Rapid detection of biofilm by wound blotting following sharp debridement of chronic pressure ulcers predicts wound healing: A preliminary study.

For optimal wound bed preparation, wound debridement is essential to eliminate bacterial biofilms. However, it is challenging for clinicians to determine whether the biofilm is completely removed. A newly developed biofilm detection method based on wound blotting technology may be useful. Thus, we aimed to investigate the effect of biofilm elimination on wound area decrease in pressure ulcers, as confirmed using the wound blotting method. In this retrospective observational study, we enrolled patients with pressure ulcers who underwent sharp debridement with pre- and post-debridement wound blotting. Biofilm was detected on the nitrocellulose membrane using ruthenium red or alcian blue staining. Patients were included if the test was positive for biofilm before wound debridement. Percent decrease in wound area after 1 week was calculated as an outcome measure. We classified the wounds into a biofilm-eliminated group and a biofilm-remaining group based on the post-debridement wound blotting result. Sixteen wound blotting samples from nine pressure ulcers were collected. The percent decrease in wound area was significantly higher in the biofilm-eliminated group (median: 14.4%, interquartile range: 4.6%-20.1%) than in the biofilm-remaining group (median: -14.5%, interquartile range: -25.3%-9.6%; P = .040). The presence of remaining biofilms was an independent predictor for reduced percent decrease in wound area (coefficient = -22.84, P = .040). Biofilm-based wound care guided by wound blotting is a promising measure to help clinicians eliminate bacterial bioburden more effectively for wound area reduction.

Prediction of therapeutic response before and during i.v. cyclophosphamide pulse therapy for interstitial lung disease in systemic sclerosis: A longitudinal observational study.

There have been no established parameters to predict responsiveness to i.v. cyclophosphamide (IVCY) pulse therapy in combination with corticosteroids in patients with interstitial lung disease (ILD) related to systemic sclerosis (SSc). This retrospective study was conducted to determine predictive factors for efficacy of IVCY at the time of before and during the treatment. Thirty-two Japanese SSc patients, ever treated for ILD with IVCY in combination with prednisolone, were analyzed retrospectively. We performed detailed time-course analyses of parameters derived from blood samples and pulmonary function tests. With the exclusion of eight unclassified patients, 24 patients were classified into 14 good responders (GR) or 10 poor responders (PR) on the basis of changes in percent predicted diffusing capacity for carbon monoxide (DLco). Pretreatment percent predicted DLco was significantly reduced in PR compared with GR. In addition, serum parameters such as Krebs von den Lungen-6 (KL-6), surfactant protein D (SP-D) and C-reactive protein were significantly higher in PR than in GR. Furthermore, our time-course analyses revealed a transient increase in serum KL-6 levels with a peak at 3 months after the first infusion of cyclophosphamide, which showed no relation to therapeutic efficacy. Moreover, continuously high serum KL-6 levels (>2000 U/mL) and rapid decrease in SP-D levels (<200 ng/mL) during IVCY were remarkably characteristic of PR and GR, respectively. ILD severity/activity before treatment and variability of serum KL-6 and SP-D levels during treatment may be useful to predict therapeutic effects of IVCY on SSc-ILD.

Japanese familial anetoderma: A report of two cases and review of the published work.

Anetoderma is a rare cutaneous disorder characterized by focal loss of dermal elastic tissue, resulting in macular atrophy or herniated saclike skin. Some families with hereditary anetoderma have been described, but there have been no reports on Japanese familial anetoderma so far. We herein report two Japanese sibling cases of primary anetoderma. A healthy 13-year-old Japanese girl and a healthy 15-year-old Japanese girl presented to our hospital with a 6-month history of small atrophic pittings on their arms and trunks. All lesions were less than 0.5 cm in diameter, which are relatively small for non-familial anetoderma. Preceding infections or skin lesions were not observed. A skin biopsy revealed a focal, complete loss of elastic tissue in the superficial to mid-dermis which was surrounded by fine, irregular or twisted elastic fibers. Based on these findings, the diagnosis of anetoderma was made. Review of published works demonstrated that the mode of inheritance of familial anetoderma is not simple, suggesting that it is important to survey any family member of the patients with anetoderma.

Progranulin overproduction due to constitutively activated c-Abl/PKC-δ/Fli1 pathway contributes to the resistance of dermal fibroblasts to the anti-fibrotic effect of tumor necrosis factor-α in localized scleroderma.

BACKGROUND: Dermal fibroblasts derived from patients with systemic sclerosis (SSc) overproduce progranulin (PGRN), an endogenous antagonist of tumor necrosis factor (TNF) receptors, due to the deficiency of transcription factor Fli1. Fli1 expression is also decreased in dermal fibroblasts derived from patients with localized scleroderma (LSc). OBJECTIVE: To investigate the expression levels of PGRN and its contribution to the induction of pro-fibrotic phenotype in LSc dermal fibroblasts. METHODS: PGRN expression levels were determined by immunohistochemistry and quantitative reverse transcription PCR in the skin of human subjects. The role of PGRN in fibroblast activation was examined with gene silencing technique. The involvement of c-Abl/protein kinase C (PKC)-δ/Fli1 pathway in the regulation of PGRN expression was investigated by immunoblotting. RESULTS: The expression levels of PGRN and TNF-α were elevated in LSc skin lesions compared with healthy control skin. LSc dermal fibroblasts were less responsive to the anti-fibrotic effect of TNF-α than normal dermal fibroblasts. Importantly, gene silencing of PGRN reversed the response to TNF-α in LSc dermal fibroblasts. Similar to SSc dermal fibroblasts, the inhibition of c-Abl/PKC-δ/Fli1 pathway by gene silencing of ABL1 or PRKCD significantly suppressed PGRN expression in LSc dermal fibroblasts. CONCLUSION: PGRN overproduction due to constitutively activated c-Abl/PKC-δ/Fli1 pathway may contribute to the resistance of LSc dermal fibroblasts to the anti-fibrotic effect of TNF-α, which may be involved in maintaining their pro-fibrotic phenotype under the pro-inflammatory condition, as is the case with SSc.

Epithelial Fli1 deficiency drives systemic autoimmunity and fibrosis: Possible roles in scleroderma.

Systemic sclerosis (SSc), or scleroderma, is a multisystem autoimmune disorder characterized by vasculopathy and fibrosis in the skin and internal organs, most frequently in the esophagus and lungs. Hitherto, studies on SSc pathogenesis centered on immune cells, vascular cells, and fibroblasts. Although dysregulated keratinocytes in SSc have been recently reported, the contribution of epithelial cells to pathogenesis remains unexplored. In this study, we demonstrated the induction of SSc-like molecular phenotype in keratinocytes by gene silencing of transcription factor Friend leukemia virus integration 1 (Fli1), the deficiency of which is implicated in SSc pathogenesis. Keratin 14-expressing epithelial cell-specific Fli1 knockout mice spontaneously developed dermal and esophageal fibrosis with epithelial activation. Furthermore, they developed remarkable autoimmunity with interstitial lung disease derived from thymic defects with down-regulation of autoimmune regulator (Aire). Importantly, Fli1 directly regulated Aire expression in epithelial cells. Collectively, epithelial Fli1 deficiency might be involved in the systemic autoimmunity and selective organ fibrosis in SSc. This study uncovers unidentified roles of dysregulated epithelial cells in SSc pathogenesis.

SIRT3 is attenuated in systemic sclerosis skin and lungs, and its pharmacologic activation mitigates organ fibrosis.

Constitutive fibroblast activation is responsible for organ fibrosis in fibrotic disorders including systemic sclerosis (SSc), but the underlying mechanisms are not fully understood, and effective therapies are lacking. We investigated the expression of the mitochondrial deacetylase sirtuin 3 (SIRT3) and its modulation by hexafluoro, a novel fluorinated synthetic honokiol analogue, in the context of fibrosis. We find that augmenting cellular SIRT3 by forced expression in normal lung and skin fibroblasts, or by hexafluoro treatment, blocked intracellular TGF-ß signaling and fibrotic responses, and mitigated the activated phenotype of SSc fibroblasts. Moreover, hexafluoro attenuated mitochondrial and cytosolic reactive oxygen species (ROS) accumulation in TGF-ß-treated fibroblasts. Remarkably, we found that the expression of SIRT3 was significantly reduced in SSc skin biopsies and explanted fibroblasts, and was suppressed by TGF-ß treatment in normal fibroblasts. Moreover, tissue levels of acetylated MnSOD, a sensitive marker of reduced SIRT3 activity, were dramatically enhanced in lesional skin and lung biopsies from SSc patients. Mice treated with hexafluoro showed substantial attenuation of bleomycin-induced fibrosis in the lung and skin. Our findings reveal a cell-autonomous function for SIRT3 in modulating fibrotic responses, and demonstrate the ability of a novel pharmacological SIRT3 agonist to attenuate fibrosis in vitro and in vivo. In light of the impaired expression and activity of SIRT3 associated with organ fibrosis in SSc, pharmacological approaches for augmenting SIRT3 might have therapeutic potential.

Tamibarotene Ameliorates Bleomycin-Induced Dermal Fibrosis by Modulating Phenotypes of Fibroblasts, Endothelial Cells, and Immune Cells.

Tamibarotene (Am80) is a synthetic retinoid that modulates the pathologic processes of various autoimmune and inflammatory diseases and their animal models. We here investigated the therapeutic potential of Am80 against systemic sclerosis using its animal models. Am80 significantly attenuated dermal and hypodermal fibrosis in bleomycin (BLM)-treated mice and tight skin 1 mice, respectively. Consistently, Am80 significantly suppressed the expression of various molecules related to tissue fibrosis, including transforming growth factor-ß1, connective tissue growth factor, IL-4, IL-10, IL-13, IL-17A, tumor necrosis factor-α, IFN-γ, and monocyte chemotactic protein 1 in the lesional skin of BLM-treated mice. Furthermore, Am80 decreased the proportion of effector T cells, while increasing that of naïve T cells among CD4+ T cells in the draining lymph nodes of BLM-treated mice. Moreover, a series of BLM-induced pathologic events, including endothelial-to-mesenchymal transition; ICAM-1 expression in endothelial cells; the infiltration of macrophages, mast cells, and lymphocytes; and M2 macrophage differentiation, were attenuated by Am80. Importantly, Am80 directly reversed the profibrotic phenotype of transforming growth factor-ß1-treated dermal fibroblasts, suppressed ICAM-1 expression in endothelial cells, and promoted M1 macrophage differentiation in vitro. Collectively, Am80 inhibits the development of experimental dermal fibrosis by reversing the profibrotic phenotype of various cell types and would be a candidate for therapeutic drugs against dermal fibrosis of systemic sclerosis.

Circulating galectin-1 concentrations in systemic sclerosis: potential contribution to digital vasculopathy.

AIM: To determine serum galectin-1 levels and their clinical associations in patients with systemic sclerosis (SSc). METHOD: Serum galectin-1 levels were examined by enzyme-linked immunosorbent assay in 66 patients with SSc and 24 healthy individuals. RESULTS: No significant differences were observed in serum galectin-1 levels between patients with SSc (9.4 ± 5.6 ng/mL), and healthy individuals (8.9 ± 1.3 ng/mL). Among patients with SSc, no significant differences were seen in serum galectin-1 levels between those with diffuse cutaneous SSc (8.8 ± 5.7 ng/mL; n = 31) and those with limited cutaneous SSc (10.0 ± 5.4 ng/mL; n = 35). Patients with SSc who had increased galectin-1 levels less often had pitting scars/digital ulcers than those with normal galectin-1 levels (17% vs. 49%; P < 0.01). Consistently, galectin-1 levels were significantly lower in SSc patients with pitting scars/digital ulcers than in those without pitting scars/digital ulcers (6.9 ± 4.8 vs. 10.9 ± 5.5 ng/mL; P < 0.01). CONCLUSION: These results suggest that galectin-1 is a protective factor against the development of digital vasculopathy in SSc. In addition, measurement of serum galectin-1 levels may be useful for risk stratification for the development of digital vasculopathy in the early phase of SSc.

Effect of ambrisentan on peripheral circulation in patients with systemic sclerosis.

Systemic sclerosis (SSc) is characterized by disturbed blood circulation. The effect of ambrisentan, an endothelin-A receptor-selective antagonist, on impaired peripheral circulation in SSc remains largely elusive. Here we show SSc patients, whose clinical symptoms such as cyanosis and Raynaud's phenomenon, were ameliorated by the treatment with ambrisentan. Additionally, objective evaluations with thermography showed improvement of hand coldness in steady-state and cold challenge tests. Ambrisentan might have a potential to improve peripheral circulation in SSc.

Progranulin Overproduction Due to Fli-1 Deficiency Contributes to the Resistance of Dermal Fibroblasts to Tumor Necrosis Factor in Systemic Sclerosis.

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.

Association of anti-RNA polymerase III antibody and malignancy in Japanese patients with systemic sclerosis.

Patients with systemic sclerosis (SSc) have an increased risk of malignancy compared with the general population. Recently, SSc patients with anti-RNA polymerase III antibody have been reported to have an increased risk of malignancy as compared with those with other disease-specific autoantibodies in US, European and Australian populations. Therefore, we studied the relationship between disease-specific autoantibodies and malignancy in 261 Japanese SSc patients. The prevalence of malignancy was significantly higher in patients with anti-RNA polymerase III antibody (7/22, 31.8%) than in those with anti-topoisomerase I antibody (2/82, 2.4%) and in those with anticentromere antibody (8/137, 5.8%). Importantly, among seven patients with anti-RNA polymerase III antibody and malignancy, three patients (42.9%) developed malignancy from 6 months before to 12 months after SSc onset. Thus, malignancy complication in Japanese SSc patients with anti-RNA polymerase III antibody is as high as that in other races, suggesting that SSc patients with anti-RNA polymerase III antibody share the same pathological process among different ethnic groups.

Endothelin receptor blockade ameliorates vascular fragility in endothelial cell-specific Fli-1-knockout mice by increasing Fli-1 DNA binding ability.

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 Thr(312) 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.

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.

Fibrosis, vascular activation, and immune abnormalities resembling systemic sclerosis in bleomycin-treated Fli-1-haploinsufficient mice.

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 ß (TGFß) bioassay was used to evaluate the activation of latent TGFß. 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 αvß3 and αvß5 integrins and activation of latent TGFß. 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.

Increased expression of chemerin in endothelial cells due to Fli1 deficiency may contribute to the development of digital ulcers in systemic sclerosis.

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 down-regulated in fibroblasts surrounded with thickened collagen bundles. The decreased expression of chemerin was significantly reversed by TGF-ß1 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-ß, while it is up-regulated 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.

Simultaneous downregulation of KLF5 and Fli1 is a key feature underlying systemic sclerosis.

Systemic sclerosis (SSc) is manifested by fibrosis, vasculopathy and immune dysregulation. So far, a unifying hypothesis underpinning these pathological events remains unknown. Given that SSc is a multifactorial disease caused by both genetic and environmental factors, we focus on the two transcription factors, which modulate the fibrotic reaction and are epigenetically suppressed in SSc dermal fibroblasts, Friend leukaemia integration 1 (Fli1) and Krüppel-like factor 5 (KLF5). In addition to the Fli1 silencing-dependent collagen induction, the simultaneous knockdown of Fli1 and KLF5 synergistically enhances expression of connective tissue growth factor. Notably, mice with double heterozygous deficiency of Klf5 and Fli1 mimicking the epigenetic phenotype of SSc skin spontaneously recapitulate all the three features of SSc, including fibrosis and vasculopathy of the skin and lung, B-cell activation and autoantibody production. These studies implicate the epigenetic downregulation of Fli1 and KLF5 as a central event triggering the pathogenic triad of SSc.

Successful experience of rituximab therapy for systemic sclerosis-associated interstitial lung disease with concomitant systemic lupus erythematosus.

Previous studies have demonstrated that B cells play critical roles in autoimmune disorders including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). However, the effectiveness of rituximab (RTX), a chimeric anti-CD20 antibody, for SSc-associated interstitial lung disease (ILD) or SLE disease activity remains controversial. We herein report an SSc patient with severely progressed ILD and concomitant SLE treated by two cycles of RTX at baseline and half a year later. This treatment improved ILD and SLE activities, along with reduction of dermal sclerosis and serum anti-topoisomerase I antibody levels. In addition, our detailed time-course data indicate that half a year may be appropriate as an interval between each cycle of RTX therapy aimed at SSc-associated ILD or SLE. Overall, the current report could pave the way to establish RTX as a disease-modifying drug for patients with SSc and/or SLE showing resistance to other already approved medications.

Bosentan reverses the pro-fibrotic phenotype of systemic sclerosis dermal fibroblasts via increasing DNA binding ability of transcription factor Fli1.

INTRODUCTION: Although the pathogenesis of systemic sclerosis (SSc) still remains unknown, recent studies have demonstrated that endothelins are deeply involved in the developmental process of fibrosis and vasculopathy associated with SSc, and a dual endothelin receptor antagonist, bosentan, has a potential to serve as a disease modifying drug for this disorder. Importantly, endothelin-1 (ET-1) exerts a pro-fibrotic effect on normal dermal fibroblasts and bosentan reverses the pro-fibrotic phenotype of SSc dermal fibroblasts. The purpose of this study was to clarify the details of molecular mechanisms underlying the effects of ET-1 and bosentan on dermal fibroblasts, which have not been well studied. METHODS: The mRNA levels of target genes and the expression and phosphorylation levels of target proteins were determined by reverse transcription real-time PCR and immunoblotting, respectively. Promoter assays were performed using a sequential deletion of human α2 (I) collagen (COL1A2) promoter. DNA affinity precipitation and chromatin immunoprecipitation were employed to evaluate the DNA binding ability of Fli1. Fli1 protein levels in murine skin were evaluated by immunostaining. RESULTS: In normal fibroblasts, ET-1 activated c-Abl and protein kinase C (PKC)-δ and induced Fli1 phosphorylation at threonine 312, leading to the decreased DNA binding of Fli1, a potent repressor of the COL1A2 gene, and the increase in type I collagen expression. On the other hand, bosentan reduced the expression of c-Abl and PKC-δ, the nuclear localization of PKC-δ, and Fli1 phosphorylation, resulting in the increased DNA binding of Fli1 and the suppression of type I collagen expression in SSc fibroblasts. In bleomycin-treated mice, bosentan prevented dermal fibrosis and increased Fli1 expression in lesional dermal fibroblasts. CONCLUSIONS: ET-1 exerts a potent pro-fibrotic effect on normal fibroblasts by activating "c-Abl - PKC-δ - Fli1" pathway. Bosentan reverses the pro-fibrotic phenotype of SSc fibroblasts and prevents the development of dermal fibrosis in bleomycin-treated mice by blocking this signaling pathway. Although the efficacy of bosentan for dermal and pulmonary fibrosis is limited in SSc, the present observation definitely provides us with a useful clue to further explore the potential of the upcoming new dual endothelin receptor antagonists as disease modifying drugs for SSc.