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Objectives. Cathepsin V (CTSV) is a proteolytic enzyme potentially modulating angiogenic processes, collagen degradation and keratinocyte differentiation. We aimed to investigate the clinical association of serum CTSV levels and the mechanism by which CTSV expression is altered in SSc. Methods. Serum CTSV levels were determined by ELISA in 51 SSc and 18 healthy subjects. CTSV expression was evaluated by immunostaining in SSc and normal skin and by RT-real-time PCR in normal and SSc dermal fibroblasts, normal dermal fibroblasts treated with TGF-β1 or Fli1 siRNA and human dermal microvascular endothelial cells (ECs) treated with Fli1 siRNA. Results. Serum CTSV levels were significantly lower in dcSSc and lcSSc patients than in healthy controls. In early-stage dcSSc, serum CTSV levels were remarkably and uniformly decreased compared with healthy controls. The decrease in serum CTSV levels in mid- and late-stage dcSSc and in lcSSc was linked to the development of proliferative vasculopathy. CTSV expression was decreased in microvascular ECs, pericytes/vascular smooth muscle cells and keratinocytes of dcSSc and lcSSc skin and in dermal fibroblasts of dcSSc skin compared with control skin. Consistently, CTSV expression was decreased in cultured dermal fibroblasts from early-stage dcSSc. Furthermore, mRNA levels of the CTSV gene were significantly decreased in normal fibroblasts treated with TGF-β1 or Fli1 siRNA and in human dermal microvascular ECs treated with Fli1 siRNA. Conclusion. Loss of CTSV expression may contribute to the development of fibrosis, vasculopathy and the altered phenotype of keratinocytes in SSc. [ABSTRACT FROM AUTHOR]

Objective Fli-1, a potential predisposing factor for systemic sclerosis (SSc), is constitutively down-regulated in the lesional skin of patients with SSc by an epigenetic mechanism. To investigate the impact of Fli-1 deficiency on the induction of an SSc phenotype in various cell types, we generated bleomycin-induced skin fibrosis in Fli-1+ /− mice and investigated the molecular mechanisms underlying its phenotypic alterations. Methods Messenger RNA (mRNA) levels and protein expression of target molecules were examined by quantitative reverse transcription-polymerase chain reaction and immunostaining. Transforming growth factor β (TGFβ) bioassay was used to evaluate the activation of latent TGFβ. The binding of Fli-1 to the target gene promoters was assessed with chromatin immunoprecipitation. Results Bleomycin induced more severe dermal fibrosis in Fli-1+/− mice than in wild-type mice. Fli-1 haploinsufficiency activated dermal fibroblasts via the up-regulation of αvβ3 and αvβ5 integrins and activation of latent TGFβ. Dermal fibrosis in Fli-1+/− mice was also attributable to endothelial-to-mesenchymal transition, which is directly induced by Fli-1 deficiency and amplified by bleomycin. Th2/Th17-skewed inflammation and increased infiltration of mast cells and macrophages were seen, partly due to the altered expression of cell adhesion molecules in endothelial cells as well as the induction of the skin chemokines. Fli-1+/− mouse macrophages preferentially differentiated into an M2 phenotype upon stimulation with interleukin-4 (IL-4) or IL-13. Conclusion Our findings provide strong evidence for the fundamental role of Fli-1 deficiency in inducing SSc-like phenotypic alterations in dermal fibroblasts, endothelial cells, and macrophages in a manner consistent with human disease. [ABSTRACT FROM AUTHOR]

Objective. Although several miRNAs have been shown to regulate autoimmune pathogenesis by affecting lymphocyte function, the roles of miRNAs in the pathogenesis of SSc remain unclear. Therefore the purpose of this study was to identify miRNAs that play a role in the pathogenesis of SSc by quantitative PCR screening of serum miRNAs.Methods. Ninety-five miRNAs that were predicted to target SSc-related genes [IL-4, TGF-β, CTGF, PDGF-B, PDGF receptor (PDGFR) α/β and COL1A2) by in silico analyses were selected. The expression of these miRNAs in sera of SSc patients and healthy controls was measured by quantitative PCR. Involvement of miR-30b, which was most strongly down-regulated in SSc patients, in the regulation of PDGFR-β expression was examined by transfection experiments and 3′-untranslated region (3′-UTR) target luciferase assays. The expression of miR-30b in skin was evaluated in a bleomycin-induced dermal fibrosis model in mice and in SSc patients.Results. Nineteen of 95 miRNAs were significantly decreased in the sera of SSc patients. Among them, miR-30b was most strongly down-regulated in SSc patients (P = 0.00006) and the levels of miR-30b were inversely correlated with modified Rodnan skin scores. Transfection of a miR-30b mimic repressed PDGFR-β expression in dermal fibroblasts and the activity of a luciferase reporter containing 3′-UTR of PDGFR-β. Moreover, the expression of miR-30b was down-regulated in bleomycin-treated sclerotic skin and in affected skin in SSc patients.Conclusion. Down-regulation of miR-30b might be involved in the pathogenesis of SSc. [ABSTRACT FROM PUBLISHER]

Interstitial lung disease (ILD) is a leading cause of death in patients with systemic sclerosis (SSc). Nonspecific immunosuppressants have been the first-line treatment for SSc-associated ILD (SSc-ILD). Nintedanib, an oral triple kinase inhibitor targeting profibrotic pathways, has been employed for the treatment of idiopathic pulmonary fibrosis and has recently received marketing approval in the United States and Japan, based on the results of a placebo-controlled randomized controlled trial. In this clinical trial, nintedanib delayed the progression of SSc-ILD compared with placebo. This review covers current pharmacotherapies for SSc-ILD, drug profiles of nintedanib, and efficacy and safety profiles of nintedanib in patients with idiopathic pulmonary fibrosis and SSc-ILD observed in randomized controlled trails. Currently, we have two treatment options for SSc-ILD, i.e., immunosuppressants and antifibrotic agents. However, appropriate utilization of antifibrotic agents in clinical practice remains challenging, i.e., in which cases they are to be used, timing of use, how to use them properly, and whether in combination with immunosuppressants. [ABSTRACT FROM AUTHOR]

Transforming growth factor β (TGFβ) plays an important role in regulating aberrant extracellular matrix (ECM) production from alveolar/epithelial cells (AECs) and fibroblasts in pulmonary fibrosis. Although the tumor suppressor gene phosphatase and tensin homologue deleted from chromosome 10 (PTEN) can negatively control many TGFβ-activated signaling pathways via the phosphatase activity, hyperactivation of the TGFβ-related signaling pathways is often observed in fibrosis. Loss of PTEN expression might cause TGFβ-induced ECM production. In addition, TGFβ was recently shown to induce loss of PTEN enzymatic activity by phosphorylating the PTEN C-terminus. Therefore, we hypothesized that exogenous transfer of unphosphorylated PTEN (PTEN4A) might lead to reduce TGFβ-induced ECM expression in not only epithelial cells but also fibroblasts. Adenovirus-based exogenous PTEN4A induction successfully reduced TGFβ-induced fibronectin expression and retained β-catenin at the cell membrane in human epithelial cells. Exogenous unphosphorylated PTEN also attenuated TGFβ-induced ECM production and inhibited TGFβ-induced β-catenin translocation in a human fibroblast cell line and in mouse primary isolated lung fibroblasts. Conversely, TGFβ-induced α-smooth muscle actin expression did not seem to be inhibited in these fibroblasts. Our data suggest that exogenous administration of unphosphorylated PTEN might be a promising strategy to restore TGFβ-induced loss of PTEN activity and reduce aberrant TGFβ-induced ECM production from epithelial cells and fibroblasts in lung fibrosis as compared with wild-type PTEN induction. [ABSTRACT FROM AUTHOR]

Objective Progranulin is a growth factor that is active in wound repair and is an antagonist of tumor necrosis factor (TNF) receptors, regulating fibroblast activation, angiogenesis, and inflammation. Because long-standing activation of gene programs related to wound healing is a hallmark of systemic sclerosis (SSc), we sought to investigate the role of progranulin in SSc. Methods Progranulin expression levels in human and murine skin samples were determined by immunohistochemical analysis and quantitative reverse transcription-polymerase chain reaction. The role of progranulin in fibroblast activation was examined using a gene-silencing technique. Progranulin levels in serum obtained from 60 patients with SSc and 16 healthy control subjects were determined by enzyme-linked immunosorbent assay. Results Progranulin expression was increased in SSc dermal fibroblasts compared with normal dermal fibroblasts, both in vivo and in vitro. Transcription factor Fli-1, a deficiency of which is involved in the activation of SSc dermal fibroblasts, served as a potent repressor of the progranulin gene, and Fli-1+/− mice and bleomycin-treated wild-type mice exhibited up-regulated expression of progranulin in dermal fibroblasts. SSc dermal fibroblasts were resistant to the antifibrotic effect of TNF, but this resistance was reversed by gene silencing of progranulin. Serum progranulin levels were elevated in patients with early diffuse cutaneous SSc (dcSSc), especially in those with inflammatory skin symptoms, and were positively correlated with the C-reactive protein level. Conclusion Progranulin overproduction due to Fli-1 deficiency may contribute to the constitutive activation of SSc dermal fibroblasts by antagonizing the antifibrotic effect of TNF. Progranulin may also be involved in the inflammatory process associated with progressive skin sclerosis in early dcSSc. [ABSTRACT FROM AUTHOR]

Presents a case report on observation of asymptomatic nodule in the center of the coccygeal area in a Japanese boy. Mild parakeratosis and marked fibrosis in the dermis revealed by skin biopsy; Anterior subluxation of the coccyx; Surgical excision of the nodule together with shaving of the underlying angular bony structure; Relation of the condition of acquired nodule on sacrococcygeal area to bicycle riding.

Objectives. Chemerin is a member of adipocytokines with a chemoattractant effect on plasmacytoid dendritic cells and macrophages and pro-angiogenic properties. We investigated the potential role of chemerin in the development of SSc. Methods. Chemerin expression was evaluated by immunostaining and/or real-time quantitative RT-PCR in human and murine skin. The mechanisms regulating chemerin expression in dermal fibroblasts and endothelial cells were examined using the gene silencing technique and chromatin immunoprecipitation. Serum chemerin levels were determined by ELISA in 64 SSc patients and 19 healthy subjects. Results. In SSc lesional skin, chemerin was up-regulated in small blood vessels, while it was downregulated in fibroblasts surrounded with thickened collagen bundles. The decreased expression of chemerin was significantly reversed by TGF-b1 antisense oligonucleotide in cultured SSc dermal fibroblasts and chemerin expression was markedly decreased in dermal fibroblasts of bleomycin-treated mice. Gene silencing of transcription factor Fli1, which binds to the chemerin promoter, induced chemerin expression in human dermal microvascular endothelial cells and Fli1+/- mice exhibited elevated chemerin expression in dermal blood vessels. Serum chemerin levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction. In SSc patients with normal renal function, patients with digital ulcers had higher serum chemerin levels than those without. Conclusion. Chemerin is down-regulated in SSc dermal fibroblasts by autocrine TGF-b, while it is upregulated in SSc dermal blood vessels through endothelial Fli1 deficiency. Increased chemerin expression in dermal blood vessels may be associated with the development of digital ulcers in SSc. [ABSTRACT FROM AUTHOR]

Objective It is generally accepted that blockade of endothelin receptors has potentially beneficial effects on vasculopathy associated with systemic sclerosis (SSc). The aim of this study was to clarify the molecular mechanism underlying these effects using endothelial cell-specific Fli-1-knockout (Fli-1 ECKO) mice, an animal model of SSc vasculopathy. Methods Levels of messenger RNA for target genes and the expression and phosphorylation levels of target proteins were determined in human and murine dermal microvascular endothelial cells by real-time quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively. The binding of Fli-1 to the target gene promoters was evaluated using chromatin immunoprecipitation. Expression levels of Fli-1 and α-smooth muscle actin in murine skin were evaluated using immunohistochemistry. Vascular structure and permeability were evaluated in mice injected with fluorescein isothiocyanate-dextran and Evans blue dye, respectively. Results In human dermal microvascular endothelial cells, endothelin 1 induced phosphorylation of Fli-1 at Thr312 through the sequential activation of c-Abl and protein kinase Cδ, leading to a decrease in Fli-1 protein levels as well as a decrease in binding of Fli-1 to the target gene promoters, whereas bosentan treatment reversed those effects. In Fli-1 ECKO mice, 4 weeks of treatment with bosentan increased endothelial Fli-1 expression, resulting in vascular stabilization and the restoration of impaired leaky vessels. Conclusion The vascular fragility of Fli-1 ECKO mice was improved by bosentan through the normalization of Fli-1 protein levels and activity in endothelial cells, which may explain, in part, the mechanism underlying the beneficial effects of endothelin receptor blockade on SSc vasculopathy. [ABSTRACT FROM AUTHOR]

ABSTRACT This study assessed whether hypertension, a circulating factor, influences local keloid severity. This retrospective cross-sectional study involved 304 consecutive patients (13-78 years old) with keloids who were surgically treated in our hospital between January 2011 and August 2013. Their blood pressure (BP), age and gender, and the size and number of their keloids before surgery were recorded. Ordinal logistic regression analyses showed that BP associated significantly with both keloid size and number (all p < 0.0001). Age also associated with keloid size ( p < 0.0001). However, a Goodness-of-fit chi-square test showed that the prevalence of hypertension was not higher among keloid patients than in the general Japanese population. This study provides epidemiological evidence for the possibility that primary hypertension may aggravate keloids. We propose that the skin, along with the heart and liver, is a target organ of hypertension. The observations of this study, which require validation with large-scale prospective interventional trials, suggest that keloid patients should be screened for hypertension and that antihypertensive treatments may be of prophylactic and therapeutic value for skin fibrosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective To clarify the role of interleukin-20 (IL-20) in the regulatory mechanism of extracellular matrix expression and to determine the contribution of IL-20 to the phenotype of systemic sclerosis (SSc). Methods Protein and messenger RNA (mRNA) levels of collagen, Fli-1, IL-20, and IL-20 receptor (IL-20R) were analyzed using polymerase chain reaction (PCR) array, immunoblotting, immunohistochemical staining, enzyme-linked immunosorbent assay, and real-time PCR. Results PCR array revealed that IL-20 decreased gene expression of α2(I) collagen (0.03-fold), Smad3 (0.02-fold), and endoglin (0.05-fold) in cultured normal dermal fibroblasts. Fli-1 protein expression was induced by IL-20 (∼2-fold). The inhibition of collagen by IL-20, the induction of Fli-1 by IL-20, and the reduction of Smad3 and endoglin by IL-20 were also observed in SSc fibroblasts. Serum IL-20 levels were reduced only slightly in SSc patients but were significantly decreased in patients with scleroderma spectrum disorders (the prodromal stage of SSc) compared with those in normal subjects (111.3 pg/ml versus 180.4 pg/ml; P < 0.05). On the other hand, IL-20 mRNA expression in SSc skin was decreased compared with that in normal skin ( P < 0.05), which may result in the induction of collagen synthesis in SSc dermal fibroblasts. IL-20R was expressed in normal and SSc fibroblasts. Moreover, IL-20 supplementation by injection into the skin reversed skin fibrosis induced by bleomycin in mice (∼0.5-fold). Conclusion IL-20 reduces basal collagen transcription via Fli-1 induction, while down-regulation of Smad3 and endoglin may cancel the effect of transforming growth factor β in SSc fibroblasts. To confirm the therapeutic value of IL-20 and IL-20R, their function and expression in vivo should be further studied. [ABSTRACT FROM AUTHOR]

Objective Patients with systemic sclerosis (SSc) exhibit enhanced production of free radicals due to ischemia and reperfusion injury following Raynaud's phenomenon, an initial clinical manifestation. Oxidative stress induces cytokine production, inflammatory cell recruitment, and tissue injury in several inflammatory diseases. The aim of this study was to examine the effect of edaravone, a free radical scavenger, on the development of fibrosis and autoimmunity in two different mouse models of SSc. Methods The bleomycin-induced SSc model in mice and the tight skin mouse model were used to evaluate the effect of edaravone on fibrosis and immunologic abnormalities. To assess the reaction of fibroblasts to stimulation with free radicals, fibroblasts from these mice were cultured with NONOate, a nitric oxide-releasing agent, and hydrogen peroxide. Results Treatment with edaravone reduced fibrosis in mice with bleomycin-induced SSc and in TSK/+ mice. The production of free radicals was also attenuated by edaravone in both models. In addition, production of fibrogenic cytokines such as interleukin-6 and transforming growth factor β1, production of anti-topoisomerase I antibody, and the degree of hypergammaglobulinemia were reduced by edaravone. Furthermore, bleomycin induced the production of H2O2 and nitric oxide from inflammatory cells, and collagen production was increased in fibroblasts cultured with H2O2 and NONOate. Conclusion This study is the first to show that edaravone has a significant inhibitory effect on fibrosis both in the bleomycin-induced SSc model and in TSK/+ mice. These results indicate that edaravone should be further evaluated for potential use as an antifibrotic agent in SSc. [ABSTRACT FROM AUTHOR]

Lesions of naevus of Ota range in colour from light brown to blue, and even greenish-black. To develop guidelines for optimal treatment, we evaluated the number of Q-switched ruby laser treatments required to eliminate the pigmentation of such lesions classified by colour. Over a period of 6 years, we evaluated 151 Japanese patients with naevus of Ota who had been treated with the Q-switched ruby laser at a low energy level (wavelength 694·3 nm; pulse duration 28 × 10-9 s; energy fluence 5 J/cm2; spot size 6·5 mm) every 2 months. Each lesion was classified by colour as brown (n = 22), brown–violet (n = 42), violet–blue (n = 81) and blue–green (n = 6). The 22 predominantly brown lesions attained an excellent (100–95%) or good (95–75%) cosmetic result following three laser treatments in all patients who received this number of treatments. In the 42 brown–violet lesions, 25 of the 29 good or excellent results were achieved after four treatments; the 13 less successful results were in patients who had one to three treatments. In the 81 violet–blue lesions, 54 of the 65 good or excellent results were achieved after four treatments and 64 of 65 after five treatments, whereas all 16 less good results were in patients who had only one to three treatments. However, in the six blue–green lesions, six or more treatments were required to achieve a similarly favourable result. At the end of treatment, the area was virtually free of scarring, and its texture resembled that of the surrounding normal skin. We have confirmed that the use of the Q-switched ruby laser at a low energy level can eliminate the pigmentation of naevus of Ota. While the desired improvement can be obtained within 1 year, the number of treatments appears to depend on the predominant colour of the lesion. [ABSTRACT FROM AUTHOR]

A 5-year-old male Siberian husky bred outdoor in Tokyo had a swollen paw with interdigital granulomatous lesions in the left hindlimb. The dog had no apparent pulmonary or gastrointestinal involvement. Histopathological analysis of the skin lesions demonstrated yeast-like organisms predominantly within macrophages. Sequence analysis of fungal ribosome RNA gene isolated from a paraffin sample revealed a 100% homology with the teleomorph of Histoplasma capsulatum. The present case may support the concept of primary cutaneous canine histoplasmosis as an endemic phenotype recognized in Japan. [ABSTRACT FROM AUTHOR]