The Contribution of LIGHT (TNFSF14) to the Development of Systemic Sclerosis by Modulating IL-6 and T Helper Type 1 Chemokine Expression in Dermal Fibroblasts.

Systemic sclerosis (SSc) is an autoimmune and vascular disease resulting in multiple organ fibrosis, in which IL-6 and T helper (Th)2/Th17 cytokines serve as critical disease drivers. LIGHT is a proinflammatory cytokine promoting IL-6 production in lung fibroblasts and Th1 chemokine expression in dermal fibroblasts (DFs) stimulated with IFN-γ. In this study, we investigated the potential contribution of LIGHT to SSc development using clinical samples and animal models. In SSc-involved skin, LIGHT was upregulated in inflammatory cells, whereas herpesvirus entry mediator (HVEM), a receptor of LIGHT, was downregulated in DFs. Similar expression profiles of LIGHT and HVEM were reproduced in bleomycin-treated mice. Transcription factor FLI1 bound to the HVEM promoter, and FLI1 small interfering RNA suppressed HVEM expression in normal DFs. In SSc DFs, LIGHT significantly increased IL-6 production, whereas IFN-γ/LIGHT-dependent Th1 chemokine induction was decreased compared with that in normal DFs. Importantly, LIGHT small interfering RNA significantly attenuated bleomycin-induced skin fibrosis, and serum LIGHT levels were elevated in patients with diffuse cutaneous SSc and positively correlated with clinical parameters reflecting skin and pulmonary fibrosis. Taken together, these results suggest that altered response of DFs to LIGHT, namely increased IL-6 production and decreased Th1 chemokine expression, contributes to the development of skin fibrosis in SSc.

Altered Properties of Endothelial Cells and Mesenchymal Stem Cells Underlying the Development of Scleroderma-like Vasculopathy in KLF5+/- ;Fli-1+/- Mice.

OBJECTIVE: In prevous studies, we established a new animal model, KLF5+/- ;Fli-1+/- mice, in which fundamental pathologic features of systemic sclerosis (SSc) are broadly recapitulated. SSc vasculopathy is believed to occur as a result of impaired vascular remodeling, but its detailed mechanism of action remains unknown. To address this, the present study investigated the properties of dermal microvascular endothelial cells (DMECs), bone marrow-derived endothelial progenitor cells (BM-EPCs), and bone marrow-derived mesenchymal stem cells (BM-MSCs), a precursor of pericytes, in KLF5+/- ;Fli-1+/- mice. METHODS: Neovascularization and angiogenesis were assessed in KLF5+/- ;Fli-1+/- mice by in vivo Matrigel plug assay and in vitro tube formation assay, respectively. The properties of mouse BM-EPCs and BM-MSCs were assessed with in vitro studies. Dermal vasculature was visualized in vivo by injecting the mice with fluorescein isothiocyanate-conjugated dextran. RESULTS: Neovascularization was diminished in skin-embedded Matrigel plugs from KLF5+/- ;Fli-1+/- mice. DMECs from KLF5+/- ;Fli-1+/- mice showed defective tubulogenic activity, decreased expression of VE-cadherin and CD31, and an imbalance in the expression of Notch1/Dll4, suggesting that angiogenesis and anastomosis are disturbed. KLF5+/- ;Fli-1+/- mouse BM-MSCs exhibited enhanced proliferation and migration and increased collagen production following stimulation with transforming growth factor ß1, indicating that these cells differentiate preferentially into myofibroblasts rather than pericytes. KLF5+/- ;Fli-1+/- mouse BM-EPCs displayed a transition toward mesenchymal cells, suggesting that vasculogenesis is impaired. Wound healing was delayed in KLF5+/- ;Fli-1+/- mice (mean ± SD healing time 15.67 ± 0.82 days versus 13.50 ± 0.84 days; P = 0.0017), and the vascular network was poorly developed in wound scar tissue. CONCLUSION: The characteristics observed in the KLF5+/- ;Fli-1+/- mouse model - specifically, impaired neovascularization and vascular maturation - are similar to those observed in human SSc, and could be at least partially attributable to the induction of SSc-like properties in DMECs, BM-EPCs, and BM-MSCs. These findings indicate the critical contribution of Klf5 and Fli1 deficiency in vascular cells and related cell precursors to the development of SSc vasculopathy.

Assessment of endothelial function during the loading phase of infliximab in psoriasis: a potential predictor of its drug survival.

BACKGROUND: Tumor necrosis factor inhibitors decrease the risk of cardiovascular events in moderate to severe psoriasis, but the association between their effects on endothelial function and those on skin lesions has not been well studied. We investigated the association between infliximab effects on endothelial function during the loading phase and those on skin lesions in patients with psoriasis. METHODS: We evaluated endothelial function with reactive hyperemia-peripheral arterial tonometry index (RHI) in 15 patients with psoriasis before the first and third infusions of infliximab. Patients were stratified into two groups; those who maintained Psoriasis Area and Severity Index (PASI) 75 response for more than 6 months (defined as responders) and the others (defined as nonresponders). RESULTS: Six weeks after the initiation of infliximab (before the third infusion), PASI scores were significantly improved compared with baseline, while RHI values were not altered in the whole patient group. However, when the responders and the nonresponders were analyzed separately, RHI values tended to be decreased before the third infusion compared with baseline in the nonresponders, while being unchanged in the responders. Importantly, the difference in ∆RHI reached a statistical significance between the two groups, and the cutoff value (mean - 2 standard deviation of RHI values in the responders) identified the nonresponders with 67% of sensitivity and 100% of specificity. CONCLUSIONS: The decrease in RHI values before the third infusion may serve as a predictor for the long-term unfavorable effect of infliximab on psoriatic skin lesions.

Increased expression of aquaporin-1 in dermal fibroblasts and dermal microvascular endothelial cells possibly contributes to skin fibrosis and edema in patients with systemic sclerosis.

BACKGROUND: Aquaporin-1 (AQP1), a water channel protein controlling the water contents of cells and tissues, exerts pleiotropic effects on various biological activities, including inflammation, angiogenesis, and extracellular matrix remodeling, by regulating cell behaviors and tissue water balance. OBJECTIVE: To investigate AQP1 roles in systemic sclerosis (SSc) which is characterized by autoimmune inflammation, vasculopathy, and tissue fibrosis. METHODS: AQP1 expression was evaluated by immunohistochemistry and quantitative reverse transcription PCR in skin samples from human and animal models and by immunoblotting in cultured cells. Fli1 binding to the AQP1 promoter was evaluated by chromatin immunoprecipitation. Cell migration was assessed by scratch assay. RESULTS: Dermal fibroblasts and endothelial cells highly expressed AQP1 in SSc lesional skin, and AQP1 expression in dermal fibroblasts and endothelial cells positively correlated with the degrees of tissue fibrosis and edema, respectively. Consistently, SSc dermal fibroblasts up-regulated AQP1 compared with normal dermal fibroblasts in vitro. Furthermore, TGF-ß stimulation induced AQP1 expression in normal dermal fibroblasts, while TGF-ß1 antisense oligonucleotide suppressed AQP1 expression in SSc dermal fibroblasts. In endothelial cells, Fli1 deficiency resulted in AQP1 up-regulation in vivo and in vitro and Fli1 bound to the AQP1 promoter. Importantly, SSc dermal fibroblasts and FLI1 siRNA-treated endothelial cells had a pro-migratory property, which was remarkably diminished by gene silencing of AQP1. CONCLUSION: AQP1 is up-regulated in SSc dermal fibroblasts and SSc endothelial cells at least partially due to autocrine TGF-ß stimulation and Fli1 deficiency, respectively, possibly contributing to inflammation, vasculopathy, and tissue fibrosis by regulating tissue edema and cell migration.

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.

CXCL13 produced by macrophages due to Fli1 deficiency may contribute to the development of tissue fibrosis, vasculopathy and immune activation in systemic sclerosis.

CXCL13, a chemokine for B cells, follicular T cells, T helper 17 cells, and regulatory T cells, is reported to contribute to the development of systemic sclerosis (SSc), reflecting aberrant activation of immune system. To better understand the role of CXCL13 in SSc, we investigated the influence of Fli1 deficiency, a potential predisposing factor of this disease, on CXCL13 expression and assessed the clinical correlation of serum CXCL13 levels by multivariate regression analysis. Haploinsufficient loss of Fli1 remarkably induced CXCL13 expression in murine peritoneal macrophages, while gene silencing of FLI1 did not affect the expression of CXCL13 in human dermal fibroblasts and human dermal microvascular endothelial cells. Serum CXCL13 levels were elevated in SSc patients compared with healthy controls and correlated positively with skin score and negatively with pulmonary function test results. SSc patients with elevated serum CXCL13 levels had longer disease duration, diffuse cutaneous involvement, interstitial lung disease (ILD), heart involvement, pulmonary arterial hypertension, Raynaud's phenomenon, pitting scars, digital ulcers, telangiectasia, and high serum IgG levels more frequently than the other patients. In particular, serum CXCL13 levels were associated with ILD and digital ulcers by multivariate regression analysis. Taken together, these results indicate that CXCL13 expression is upregulated by Fli1 deficiency in macrophages, potentially contributing to the development of tissue fibrosis, vasculopathy and immune activation in SSc, especially ILD and digital ulcers.

Fli1-haploinsufficient dermal fibroblasts promote skin-localized transdifferentiation of Th2-like regulatory T cells.

BACKGROUND: Friend leukemia virus integration 1 (Fli1) deficiency, a predisposing factor of systemic sclerosis (SSc), induces SSc-like phenotypes in various cell types. A recent study demonstrated the transdifferentiation of T helper type 2 cell (Th2)-like regulatory T cells (Tregs) in SSc lesional skin through interleukin (IL)-33 produced by fibroblasts. Therefore, we investigated the role of Fli1 deficiency in dermal fibroblast-mediated transdifferentiation of Tregs. METHODS: Cytokine expression was assessed in Tregs by flow cytometry and in skin samples and cultivated cells by immunostaining, immunoblotting, and/or qRT-PCR. Fli1 binding to the target gene promoters was examined by chromatin immunoprecipitation. Murine dermal fibroblasts and Tregs were cocultured with or without blocking antibodies against target cytokines. RESULTS: Th2- and Th17-like cell proportions in skin-homing Tregs were increased in bleomycin-treated Fli1 +/- mice compared with bleomycin-treated wild-type mice, whereas Th1-, Th2-, and Th17-like cell proportions in splenic Tregs were comparable. Fli1+/- fibroblasts overproduced IL-33 and IL-6, in particular IL-33, and Fli1 occupied the IL33 and IL6 promoters in dermal fibroblasts. Importantly, the IL-4-producing cell proportion was significantly higher in wild-type Tregs cocultured with Fli1+/- fibroblasts than in those cocultured with wild-type fibroblasts, which were canceled by neutralizing anti-IL-33 antibody. Under the same coculture condition, an increased tendency of IL-17A-producing cell proportion, which was possibly mediated by IL-6, was evident. CONCLUSIONS: Fli1 haploinsufficiency increases the proportions of Th2- and Th17-like Tregs in bleomycin-induced profibrotic skin conditions, in which IL-33-producing dermal fibroblasts contribute to Th2-like Treg transdifferentiation, suggesting a critical role of Fli1 deficiency in the interaction of dermal fibroblasts with immune cells in pathological skin fibrosis.

Possible pro-inflammatory role of heparin-binding epidermal growth factor-like growth factor in the active phase of systemic sclerosis.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family growth factors, which affects multiple aspects of the wound healing process such as epithelialization, wound contraction and angiogenesis. In our study, we measured the serum HB-EGF levels of 51 systemic sclerosis (SSc) patients, which showed a significant increase compared with those of 20 normal subjects. Further analysis revealed a positive correlation between the HB-EGF level and pulmonary ground-glass score but no correlation between the former and pulmonary fibrosis score. Other findings include: a significant increase of serum sialylated carbohydrate antigen KL-6 levels and significant shortness of disease duration in the diffuse cutaneous SSc patients with elevated HB-EGF levels; and significantly higher HB-EGF levels in the presence of Raynaud's phenomenon, in that of telangiectasia, and in the absence of contracture of phalanges in all SSc patients. We then evaluated HB-EGF mRNA levels of fibroblasts harvested from skin samples of the SSc patients and those of foreskin-derived fibroblasts treated with transforming growth factor-ß, both of which were significantly higher than each control. In conclusion, we speculate that HB-EGF plays a pro-inflammatory role in the active skin and lung lesions of SSc.

A possible implication of reduced levels of LIF, LIFR, and gp130 in vasculopathy related to systemic sclerosis.

Leukemia inhibitory factor (LIF) is a member of IL-6 family, which serves as a potent chemoattractant for neutrophils as well as a potent angiostatic factor. LIF has been implicated in various autoimmune inflammatory diseases, but its role still remains elusive in systemic sclerosis (SSc). Therefore, we investigated the potential role of LIF in the development of SSc by evaluating the clinical correlation of serum LIF levels, the expression of LIF and its receptors in skin samples, and in vitro experiments with human dermal microvascular endothelial cells. Serum LIF levels were significantly decreased in patients with SSc, especially in those with disease duration of < 1 year compared with healthy controls. As for clinical correlation, SSc patients with digital ulcers exhibited serum LIF levels significantly lower than those without. In immunohistochemistry, the expression of LIF and its receptors, LIF receptor and gp130, was remarkably decreased in dermal blood vessels of SSc lesional skin relative to those of healthy control skin. Furthermore, gene silencing of transcription factor Fli1, whose deficiency is involved in the development of SSc vasculopathy, suppressed the expression of LIF, LIF receptor, and gp130 and Fli1 bound to the promoters of those genes in human dermal microvascular endothelial cells. Collectively, these results suggest that decreased serum LIF levels may be associated with vasculopathy in SSc and that Fli1 deficiency may contribute to the inhibition of LIF-dependent biological effects on SSc endothelial cells by suppressing the expression of LIF, LIF receptor, and gp130.

Systemic Sclerosis Dermal Fibroblasts Suppress Th1 Cytokine Production via Galectin-9 Overproduction due to Fli1 Deficiency.

Dermal fibroblasts promote skin-localized transdifferentiation of regulatory T cells to T helper (Th) type 2-like cells in systemic sclerosis (SSc). However, the entire effect of SSc dermal fibroblasts on immune cells still remains unknown. Because galectin-9 induces Th2 cytokine-predominant immune imbalance by negatively regulating Th1/Th17 cells in inflammatory diseases, we investigated the contribution of galectin-9 to Th immune balance in SSc lesional skin. We used human clinical samples and Fli1+/- mice because Fli1 deficiency induces SSc-like phenotypes in various cell types. Galectin-9 was overexpressed in SSc dermal fibroblasts in vivo and in vitro. Serum galectin-9 levels were significantly elevated in SSc patients and positively correlated with skin score. Galectin-9 was up-regulated by autocrine endothelin stimulation and Fli1 deficiency, and Fli1 occupied the LGALS9 promoter in dermal fibroblasts. Co-culture of splenic CD4+ T cells with Fli1+/- dermal fibroblasts significantly increased IL-4-producing cell proportion, and this effect was cancelled in parallel with the increased interferon-γ production when Fli1+/- dermal fibroblasts were transfected with Lgals9 small interfering RNA. Furthermore, Lgals9 small interfering RNA suppressed dermal collagen deposition by increasing interferon-γ production of skin-infiltrating CD4+ T cells in bleomycin-treated mice. These results suggest that SSc dermal fibroblasts suppress interferon-γ expression of skin-infiltrating CD4+ T cells through galectin-9 overproduction, promoting skin fibrosis development.

Contribution of Soluble Forms of Programmed Death 1 and Programmed Death Ligand 2 to Disease Severity and Progression in Systemic Sclerosis.

OBJECTIVE: To determine the function and serum levels of soluble forms of programmed death 1 (sPD-1) and one of its ligands, soluble PD ligand 2 (sPD-L2), in patients with systemic sclerosis (SSc) and in a mouse model of topoisomerase I (topo I)-induced SSc. METHODS: Serum levels of sPD-1 and sPD-L2 in 91 patients with SSc were examined by enzyme-linked immunosorbent assay (ELISA). Expression of PD-1 and PD-L2 on T cells, B cells, and macrophages was quantified by flow cytometry. The effects of blockade of PD-1 and PD-L2 were analyzed by microfluidic ELISA (micro-ELISA), a technique that can measure very low amounts of cytokines. In addition, the effects of sPD-1 and sPD-L2 on disease progression were assessed in mice with topo I-induced SSc. RESULTS: Serum levels of sPD-1 and sPD-L2 were elevated in patients with SSc and correlated with the extent of fibrosis and immunologic abnormalities. Expression levels of PD-1 and PD-L2 were significantly elevated on SSc T cells, B cells, and macrophages. Micro-ELISA analysis of serum samples from patients with SSc showed that PD-L2high B cells had higher levels of interleukin-10 (IL-10) production compared with PD-L2low B cells, indicating that PD-L2 acts as a regulator of T cell cytokine production via cognate interactions with T cells and B cells. In mice with topo I-induced SSc, production of IL-10 by topo I-specific B cells in cultures with T cells and topo I protein was significantly higher than that by conventional B cells, and intraperitoneal injection of recombinant chimeric PD-1-Fc and PD-L2-Fc canceled these enhanced effects. CONCLUSION: These results suggest that sPD-1 and sPD-L2 contribute to disease development in SSc via the regulation of cognate interactions with T cells and B cells.

Fli1 Deficiency Induces CXCL6 Expression in Dermal Fibroblasts and Endothelial Cells, Contributing to the Development of Fibrosis and Vasculopathy in Systemic Sclerosis.

OBJECTIVE: CXCL6, a chemokine with proangiogenic property, is reported to be involved in vasculopathy associated with systemic sclerosis (SSc). We investigated the contribution of CXCL6 to SSc development by focusing on the association of friend leukemia virus integration 1 (Fli1) deficiency, a potential predisposing factor of SSc, with CXCL6 expression and clinical correlation of serum CXCL6 levels. METHODS: mRNA levels of target genes and the binding of Fli1 to the CXCL6 promoter were evaluated by quantitative reverse transcription-PCR and chromatin immunoprecipitation, respectively. Serum CXCL6 levels were determined by ELISA. RESULTS: FLI1 siRNA significantly enhanced CXCL6 mRNA expression in human dermal fibroblasts and human dermal microvascular endothelial cells, while Fli1 haploinsufficiency significantly suppressed CXCL6 mRNA expression in murine peritoneal macrophages stimulated with lipopolysaccharide. Supporting a critical role of Fli1 deficiency to induce SSc-like phenotypes, CXCL6 mRNA expression was higher in SSc dermal fibroblasts than in normal dermal fibroblasts. Importantly, Fli1 bound to the CXCL6 promoter in dermal fibroblasts, endothelial cells, and THP-1 cells. In patients with SSc, serum CXCL6 levels correlated positively with the severity of dermal and pulmonary fibrosis and were elevated in association with cardiac and pulmonary vascular involvement and cutaneous vascular symptoms, including Raynaud phenomenon, digital ulcers (DU)/pitting scars, and telangiectasia. Especially, serum CXCL6 levels were associated with DU/pitting scars and heart involvement by multiple regression analysis. CONCLUSION: CXCL6 expression is upregulated by Fli1 deficiency in fibroblasts and endothelial cells, potentially contributing to the development of fibrosis and vasculopathy in the skin, lung, and heart of SSc.

The impact of transcription factor Fli1 deficiency on the regulation of angiogenesis.

The insufficiency of Friend leukaemia virus integration 1 (Fli1), a member of the Ets family transcription factors, is implicated in the pathogenesis of vasculopathy associated with systemic sclerosis (SSc). Fli1 deficiency accelerates early steps of angiogenesis, including detachment of pre-existing pericytes and extracellular matrix degradation by endothelial proteinases, but the impact of Fli1 deficiency on the other steps of angiogenesis has not been investigated. Therefore, we evaluated the effect of Fli1 deficiency on migration, proliferation, cell survival and tube formation of human dermal microvascular endothelial cells (HDMECs). HDMECs transfected with FLI1 siRNA exhibited a greater migratory property in scratch assay and transwell migration assay and a higher proliferation rate in BrdU assay than HDMECs transfected with non-silencing scrambled RNA. In flow cytometry-based apoptosis assay, FLI1 siRNA-transduced HDMECs revealed the decreased number of annexin and propidium iodide-double-positive apoptotic cells compared with control cells, reflecting the promotion of cell survival. On the other hand, tubulogenic activity on Matrigel was remarkably suppressed in Fli1-deficient HDMECs relative to control cells. These results indicate that Fli1 deficiency promotes migration, proliferation and cell survival, while abating tube formation of endothelial cells, suggesting that Fli1 deficiency is potentially attributable to the development of both proliferative obliterative vasculopathy (occlusion of arterioles and small arteries) and destructive vasculopathy (loss of small vessels) characteristic of SSc vasculopathy.

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.

A potential contribution of altered cathepsin L expression to the development of dermal fibrosis and vasculopathy in systemic sclerosis.

Cathepsin L (CTSL) is a lysosomal proteolytic enzyme involved in inflammation and vascular and extracellular matrix remodelling, which are the three cardinal pathological events associated with systemic sclerosis (SSc). To elucidate the potential role of CTSL in the development of SSc, we here investigated CTSL expression in the lesional skin of patients with SSc and SSc animal models and the clinical correlation of serum CTSL levels. CTSL expression was elevated in dermal small vessels of SSc patients compared with those of healthy controls. Consistently, CTSL mRNA levels were increased in SSc lesional skin samples, but not in cultivated SSc dermal fibroblasts, compared with corresponding control samples from healthy individuals. Serum CTSL levels were significantly higher in SSc patients than in healthy controls and inversely correlated with skin score. Furthermore, the elevation of serum CTSL levels was linked to SSc vasculopathy. Supporting these results, Ctsl mRNA levels were decreased in the skin of bleomycin-treated mice, an SSc animal model recapitulating its fibrotic aspect, and CTSL expression was enhanced in dermal small vessels of endothelial cell-specific Fli1 knockout mice, reminiscent of SSc vasculopathy. Importantly, gene silencing of FLI1 induced CTSL mRNA expression and Fli1 occupied the CTSL promoter in human dermal microvascular endothelial cells. Collectively, these results suggest that endothelial CTSL up-regulation partially due to Fli1 deficiency may contribute to the development of vasculopathy, while the decrease in dermal CTSL expression is likely associated with dermal fibrosis 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.