymes that catalyze addition of phosphate to tyrosine residues, are known players in systemic sclerosis, and tyrosine kinase inhibitors are undergoing clinical trials for treatment of this disease. Until recently, the role of tyrosine phosphatases-the enzymes that counteract the action of tyrosine kinases by removing phosphate from tyrosine residues-in systemic sclerosis has remained largely unknown. Here, we review the function of tyrosine phosphatases in pathways relevant to the pathogenesis of systemic sclerosis and their potential promise as therapeutic targets to halt progression of this debilitating rheumatic disease. RECENT FINDINGS: Protein tyrosine phosphatases are emerging as important regulators of a multitude of signaling pathways and undergoing validation as molecular targets for cancer and other common diseases. Recent advances in drug discovery are paving the ways to develop new classes of tyrosine phosphatase modulators to treat human diseases. Although so far only few reports have focused on tyrosine phosphatases in systemic sclerosis, these enzymes play a role in multiple pathways relevant to disease pathogenesis. Further studies in this field are warranted to explore the potential of tyrosine phosphatases as drug targets for systemic sclerosis.

Low heme oxygenase-1 levels in patients with systemic sclerosis are associated with an altered Toll-like receptor response: another role for CXCL4?

OBJECTIVE: SSc is a disease characterized by inflammation and fibrosis. Heme Oxygenase-1 (HO-1) is a haem-degrading enzyme that mediates resolution of inflammation and is induced upon mediators abundantly present in SSc. We aimed to assess whether HO-1 expression/function is disturbed in SSc patients and could therefore be contributing to the ongoing inflammation. METHODS: In total, 92 SSc patients and 48 healthy controls were included. By measuring total bilirubin in plasma in vivo, HO-activity was assessed. HO-1 expression levels were determined with western blot in monocytes before and after induction of HO-1 with cobalt protoporphyrin (CoPP) with or without CXCL4. Monocyte-derived dendritic cells (DCs) were stimulated with several Toll-like receptor (TLR) ligands with or without pre-stimulation with CoPP for 24 h. Cytokine levels were measured in the supernatants using the Luminex Bead Array. RESULTS: SSc patients have lower plasma levels of bilirubin, suggestive of an aberrant HO-1 function. We demonstrated low HO-1 expression in immune cells from SSc patients, whereas induction with CoPP was able to restore HO-1 levels in DCs from SSc patients, almost normalizing the increased TLR response observed in SSc. Co-exposure to CXCL4 completely abrogated CoPP-induced HO-1 expression, suggesting that the high CXCL4 levels present in SSc patients block the normal induction of HO-1 and its function. CONCLUSION: We demonstrate that HO activity in SSc patients is decreased and show its functional consequences. Since CXCL4 blocks the induction of HO-1 expression, neutralization of CXCL4 in SSc patients could have clinical benefits by diminishing overactivation of immune cells and other anti-inflammatory effects of HO-1.

Epigenetic regulation of cyclooxygenase-2 by methylation of c8orf4 in pulmonary fibrosis.

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.

Global DNA hypomethylation and hypoxia-induced expression of the ten eleven translocation (TET) family, TET1, in scleroderma fibroblasts.

The precise mechanisms of tissue fibrosis have not yet been elucidated in systemic sclerosis (SSc). However, studies of the regulation of DNA methylation, the most widely studied epigenetic mechanism, have confirmed the involvement of the TET family proteins, recently identified DNA demethylases, in the pathogenesis of SSc. The mRNA levels of TET family members were compared in normal and SSc fibroblasts. The effects of hypoxia and siRNA specific to HIF-1α on TET expression were also examined. Global methylation status was analysed by LUMA. The presence of 5-hydroxymethylcytosine (5hmC) in SSc was examined by immunohistochemistry. The level of TET1 mRNA in SSc fibroblasts was elevated by 1.68 fold compared with that of normal fibroblasts, but the expression levels of TET2 and TET3 were comparable between both cell types. The expression levels of DNMT1 and DNMT3B mRNA have a tendency to elevate in SSc fibroblasts. Among TET family members, the expression of TET1 was exclusively induced by hypoxia via HIF-1α-independent pathways in SSc fibroblasts, but not in normal fibroblasts. The methylation level was decreased in SSc fibroblasts relative to normal fibroblasts, and 5hmC was present in dermal fibroblasts of skin sections from patients with SSc. TET1 expression in SSc fibroblasts was abnormally regulated in the hypoxic environment and accompanied by global DNA hypomethylation, suggesting the involvement of aberrant DNA methylation in the pathogenesis of SSc.

Oligosaccharide modification by N-acetylglucosaminyltransferase-V in macrophages are involved in pathogenesis of bleomycin-induced scleroderma.

Oligosaccharide modification by N-acetylglucosaminyltransferase-V (GnT-V), which catalyses the formation of ß1,6 GlcNAc (N-acetylglucosamine) branches on N-glycans, is associated with various pathologies, such as cancer metastasis, multiple sclerosis and liver fibrosis. In this study, we demonstrated the involvement of GnT-V in the pathophysiology of scleroderma. High expression of GnT-V was observed in infiltrating cells in skin section samples from systemic and localized patients with scleroderma. Most of the infiltrating cells were T cells and macrophages, most of which were CD163(+) M2 macrophages. To determine the role of GnT-V in scleroderma, we next investigated skin sclerosis in GnT-V knockout (MGAT5(-/-) ) mice. Expression of GnT-V was also elevated in bleomycin (BLM)-injected sclerotic skin, and MGAT5(-/-) mice were resistant to BLM-induced skin sclerosis with reduced collagen type 1 α1 content, suggesting the biological significance of GnT-V in skin sclerosis. Furthermore, the number of CD163(+) M2 macrophages and CD3-positive T cells in BLM-induced skin sclerosis was significantly fewer in MGAT5(-/-) mice. In bone marrow-derived macrophages (BMDMs), IL-4-induced expressions of Fizz1 and Ym1 were significantly reduced in MGAT5(-/-) mice-derived BMDMs. Taken together, these results suggest the induction of GnT-V in skin sclerosis progression is possibly dependent on increased numbers of M2 macrophages in the skin, which are important for tissue fibrosis and remodelling.

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.

Diagnostic criteria of systemic sclerosis.

Systemic sclerosis (SSc) is a multisystem disease characterized by vascular abnormalities, immune system activation manifested by SSc-specific autoantibodies and disturbances in fibroblast function. The clinical manifestations are highly heterogeneous and commonly include skin thickening, Raynaud's phenomenon, digital ulcers, gastroesophageal reflux disease, interstitial lung disease and cardiac diastolic dysfunction. The diagnosis of SSc in a patient with typical end-organ disease is relatively straight-forward, but is unsatisfactory because it implies that the diagnosis is delayed until irreversible tissue damage is present. Diagnostic criteria are generally designed to facilitate the clinical process and to allow early institution of therapy to relieve symptoms and possibly prevent irreversible damage. Several attempts at defining diagnostic criteria for SSc have been made in the past. Raynaud's phenomenon, SSc-specific autoantibodies and nailfold capillary abnormalities are among the most promising items likely to be retained in a final set of diagnostic criteria. The EULAR Scleroderma Trial and Research group (EUSTAR) is currently in the process of prospectively validating a set of diagnostic criteria for the very early diagnosis of SSc and results are expected in 2015.

Chitinase 1 is a biomarker for and therapeutic target in scleroderma-associated interstitial lung disease that augments TGF-ß1 signaling.

Interstitial lung disease (ILD) with pulmonary fibrosis is an important manifestation in systemic sclerosis (SSc, scleroderma) where it portends a poor prognosis. However, biomarkers that predict the development and or severity of SSc-ILD have not been validated, and the pathogenetic mechanisms that engender this pulmonary response are poorly understood. In this study, we demonstrate in two different patient cohorts that the levels of chitotriosidase (Chit1) bioactivity and protein are significantly increased in the circulation and lungs of SSc patients compared with demographically matched controls. We also demonstrate that, compared with patients without lung involvement, patients with ILD show high levels of circulating Chit1 activity that correlate with disease severity. Murine modeling shows that in comparison with wild-type mice, bleomycin-induced pulmonary fibrosis was significantly reduced in Chit1⁻/⁻ mice and significantly enhanced in lungs from Chit1 overexpressing transgenic animals. In vitro studies also demonstrated that Chit1 interacts with TGF-ß1 to augment fibroblast TGF-ß receptors 1 and 2 expression and TGF-ß-induced Smad and MAPK/ERK activation. These studies indicate that Chit1 is potential biomarker for ILD in SSc and a therapeutic target in SSc-associated lung fibrosis and demonstrate that Chit1 augments TGF-ß1 effects by increasing receptor expression and canonical and noncanonical TGF-ß1 signaling.

Targeting the TGF-ß receptor with kinase inhibitors for scleroderma therapy.

Scleroderma (systemic sclerosis) is a connective tissue disease that affects various organ systems; the treatment of scleroderma is still difficult and remains a challenge to the clinician. Recently, kinase inhibitors have shown great potential against fibrotic diseases and, specifically, the transforming growth factor-ß receptor (TGF-ßR) was found as a new and promising target for scleroderma therapy. In the current study, we propose that the large pool of existing kinase inhibitors could be exploited for inhibiting the TGF-ßR to suppress scleroderma. In this respect, we developed a modeling protocol to systematically profile the inhibitory activities of 169 commercially available kinase inhibitors against the TGF-ßR, from which five promising candidates were selected and tested using a standard kinase assay protocol. Consequently, two molecular entities, namely the PKB inhibitor MK-2206 and the mTOR C1/C2 inhibitor AZD8055, showed high potency when bound to the TGF-ßR, with IC50 values of 97 and 86 nM, respectively, which are close to those of the recently developed TGF-ßR selective inhibitors SB525334 and LY2157299 (IC50 = 14.3 and 56 nM, respectively). We also performed atomistic molecular dynamics simulations and post-molecular mechanics/Poisson-Boltzmann surface area analyses to dissect the structural basis and energetic properties of intermolecular interactions between the TGF-ßR kinase domain and these potent compounds, highlighting intensive nonbonded networks across the tightly packed interface of non-cognate TGF-ßR-inhibitor complexes.

Inactivation of tankyrases reduces experimental fibrosis by inhibiting canonical Wnt signalling.

OBJECTIVES: Canonical Wnt signalling has recently emerged as a key mediator of fibroblast activation and tissue fibrosis in systemic sclerosis. Here, we investigated tankyrases as novel molecular targets for inhibition of canonical Wnt signalling in fibrotic diseases. METHODS: The antifibrotic effects of the tankyrase inhibitor XAV-939 or of siRNA-mediated knockdown of tankyrases were evaluated in the mouse models of bleomycin-induced dermal fibrosis and in experimental fibrosis induced by adenoviral overexpression of a constitutively active TGF-ß receptor I (Ad-TBRI). RESULTS: Inactivation of tankyrases prevented the activation of canonical Wnt signalling in experimental fibrosis and reduced the nuclear accumulation of ß-catenin and the mRNA levels of the target gene c-myc. Treatment with XAV-939 or siRNA-mediated knockdown of tankyrases in the skin effectively reduced bleomycin-induced dermal thickening, differentiation of resting fibroblasts into myofibroblasts and accumulation of collagen. Potent antifibrotic effects were also observed in Ad-TBRI driven skin fibrosis. Inhibition of tankyrases was not limited by local or systemic toxicity. CONCLUSIONS: Inactivation of tankyrases effectively abrogated the activation of canonical Wnt signalling and demonstrated potent antifibrotic effects in well-tolerated doses. Thus, tankyrases might be candidates for targeted therapies in fibrotic diseases.