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.

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.

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.

Serum angiopoietin-like protein 3 levels: possible correlation with progressive skin sclerosis, digital ulcers and pulmonary vascular involvement in patients with systemic sclerosis.

Angiopoietin-like protein 3 (ANGPTL3), which is part of a family of secreted glycoproteins that are structurally similar to angiopoietins, is principally expressed in the liver and is involved in lipid metabolism and angiogenesis. The aim of this study was to determine the clinical significance of serum ANGPTL3 levels, measured with a specific enzyme-linked immunosorbent assay, in patients with systemic sclerosis. Serum ANGPTL3 levels correlated positively with skin score in diffuse cutaneous systemic sclerosis with a disease duration ≤ 6 years. Furthermore, the prevalence of digital ulcers was significantly higher in patients with elevated serum ANGPTL3 levels than in other patients. Moreover, among patients excluding diffuse cutaneous systemic sclerosis with disease duration ≤ 6 years, serum ANGPTL3 levels correlated positively with estimated right ventricular systolic pressure. In conclusion, ANGPTL3 may contribute to the development of progressive skin sclerosis and proliferative obliterative vasculopathy, such as digital ulcers and pulmonary vascular involvement leading to pulmonary arterial hypertension, in systemic sclerosis.

Serum resistin levels: a possible correlation with pulmonary vascular involvement in patients with systemic sclerosis.

Our latest studies demonstrated the potential role of adipocytokines, including adiponectin, visfatin, retinol binding protein-4, and apelin, in the pathogenesis of systemic sclerosis (SSc). Given that resistin is another member of adipocytokines with pro-inflammatory and pro-angiogenic properties, we measured serum resistin levels by enzyme-linked immunosorbent assay in 52 SSc and 19 control subjects and evaluated their clinical correlation. Since serum resistin levels greatly and inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction [r = -0.78, p < 0.05 (n = 9)], we evaluated the clinical correlation of serum resistin levels in SSc patients with normal renal function (n = 43). Although serum resistin levels were comparable between diffuse cutaneous SSc (n = 22), limited cutaneous SSc (n = 21), and control subjects (n = 19) [median (25-75 percentiles); 18.7 ng/ml (13.3-48.0), 23.3 ng/ml (12.9-54.1), and 22.9 ng/ml (9.4-36.7), respectively], the prevalence of elevated right ventricular systolic pressure (RVSP) was significantly higher in SSc patients with elevated serum resistin levels than in those with normal levels [67 % (4/6) vs. 16 % (6/37), p < 0.05], and serum resistin levels were significantly increased in SSc patients with elevated RVSP (n = 10) as compared to those with normal RVSP (n = 33) [52.1 ng/ml (20.8-117.5) vs. 18.5 ng/ml (12.2-46.2), p < 0.05]. Thus, serum resistin levels may serve as a useful marker for pulmonary vascular involvement in SSc, suggesting a possible contribution of resistin to the pathogenesis of pulmonary arterial hypertension associated with SSc.

Decreased cathepsin V expression due to Fli1 deficiency contributes to the development of dermal fibrosis and proliferative vasculopathy in systemic sclerosis.

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-ß1 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-ß1 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.

A possible contribution of visfatin to the resolution of skin sclerosis in patients with diffuse cutaneous systemic sclerosis via a direct anti-fibrotic effect on dermal fibroblasts and Th1 polarization of the immune response.

OBJECTIVE: Visfatin is a member of the adipocytokines with pro-fibrotic, pro-inflammatory and immunomodulating properties potentially implicated in the pathogenesis of certain fibrotic and inflammatory autoimmune diseases. In this study, we investigated THE CLINICAL SIGNIFICANCE OF SERUM VISFATIN LEVELS AND ITS CONTRIBUTION TO THE DEVELOPMENTAL PROCESS IN SSC. METHODS: Serum visfatin levels were determined by a specific ELISA in 57 SSc patients and 19 healthy controls. The mRNA levels of target genes were determined in normal and SSc fibroblasts by real-time RT-PCR. The levels of IL-12p70 produced by THP-1 cells were measured by a specific ELISA. RESULTS: Serum visfatin levels were comparable among total SSc, diffuse cutaneous SSc (dcSSc), limited cutaneous SSc and healthy controls. The only finding in a series of analyses regarding the correlation of serum visfatin levels with clinical symptoms and laboratory data was the significantly longer disease duration in dcSSc with elevated serum visfatin levels than in those with normal levels. Consistently, serum visfatin levels were significantly elevated in late-stage dcSSc (disease duration >6 years), but not in early and mid-stage dcSSc compared with healthy controls. In in vitro experiments, visfatin reversed the pro-fibrotic phenotype of SSc dermal fibroblasts and induced the expression of IL-12p70 in THP-1 cells treated with IFN-γ plus lipopolysaccharide. CONCLUSION: Visfatin may contribute to the resolution of skin sclerosis in late-stage dcSSc via a direct anti-fibrotic effect on dermal fibroblasts and Th1 polarization of the immune response.

Dynamics of serum angiopoietin-2 levels correlate with efficacy of intravenous pulse cyclophosphamide therapy for interstitial lung disease associated with systemic sclerosis.

OBJECTIVE: Angiopoietin-2 (Ang2) regulates the transition between vascular quiescence and angiogenesis in a context-dependent manner. In systemic sclerosis (SSc), serum Ang2 levels correlate with its disease activity. Therefore, we investigated the clinical significance of monitoring serum Ang2 levels during intravenous pulse cyclophosphamide (IVCY) therapy in SSc patients with interstitial lung disease (ILD). METHODS: Serum Ang2 levels were determined by a specific enzyme-linked immunosorbent assay in seven SSc patients treated with IVCY and 20 healthy controls. In the patient group, serum samples were drawn the day before each IVCY therapy. RESULTS: Serum Ang2 levels tended to be higher in SSc patients before IVCY than in healthy controls and significantly correlated with KL-6, surfactant protein D, erythrocyte sedimentation rate, and C-reactive protein in SSc patients with ILD. In sera drawn before the last IVCY, Ang2 levels were significantly decreased compared with initial levels. Notably, Δ serum Ang2 levels between baseline and after the first IVCY significantly correlated with Δ ILD score between before and after the entire IVCY therapy (r = 0.90, p < 0.01). CONCLUSION: Monitoring Ang2 levels during IVCY treatment may be useful to evaluate and predict the efficacy of this treatment for SSc-ILD.

Clinical significance of monitoring serum adiponectin levels during intravenous pulse cyclophosphamide therapy in interstitial lung disease associated with systemic sclerosis.

OBJECTIVE: To investigate the clinical significance of monitoring serum adiponectin levels during intravenous pulse cyclophosphamide (IVCY) in systemic sclerosis (SSc) patients with interstitial lung disease (ILD). METHODS: Serum adiponectin levels were determined by a specific enzyme-linked immunosorbent assay in eight SSc patients with active ILD who underwent IVCY and 27 healthy controls. In patients, serum samples were drawn the day before each IVCY. RESULTS: Serum adiponectin levels were significantly decreased in SSc patients with active ILD before the first IVCY compared with healthy controls [median (25-75 percentile): 3.21 (2.70-4.19) vs. 7.42 (6.06-10.82) µg/ml; P < 0.01). After the completion of whole IVCY, serum adiponectin levels were significantly increased [17.55 (6.47-39.45) µg/ml; P < 0.05] compared with the initial levels, and this increase significantly correlated with the decrease in ILD scores. Importantly, the dynamics of serum adiponectin levels during the IVCY therapy reflected its efficacy against SSc-ILD over the treatment and the follow-up period. CONCLUSION: The monitoring of serum adiponectin levels during the IVCY treatment may be useful to identify SSc patients with ILD refractory to the treatment and at high risk for exacerbations during the follow-up period.

Clinical significance of serum decoy receptor 3 levels in patients with systemic sclerosis.

Decoy receptor 3 (DcR3) is associated with autoimmunity and altered angiogenesis in certain pathological conditions. We herein measured serum DcR3 levels in 51 patients with systemic sclerosis (SSc) and 19 healthy controls and evaluated their clinical significance in this disorder. Serum DcR3 levels were significantly higher in diffuse cutaneous SSc (dcSSc) patients than in limited cutaneous SSc patients and in healthy controls. In dcSSc, serum DcR3 levels were significantly elevated in patients with disease duration of ≤6 years compared with healthy controls, but not in those with disease duration of >6 years. Serum DcR3 levels correlated negatively with the percentage of predicted diffusion lung capacity for carbon monoxide and positively with right ventricular systolic pressure. Furthermore, serum DcR3 levels positively correlated with C-reactive protein, erythrocyte sedimentation rate and immunoglobulin G. Collectively, the elevation of serum DcR3 levels is associated with the development of pulmonary arterial hypertension and systemic inflammation in SSc.

Clinical significance of serum levels of secretory leukocyte protease inhibitor in patients with systemic sclerosis.

We aimed to investigate the clinical significance of serum levels of secretory leukocyte protease inhibitor (SLPI), which is widely expressed in lung tissues and serves as a useful marker reflecting the activity of various lung diseases, in patients with systemic sclerosis (SSc). Serum SLPI levels were measured by a specific enzyme-linked immunosorbent assay (ELISA) in 58 SSc patients and 16 healthy controls. Serum SLPI levels in diffuse cutaneous SSc and in limited cutaneous SSc with interstitial lung disease (ILD) were significantly higher than those in healthy controls (43.1 ± 18.4 vs. 30.9 ± 3.76 ng/ml, p < 0.05 and 39.8 ± 10.3 vs. 30.9 ± 3.76 ng/ml, p < 0.01, respectively). The incidences of decreased percent diffusing capacity for carbon monoxide (%DLco) and decreased percent vital capacity (%VC) were significantly greater in SSc patients with elevated SLPI levels than in those with normal levels (73 vs. 31%, p < 0.01 and 24 vs. 4%, p < 0.05, respectively). Furthermore, serum SLPI levels were inversely correlated with %DLco (r = -0.40, p < 0.01), while they were positively correlated with surfactant protein D (r = 0.28, p < 0.05). Longitudinal study revealed the association of serum SLPI levels with the disease activity of SSc-ILD. SLPI serves as a useful serum marker for evaluating 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.

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.

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.

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.

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.

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.

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.