transforming-growth-factor-beta and Scleroderma--Limited

Endothelial cell (EC) dysfunction has been associated with inflammatory and autoimmune diseases; however, the factors contributing to this dysfunction have not been fully explored. Because activation of TLRs has been implicated in autoimmune diseases, the goal of this study was to determine the effects of TLR ligands on EC function. Human dermal microvascular ECs (HDMECs) treated with TLR3 [Poly(I:C)], TLR4 (LPS), and TLR7 (imiquimod) agonists showed decreased proliferation and a reduced total number of branching tubules in three-dimensional human dermal organoid ex vivo culture. In contrast, the TLR9 ligand class C, ODN2395, increased angiogenesis. The antiproliferative effects of TLR3, TLR4, and TLR7 ligands correlated with significant downregulation of a key regulator of vascular homeostasis, Fli1, whereas TLR9 increased Fli1 levels. Furthermore, Poly(I:C) and LPS induced endothelial to mesenchymal transition that was reversed by the pretreatment with TGF-β neutralizing Ab or re-expression of Fli1. We showed that Fli1 was required for the HDMEC proliferation by transcriptionally repressing FOXO3A. In contrast to TLR9, which suppressed activation of the FOXO3A pathway, TLR3, TLR4, and TLR7 ligands activated FOXO3A as indicated by decreased phosphorylation and increased nuclear accumulation. The inverse correlation between Fli1 and FOXO3A was also observed in the vasculature of scleroderma patients. This work revealed opposing effects of TLR9 and TLR3, TLR4, and TLR7 on the key angiogenic pathways, Fli1 and FOXO3A. Our results provide a mechanistic insight into the regulation of angiogenesis by TLRs and confirm a central role of Fli1 in regulating vascular homeostasis.

Topics: Adult; Aminoquinolines; Cell Line; Dermis; Endothelium, Vascular; Female; Forkhead Box Protein O3; Humans; Imiquimod; Lipopolysaccharides; Male; Microvessels; Middle Aged; Oligodeoxyribonucleotides; Poly I-C; Proto-Oncogene Protein c-fli-1; RNA, Small Interfering; Scleroderma, Limited; Signal Transduction; Toll-Like Receptors; Transforming Growth Factor beta

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

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

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

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

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

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