transforming-growth-factor-beta and Scleroderma--Diffuse

Pulmonary fibrosis occurs in up to 70% of scleroderma patients and progresses to cause severe restrictive lung disease in about 15% of patients. The mechanisms that cause pulmonary fibrosis in scleroderma remain incompletely understood. Increased amounts of mRNA or protein for multiple profibrotic cytokines and chemokines have been identified in lung tissue or broncholveolar lavage samples from scleroderma patients, when compared to healthy controls. These cytokines include transforming growth factor (TGF)-beta, connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), oncostatin M (OSM), monocyte chemotactic factor-1 and pulmonary and activation-regulated chemokine (PARC). Potential cellular sources of these profibrotic cytokines and chemokines in scleroderma lung disease include alternatively activated macrophages, activated CD8+ T cells, eosinophils, mast cells, epithelial cells and fibroblasts themselves. This review summarizes the literature on involvement of cytokines and chemokines in the development of pulmonary fibrosis in scleroderma.

Topics: Adult; CD8-Positive T-Lymphocytes; Chemokine CCL2; Chemokines; Connective Tissue Growth Factor; Cytokines; DNA Topoisomerase IV; Eosinophils; Epithelial Cells; Female; Fibroblasts; Fibrosis; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Interleukin-1; Lung; Male; Mast Cells; Oncostatin M; Peptides; Platelet-Derived Growth Factor; Pulmonary Fibrosis; RNA, Messenger; Scleroderma, Diffuse; Transforming Growth Factor beta

Imatinib mesylate is a potent inhibitor of platelet-derived growth factor and transforming growth factor-β signalling pathways which may play a role in systemic sclerosis (SSc)-associated skin changes.. We aimed primarily at assessing the efficacy of imatinib mesylate in scleroderma skin fibrosis.. We performed a phase II double-blinded trial on patients with scleroderma with either morphoea involving > 20% of body surface area or SSc with extensive skin involvement: modified Rodnan Skin Score (mRSS) ≥ 20/51. Each patient was randomized to receive either imatinib mesylate 400 mg or placebo daily for a total of 6 months, and then was followed up 6 months after therapy discontinuation. Skin fibrosis was assessed by mRSS and measurement of the dermal thickness using skin biopsies performed at inclusion and at 6 months of treatment. In addition, quality of life (Dermatology Life Quality Index and modified Health Assessment Questionnaire for Scleroderma) was recorded at each visit, and pulmonary function before and after intervention.. Twenty-eight patients were included in the study with a mean age of 48·9 years (range 30-71): 25 had a diagnosis of a SSc and three of diffuse cutaneous scleroderma. Demographic data, frequency of organ involvement of SSc and mRSS were comparable between groups. At 6 months, the proportion of variation of mRSS from inclusion was not statistically significantly different between the two groups (median +0·10 in imatinib group vs. -0·16 in placebo group, P = 0·098). Similarly, changes in dermal thickness, quality of life and diffusion capacity for carbon monoxide were not significantly different between groups.. This study failed to demonstrate the efficacy of imatinib 400 mg daily to improve skin fibrosis of diffuse scleroderma after 6 months of treatment based on validated outcome measurements.

Topics: Adult; Aged; Benzamides; Double-Blind Method; Female; Fibrosis; Humans; Imatinib Mesylate; Male; Middle Aged; Piperazines; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Pyrimidines; Quality of Life; Scleroderma, Diffuse; Skin; Transforming Growth Factor beta; Treatment Outcome

Treg abnormalities have been implicated in the pathogenesis of systemic sclerosis (SSc). Treg subpopulations and their cytokines, IL-10 and TGF-β in the peripheral blood of early stage SSc patients were investigated. We hypothesized that epigenetically regulated methylation of the FOXP3 promoter and enhancer regions are altered in Tregs of SSc patients, which might be involved in the T cell imbalance. CD4+CD25+Foxp3+CD127- Treg cells were significantly elevated in patients with diffuse cutaneous SSc and in patients with anti-Scl-70/RNA-Pol-III autoantibody positivity and with lung fibrosis. Increased CD62L+ Treg cells were present in all SSc subgroups. The production of immunosuppressive cytokines by both CD127- and CD62L+ Tregs was diminished. We observed reduced methylation of Treg specific FOXP3 enhancer regions, and elevated FOXP3 gene expression in active SSc cases with negative correlation in the frequency of CD62L+IL-10+ Tregs. Our data indicate an inappropriate distribution and cytokine production of Treg cells in early form SSc.

Topics: Adult; Aged; Antibodies, Antinuclear; DNA Methylation; DNA Topoisomerases, Type I; Epigenesis, Genetic; Female; Forkhead Transcription Factors; Gene Expression; Gene Expression Regulation; Humans; Interleukin-10; Middle Aged; Nuclear Proteins; Promoter Regions, Genetic; Pulmonary Fibrosis; RNA Polymerase III; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect.. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression.. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes.. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy.

Topics: Actins; Adult; Aged; Cell Differentiation; Cells, Cultured; Chemokine CCL5; Collagen Type I; Collagen Type I, alpha 1 Chain; Culture Media, Conditioned; Down-Regulation; Female; Fibroblasts; Fibrosis; Gene Expression Profiling; Humans; Immunohistochemistry; Keratinocytes; Male; Middle Aged; NF-kappa B; PPAR gamma; Real-Time Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Diffuse; Scleroderma, Localized; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Up-Regulation

To study the role of B lymphocytes in systemic sclerosis (SSc).. Peripheral B cell subpopulations and the production of interleukin-6 (IL-6) and transforming growth factor β (TGFβ) were analyzed using flow cytometry and multiplex assay. The fibroblast proliferation rate upon incubation with supernatants from B cells isolated from SSc patients or healthy controls was assessed using XTT, bromodeoxyuridine, and Ki-67. Collagen production was assessed using a collagen assay.. Ninety untreated patients (12 males) fulfilling the American College of Rheumatology/European League Against Rheumatism criteria for SSc (23 with diffuse cutaneous SSc [dcSSc] and 67 with limited cutaneous SSc [lcSSc]) and 30 healthy controls were recruited. Increased proportions of B cells expressing CD69 and CD95 were identified among the patients with SSc. B lymphocytes from dcSSc patients versus lcSSc patients and healthy controls expressed increased proportion of cells positive for CD5 (mean ± SD 24.12 ± 7.93% versus 14.09 ± 6.58% [P = 0.03] and 14.21 ± 5.34% [P = 0.01]), CD86 (39.89 ± 22.11% versus 17.72 ± 13.98% [P = 0.0007] and 11.68 ± 11.09% [P < 0.001]), IL-6 receptor (IL-6R; 33.64 ± 23.12% versus 17.91 ± 13.62% [P < 0.0001] and 12.08 ± 8.68% [P = 0.0009]), or IL-21R (32.55 ± 20.19% versus 5.76 ± 4.40% [P < 0.0001] and 5.93 ± 3.29% [P < 0.0001]). In addition, the levels of IL-6 (mean ± SD 314.3 ± 317.8 pg/ml versus 6.10 ± 2.58 pg/ml; P = 0.0007) and TGFβ (mean ± SD 1,020 ± 569 pg/ml versus 163.8 ± 98.69 pg/ml; P = 0.001) secreted by B lymphocytes from patients with SSc were increased compared to healthy controls. Fibroblast proliferation and collagen production were also significantly increased in the presence of B cell supernatant from SSc patients as compared to healthy controls.. The numbers of activated B cells were increased in SSc patients, and the up-regulation of CD5, CD86, IL-6R, and IL-21R discriminated between patients with dcSSc and those with lcSSc. Peripheral B lymphocytes from SSc patients secreted both IL-6 and TGFβ, and they activated fibroblasts in vitro.

Topics: B-Lymphocytes; B7-2 Antigen; Case-Control Studies; CD5 Antigens; Cell Proliferation; Cells, Cultured; Collagen; Female; Fibroblasts; Humans; Immunohistochemistry; Interleukin-6; Male; Receptors, Interleukin-21; Receptors, Interleukin-6; Scleroderma, Diffuse; Scleroderma, Limited; Transforming Growth Factor beta

Microvascular damage is pivotal in the pathogenesis of systemic sclerosis (SSc), preceding fibrosis, and whose trigger is not still fully understood. Perivascular progenitor cells, with profibrotic activity and function, are identified by the expression of the isoform 12 of ADAM (ADAM12) and this molecule may be upregulated by transforming growth factor-β (TGF-β). The goal of this work was to evaluate whether pericytes in the skin of patients with diffuse cutaneous SSc (dcSSc) expressed ADAM12, suggesting their potential contribution to the fibrotic process, and whether TGF-β might modulate this molecule.. After ethical approval, mesenchymal stem cells (MSC) and fibroblasts (FB) were isolated from bone marrow and skin samples collected from 20 patients with dcSSc. ADAM12 expression was investigated in the skin and in isolated MSC and FB treated with TGF-β by immunofluorescence, quantitative real-time PCR, and western blot. Further, we silenced ADAM12 expression in both dcSSc-MSC and -FB to confirm the TGF-β modulation.. Pericytes and FB of dcSSc skin showed an increased expression of ADAM12 when compared with healthy control skin. TGF-β in vitro treatment induced a significant increase of ADAM12 in both SSc-MSC and -FB, with the higher levels observed in dcSSc cells. After ADAM12 silencing, the TGF-β ability to upregulate α-smooth muscle actin in both SSc-MSC and SSc-FB was inhibited.. Our results suggest that in SSc, pericytes that transdifferentiate toward activated FB are present in the vascular tree, and TGF-β, while increasing ADAM12 expression, may modulate this transdifferentiation.

Topics: Actins; ADAM12 Protein; Adult; Cell Transdifferentiation; Female; Fibrosis; Humans; Male; Mesenchymal Stem Cells; Middle Aged; Myofibroblasts; Pericytes; Scleroderma, Diffuse; Skin; Transforming Growth Factor beta; Up-Regulation; Young Adult

Pulmonary arterial hypertension (PAH) can be idiopathic or secondary to autoimmune diseases, and it represents one of the most threatening complications of systemic sclerosis (SSc). Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with proinflammatory functions that appears to be involved in the pathogenesis of hypoxia-induced PH. In SSc patients, high serum levels of MIF have been associated with the development of ulcers and PAH. Stem cell growth factor β (SCGF β) is a human growth factor that, together with MIF, is involved in the pathogenesis of chronic spinal cord injury. The aim of our study was to measure serum levels of MIF in patients with idiopathic and SSc-associated PAH. We enrolled 13 patients with idiopathic PAH and 15 with SSc-associated PAH. We also selected 14 SSc patients without PAH and 12 normal healthy controls, matched for sex and age. PAH was confirmed by right hearth catheterism (mPAP>25 mmHg). MIF and SCGF β levels were measured by ELISA. We found significantly higher circulating levels of MIF and of SCGF β in patients with idiopathic PAH (P=0.03 and P=0.004) and with PAH secondary to SSc (P=0.018 and P=0.023) compared to SSc patients without PAH. Higher levels of MIF were found in those patients with an higher New York Heart Association (NYHA) class (P=0.03). We can hypothesize that MIF and SCGF β are able to play a role in PAH, both idiopathic or secondary, and in the future they may be evaluated as useful biomarkers and prognostic factors for this serious vascular disease.

Topics: Aged; Biomarkers; Case-Control Studies; Female; Humans; Hypertension, Pulmonary; Macrophage Migration-Inhibitory Factors; Middle Aged; Predictive Value of Tests; Prognosis; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; Sensitivity and Specificity; Transforming Growth Factor beta

Topics: Biomarkers; Biopsy, Needle; Cells, Cultured; Fibroblasts; Fibrosis; Humans; Imidazoles; Immunohistochemistry; Molecular Targeted Therapy; Phosphorylation; Polymerase Chain Reaction; Scleroderma, Diffuse; Sensitivity and Specificity; Sirolimus; Smad2 Protein; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Triazines

The present pilot study aims to evaluate the frequency and the function of regulatory T (Treg) cells in patients with diffuse cutaneous SSc (dcSSc) before and after autologous hematopoietic SCT (aHSCT). Peripheral blood lymphocytes from seven dcSSc patients were analyzed before and 24 months after aHSCT and were compared with those from seven healthy donors (controls). Immunophenotyping of CD4(+)CD25(high)FoxP3(+) natural Treg (nTreg), CD4(+)CD25(+)TGF-β(+) and CD4(+)CD25(+)IL-10(+) adaptive Treg (aTreg) cell subsets was performed using four-color flow cytometry. Treg-suppressive capability was measured after coculture with autologous T effector cells by evaluation of T-cell proliferation using (3)H-thymidine incorporation. Peripheral CD4(+)CD25(high)FoxP3(+) (2±0.5 vs 4.2±1.1, P<0.01), CD4(+)CD25(+)TGF-β(+) (6.9±1.8 vs 14.6±5.0, P<0.05) and CD4(+)CD25(+)IL-10(+) (10.7±0.5 vs 16.1±3.2, P<0.01) Tregs as well as CD4(+)CD25(high)CD127(low) Tregs suppressive capacity (P<0.05) were decreased in dcSSc patients vs controls. After aHSCT (n=7), the percentages of CD4(+)CD25(high)FoxP3(+) (4.1±1.8) and CD4(+)CD25(+)IL-10(+) (15.7±2.2) Treg cells and the suppressive activity of CD4(+)CD25(high)CD127(low) were restored to the levels in controls. The decreased frequency and the functional defect of peripheral Treg cells from patients with dcSSc are reversed following aHSCT to reach those observed in controls. This pilot study brings evidence of an effective restoration of nTreg and aTreg subsets, and recovery of nTreg suppressive function following aHSCT.

Topics: Adult; CD4-Positive T-Lymphocytes; Cell Proliferation; Coculture Techniques; Female; Forkhead Transcription Factors; Hematopoietic Stem Cell Transplantation; Humans; Immunophenotyping; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Interleukin-7 Receptor alpha Subunit; Lymphocytes; Male; Middle Aged; Pilot Projects; Scleroderma, Diffuse; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Transplantation, Autologous

To assess if an impaired cross-talk between endothelial cells (ECs) and perivascular/multipotent mesenchymal stem cells (MSCs) might induce a perturbation of vascular repair and leading to a phenotypic switch of MSC toward myofibroblast in Systemic Sclerosis (SSc).. We investigated different angiogenic and profibrotic molecules in a tridimentional matrigel assay, performing co-cultures with endothelial cells (ECs) and bone marrow derived MSCs from patients and healthy controls (HC). After 48 hours of co-culture, cells were sorted and analyzed for mRNA and protein expression.. ECs-SSc showed a decreased tube formation ability which is not improved by co-cultures with different MSCs. After sorting, we showed: i. an increased production of vascular endothelial growth factor A (VEGF-A) in SSc-MSCs when co-cultured with SSc-ECs; ii. an increased level of transforming growth factor beta (TGF-β) and platelet growth factor BB (PDGF-BB) in SSc-ECs when co-cultured with both HC- and SSc-MSCs; iii. an increase of TGF-β, PDGF-R, alpha smooth muscle actin (α-SMA) and collagen 1 (Col1) in both HC- and SSc-MSCs when co-cultured with SSc-ECs.. We showed that during SSc, the ECs-MSCs crosstalk resulted in an altered expression of different molecules involved in the angiogenic processes, and mainly SSc-ECs seem to modulate the phenotypic switch of perivascular MSCs toward a myofibroblast population, thus supporting the fibrotic process.

Topics: Actins; Adult; Becaplermin; Blotting, Western; Cell Communication; Cells, Cultured; Coculture Techniques; Collagen Type I; Collagen Type I, alpha 1 Chain; Endothelial Cells; Endothelium, Vascular; Female; Gene Expression; Humans; Male; Mesenchymal Stem Cells; Middle Aged; Myofibroblasts; Neovascularization, Physiologic; Proto-Oncogene Proteins c-sis; Receptors, Platelet-Derived Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Diffuse; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Young Adult

Uncontrolled fibrosis in multiple organs is the main cause of death in systemic sclerosis (SSc), and transforming growth factor-β (TGF-β) activation plays a fundamental role in the process. Our previous study demonstrated that miR-21 was significantly up-regulated in SSc fibroblasts. Here, we found that TGF-β regulated the expression of miR-21 and fibrosis-related genes, and decreased Smad7 expression. Over-expression of miR-21 in fibroblasts decreased the levels of Smad7, whereas knockdown of miR-21 increased its expression. Further study using a reporter gene assay demonstrated Smad7 was a direct target of miR-21. Similar to human SSc, the expression of miR-21 increased in the bleomycin induced skin fibrosis. Inhibition of fibrosis by treatment with anti-fibrosis drug bortezomib restored the levels of miR-21 and Smad7. MiR-21 may function in an amplifying circuit to enhance TGF-β signaling events in SSc fibrosis, and suggesting that miR-21 may act as a potential therapeutic target.

Topics: Animals; Bleomycin; Boronic Acids; Bortezomib; Cells, Cultured; Female; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Mice; Mice, Inbred DBA; MicroRNAs; Molecular Targeted Therapy; Pyrazines; RNA, Small Interfering; Scleroderma, Diffuse; Signal Transduction; Skin; Smad7 Protein; Transforming Growth Factor beta

Systemic sclerosis (SSc) is still an enigmatic disease of unknown etiology, although the pathophysiology is thought to be based on vascular alterations as well as immunological and fibrotic processes. Here we present the case of a female patient with diffuse SSc (dSSc), who developed multiple subcutaneous nodules. Histologic evaluation confirmed the diagnosis of nodular scleroderma, a very rare condition. Histological analysis of biopsies was combined with ultrastructural analysis by transmission electron microscopy and immunohistochemistry/immunofluorescence, using antibodies against different collagens and non-collagenous ECM proteins. Collagen fibrils were deposited at very high density in nodules as well as in adjacent extra nodular skin. Within nodules, a large fraction of immature collagen fibrils was detected with smaller and highly variable diameter. Activated fibroblasts were present, however no myofibroblasts were identified. Cartilage Oligomeric Matrix Protein (COMP), collagen XII and fibrillin-1 were all deposited at increased amounts within nodules and their distribution differed markedly from that in healthy skin. The excessive deposition of COMP within nodules closely resembled the distribution of COMP in keloids. Nodules-like keloids-were characterized by lack of myofibroblasts. By virtue of its structural properties and the capacity to avidly bind collagen I and XII, COMP is thought to reorganize and compact the collagen network, leading to a tissue with locally increased biomechanical tension acting on fibroblasts. In addition, COMP may present active TGFβ to fibroblasts. Both mechanisms in concert can activate fibroblast proliferation and production of extracellular matrix, resulting in a sustained activation loop.

Topics: Cartilage Oligomeric Matrix Protein; Cells, Cultured; Collagen Type XII; Extracellular Matrix; Female; Fibrillin-1; Fibrillins; Fibroblasts; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Keloid; Microfilament Proteins; Middle Aged; Scleroderma, Diffuse; Skin; Transforming Growth Factor beta

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.. 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.. 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.. Down-regulation of miR-30b might be involved in the pathogenesis of SSc.

Topics: Adult; Aged; Animals; Bleomycin; Case-Control Studies; Cells, Cultured; Down-Regulation; Female; Fibroblasts; Gene Expression Regulation; Humans; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Middle Aged; Receptor, Platelet-Derived Growth Factor beta; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; Sclerosis; Skin; Transfection; Transforming Growth Factor beta

To explore the expression of thymic stromal lymphopoietin (TSLP) in patients with diffuse cutaneous systemic sclerosis (dcSSc) and compare its effects in vivo and in vitro with those of interleukin-13 (IL-13) and transforming growth factor β (TGFβ).. Skin biopsy specimens from patients with dcSSc (n = 14) and healthy controls (n = 13) were analyzed by immunohistochemistry and immunofluorescence for TSLP, TSLP receptor, CD4, CD8, CD31, and CD163 markers. Wild-type, IL-4Rα1-, and TSLP-deficient mice were treated with TGFβ, IL-13, poly(I-C), or TSLP by osmotic pump. Human fibroblasts and peripheral blood mononuclear cells (PBMCs) were stimulated with TGFβ, IL-13, poly(I-C), or TSLP. Microarray analysis and quantitative polymerase chain reaction were performed to determine gene expression, and protein levels of phospho-Smad2 and macrophage marker CD163 were tested.. TSLP was highly expressed in the skin of dcSSc patients, more strongly in perivascular areas and in immune cells, and was produced mainly by CD163+ cells. The skin of TSLP-treated mice showed up-regulated clusters of gene expression that overlapped strongly with those in IL-13- and TGFβ-treated mice. TSLP up-regulated specific genes, including CXCL9, proteasome, and interferon (IFN)-regulated genes. TSLP treatment in IL-4Rα1-deficient mice promoted similar cutaneous inflammation as in wild-type mice, though TSLP-induced arginase 1, CCL2, and matrix metalloproteinase 12 messenger RNA levels were blocked. In PBMCs, TSLP up-regulated tumor necrosis factor α, Mx-1, IFNγ, CXCL9, and mannose receptor 1 gene expression. TSLP-deficient mice treated with TGFβ showed less fibrosis and blocked expression of plasminogen activator inhibitor 1 and osteopontin 1. Poly(I-C)-treated mice showed high levels of cutaneous TSLP.. TSLP is highly expressed in the skin of dcSSc patients and interacts in a complex manner with 2 other profibrotic cytokines, TGFβ and IL-13, strongly suggesting that it might promote SSc fibrosis directly or indirectly by synergistically stimulating profibrotic genes, or production of these cytokines.

Topics: Animals; Biomarkers; Cytokines; Fibroblasts; Fibrosis; Gene Expression; Humans; Interleukin-13; Intracellular Signaling Peptides and Proteins; Leukocytes, Mononuclear; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Poly I-C; RNA, Messenger; Scleroderma, Diffuse; Skin; Thymic Stromal Lymphopoietin; Transforming Growth Factor beta; Up-Regulation

Scleroderma (systemic sclerosis, SSc) is a complex autoimmune disease caused by progressive fibrotic replacement of normal tissue architecture, a progressive and ultimately fatal process that currently has no cure. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in SSc is unclear. In comparison with the normal skin tissues, miRNAs were aberrantly expressed in limited cutaneous scleroderma and diffuse cutaneous scleroderma skin tissues. We also identified miRNAs whose expressions were correlated with SSc fibrosis: miR-21, miR-31, miR-146, miR-503, miR-145, and miR-29b were predicted to be involved. This study further confirmed that miR-21 was increased whereas miR-145 and miR-29b were decreased both in the skin tissues and fibroblasts. As predicted target genes, SMAD7, SAMD3, and COL1A1 were regulated by these miRNAs. After stimulation with transforming growth factor β, the expression of miR-21 was increased and that of SMAD7 mRNA was decreased. MiR-145 was upregulated whereas the mRNA level of SMAD3 was downregulated. The downregulation of miR-29b was correlated with the upregulation of COL1A1 mRNA. MiRNAs might play an important role in the pathogenesis of SSc and suggest a potential therapy.

Topics: Adult; Collagen Type I; Collagen Type I, alpha 1 Chain; Female; Humans; Male; MicroRNAs; Middle Aged; RNA, Messenger; Scleroderma, Diffuse; Scleroderma, Limited; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta; Young Adult

microRNAs (miRNAs) play a part in various cellular activities. However, the role of miRNA in SSc is not fully understood. This study investigated the expression and role of miR-92a in SSc patients and evaluated the possibility that miR-92a is involved in the pathogenesis of this disease.. Serum samples were obtained from 61 SSc patients. mRNAs were purified from serum and levels of miR-92a and miR-135 were measured with quantitative real-time PCR. miR-92a expression in dermal fibroblasts was also determined by quantitative real-time PCR. Immunoblotting was performed to detect MMP-1 protein.. The median serum levels of miR-92a, not miR-135, were significantly higher in SSc patients than normal subjects. The constitutive up-regulated miR-92a expression was also found in cultured dermal fibroblasts from SSc skin, which was decreased by the transfection with siRNA of TGF-β. Furthermore, the forced overexpression of miR-92a in normal dermal fibroblasts using miR-92a mimic resulted in the down-regulation of MMP-1 expression.. The increase of miR-92a in SSc may be due to the stimulation of intrinsic TGF-β activation seen in this disease. There is also a possibility that MMP-1 is the target of miR-92a and that increased miR-92a expression therefore plays a role in excessive collagen accumulation in SSc via the down-regulation of MMP-1. Clarifying the role of miRNAs in SSc may result in a better understanding of this disease and the development of new therapeutic approaches.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Cells, Cultured; Dermatomyositis; Dermis; Female; Fibroblasts; Gene Expression Regulation; Gene Silencing; Humans; Integrin alphaVbeta3; Lupus Erythematosus, Systemic; Male; Matrix Metalloproteinase 1; MicroRNAs; Middle Aged; RNA, Small Interfering; Scleroderma, Diffuse; Scleroderma, Localized; Transforming Growth Factor beta

TGF-β is the primary inducer of extracellular matrix proteins in scleroderma (systemic sclerosis, SSc). Previous studies indicate that in a subset of SSc fibroblasts TGF-β signaling is activated via elevated levels of activin receptor-like kinase (ALK) 1 and phosphorylated Smad1 (pSmad1). The goal of this study was to determine the role of endoglin/ALK1 in TGF-β/Smad1 signaling in SSc fibroblasts. In SSc fibroblasts, increased levels of endoglin correlated with high levels of pSmad1, collagen, and connective tissue growth factor (CCN2). Endoglin depletion via siRNA in SSc fibroblasts inhibited pSmad1 but did not affect pSmad2/3. Following endoglin depletion mRNA and protein levels of collagen and CCN2 were significantly decreased in SSc fibroblasts but remained unchanged in normal fibroblasts. ALK1 was expressed at similar levels in SSc and normal fibroblasts. Depletion of ALK1 resulted in inhibition of pSmad1 and a moderate but significant reduction of mRNA and protein levels of collagen and CCN2 in SSc fibroblasts. Furthermore, constitutively high levels of endoglin were found in complexes with ALK1 in SSc fibroblasts. Overexpression of constitutively active ALK1 (caALK1) in normal and SSc fibroblasts led to a moderate increase of collagen and CCN2. However, caALK1 potently induced endothelin 1 (ET-1) mRNA and protein levels in SSc fibroblasts. Additional experiments demonstrated that endoglin and ALK1 mediate TGF-β induction of ET-1 in SSc and normal fibroblasts. In conclusion, this study has revealed an important profibrotic role of endoglin in SSc fibroblasts. The endoglin/ALK1/Smad1 pathway could be a therapeutic target in patients with SSc if appropriately blocked.

Topics: Activin Receptors, Type II; Antigens, CD; Collagen; Connective Tissue Growth Factor; Endoglin; Endothelin-1; Enzyme Activation; Fibroblasts; Fibrosis; HEK293 Cells; Humans; Mutation; Phenotype; Phosphorylation; Receptors, Cell Surface; RNA Interference; RNA, Messenger; Scleroderma, Diffuse; Signal Transduction; Skin; Smad1 Protein; Smad2 Protein; Smad3 Protein; Transfection; Transforming Growth Factor beta; Up-Regulation

To determine the skin and fibroblast expression of ephrins (EphB4 and EphrinB2) and thrombospondins (TSPs: TSP1 and TSP2) in patients with SSc.. All experiments were performed in skin sections and dermal fibroblasts issued from control and clinically involved/non-involved SSc skin biopsies. Dermal fibroblasts were stimulated with hypoxia or TGF-β, or treated with TGF-β-neutralizing antibodies. Ephrin and TSP mRNA levels were assessed in skin tissue and dermal fibroblasts by in situ hybridization and quantitative RT-PCR, respectively, and protein levels were assessed by immunohistochemistry and western blots, respectively.. Enhanced ephrin and TSP mRNA and protein levels were observed in clinically involved SSc skin. EphrinB2, TSP1 and TSP2 mRNA and protein levels were also up-regulated in non-involved SSc skin. Similar mRNA and protein levels of ephrinB2 and EphB4 were detected in unstimulated and stimulated control and SSc dermal fibroblasts. TSP1 and TSP2 mRNA and protein levels were significantly increased in fibroblasts issued from involved and non-involved SSc skin. This up-regulation was not modified by hypoxic exposure, but was markedly reduced by the addition of TGF-β-neutralizing antibodies. Stimulation of healthy fibroblasts with TGF-β significantly increased TSP1 and TSP2 mRNA and protein levels.. EphB4 and EphrinB2 are up-regulated in clinically involved skin of SSc patients, suggesting their participation in SSc-perturbed angiogenesis. TSP1 and TSP2 are up-regulated in both clinically involved and non-involved SSc skin and are constitutively overexpressed in a TGF-β-dependent and hypoxia-independent manner in SSc dermal fibroblasts, suggesting their potential early contribution in SSc pathogenesis.

Topics: Antibodies, Neutralizing; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cell Hypoxia; Cells, Cultured; Dermis; Ephrin-B2; Female; Fibroblasts; Fibrosis; Gene Expression; Humans; In Situ Hybridization; Male; Middle Aged; Neovascularization, Pathologic; Receptor, EphB4; Recombinant Proteins; Scleroderma, Diffuse; Thrombospondins; Transforming Growth Factor beta

Systemic sclerosis is a complex disease with widespread skin fibrosis and variable visceral organ involvement. Since transforming growth factor-beta (TGFbeta) has been implicated in driving fibrosis in systemic sclerosis, a mechanism-derived gene expression signature was used to assay TGFbeta-responsive gene expression in the skin of patients with systemic sclerosis (SSc). Primary dermal fibroblasts from patients with diffuse SSc (dSSc) and healthy controls were treated with TGFbeta, and the genome-wide gene expression was measured on DNA microarrays over a time course of 24 hours. Eight hundred and ninety-four probes representing 674 uniquely annotated genes were identified as TGFbeta responsive. Expression of the TGFbeta-responsive signature was examined in skin biopsies from 17 dSSc, seven limited SSc (lSSc), three morphea patients, and six healthy controls. The TGFbeta-responsive signature was expressed in 10 out of 17 dSSc skin biopsies, but was not found in lSSc, morphea, or healthy control biopsies. Expression of dSSC the TGFbeta-responsive signature stratifies patients into two major groups, one of which corresponds to the "diffuse-proliferation" intrinsic subset that showed higher modified Rodnan skin score and a higher likelihood of scleroderma lung disease. The TGFbeta-responsive signature is found in only a subset of dSSc patients who could be targeted by specific therapies.

Topics: Adult; Biopsy; Cell Division; Cells, Cultured; Dermis; Fibroblasts; Gene Expression Regulation; Genome, Human; Humans; Lung Diseases; Oligonucleotide Array Sequence Analysis; Scleroderma, Diffuse; Severity of Illness Index; Transforming Growth Factor beta

It has been demonstrated that the endocannabinoid system is up-regulated in pathologic fibrosis and that modulation of the cannabinoid receptors might limit the progression of uncontrolled fibrogenesis. The aim of this study was to investigate whether the synthetic cannabinoid receptor agonist WIN55,212-2 could modulate fibrogenesis in an in vitro model of dcSSc.. The expression of cannabinoid receptors CB1 and CB2 was assessed in dcSSc fibroblasts and healthy control fibroblasts. To investigate the effect of WIN55,212-2 on dcSSc fibrogenesis, we studied type I collagen, profibrotic cytokines, fibroblast transdifferentiation into myofibroblasts, apoptotic processes and activation of the extracellular signal-related kinase 1/2 pathway prior to and after the treatment with the synthetic cannabinoid at increasing concentrations.. Both CB1 and CB2 receptors were over-expressed in dcSSc fibroblasts compared with healthy controls. WIN55,212-2 caused a reduction in extracellular matrix deposition and counteracted several behavioural abnormalities of scleroderma fibroblasts including transdifferentiation into myofibroblasts and resistance to apoptosis. The anti-fibrogenic effect of WIN55,212-2 was not reverted by selective cannabinoid antagonists.. Our preliminary findings suggest that cannabinoids are provided with an anti-fibrotic activity, thereby possibly representing a new class of agents targeting fibrosis diseases.

Topics: Aged; Apoptosis; Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoids; Cell Survival; Cells, Cultured; Collagen Type I; Connective Tissue Growth Factor; Drug Evaluation, Preclinical; Female; Fibroblasts; Gene Expression Regulation; Humans; Interleukin-6; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Naphthalenes; Phosphorylation; Receptors, Cannabinoid; RNA, Messenger; Scleroderma, Diffuse; Skin; Transforming Growth Factor beta

A clinico-pathological study in diffuse systemic sclerosis (SSc) patients was performed to analyse whether the skin histological organization and the pro-fibrotic signals elicited by TGF-beta in fibroblasts vary according to the modified Rodnan skin score (mRSS).. Twenty-seven SSc patients underwent 45 skin biopsies with simultaneous measure of mRSS before or after treatment by immunosuppressive drugs, with or without autologous peripheral haematopoietic stem cell transplantation (HSCT).. Double-blind optic microscopy analysis of the biopsies standard extracellular matrix stains allowed to define three histological subgroups: 6 with grade 1 weak fibrosis, 30 with grade 2 moderate fibrosis and 9 with grade 3 severe fibrosis. A significant (P < 0.0001) was identified between the grades of fibrosis and the mRSS. In skin fibroblast cultures, Smad3 phosphorylation levels, as well as mRNA steady-state levels of two transforming growth factor (TGF)-beta/Smad3 targets, COL1A2 and PAI-1, increased in parallel with the mRSS. When compared with pre-transplant values the degree of fibrosis observed after HSCT in the papillary and in the reticular dermis decreased in parallel with the fall in mRSS (n = 5 consecutive patients with repeated biopsies).. The histological extent of skin fibrosis correlates closely with the mRSS. Both parameters appeared to regress after HSCT. The extent of TGF-beta signalling activation in SSc skin fibroblasts appears to parallel the severity of disease.

Topics: Adult; Aged; Biopsy; Cells, Cultured; Collagen; Collagen Type I; Combined Modality Therapy; Double-Blind Method; Female; Fibroblasts; Fibrosis; Humans; Immunoenzyme Techniques; Immunosuppressive Agents; Male; Middle Aged; Peripheral Blood Stem Cell Transplantation; Phosphorylation; Plasminogen Activator Inhibitor 1; RNA, Messenger; Scleroderma, Diffuse; Severity of Illness Index; Signal Transduction; Skin; Smad Proteins; Smad3 Protein; Transforming Growth Factor beta

To determine the relationship between clinical features and circulating levels of active transforming growth factor (TGF) beta1 in the major subsets of systemic sclerosis (SSc).. In a cross-sectional study cases of diffuse cutaneous SSc (dose) (n = 27) or limited cutaneous SSc (dose) (n = 20) were compared with healthy controls (n = 22). Active and total TGFbeta1 was measured in serum and plasma by a high-sensitivity enzyme-linked immunosorbent assay.. There were no significant differences between levels of total serum TGFbeta1. However, cases of dcSSc had lower levels of active TGFbeta1 than cases of lcSSc or controls. In addition, more cases of dcSSc (18/27; 66%, P < 0.025) had no detectable active TGFbeta1 than controls (7/22, 32%) or lcSSc (7/20, 35%). In dcSSc, serum active TGFbeta1 levels correlated negatively with skin score and positively with disease duration.. Contrary to expectation, levels of active TGFbeta1 are reduced in dcSSc and this correlates with two variables known to associate with disease activity, shorter duration and more extensive skin sclerosis. This suggests that active TGFbeta1 may be sequestered in active involved SSc skin and that serum levels are reduced despite strong evidence implicating TGFbeta isoforms in the pathogenesis of fibrosis. Our findings may have implications for systemic TGFbeta-trapping therapies in this disease.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Cross-Sectional Studies; Female; Humans; Male; Middle Aged; Scleroderma, Diffuse; Scleroderma, Limited; Severity of Illness Index; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

We sought to determine whether the spontaneous production of transforming growth factor-beta (TGF-beta) by peripheral blood mononuclear cells (PBMC) is increased in patients with systemic sclerosis (SSc). Culture supernatants of PBMC from SSc patients (n = 88) and healthy controls (n = 44) were analyzed by enzyme-linked immunosorbent assay. The production of active TGF-beta1 and total (active and latent) TGF-beta1 by PBMC from patients with limited cutaneous SSc (lSSc) and by PBMC from patients with diffuse cutaneous SSc (dSSc) was significantly elevated compared to the production by PBMC from normal controls. Production of active TGF-beta1 by dSSc PBMC was higher than that by lSSc PBMC, although not significantly. Patients with PBMC with increased active or total TGF-beta1 production showed significantly shorter disease duration than patients with PBMC with normal production levels. PBMC from patients without anticentromere antibody showed enhanced active TGF-beta1 production more frequently than those from patients with anticentromere antibody. PBMC from SSc patients more frequently showed enhanced total TGF-beta2 production than PBMC from normal controls. Among each leukocyte subset, spontaneous production of total TGF-beta1 was significantly higher in cultured peripheral monocytes/macrophages, but not in T cells, B cells, or NK cells, from patients than from normal controls. Thus, the enhanced production of TGF-beta by PBMC may contribute to the disease process in SSc

Topics: Adult; Case-Control Studies; Cells, Cultured; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Prednisolone; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

TGF-beta is implicated in the pathogenesis of fibrotic disorders. It has been shown that Smad3 promotes the human alpha2(I) collagen (COL1A2) gene expression by TGF-beta1 in human dermal fibroblasts. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K) in the COL1A2 gene expression in normal and scleroderma fibroblasts. In normal fibroblasts, the PI3K inhibitor, LY294002, significantly decreased the basal and the TGF-beta1-induced increased stability of COL1A2 mRNA. The TGF-beta1-induced COL1A2 promoter activity, but not the basal activity, was significantly attenuated by LY294002 or the dominant negative mutant of p85 subunit of PI3K, while the constitutive active mutant of p110 subunit of PI3K did not affect the basal or the TGF-beta1-induced COL1A2 promoter activity. LY294002 significantly decreased the phosphorylation of Smad3 induced by TGF-beta1. Furthermore, the transient overexpression of 2xFYVE, which induces the mislocalization of FYVE domain proteins, decreased the TGF-beta1-induced Smad3 phosphorylation to a similar extent to LY294002. In scleroderma fibroblasts, the blockade of PI3K significantly decreased the mRNA stability and the promoter activity of the COL1A2 gene. Furthermore, LY294002 and the transient overexpression of 2xFYVE completely diminished the constitutive phosphorylation of Smad3. These results indicate that 1) the basal activity of PI3K is necessary for the COL1A2 mRNA stabilization in normal and scleroderma fibroblasts, 2) there is an unidentified FYVE domain protein specifically interacting with Smad3, and 3) the basal activity of PI3K and the FYVE domain protein are indispensable for the efficient TGF-beta/Smad3 signaling in normal fibroblasts and for the establishment of the constitutive activation of TGF-beta/Smad3 signaling in scleroderma fibroblasts.

Topics: Cells, Cultured; Chromones; Collagen Type I; DNA; DNA-Binding Proteins; Fibroblasts; Gene Expression; Humans; Morpholines; Phosphatidylinositol 3-Kinases; Phosphorylation; Promoter Regions, Genetic; Scleroderma, Diffuse; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta