endothelin-1 and Scleroderma--Systemic

Systemic sclerosis (SSc) is a disease of connective tissue with high rate of morbidity and mortality highlighted by extreme fibrosis affecting various organs such as the dermis, lungs, and heart. Until now, there is no specific cure for the fibrosis occurred in SSc disease. The SSc pathogenesis is yet unknown, but transforming growth factor beta (TGF-β), endothelin-1 (ET-1), and Ras-ERK1/2 cascade are the main factors contributing to the tissue fibrosis through extracellular matrix (ECM) accumulation. Several studies have hallmarked the association of ET-1 with or without TGF-β and Ras-ERK1/2 signaling in the development of SSc disease, vasculopathy, and fibrosis of the dermis, lungs, and several organs. Accordingly, different clinical and experimental studies have indicated the potential therapeutic role of ET-1 and Ras antagonists in these situations in SSc. In addition, ET-1 and connective tissue growth factor (CTGF) as a cofactor of the TGF-β cascade play a substantial initiative role in inducing fibrosis. Once initiated, TGF-β alone or in combination with ET-1 and CTGF can activate several kinase proteins such as the Ras-ERK1/2 pathway that serve as the fundamental factor for developing fibrosis. Furthermore, Salirasib is a synthetic small molecule that is able to inhibit all Ras forms. Therefore, it can be used as a potent therapeutic factor for fibrotic disorders. So, this review discusses the role of TGF-β/ET-1/Ras signaling and their involvement in SSc pathogenesis, particularly in its fibrotic situation.

Topics: Connective Tissue Growth Factor; Endothelin-1; Fibroblasts; Fibrosis; Humans; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

There are important knowledge gaps concerning scleroderma renal crisis (SRC), in large part because its rarity has hampered research. Many studies to date share limitations, in particular small samples, prevalent cases, and retrospective study designs. This review features some of the most recent studies that attempt to shed new insights into SRC while trying to overcome those limitations.. The most important recent progress in the understanding of the pathophysiology of SRC includes identification of novel genetic and serological biomarkers. Outcomes of SRC remain poor and there are also ongoing efforts to identify novel therapeutic strategies, in particular targeting the endothelin-1 pathway.. Meaningful improvement in outcomes of SRC will be predicated on greater understanding of the underlying mechanisms of disease and identification of novel therapeutic and preventive strategies. Some efforts are ongoing but ultimately, international cooperation will be necessary to achieve this for a rare complication of a rare disease.

Topics: Endothelin-1; Humans; Kidney Diseases; Risk Factors; Scleroderma, Systemic

Systemic sclerosis (SSc) is a multifactorial connective tissue disease characterized by excessive and progressive fibrosis along with microvasculopathy due to poor vascular formation and repair. Despite a general increase in many potent angiogenic factors, the vasculopathy compensatory angiogenesis and vasculogenesis are impaired. In this review, we discuss the role of proangiogenic factors--VEGF, PlGF, endoglin, PDGF, endothelin-1, angiopoietins, SDF-1, uPAR--and the paradoxical paucity of an inadequate angiogenic response in SSc.

Topics: Angiopoietins; Chemokine CXCL12; Cytokines; Endothelin-1; Humans; Placenta Growth Factor; Pregnancy Proteins; Scleroderma, Systemic; Wound Healing

Scleroderma (systemic sclerosis, SSc) is an autoimmune disease of unknown etiology characterized by organ fibrosis. There is no therapy for SSc. However, a recent body of evidence strongly implicates endothelin-1 (ET-1) in the pathogenesis of SSc. ET-1 is found in abundance in SSc patients. ET-1 directly induces fibrogenic effects in vitro, and is required for the ability of TGFβ to induce fibrogenic effects both in vitro and in vivo. Moreover, endothelin receptor antagonism reverses key features of the persistent fibrotic phenotype of fibroblasts isolated from lesions of SSc patients. However, clinically, endothelin receptor antagonism alone has had mixed results. This minireview summarizes these observations.

Topics: Animals; Endothelin-1; Fibrosis; Humans; Mice; Molecular Targeted Therapy; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Systemic sclerosis (SSc) is an autoimmune disorder with clinical manifestations resulting from immune activation, fibrosis development and damage of small blood vessels. Although there have been no established treatments for SSc, lots of new treatments targeting organ and pathogenesis are in the process of development. Transforming growth factor (TGF)-beta is a major cytokine involved in the pathogenesis of fibrosis in SSc. The blockade of cell surface molecules capable of activating latent TGF-beta, blockade of ligand by the pan-isoform-specific antibody, soluble TGF-beta receptors and a recombinant latency associated peptide, as well as inhibitors for ALK5 and Smad3 are the potential strategies to abolish the pathological activation of TGF-beta signaling in SSc fibroblasts. Besides TGF-beta, connective tissue growth factor (CTGF)/CCN2, platelet-derived growth factor (PDGF) and endothelin-1 are the candidates for the new therapeutic targets. As for immune dysfunction in SSc, i.v. immunoglobulin infusion, stem cell transplantation and B-cell depletion are potential new therapies under or awaiting a randomized, double-blind, placebo-controlled trial, although their efficacies are still controversial. Phosphodiesterase-5 inhibitors, endothelin receptor antagonists and inhibitors for serotonin signaling are the new therapeutic targets for Raynaud's phenomenon, digital ulceration and pulmonary arterial hypertension in SSc. Imatinib mesylate may be a novel new therapy for fibrosis and vasculopathy in SSc because it reverses the expression levels of Fli1, which is a transcription factor downregulated in SSc through an epigenetic mechanism and is likely to be involved in the development of fibrosis and vasculopathy in this disease. Potential therapeutic targets other than those described above are also reviewed.

Topics: Animals; Autoimmune Diseases; B-Lymphocytes; Benzamides; Connective Tissue Growth Factor; Endothelin Receptor Antagonists; Endothelin-1; Humans; Imatinib Mesylate; Immunoglobulins; Mice; Phosphodiesterase 5 Inhibitors; Piperazines; Platelet-Derived Growth Factor; Pyrimidines; Randomized Controlled Trials as Topic; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Selective Serotonin Reuptake Inhibitors; Smad3 Protein; Transforming Growth Factor beta

Recent years have seen the advent and progress in our understanding of fibrosis and vasculopathy in systemic sclerosis, scleroderma (SSc) largely mediated through the development and study of novel animal models. The most well studied animal models of SSc involve the bleomycin model of induced fibrosis and the Tsk/+ model. However, even though these models provide useful insights into the pathogenesis of fibrosis and vasculopathy, they do not mimic the disease accurately.. Several mouse models have been developed that have specifically focused on the vasculopathy of SSc and have yielded relevant insights into this disorder further highlighting the novel mechanisms that may be responsible for this pathological feature. Furthermore, the contribution of the innate immune system mediated by the inflammasome in the induction of fibrosis has also demonstrated significant insights, possibly implicating an etiological mechanism of SSc. And recent transgenic or knockout animal models have emphasized the relevance of macrophage chemoattractant protein-1 (MCP-1), alpha-melanocyte stimulating hormone (α-MSH), and peroxisome proliferator-activated receptor-gamma (PPARγ) in fibrosis.. Recent advances in animal models of SSc have elucidated the involvement of relevant proteins that appear to mediate vasculopathy and also implicated the involvement of the innate immune system in fibrosis. These models have identified novel therapeutic targets that may lead to more effective treatments for this incurable disease.

Topics: Animals; Disease Models, Animal; Endothelin-1; Fibrosis; Fos-Related Antigen-2; Humans; Mice; Mice, Transgenic; Proto-Oncogene Protein c-fli-1; Scleroderma, Systemic

Systemic sclerosis-related pulmonary arterial hypertension (PAH) is a severe disease affecting about 1000 patients in France. In 2008, all scleroderma patients are screened for PAH by a yearly cardiac Doppler ultrasonography. The pathogenesis of systemic sclerosis-related PAH is poorly known but it seems that besides common arteriolar remodeling (media hypertrophy, intimal thickening, endothelial proliferation), venular lesions suggesting obstructive venous disease and inflammatory lesions may be also be involved. Prostacyclin and analogues, phosphodiesterase-5 inhibitors (sildenafil) and endothelin-1 receptor antagonists are proposed as specific treatments for systemic sclerosis-related PAH. Unlike bosentan, which is non-selective, inhibiting both ETA and ETB receptors, sodium sitaxentan is highly selective for ETA receptors; this could favor pulmonary vasodilation.

Topics: Altitude; Antihypertensive Agents; Bosentan; Echocardiography; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; France; Humans; Hypertension, Pulmonary; Hypoxia; Oxygen; Phosphodiesterase 5 Inhibitors; Prognosis; Scleroderma, Systemic; Sulfonamides

Systemic sclerosis (SSc), the focus of this review, is a generalized connective tissue disease that involves sclerotic changes in the skin and sometimes various other organ systems. Clinical outcomes have improved probably due to better management of the complications, but SSc is still considered to be incurable and diffuse cutaneous SSc carries high risk of fatality. Although the pathogenesis of SSc is still unknown, the basic mechanism appears to involve endothelial cell injury, overproduction of extracellular matrix (ECM), and aberrant immune activation. This review discusses recent studies that investigated the cellular and molecular mechanisms in the pathogenesis of SSc.

Topics: Antibodies, Antinuclear; Collagen; Endothelial Cells; Endothelin-1; Extracellular Matrix; Fibroblasts; Humans; Lymphocytes; Platelet-Derived Growth Factor; Scleroderma, Systemic; Transcription Factors; Transforming Growth Factor beta

Scleroderma [systemic sclerosis (SSc)] is a spectrum of connective tissue diseases characterized by micro- and macro-vasculopathy, inflammation and autoimmunity and tissue remodelling that often leads to excessive scarring and fibrosis in both interstitial and vascular compartments. Pre-clinical investigations and gene association studies have led to improved understanding of the cell and molecular mechanisms underlying disease pathogenesis and to the identification of key molecular candidates that may represent potentially useful disease biomarkers and effective therapeutic targets. Studies on the endothelin (ET) system, pre-dominantly ET-1 and the cell surface receptors [type A (ET(A))] and type B (ET(B))], have provided evidence for an important role of this system in the vascular and fibrotic pathologies in SSc. To date, promising clinical results, utilizing dual/mixed ET receptor antagonism have been obtained in two of the vascular complications associated with SSc, ischaemic digital ulceration and pulmonary arterial hypertension. Evidence suggests that ET-1 is able to activate and re-program the functional phenotypes of vascular smooth muscle cells, microvascular pericytes and tissue fibroblasts into pro-fibrogenic cell populations with myofibroblasts-like properties. The impact of receptor-selective, over mixed-receptor, antagonism has also been studied in vitro with respect to cell differentiation and proliferation, extracellular matrix synthesis, production and deposition and in pathological cellular contraction. However, the complexity of the ET system, potential for receptor cross-talk, interactions with down-stream signal transduction cascades, as well as the potent inter-relationships with other important ligand-receptor pathways have made in vivo studies difficult to unravel. Moreover, little information is available on the role of the ET system and receptor selectivity in the recruitment and activation of mesenchymal progenitor cells in tissue remodelling and fibrosis or on the early inflammatory response. Here, we discuss the available pre-clinical evidence for the role of the ET system in tissue repair, scarring and fibrosis, using the connective tissue diseases SSc and model systems of fibrogenesis.

Topics: Animals; Connective Tissue; Disease Models, Animal; Endothelin-1; Fibrosis; Humans; Intercellular Adhesion Molecule-1; Receptors, Endothelin; Scleroderma, Systemic; Signal Transduction

Fibrosis affects organs such as the skin, liver, kidney and lung and is a cause of significant morbidity. There is no therapy for fibrosis. Recent significant molecular insights into the signaling underlying fibrosis have been made. Transforming growth factor beta (TGF beta) signaling is a major contributor to fibrogenesis. The signaling mechanisms through which TGF beta induces fibrogenic responses have been under intense scrutiny. Moreover, the potent pro-fibrotic proteins endothelin-1 (ET-1) and CCN2 (connective tissue growth factor, CTGF) are believed to play an essential role in this process as downstream regulators or co-factors of TGF beta signaling. This review summarizes these recent crucial observations with emphasis on the disease scleroderma.

Topics: Animals; Connective Tissue Growth Factor; Endothelin-1; Fibrosis; Humans; Rats; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Although being classified as autoimmune connective tissue disease, dominant components of the pathophysiology of systemic sclerosis (SSc) consists of mechanisms of vascular damage, which can occur early in the course of the disease. Amongst them are abnormal vasoreactivity, hypoxia, insufficient neoangiogenesis and direct damage of vascular and perivascular cells. They result in a decreased capillary blood flow, and subsequently in clinically overt symptoms such as Raynaud's syndrome and fingertip ulcers. In addition, in active disease vascular pathology can affect various other organs, predominantly the lung, the kidney, the heart but also the gastrointestinal tract. Vascular pathology contributes also significantly to overall morbidity and mortality in SSc patients and reduces life expectancy by at least a decade. Fortunately, molecular biology has revealed a number of underlying pathways on the cellular and subcellular levels, including key factors of the aberrant function of (peri)vascular cells and autoimmune effector cells, the dysregulation of vasoconstrictive molecules and their receptors, the upregulation of intracellular signaling kinases and the altered balance of hypoxia-induced vascular growth factors. This increasing knowledge of vascular pathology in SSc has also resulted in novel therapeutic approaches ranging from endothelin antagonists to application of progenitor cells to counteract this aberrant vascular pathology and to support the repair of the dysfunctional vasculature.

Topics: Apoptosis; Autoantibodies; Blood Vessels; Endothelin-1; Endothelium, Vascular; Epigenesis, Genetic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intercellular Adhesion Molecule-1; Neovascularization, Pathologic; Polymorphism, Single Nucleotide; Scleroderma, Systemic; Vascular Diseases; Vascular Endothelial Growth Factor A

Scleroderma-associated interstitial lung disease (SSc-ILD) occurs frequently and for many patients SSc-ILD is a significant complication of their disease. SSc-ILD is now one of the leading causes of death among patients with SSc. SSc-ILD, classified most often as a non-specific interstitial pneumonia, may culminate in interstitial pulmonary fibrosis and end-stage lung disease. Fibrosis in the lung is the net result of fibroblast proliferation and deposition of excessive amounts of extracellular matrix proteins. Among the many cytokines and growth factors involved in the pathogenesis of SSc-ILD, ET-1 may be a central mediator. In vitro and in vivo studies of animals and SSc patients support the notion that ET-1 can enhance the proliferation of pulmonary mesenchymal cells and may also enhance the fibrogenic effects of TGF-beta. Two well-designed randomized controlled trials of the dual ET receptor blocker bosentan were negative in their primary (and for SSc also secondary) endpoints, although there might be explanations for this apparent lack of efficacy.

Topics: Animals; Antihypertensive Agents; Bosentan; Endothelin Receptor Antagonists; Endothelin-1; Humans; Lung Diseases, Interstitial; Randomized Controlled Trials as Topic; Scleroderma, Systemic; Sulfonamides; Transforming Growth Factor beta; Treatment Failure

SSc is a CTD, which may cause critical organ fibrosis. It has a highly variable clinical presentation and course. While it is more common in females, this heterogeneity has led to significant problems with classification. Biomedical (clinical) and biomolecular markers to identify diagnostic, prognostic and therapeutic response have been elusive in part as a result of difficulties with classification and also due to the rarity of the disease. Existing biomarkers have been identified largely in small cohorts and larger cross-sectional or occasional longitudinal observational cohorts. The nature of biomarkers requires well-defined clinical characteristics and/or defined clinical outcomes and this has been extremely challenging to the international SSc research community. This brief review summarizes the current level of knowledge; however, it most importantly highlights the potential now to find biomarkers through a large, multicentre, international collaborative group approach.

Topics: Acute-Phase Proteins; Biomarkers; Blood Sedimentation; CD40 Ligand; Endothelin-1; Hemoglobins; Humans; Interleukins; Natriuretic Peptide, Brain; Peptide Fragments; Receptors, Interleukin; Scleroderma, Systemic

The endothelins (ET) are the family of 21 amino acid endogenous peptides with potent vasoconstriction function. There are 3 isoforms of the endothelin protein (ET-1, ET-2 and ET-3) encoded by separate genes and exhibit distinct tissue distribution and function. Endothelin 1 is the significant isoform in humans. Endothelin 1 is the most abundant, best characterized isoform with truly pluripotent properties. Endothelin 1 is involved in physiological processes of vascular tone and mitogenesis, whereas under pathological conditions fibrosis, vascular hypertension and inflammation are induced. In human body there are 2 separate ET receptors, ET(A)R and ET(B)R belonging to the G-protein family which produce differing, sometimes opposite effects. Both receptors are differentially expressed by different cell types as well as in different disease entities, In fibroblast cell culture in vitro ET-1 through its receptors modulates cell proliferation, differentiation, contraction and migration. Endothelin 1 is implicated in extracellular matrix (ECM) components synthesis. The dual regulatory role of ET-1 consist on stimulation of collagen I and III synthesis and simultaneously on inhibition of MMP-1 expression through inhibition of tissue inhibitors of metalloproteinase: TIMP-1 and TIMP-3. Endothelin 1 promotes the differentiation of fibroblasts into myofibroblast's phenotype via elevated expression of procontractile proteins alpha-SMA, ezrin, paxillin and moesin. The elevated level of endogenous ET-1 expression cause deficient of myofibroblast apoptosis and increased ECM components deposition. Endothelin 1 is a potent vasoconstrictor, a potent mitogen for fibroblast and smooth muscle cells, a strong stimulant of matrix biosynthesis and is a survival factor for myofibroblasts. Endothelin 1 plays a key role in inflammatory disease and in the connective tissue fibrosis. Elevated level of ET-1, TGF-beta and their receptors has been reported in the pathogenesis of systemic sclerosis.

Topics: Apoptosis; Biomarkers; Cell Differentiation; Cell Proliferation; Cells, Cultured; Connective Tissue; Endothelin-1; Endothelins; Fibrosis; Humans; Scleroderma, Systemic; Transforming Growth Factor beta

Fibrosis affects organs such as the skin, liver, kidney and lung and is a cause of significant morbidity. There is no therapy for fibrosis. Recent significant molecular insights into the signaling underlying the fibrosis in the autoimmune connective tissue disease scleroderma (systemic sclerosis, SSc) have been made. Transforming growth factor beta (TGFbeta) signaling is a major contributor to fibrogenesis, including in SSc. However, it is now appreciated that TGFbeta-dependent and TGFbeta-independent mechanisms play key roles in the pathological fibrosis in SSc. In particular the potent pro-fibrotic proteins endothelin-1 (ET-1) and CCN2 (connective tissue growth factor, CTGF) are believed to play an essential role in this process. This review summarizes these recent crucial observations.

Topics: Connective Tissue Growth Factor; Endothelin-1; Fibrosis; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

A considerable amount of research time has been invested in studies aimed at elucidating pathogenic processes in systemic sclerosis (SSc). Despite this, major challenges for biomedical science remain, such as identification of the key factors that determine susceptibility to SSc, and elucidation of the precise nature of the initiating event that causes endothelial cell injury and ultimately brings about the biological cascade(s) that lead to the pathologic vascular changes. Involved factors are likely to include genetic perturbations, environmental cues, tissue injury, infection and hypoxia/oxidative stress. As important as determining the initiating events are the identification and characterization of key factors that are functionally important in driving vascular disease progression, because these factors are potential targets for therapeutic intervention. This article reviews the role of endothelin as an example of a pleiotropic mediator with effects on various aspects of SSc pathogenesis, such as inflammation, vasculopathy and tissue remodelling.

Topics: Animals; Endothelial Cells; Endothelin-1; Endothelins; Humans; Scleroderma, Systemic

Systemic scleroderma is characterized by a chronic inflammatory process of unknown etiology resulting in an increased deposition of connective tissue proteins in the involved organs. Involvement of the vascular system and the resulting fibrosis lead to atrophy and malfunction of the involved internal organs and the skin. Due to the development of new therapeutic concepts in particular with regard to the vascular involvement, the interaction between the vascular system and the connective tissue moves increasingly into focus. This review describes the major advancemades during recent years for the understanding of the pathophysiology of systemic scleroderma.

Topics: Animals; Antibodies, Antinuclear; Cell Hypoxia; Collagen; Connective Tissue; Disease Models, Animal; Endostatins; Endothelial Cells; Endothelin-1; Extracellular Matrix; Fibroblasts; Humans; Mice; Scleroderma, Systemic; Vasoconstriction

Endothelin-1 is a naturally occurring polypeptide which possesses a broad range of activities including vasospastic, proinflammatory and profibrotic properties. Systemic sclerosis is a multisystem connective tissue disease characterized by vascular damage, inflammatory infiltrates and progressive fibrosis of the skin and internal organs. The results of the recent studies indicate that endothelin-1 may be a key element of the pathogenesis of systemic sclerosis. Accordingly, new class of drugs, endothelin receptor antagonists have been introduced for treatment of patients with systemic sclerosis. This article reviews the role of endothelin-1 in the pathogenesis of systemic sclerosis and the implications of endothelin receptor antagonism in the treatment of systemic sclerosis.

Topics: Endothelin Receptor Antagonists; Endothelin-1; Humans; Receptors, Endothelin; Scleroderma, Systemic

The pathological changes in systemic sclerosis are characterised by severe organ fibrosis, obliteration of arteries and arterioles and disturbance of immunological functions. Recent research has yielded new insight into the complex pathogenesis of systemic sclerosis, with new therapeutic options as a possible consequence.. Relevant medical literature has been reviewed.. The pathological deposition of extracellular matrix in systemic sclerosis is most probably caused by changes in the regulation of dermal fibroblasts. The review focuses on three of the many molecules involved in the regulation of fibrosis; Transforming growth factor beta, Connective tissue growth factor and Endothelin-1.. Treatment of organ-specific disease complication has for long remained the only therapeutic option in systemic sclerosis. Based on current knowledge of the process of fibrosis, new therapeutic trials employing substances directed towards pro-fibrotic molecules are now in progress.

Topics: Connective Tissue Growth Factor; Endothelin-1; Endothelins; Fibroblasts; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Scleroderma, Systemic; Transforming Growth Factor beta

Increasing evidence suggests that the vasculopathy of scleroderma is mediated by a number of soluble factors and involves a complex interaction between endothelial cells, smooth muscle cells, extracellular matrix, intravascular coagulation factors, and circulating cells. Novel therapeutic approaches beyond vasodilator therapy are being developed by recognizing important molecular pathways involved in scleroderma vascular disease. The success of this strategy is most evident in pulmonary hypertension, an often fatal complication of scleroderma. In this article, the authors explore therapies for scleroderma that target endothelial cells, smooth muscle cells, reactive oxygen species, and circulating blood cells. The authors highlight clinical trials that have investigated the role of prostacyclin (and its analogues) and bosentan in managing scleroderma-related pulmonary hypertension. Finally, the authors look at the potential role of biomarkers as surrogate indicators of active vascular disease in scleroderma.

Topics: Antihypertensive Agents; Biomarkers; Bosentan; Clinical Trials as Topic; Endothelin-1; Endothelium, Vascular; Epoprostenol; Humans; Hypertension, Pulmonary; Muscle, Smooth, Vascular; Reactive Oxygen Species; Scleroderma, Systemic; Sulfonamides

Digital ulcers in patients with systemic sclerosis are associated with pain and poor quality of life. Endothelin-1 promotes vasculopathy in systemic sclerosis after macitentan, an endothelin-1 blocker.. To evaluate the efficacy of macitentan in reducing the number of new digital ulcers in patients with systemic sclerosis.. Two international, randomized, double-blind, placebo-controlled trials (DUAL-1, DUAL-2) were conducted between January 2012 and February 2014. Participants were patients with systemic sclerosis and active digital ulcers at baseline. Target enrollment for each study was 285 patients.. Patients were randomized (1:1:1) to receive oral doses of 3 mg of macitentan, 10 mg of macitentan, or placebo once daily and stratified according to number of digital ulcers at baseline (≤3 or >3).. The primary outcome for each trial was the cumulative number of new digital ulcers from baseline to week 16. Treatment effect was expressed as the ratio between treatment groups.. In DUAL-1, among 289 randomized patients (mean age 51.2 years; 85.8% women), 226 completed the study. The adjusted mean number of new digital ulcers per patient over 16 weeks was 0.94 in the 3-mg macitentan group (n = 95) and 1.08 in the 10-mg macitentan group (n = 97) compared with 0.85 in the placebo group (n = 97) (absolute difference, 0.09 [95% CI, -0.37 to 0.54] for 3 mg of macitentan vs placebo and 0.23 [-0.27 to 0.72] for 10 mg of macitentan vs placebo). Among 265 patients randomized in DUAL-2 (mean age 49.6 years; 81.9% women), 216 completed the study. In DUAL-2, the adjusted mean number of new digital ulcers was 1.44 in the 3-mg macitentan group (n = 88) and 1.46 in the 10-mg macitentan group (n = 88) compared with 1.21 in the placebo group (n = 89) (absolute difference, 0.23 [95% CI, -0.35 to 0.82] for 3 mg of macitentan vs placebo and 0.25 [95% CI, -0.34 to 0.84] for 10 mg of macitentan vs placebo). Adverse events more frequently associated with macitentan than with placebo were headache, peripheral edema, skin ulcer, anemia, upper respiratory tract infection, diarrhea, and nasopharyngitis.. Among patients with systemic sclerosis and active ischemic digital ulcers, treatment with macitentan did not reduce new digital ulcers over 16 weeks. These results do not support the use of macitentan for the treatment of digital ulcers in this patient population.. clinicaltrials.gov Identifiers: NCT01474109, NCT01474122.

Topics: Administration, Oral; Double-Blind Method; Endothelin-1; Female; Fingers; Humans; Male; Middle Aged; Outcome Assessment, Health Care; Pyrimidines; Scleroderma, Systemic; Skin Ulcer; Sulfonamides

Endothelial and vascular damage are main leading disability in systemic sclerosis (SSc). Raynaud's phenomenon is the early symptom that presents vascular damage. Nailfold capillaroscopy (NFC) is an easily accessible diagnostic tool in secondary Raynaud's phenomenon. Considering the endothelial damage, clinical manifestations, and plasma cytokines was compared with traditionally used NFC parameter for, which to observe the number of capillaries, deletions in 3 mm, apical limb width and the capillary width itself. We hypothesize that a computer-based NFC system can generate a new powerful parameter which predicts the capillary dimension. We investigated the relationship among the plasma endothelin-1 (ET-1), clinical manifestations and quantitative analysis of computerized NFC, and to assess the optimal method in SSc. The level of ET-1 in 60 SSc patients, 30 healthy, and 23 disease controls were measured by enzyme-linked immunosorbent assay (ELISA) kit. We present a significant difference in all parameters of NFC between SSc patients and control groups. ET-1 level was increased in patients with SSc. In SSc group, capillary dimension and loss of capillaries were strongly associated with digital ulceration (p < 0.01) and pulmonary hypertension (p < 0.05). Capillary dimension and ET-1 level was in correlation with skin-hardening grade, and was higher in SSc patients with pulmonary hypertension or digital ulcer. Capillary dimension showed strong correlation with the endothelin-1 in SSc, healthy and disease control groups. (Rs = 0.31/p < 0.05, Rs = 0.82/p < 0.001, Rs = 0.83/p < 0.001). The results suggest that computer-based microscopic analysis of NFC is a useful method that potentially provides information on organ involvement and plasma ET-1. Capillary dimension maybe a powerful parameter possibly applicable in outpatient clinic for assessing SSc patients.

Topics: Adolescent; Adult; Aged; Biomarkers; Capillaries; Endothelin-1; Female; Humans; Image Processing, Computer-Assisted; Male; Microscopic Angioscopy; Middle Aged; Nails; Predictive Value of Tests; Raynaud Disease; Scleroderma, Systemic; Young Adult

Increased endothelin activity may play a role in the pathogenesis of vascular injury, a primary feature of systemic sclerosis (SSc; scleroderma). Our goal was to test the hypothesis that treatment with the oral endothelin receptor antagonist bosentan might improve vascular endothelial function in SSc patients.. A 4-week, prospective, parallel-group study compared 12 SSc patients who did not receive bosentan treatment with 12 patients who did receive treatment (125 mg/day) for pulmonary hypertension and/or digital ulcers. There were no differences in demographic and clinical characteristics or medications between the 2 groups. Baseline endothelial dysfunction was documented by decreased brachial artery ultrasound-derived flow-mediated dilation (FMD%; <5.5). Pulse wave analysis, venous occlusion plethysmography, and measurement of serum vascular markers were performed in parallel.. FMD%, the main end point, increased significantly from a mean +/- SD of 3.1 +/- 1.3% to 8.4 +/- 2.6% after 4 weeks of bosentan treatment (P < 0.001, compared with a change from 2.4 +/- 1.6% to 2.4 +/- 2.2% in control patients). Arterial blood pressure, endothelium-independent vascular function, augmentation index, peripheral flow reserve, as well as circulating intercellular adhesion molecule 1, E-selectin, vascular endothelial growth factor, and endothelin 1 were not significantly affected by bosentan treatment. In patients continuously treated for 4 months, during which the dosage of bosentan remained at 125 mg/day (n = 5) or increased to 250 mg/day (n = 5), the 4-week results remained unchanged.. Small doses of bosentan improve endothelial function without affecting hemodynamic parameters or endothelial activation-related processes, thus supporting a direct, reversible effect of endothelin in SSc-associated vascular injury. A long-term, controlled trial to examine the potentially global clinical benefit of endothelin receptor blockade in patients with early SSc may be warranted.

Topics: Administration, Oral; Adult; Aged; Bosentan; Brachial Artery; Dose-Response Relationship, Drug; E-Selectin; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Female; Humans; Hypertension, Pulmonary; Intercellular Adhesion Molecule-1; Male; Middle Aged; Prospective Studies; Regional Blood Flow; Scleroderma, Systemic; Sulfonamides; Ultrasonography; Vascular Endothelial Growth Factor A; Vasodilation

To investigate the effects of prolonged inhibition of phosphodiesterase type-5, using once-daily long-acting phosphodiesterase type-5 inhibitor (tadalafil) on erectile function and biomarkers of endothelial function in male patients with systemic sclerosis (SSc) and erectile dysfunction (ED).. In an open-label study, 14 nonconsecutive male patients with SSc with different degrees of ED were enrolled into the study irrespective of their clinical response to tadalafil, and received once-daily tadalafil 10 mg for 12 weeks. Primary endpoints were variations from baseline of penile arterial inflow [peak systolic velocity (PSV, cm/s); measured with dynamic color duplex ultrasound] and the erectile function domain score (measured with the International Index of Erectile Function questionnaire). Secondary endpoints were variations from baseline of morning erections (determined by modified question 13 of the Structured Interview on Erectile Dysfunction questionnaire) and plasma concentrations of endothelin-1 (ET1).. The PSV and the erectile function domain score were significantly improved by once-daily tadalafil (from 21.3 +/- 6.4 to 30.0 +/- 7.0 cm/s and from 13.0 +/- 6.8 to 17.0 +/- 9.0 vs baseline, respectively; p <0.05). Question 13 scores decreased dramatically after treatment compared with baseline (from 2.2 +/- 0.2 to 0.8 +/- 0.5 arbitrary units; p < 0.001), and plasma ET1 levels decreased (from 24 +/- 15 to 9.8 +/- 7.4 pg/ml; p < 0.05).. In men with SSc-related ED, once-daily tadalafil improved both erectile function and vascular measures of cavernous arteries. Increases in morning erections and decreases in plasma ET1 levels were found, which may play a potential role in preventing progression of penile fibrosis and erectile dysfunction.

Topics: Adult; Carbolines; Disease Progression; Endothelin-1; Erectile Dysfunction; Health Surveys; Humans; Male; Middle Aged; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Scleroderma, Systemic; Tadalafil

Endothelial vascular tone modulators are thought to be involved in aetiopathogenesis of systemic sclerosis (SS) and of peripheral artery occlusive disease (PAOD). Iloprost, a prostacyclin (PGI2) analogue, induces clinical benefit in patients suffering from peripheral ischaemia. This study was performed to investigate the effect of this drug on endothelial function in vivo to elucidate the role of vascular tone modulators.. Fourteen PAOD and 15 SS patients were treated for 24 and 10 days respectively. On the first day, before and after therapy, nitric oxide metabolites (NO2-/NO3-) and endothelin-1 (ET-1) plasma concentrations were detected; moreover, the endothelium-dependent vasodilatation in response to artificial ischaemia was evaluated by means of an echo-Doppler device.. The echo-Doppler evaluation showed that the percentage of arterial reactive dilatation was not modified by placebo or by iloprost, and that the increase in blood velocity flow lasted for a significant longer time after drug infusion (226.79 +/- 17.49 vs. 310.71 +/- 36.32 s; P > 0.04). NO2-/NO3- and ET-1 plasma concentration were higher in patients than in control subjects (P < 0.004). After 6 h of iloprost infusion, no significant modifications were detected.. This study provides evidence that iloprost enhances the microvascular functional capacity and clinical benefit for patients. The effects of the drug seem to be independently or not directly correlated with its interactions with vascular tone modulators such as NO2-/NO3- or ET-1.

Topics: Adult; Aged; Arterial Occlusive Diseases; Chronic Disease; Endothelin-1; Endothelium, Vascular; Female; Humans; Iloprost; Infusions, Intravenous; Ischemia; Male; Microcirculation; Middle Aged; Nitrates; Nitric Oxide; Nitrites; Scleroderma, Systemic; Ultrasonography; Vasodilator Agents

In all 11 patients with progressive systemic sclerosis (PSS; Barnett type I, 2; type II, 5; type III, 4 cases; male 1; female 10 cases; 45.2 +/- 10.2 years-old), 6 cases of scleroderma spectrum disorders (SSD, male 1; female 5 cases; 51.2 +/- 13.2 years-old) and 7 healthy controls (HC, male 1; female 6 cases; 43.1 +/- 8.4 years-old) were entered to be examined. The plasma endothelin-1 (ET-1) levels of PSS, SSD and HC were 1.98 +/- 0.69, 1.76 +/- 0.39 and 1.15 +/- 0.38 pg/ml, respectively. After the stimulation with the low frequency electrical current, electrical acupuncture, for unilateral side of hand/arm (30 min), the plasma ET-1 levels decreased in 10 cases of PSS treated (1.61 +/- 0.45 pg/ml), but no change of plasma serotonin levels. In 4 of 6 cases of SSD, plasma ET-1 levels increased (2.06 +/- 0.39 pg/ml), however, nitrate levels increased and serotonin decreased in 3 of 5 cases of SSD. In 6 cases of HC treated with the electrical acupuncture, the plasma ET-1 levels increased (1.72 +/- 0.58 pg/ml). Thermographically, 9 of 11 cases of PSS and 5 of 6 cases of SSD showed temporally temperature-elevation of hand/fingers not only in treated sides, but also in non treated sides, although none of 7 HC showed temperature-elevation of hands/fingers. The decrease in plasma ET-1 levels due to the electrical acupuncture was thought to induce the vasodilatation and elevate the surface temperature in patients with PSS. These results will provide an excellent basis to study the efficacy of electrical acupunctural stimulation.

Topics: Acupuncture Therapy; Adult; Aged; Electric Stimulation Therapy; Endothelin-1; Female; Humans; Male; Middle Aged; Nitrates; Nitrites; Scleroderma, Systemic; Serotonin; Skin Temperature

Inflammation is a major pathological feature of pulmonary arterial hypertension (PAH), particularly in the context of inflammatory conditions such as systemic sclerosis (SSc). The endothelin system and anti-endothelin A receptor (ET. Serum levels of anti-ET. Anti-ET. This study provides evidence for an anti-inflammatory role of ET

Topics: Animals; Autoantibodies; Endothelin-1; Familial Primary Pulmonary Hypertension; Humans; Hypertrophy, Right Ventricular; Inflammation; Mice; Pulmonary Arterial Hypertension; Receptor, Endothelin B; Scleroderma, Systemic

The aim of this study was to investigate the possible link between different types of systemic sclerosis-specific antinuclear antibodies, adipokines and endothelial molecules which were recently found to have a pathogenic significance in systemic sclerosis.. Serum concentration of adiponectin, resistin, leptin, endothelin-1, fractalkine and galectin-3 were determined in the sera of patients with systemic sclerosis (n ​= ​100) and healthy controls (n ​= ​20) using ELISA.. The following associations between antinuclear antibodies and increased serum concentrations were identified: anticentromere antibodies with endothelin-1 (p ​< ​0.0001; mean level in patients 2.21 vs control group 1.31 ​pg/ml), anti-topoisomerase I antibodies with fractalkine (p ​< ​0.0001; 3.68 vs 1.68 ​ng/ml) and galectin-3 (p ​= ​0.0010, 6.39 vs 3.26 ​ng/ml). Anti-RNA polymerase III antibodies were associated with increased resistin (p ​< ​0.0001; 15.13 vs 8.54 ​ng/ml) and decreased adiponectin (p ​< ​0.0001; 2894 vs 8847 ​ng/ml).. In systemic sclerosis metabolic and vascular factors may serve as mediators between immunological abnormalities and non-immune driven clinical symptoms.

Topics: Adipokines; Adiponectin; Antibodies, Antinuclear; Biomarkers; Blood Proteins; Case-Control Studies; Chemokine CX3CL1; Endothelin-1; Female; Follow-Up Studies; Galectins; Humans; Leptin; Male; Middle Aged; Prognosis; Resistin; Scleroderma, Systemic

Systemic sclerosis (SSc) is a chronic connective tissue disease characterized by progressive fibrosis, vascular impairment and immune abnormalities. In recent years, adipokines (mediators synthetized by adipose tissue) have been indicated as a possible missing link in the pathogenesis of SSc. The aim of this study was to investigate the serum concentration of metabolic adipose tissue factors: adiponectin, resistin, leptin and endothelial proteins: endothelin-1, fractalkine and galectin-3 in patients with systemic sclerosis. The study included 100 patients with confirmed SSc diagnosis and 20 healthy individuals. The concentration of respective proteins was determined by enzyme-linked immunosorbent assay. The following markers showed statistically significant increased mean concentrations in patients with SSc in comparison to healthy control: resistin (13.41 vs 8.54 ng/mL; P = 0.0012), endothelin-1 (1.99 vs 1.31 pg/mL; P = 0.0072) and fractalkine (2.93 vs 1.68 ng/mL; P = 0.0007). Elevated serum levels of galectin-3 (4.54 vs 3.26 ng/mL; P = 0.0672) and leptin (19,542 vs 14,210 pg/mL; P = 0.1817) were observed. Decreased concentration of adiponectin was found in patients with SSc (5150 vs 8847 pg/mL; P = 0.0001). Fractalkine and galectin-3 levels were significantly higher in diffuse cutaneous SSc than limited cutaneous SSc subset (3.93 ng/mL vs 2.58 ng/mL, P = 0.0018; 6.86 ng/mL vs 3.78 ng/mL, P = 0.0008, respectively) and correlated positively with modified Rodnan Skin Score in total SSc patients (r = 0.376, P = 0.0009; r = 0.236, P = 0.018, respectively). In conclusion, an increased serum level of resistin associated with increased endothelin-1 and fractalkine level and decreased adiponectin level may indicate a significant role of the adipose tissue in the development and progression of vascular abnormalities in patients with systemic sclerosis. Fractalkine and galectin-3 may participate in promoting and exacerbating the fibrotic process in SSc.

Topics: Adipokines; Adipose Tissue; Adult; Aged; Aged, 80 and over; Biomarkers; Chemokine CX3CL1; Endothelin-1; Female; Fibrosis; Galectin 3; Humans; Male; Middle Aged; Resistin; Scleroderma, Systemic; Up-Regulation; Vascular Diseases

Fibrosis is the most characteristic pathological hallmark of SSc, a connective tissue disease characterized by vascular and immunological abnormalities, inflammation and enhanced extracellular matrix production, leading to progressive fibrosis of skin and internal organs. We previously demonstrated that parvovirus B19 (B19V) can infect normal human dermal fibroblasts (NHDFs) and that B19V persists in SSc fibroblasts. In this study, we investigated whether parvovirus B19V is able to activate in vitro NHDFs and to induce in these cells some phenotypic features similar to that observed in the SSc fibroblasts.. We preliminarily analysed the time course of B19V infection in cultured NHDFs, then we investigated the ability of B19V to induce cell migration, invasive phenotype and mRNA expression of some profibrotic and/or proinflammatory genes.. We confirmed our previous findings that B19V infects NHDFs, but the infection is not productive. After incubation with B19V, NHDFs showed a significant increase of both migration and invasiveness, along with mRNA expression of different profibrotic genes (α-SMA, EDN-1, IL-6, TGF-β1 receptors 1 and 2, Col1α2), some genes associated with inflammasome platform (AIM2, IFI16, IL-1β, CASP-1) and genes for metalloprotease (MMP 2, 9 and 12).. These data suggest that B19V can activate dermal fibroblasts and may have a role in the pathogenesis of fibrosis. B19V-induced fibroblast migration and invasiveness could be due to the B19V-associated MMP9 overexpression and activation. Moreover, the up-regulation of MMP12, typical of SSc, could link the B19V infection of fibroblasts to the anti-angiogenic process.

Topics: Actins; Caspase 1; Cell Movement; Cells, Cultured; Collagen Type I; DNA-Binding Proteins; Endothelin-1; Fibroblasts; Fibrosis; Humans; In Vitro Techniques; Inflammation; Interleukin-1beta; Interleukin-6; Matrix Metalloproteinase 12; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Nuclear Proteins; Parvoviridae Infections; Parvovirus B19, Human; Phosphoproteins; Receptors, Transforming Growth Factor beta; RNA, Messenger; Scleroderma, Systemic; Skin; Transcriptome

Systemic sclerosis (SSc) is a connective tissue disorder which key feature is a fibrotic process. The role of Endothelin-1 (ET-1) and T-helper (Th)-1 cells in lung and skin fibrosis is well known, although Th17- and Treg-cells were found to be involved. However, no studies analyzed cytokines expression in gastric-juice of SSc patients. Our study aimed to evaluate proinflammatory and profibrotic cytokines in gastric-juice of SSc patients and to investigate their correlations with esophageal dysmotility.. Patients performed upper-gastrointestinal-endoscopy with gastric-juice collection, esophageal manometry and thoracic CT-scan. GM-CSF, ET-1, Th-1 (IFN-γ, IL-1β, TNF-α, IL-2, IL-6, IL-9), Th-17 (IL-17, IL-21, IL-22, IL-23) and T-reg (IL-10, TGF-β) related cytokines were measured in 29 SSc-patients and 20 healthy-controls.. Patients showed significant lower levels of IL-6, IL-17, IL-22 and ET-1 (

Topics: Adult; Case-Control Studies; Endothelin-1; Esophagus; Female; Gastric Juice; Gene Expression; Humans; Interleukin-17; Interleukin-1beta; Interleukin-2; Interleukin-22; Interleukin-23; Interleukin-6; Interleukin-9; Interleukins; Lung; Male; Middle Aged; Scleroderma, Systemic; Skin; Stomach; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Tomography, X-Ray Computed

The disruption of endothelial homeostasis is a major determinant in the pathogenesis of systemic sclerosis (SSc) and is reflected by soluble and cellular markers of activation, injury and repair. We aimed to provide a combined assessment of endothelial markers to delineate specific profiles associated with SSc disease and its severity.. We conducted an observational, single-centre study comprising 45 patients with SSc and 41 healthy control subjects. Flow cytometry was used to quantify circulating endothelial microparticles (EMPs) and CD34. Enhanced numbers of EMPs, CFU-ECs and non-haematopoietic CD34. This study identifies the mobilisation of CD34

Topics: Aged; Antigens, CD34; Biomarkers; Cell Count; Cell Movement; Cell-Derived Microparticles; Chemokine CX3CL1; Endothelial Progenitor Cells; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Leukocyte Common Antigens; Logistic Models; Male; Middle Aged; Multivariate Analysis; Scleroderma, Systemic; Severity of Illness Index; Vascular Endothelial Growth Factor A

Endothelin-1 (ET-1) is important in the pathogenesis of systemic sclerosis (SSc). ET-1 binds two receptors, endothelin type A (ETA) and endothelin type B (ETB). Dual ETA/ETB receptor antagonists and a selective ETA receptor antagonist are used clinically to treat SSc, and the effect of these antagonists on fibroblast activation has been described. However, the role of ETB receptor signaling in fibrogenesis is less clear. This study was conducted to evaluate the profibrotic function of ETB receptor signaling in a murine model of bleomycin (BLM)-induced scleroderma.. We used ETB receptor-knockout (ETBKO) mice, which are genetically rescued from lethal intestinal aganglionosis by an ETB receptor transgene driven by the human dopamine β-hydroxylase (DβH)-gene promoter, and wild-type mice with DβH-ETB (WT). BLM or phosphate-buffered saline (PBS) was administered subcutaneously by osmotic minipump, and skin fibrosis was assessed by dermal thickness, subcutaneous fat atrophy, and myofibroblast count in the dermis. Dermal fibroblasts isolated from ETBKO and WT mice were cultured in vitro, stimulated with BLM or ET-1, and the expression of profibrotic genes was compared by quantitative PCR.. Dermal thickness, subcutaneous fat atrophy, and myofibroblast counts in the dermis were significantly reduced in ETBKO mice compared to WT mice, after BLM treatment. Compared with wild-type, dermal fibroblasts isolated from ETBKO mice showed lower gene expressions of α-smooth muscle actin and collagen 1α1 in response to BLM or ET-1 stimulation in vitro.. ET-1-ETB receptor signaling is involved in skin sclerosis and in collagen synthesis by dermal fibroblasts.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Collagen Type I; Disease Models, Animal; Endothelin-1; Fibroblasts; Fibrosis; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Polymerase Chain Reaction; Receptor, Endothelin B; Scleroderma, Systemic; Signal Transduction; Skin

To evaluate the morphological and functional abnormalities of the microcirculation associated with markers of vascular injury in patients with early systemic sclerosis (SSc).. Forty-six patients with early SSc were compared with 80 patients with definite SSc, 40 patients with primary Raynaud's phenomenon (PRP), and 45 healthy subjects. Widefield nailfold capillaroscopy (NFC) (10-25 × magnification), videocapillaroscopy (200 × magnification), and laser Doppler imaging (LDI) assessment were performed in all participants. The number of capillaries/mm, enlarged, giant and ramified capillaries, microhaemorrhages, and the avascular score were determined by widefield NFC and videocapillaroscopy. Fingertip blood flow (FBF) was measured using LDI before and after cold stimulus (CS). Serum endothelin-1 (ET-1), von Willebrand factor (vWF), and transforming growth factor beta-1 (TGF-β1) were measured by enzyme-linked immunosorbent assay (ELISA).. Upon both widefield NFC and videocapillaroscopy, patients with early SSc showed significantly higher numbers of capillaries/mm, lower enlarged and giant capillaries, and a lower avascular score than definite SSc patients (p < 0.001). They also had more enlarged capillaries, microhaemorrhages and a higher avascular score compared to PRP and controls (p < 0.001). FBF before and after CS were significantly higher in controls than in PRP, early SSc, and definite SSc patients (p < 0.001), with no difference between early and definite SSc. Serum levels of ET-1, vWF, and TGF-β1 were similar between early and definite SSc patients.. Early SSc patients showed functional changes and vascular injury marker levels similar to patients with established disease. Nonetheless, the morphological changes were less severe in early SSc, thus providing an opportunity for further prevention of vasculopathy progression.

Topics: Adult; Aged; Autoantibodies; Biomarkers; Cross-Sectional Studies; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Fingers; Humans; Male; Microcirculation; Microscopic Angioscopy; Microvessels; Middle Aged; Raynaud Disease; Scleroderma, Systemic; Severity of Illness Index; Transforming Growth Factor beta1; von Willebrand Factor

It is generally accepted that blockade of endothelin receptors has potentially beneficial effects on vasculopathy associated with systemic sclerosis (SSc). The aim of this study was to clarify the molecular mechanism underlying these effects using endothelial cell-specific Fli-1-knockout (Fli-1 ECKO) mice, an animal model of SSc vasculopathy.. Levels of messenger RNA for target genes and the expression and phosphorylation levels of target proteins were determined in human and murine dermal microvascular endothelial cells by real-time quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively. The binding of Fli-1 to the target gene promoters was evaluated using chromatin immunoprecipitation. Expression levels of Fli-1 and α-smooth muscle actin in murine skin were evaluated using immunohistochemistry. Vascular structure and permeability were evaluated in mice injected with fluorescein isothiocyanate-dextran and Evans blue dye, respectively.. In human dermal microvascular endothelial cells, endothelin 1 induced phosphorylation of Fli-1 at Thr(312) through the sequential activation of c-Abl and protein kinase Cδ, leading to a decrease in Fli-1 protein levels as well as a decrease in binding of Fli-1 to the target gene promoters, whereas bosentan treatment reversed those effects. In Fli-1 ECKO mice, 4 weeks of treatment with bosentan increased endothelial Fli-1 expression, resulting in vascular stabilization and the restoration of impaired leaky vessels.. The vascular fragility of Fli-1 ECKO mice was improved by bosentan through the normalization of Fli-1 protein levels and activity in endothelial cells, which may explain, in part, the mechanism underlying the beneficial effects of endothelin receptor blockade on SSc vasculopathy.

Topics: Actins; Animals; Bosentan; Capillary Permeability; Cell Line; Cells, Cultured; Chromatin Immunoprecipitation; Endothelial Cells; Endothelin Receptor Antagonists; Endothelin-1; Gene Expression; Humans; Immunoblotting; Immunohistochemistry; Mice; Mice, Knockout; Microvessels; Phosphorylation; Promoter Regions, Genetic; Protein Kinase C-delta; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins c-abl; Receptors, Endothelin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Systemic; Sulfonamides; Vascular Diseases

Topics: Aged; Endothelin-1; Female; Follow-Up Studies; Hemodynamics; Humans; Hypertension, Pulmonary; Male; Middle Aged; Peptide Fragments; Pilot Projects; Prognosis; Scleroderma, Systemic

The activated NLRP3 inflammasome is associated with the etiology of fibrotic diseases. The role of inflammasomes in SSc is still poorly understood.. To determine the expression of NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) in the skin of patients with systemic sclerosis (SSc) and its relationship with pro-inflammatory cytokines and vascular mediators expression.. Skin biopsies were taken from 42 patients with either limited or diffuse SSc (21 lcSSc and 21 dcSSc), and from 13 healthy individuals. Using real-time polymerase chain reaction (PCR), the relative expression of caspase-1, IL-1β, IL-18, IL-33, TGF-β, ET-1, iNOS and eNOS genes, were measured. The location of NLRP3 and IL-1β were also determined by immunohistochemistry. Clinical characteristics were evaluated.. The mean age of the patients was 49.3 ± 12.9 (lcSSc), 44.6 ± 1 3.8 (dcSSc), and 45 ± 14.1 (healthy individuals). Compared to healthy individuals, the skin of both subtypes of SSc showed a significant increase (P < 0.05) in NLRP3, caspase-1, IL-1β, IL-18 and ET-1. Samples of lcSSc also showed a significant increase of eNOS (P < 0.029), iNOS (P < 0.04) and TGF-β (P < 0.05). Dermal fibrosis evaluated by modified Rodnan skin score (MRSS) had significant correlation with NLRP3, IL-1β, IL-18, and ET-1. Immunohistochemical analysis showed stronger staining of NLRP3 and IL-1β cytoplasmic expression in the keratinizing squamous epithelium of skin from SSc patients compared to controls.. This study identified NLRP3 over-expression in skin of patients with SSc. Skin thickness correlates positively with the NLRP3 inflammasome gene expression and with the vascular mediator and pro-fibrotic ET-1, suggesting that NLRP3 inflammasome plays a role in the pathophysiology of skin fibrosis in human SSc.

Topics: Adult; Biopsy; Carrier Proteins; Cytokines; Endothelin-1; Female; Fibrosis; Gene Expression Regulation; Humans; Inflammasomes; Inflammation Mediators; Male; Middle Aged; NLR Family, Pyrin Domain-Containing 3 Protein; Real-Time Polymerase Chain Reaction; Scleroderma, Systemic; Skin

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin and internal organ fibrosis, caused by microvascular dysfunction. In recent years, the hypothesis that anti-endothelial cell antibodies (AECA) play a key role in microvascular damage seems to be increasingly convincing. In fact, AECA can induce antibody-dependent cellular apoptosis and stimulate the microvasculature to release pro-inflammatory and pro-fibrotic cytokines. Human-microvascular-endothelial-cells (MVECs) were stimulated with SSc sera (with and without AECA) and with sera from healthy donors. The conditioned MVEC culture media were then added to fibroblast cultures obtained from control skin (CTR), non-affected skin of SSc patients (NA), and affected skin of the same sclerodermic (SSc) patients, respectively. AECA contributed to the MVEC increased release of endothelin-1 (ET-1) in the culture medium and to MVEC apoptosis. Fibroblast (CTR, NA, and SSc) proliferation was increased after treatment with AECA-positive conditioned media, compared to AECA-negative and control conditioned media. Furthermore, both AECA-positive (in major contribution) and AECA-negative conditioned media were responsible for alpha-smooth-muscle-actin (αSMA) over-expression in all fibroblast cultures, compared to control conditioned media. Fibroblast type I collagen synthesis was upregulated by both SSc conditioned media (with and without AECA). Finally, the synthesis of fibroblast transforming-growth-factor-beta (TGF-β) was statistically higher in AECA-positive conditioned media, compared to AECA-negative and control conditioned media. These findings support the concept that AECA may mediate the crosstalk between endothelial damage and dermal-fibroblast activation in SSc.

Topics: Actins; Autoantibodies; Cells, Cultured; Collagen Type I; Culture Media, Conditioned; Endothelial Cells; Endothelin-1; Endothelium; Female; Fibroblasts; Humans; Microvessels; Middle Aged; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Endothelin-1 (ET-1) plays a pivotal role in vasoconstriction, fibrosis, and inflammation, the key features of systemic sclerosis (SSc). ET-1 receptors (ETA and ET(B)) are expressed on endothelial cells, smooth muscle cells, and fibroblasts, but their presence on immune cells has not been deeply investigated so far. Endothelin receptors antagonists such as bosentan have beneficial effects on vasoconstriction and fibrosis, but less is known about their potential anti-inflammatory effects. We studied the expression of ET-1 receptors on immune cells (T and B lymphocytes, monocytes, and neutrophils) and the link between ET-1 and inflammation in patients with SSc. We show here that ET-1 exerts a proinflammatory effect in CD4+ T cells, since it induces an increased IFN-γ production; preincubation with antagonists of both receptors reduces IFN-γ production. Moreover, following ET-1 stimulation, neutrophils produce proinflammatory mediators, thus amplifying the effects of activated CD4+ T cells. Our data indicate that ET-1 system is involved in the pathogenesis of inflammation and fibrosis typical of SSc, through the activation of T lymphocytes and neutrophils and the consequent release of proinflammatory and profibrotic cytokines. These findings suggest that dual ET-1 receptors antagonist therapy, besides its effect on vasculopathy, has a profound impact on the immune system favouring antiinflammatory and antifibrogenic effects.

Topics: Anti-Inflammatory Agents; Bosentan; CD4-Positive T-Lymphocytes; Endothelin Receptor Antagonists; Endothelin-1; Female; Fibroblasts; Fibrosis; Humans; Inflammation; Interferon-gamma; Male; Middle Aged; Monocytes; Receptors, Endothelin; Scleroderma, Systemic; Sulfonamides

Raynaud's phenomenon and digital ulcers (DUs) are frequent among systemic sclerosis (SSc) patients. Our aim was to investigate the diagnostic and predictive value for DU of endothelial dysfunction biomarkers (flow-mediated dilatation (FMD), serum levels of endothelin-1 (ET-1), and ADMA), angiogenic/angiostatic biomarkers (vascular endothelial growth factor (VEGF), endoglin, and endostatin), and nailfold videocapillaroscopy (NVC). We compared our results with a literature review. In a cohort study of 77 SSc patients, we followed two groups of patients: (i) naïve DU patients (39) and (ii) active DU at baseline (38 patients) for 3 years. Telangiectasia (p < 0.001) and diffuse disease subset (p = 0.001) were significantly more frequent in patients with active DU at enrolment. Additionally, NVC late scleroderma pattern (AUC 0.846, 95%CI 0.760-0.932), lower values of FMD (AUC 0.754, 95%CI 0.643-0.864), increased serum levels of ET-1 (AUC 0.758, 95%CI 0.649-0.866), ADMA (AUC 0.634, 95%CI 0.511-0.757), and endoglin as well as low VEGF serum levels (AUC 0.705, 95%CI 0.579-0.830) were significantly associated to new DU events in the 3-year follow-up. Cox regression analysis showed that FMD > 9.41 % (HR 0.37, 95%CI 0.14-0.99); ET-1 >11.85 pmol/L (HR 3.81, 95%CI 1.41-10.26) and late NVC pattern (HR 2.29, 95%CI 0.97-5.38) were independent predictors of DU recurrence. When estimating the probability of occurrence of first DU in naïve DU patients, only late NVC pattern (HR 12.66, 95%CI 2.06-77.89) was an independent predictor factor. In conclusion, late scleroderma patterns in NVC are the best independent predictors of SSc patients who are at risk of developing DU. Endothelial dysfunction assessed by FMD and ET-1 was also found to be an independent predictor of DU recurrence in a 3-year follow-up.

Topics: Adolescent; Adult; Aged; Antigens, CD; Biomarkers; Cohort Studies; Endoglin; Endothelin-1; Endothelium; Female; Fingers; Follow-Up Studies; Humans; Male; Microscopic Angioscopy; Middle Aged; Predictive Value of Tests; Prognosis; Prospective Studies; Raynaud Disease; Receptors, Cell Surface; Scleroderma, Systemic; Ulcer; Vascular Endothelial Growth Factor A; Young Adult

High endothelin-1 (ET-1) and transforming growth factor-β (TGF-β) levels may induce in healthy endothelial cells (EC) an endothelial-to-mesenchymal transition (EndMT). The same cytokines are associated with fibrosis development in systemic sclerosis (SSc). Although EndMT has not been definitively shown in SSc, this process, potentially induced by a stimulatory loop involving these 2 cytokines, overexpressed in this disease might contribute to fibroblast accumulation in affected tissues. Macitentan (MAC), an ET-1 receptor antagonist interfering with this loop, might prevent EndMT and fibroblast accumulation.. EC, isolated from healthy controls (HC) and patients with SSc, were treated with ET-1 and TGF-β and successively analyzed for gene and protein expressions of endothelial and mesenchymal markers, and for Sma- and Mad-related (SMAD) phosphorylation. Further, in the supernatants, we evaluated ET-1 and TGF-β production by ELISA assay. In each assay we evaluated the ability of MAC to inhibit both the TGF-β and ET-1 effects.. We showed that both TGF-β and ET-1 treatments induced an activation of the EndMT process in SSc-EC as reported in HC cells. The ELISA assays showed a mutual TGF-β and ET-1 induction in both SSc-EC and HC-EC. A statistically significant increase of SMAD phosphorylation after treatment was observed in SSc-EC. In each assay, MAC inhibited both TGF-β and ET-1 effects.. Our work is the first demonstration in literature that SSc-EC, under the synergistic effect of TGF-β and ET-1, may transdifferentiate toward myofibroblasts, thus contributing to fibroblast accumulation. MAC, interfering with this process in vitro, may offer a new potential therapeutic strategy against fibrosis.

Topics: Adult; Biomarkers; Case-Control Studies; Cell Transdifferentiation; Cells, Cultured; Endothelial Cells; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Humans; Italy; Male; Pyrimidines; Reference Values; Scleroderma, Systemic; Sensitivity and Specificity; Statistics, Nonparametric; Sulfonamides; Transforming Growth Factor beta; Young Adult

Systemic sclerosis (SSc) is a complex and not fully understood autoimmune disease associated with fibrosis of multiple organs. The main effector cells, the myofibroblasts, are collagen-producing cells derived from the activation of resting fibroblasts. This process is regulated by a complex repertoire of profibrotic cytokines, and among them transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) play a major role. In this paper we show that TGF-β and ET-1 receptors co-operate in myofibroblast activation, and macitentan, an ET-1 receptor antagonist binding ET-1 receptors, might interfere with both TGF-β and ET-1 pathways, preventing myofibroblast differentiation.. Fibroblasts isolated from healthy controls and SSc patients were treated with TGF-β and ET-1 and successively analyzed for alpha smooth muscle actin (α-SMA) and collagen (Col1A1) expression and for the Sma and Mad Related (SMAD) phosphorylation. We further tested the ability of macitentan to interfere with these process. Furthermore, we silenced ET-1 and endothelin-1 receptor A expression and evaluated the formation of an ET-1/TGF-β receptor complex by immunoprecitation assay.. We showed myofibroblast activation in SSc fibroblasts assessing the expression of α-SMA and Col1A1, after stimulation with TGF-β and ET-1. Macitentan interfered with both ET-1- and TGF-β-induced fibroblast activation. To explain this unexpected inhibitory effect of macitentan on TGF-β activity, we silenced ET-1 expression on SSc fibroblasts and co-immunoprecipitated these two receptors, showing the formation of an ET-1/TGF-β receptor complex.. During SSc, ET-1 produced by activated endothelia contributes to myofibroblast activation using TGF-β machinery via an ET-1/TGF-β receptor complex. Macitentan interferes with the profibrotic action of TGF-β, blocking the ET-1 receptor portion of the ET-1/TGF-β receptor complex.

Topics: Actins; Adult; Blotting, Western; Collagen Type I; Collagen Type I, alpha 1 Chain; Dermis; Endothelin A Receptor Antagonists; Endothelin-1; Female; Fibroblasts; Humans; Male; Middle Aged; Multiprotein Complexes; Phosphorylation; Pyrimidines; Receptor, Endothelin A; Receptors, Transforming Growth Factor beta; RNA Interference; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Sulfonamides; Transforming Growth Factor beta1; Up-Regulation; Young Adult

Heart and pulmonary involvement is a leading cause of systemic sclerosis (SSc)-related deaths.. The aim of our study was to assess if biochemical markers of right ventricular (RV) overload, endothelial function and collagen metabolism can predict RV dysfunction assessed by Doppler echocardiography in SSc patients.. We prospectively studied 111 consecutive patients (101 F, 10 M, age 54.2 ± 13.8 years) with diagnosed SSc (mean disease duration 9.4 ± 11.4 years) and a group of 21 age-matched subjects (18 F, 3 M, age 49.3 + 10.5 years). We performed transthoracic echocardiography (Phillips iE 33) and measured serum endothelin-1 (ET-1), N-terminal pro-brain natriuretic peptide (NT-proBNP), asymmetric dimethylarginine (ADMA), endoglin and human tissue inhibitor of matrix metalloproteinase (TIMP-1) concentration.. Median serum NT-proBNP level in SSc patients was 133.5 (range 21.86-17,670 pg/ml) and was significantly higher than in controls (p = 0.0002). Moreover, the median serum ET-1 level of 1.49 (range 0.26-8.75 pg/ml) was higher in SSc patients (p = 0.002). However, no significant differences in ADMA, TIMP-1 and endoglin serum concentration between SSc patients and controls were observed. Serum NT-proBNP concentration correlated positively with echocardiographic signs of RV overload: tricuspid regurgitation pressure gradient (r = 0.38, p = 0.0004) and RV Tei index (r = 0.25, p = 0.01). ET-1 serum level correlated negatively with tricuspid annular plane systolic excursion (r = -0.4, p = 0.01) and positively with inferior vena cava diameter measured at expiration (r = 0.38, p = 0.0002). The echocardiographic signs of RV overload were significantly more pronounced in the highest NT-proBNP tertile (>195 pg/ml) group than in the lowest one (<88 pg/ml).. Serum ET-1 and NT-proBNP, but not endoglin, ADMA and TIMP-1 levels correlating with the echocardiographic parameters of RV overload, can be considered as noninvasive indicators of RV dysfunction in SSc patients.

Topics: Adult; Aged; Antigens, CD; Arginine; Biomarkers; Case-Control Studies; Collagen; Echocardiography; Echocardiography, Doppler; Endoglin; Endothelin-1; Endothelium, Vascular; Female; Fibrosis; Humans; Lung Diseases; Male; Middle Aged; Natriuretic Peptide, Brain; Peptide Fragments; Prospective Studies; Receptors, Cell Surface; Scleroderma, Systemic; Tissue Inhibitor of Metalloproteinase-1; Ventricular Function, Right

The aim of this study was to evaluate the effects of bosentan on plasma endothelin-1 (ET-1) and nitric oxide (NO) as pulmonary hypertension (PH)-associated biochemical markers in patients with systemic sclerosis (SSc). Twenty-four SSc patients receiving bosentan for 24 weeks were registered in this prospective observational study. Ten patients were complicated with clinically suspected PH. Plasma levels of ET-1 and NO were assessed at baseline and after 24 weeks of treatment in SSc patients and in 15 healthy controls. Plasma levels of ET-1 and NO at baseline were significantly higher in SSc patients than in healthy controls (p < 0.000), and they were also significantly higher in SSc patients with PH than in those without PH (p < 0.01). Plasma ET-1 levels were significantly decreased after 24 weeks of bosentan therapy (p < 0.0001), and ET-1 levels of SSc patients with PH decreased to a level comparable to that in patients without PH. In the 10 SSc patients with PH, changes in plasma ET-1 levels during the 24 weeks of the study were significantly larger in the 5 patients whose functional class (FC) improved than in the 5 patients whose FC was unchanged (p < 0.05). Plasma NO levels were also slightly decreased in SSc patients after 24 weeks of bosentan therapy. Plasma ET-1 levels could reflect the presence and severity of PH in SSc patients. Additionally, changes in plasma ET-1 levels may indicate the response to bosentan therapy in SSc patients with PH.

Topics: Aged; Antihypertensive Agents; Biomarkers; Bosentan; Endothelin-1; Humans; Hypertension, Pulmonary; Male; Middle Aged; Nitric Oxide; Prospective Studies; Scleroderma, Systemic; Severity of Illness Index; Sulfonamides; Time Factors; Treatment Outcome

Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein-Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV-Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-β1 (TGFβ1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.

Topics: Actins; Endothelin-1; Epstein-Barr Virus Infections; Fibroblasts; Humans; Immunity, Innate; In Situ Hybridization; Inflammation; Interferon Regulatory Factors; Monocytes; Muscle, Smooth; Myofibroblasts; Phenotype; RNA, Small Untranslated; Scleroderma, Systemic; Toll-Like Receptor 7; Toll-Like Receptor 9; Toll-Like Receptors; Transforming Growth Factor beta1

Evidence is increasing of a link between interferon (IFN) and pulmonary arterial hypertension (PAH). Conditions with chronically elevated endogenous IFNs such as systemic sclerosis are strongly associated with PAH. Furthermore, therapeutic use of type I IFN is associated with PAH. This was recognized at the 2013 World Symposium on Pulmonary Hypertension where the urgent need for research into this was highlighted.. To explore the role of type I IFN in PAH.. Cells were cultured using standard approaches. Cytokines were measured by ELISA. Gene and protein expression were measured using reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemistry. The role of type I IFN in PAH in vivo was determined using type I IFN receptor knockout (IFNAR1(-/-)) mice. Human lung cells responded to types I and II but not III IFN correlating with relevant receptor expression. Type I, II, and III IFN levels were elevated in serum of patients with systemic sclerosis associated PAH. Serum interferon γ inducible protein 10 (IP10; CXCL10) and endothelin 1 were raised and strongly correlated together. IP10 correlated positively with pulmonary hemodynamics and serum brain natriuretic peptide and negatively with 6-minute walk test and cardiac index. Endothelial cells grown out of the blood of PAH patients were more sensitive to the effects of type I IFN than cells from healthy donors. PAH lung demonstrated increased IFNAR1 protein levels. IFNAR1(-/-) mice were protected from the effects of hypoxia on the right heart, vascular remodeling, and raised serum endothelin 1 levels.. These data indicate that type I IFN, via an action of IFNAR1, mediates PAH.

Topics: Animals; Cells, Cultured; Chemokine CXCL10; Disease Models, Animal; Endothelial Cells; Endothelin-1; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Interferon-alpha; Interferon-beta; Interferon-gamma; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Culture Techniques; Receptor, Interferon alpha-beta; Scleroderma, Systemic; Signal Transduction

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

Topics: Animals; Bosentan; Chromatin Immunoprecipitation; Collagen Type I; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Fibroblasts; Fibrosis; Humans; Immunoblotting; Mice; Phenotype; Proto-Oncogene Protein c-fli-1; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Systemic; Signal Transduction; Skin; Sulfonamides

To determine serum and sputum Caveolin-1 (Cav-1) levels and their associations with transforming growth factor- ß (TGF-ß) and interstitial lung disease (ILD) in systemic sclerosis (SSc).. Serum and induced sputum samples from 55 patients with SSc, 25 asthma patients and 16 healthy volunteers (HC) were tested for Cav-1 and TGF-ß by the ELISA technique. As a possible downstream signaling regulator of TGF-ß, Endothelin-1 (ET-1), a potent profibrotic protein, was also measured in all serum and sputum samples and relations with Cav-1 and TGF-ß were sought. All scleroderma patients were evaluated for their clinical and laboratory parameters. Pulmonary function tests (PFT) and high resolution computerized tomography (HRCT) were performed for the diagnosis of ILD. The alveolitis-fibrosis index and the SSc disease severity scores were noted for each patient.. Serum Cav-1 levels were lower in SSc compared to HC (p<0.01). Cav-1 levels were significantly lower in the sputum of SSc patients compared to both control groups (p<0.001). It was also found significantly lower in SSc-ILD compared to those without ILD (0.19±0.04 vs 0.25±0.07, respectively, p<0.01). Although no difference was found in the serum TGF-ß levels among the groups, sputum TGF-ß levels correlated positively with the alveolitis index (r=0.34) and correlated inversely with FVC measurements (r=-0.44, p<0.05) among SSc patients. Serum ET-1 was significantly higher in SSc patients (p<0.01) but no association was found between ET-1 and Cav-1 or TGF-ß.. These results suggest that decreased sputum Cav-1 levels is associated with SSc related-ILD and may be used as a marker for the detection of SSc-ILD.

Topics: Adult; Aged; Case-Control Studies; Caveolin 1; Endothelin-1; Female; Humans; Lung Diseases, Interstitial; Male; Middle Aged; Prospective Studies; Scleroderma, Systemic; Sputum; Transforming Growth Factor beta

We have studied endothelin-1 (ET-1) levels and ET-1 ligand and receptor tissue expression in scleroderma renal crisis (SRC) and undertaken a pilot open label safety study of bosentan, a non-selective ET-1 receptor antagonist, in SRC [Bosentan in Renal Disease-1 (BIRD-1)].. Serum levels of ET-1 were measured in healthy controls (n = 20) or systemic sclerosis (SSc) (n = 80) with or without SRC, including cases of pulmonary arterial hypertension (PAH). Renal biopsies (n = 27) from patients with SRC were stained for endothelin ligand and receptors. Six cases of SRC received 6 months bosentan. Outcome measures were compared with SRC cases managed at our centre from 2000 to 2004 (n = 49).. Serum ET-1 was elevated in SRC but less than in PAH. ET-1 and both endothelin A and endothelin B receptor expression was increased in SRC biopsies in glomeruli, interstitium and hallmark vascular lesions of SRC. In the BIRD-1 cohort, serum ET-1 was elevated in all cases at SRC (median healthy controls 0.50 pg/ml; SRC 1.48 pg/ml; P < 0.0005), and increased further with bosentan therapy (1.46 vs. 3.05 pg/ml; t-test P < 0.05). Bosentan was well tolerated with no significant drug-related serious adverse events and long-term outcomes were favourable compared with historic cases. Three patients developed rebound hypertension on withdrawal of bosentan and one appeared to further benefit from maintenance therapy.. Upregulation of ET-1 ligand axis suggests that ET-1 receptor blockade is logical and treatment with bosentan appears to be safe in SRC. Future studies to assess therapeutic benefit and compare selective or non-selective receptor antagonists are justified.

Topics: Acute Kidney Injury; Adult; Aged; Antihypertensive Agents; Bosentan; Case-Control Studies; Endothelin-1; Female; Humans; Hypertension; Male; Middle Aged; Pilot Projects; Receptor, Endothelin A; Scleroderma, Systemic; Sulfonamides; Treatment Outcome

To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice.. Skin fibroblasts that were isolated from three systemic scleroderma (SSc) patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 μM). Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I) procollagen (COL1A1), alpha 1 (III) procollagen (COL3A1), matrix metalloproteinase (MMP)-1 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d) was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson's trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA). Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR.. Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3).. Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc mouse model, in part due to a reduction in ET-1.

Topics: Animals; Antibiotics, Antineoplastic; Anticarcinogenic Agents; Bleomycin; Carotenoids; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type III; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Fibrosis; Immunohistochemistry; Lung; Matrix Metalloproteinase 1; Mice; Mice, Inbred C3H; Real-Time Polymerase Chain Reaction; Scleroderma, Systemic; Skin; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Vitamin A

Systemic sclerosis (SSc) is a disease characterized by skin and internal organ involvement. There is progressive accumulation of extracellular matrix components in the skin and involved organs. Tissue fibrosis is the prominent reason for mortality, and still, there is no satisfactory treatment. The aim of this study was to evaluate the effects of urotensin-II (U-II) antagonist palosuran in an animal model of scleroderma. We also planned to measure U-II, endothelin-1 (ET-1), and transforming growth factor-β1 (TGF-β1) levels, as well as the association of these levels with dermal thickness. Twenty-four male mice were included in this study and they were divided into three groups--group 1: control group, group 2: fibrosis group, and group 3: fibrosis + palosuran treatment group. Fibrosis + palosuran treatment in group 3 reduced ET-1, U-II, and TGF-β1 levels. In total, the diminished values were statistically significant in the ET-1 and TGF-β1 levels (p < 0.05). Dermal thickness was higher in the fibrosis group, when compared with the other groups. There was no significant relationship between dermal thickness and ET-1, U-II, or TGF-β1 levels (p > 0.05). It is believed that U-II is an important mediator in SSc, and its antagonism with palosuran could be a new treatment choice in SSc.

Topics: Animals; Bleomycin; Endothelin-1; Extracellular Matrix; Fibrosis; Male; Mice; Mice, Inbred BALB C; Quinolines; Scleroderma, Systemic; Skin; Transforming Growth Factor beta1; Urea; Urotensins

Systemic sclerosis (SSc) is a complex disease characterized by vascular alterations, activation of the immune system and tissue fibrosis. Previous studies have implicated activation of the interferon pathways in the pathogenesis of SSc. The goal of this study was to determine whether interferon type I and/or type II could play a pathogenic role in SSc vasculopathy. Human dermal microvascular endothelial cells (HDMVECs) and fibroblasts were obtained from foreskins of healthy newborns. The RT Profiler PCR Array System was utilized to screen for EndoMT genes. Treatment with IFN-α or IFN-γ downregulated Fli1 and VE-cadherin. In contrast, IFN-α and IFN-γ exerted opposite effects on the expression of α-SMA, CTGF, ET-1, and TGFβ2, with IFN-α downregulating and IFN-γ upregulating this set of genes. Blockade of TGFβ signaling normalized IFN-γ-mediated changes in Fli1, VE-cadherin, CTGF, and ET-1 levels, whereas upregulation of α-SMA and TGFβ2 was not affected. Bosentan treatment was more effective than TGFβ blockade in reversing the actions of IFN-γ, including downregulation of α-SMA and TGFβ2, suggesting that activation of the ET-1 pathway plays a main role in the IFN-γ responses in HDMECs. IFN-γ induced expression of selected genes related to endothelial-to-mesenchymal transition (EndoMT), including Snail1, FN1, PAI1, TWIST1, STAT3, RGS2, and components of the WNT pathway. The effect of IFN-γ on EndoMT was mediated via TGFβ2 and ET-1 signaling pathways. This study demonstrates distinct effects of IFN-α and IFN-γ on the biology of vascular endothelial cells. IFN-γ may contribute to abnormal vascular remodeling and fibrogenesis in SSc, partially via induction of EndoMT.

Topics: Cells, Cultured; Endothelial Cells; Endothelin-1; Endothelium, Vascular; Fibrosis; Humans; Interferon-gamma; Scleroderma, Systemic; Transforming Growth Factor beta2; Up-Regulation

Circulating monocytes are a highly plastic and functionally heterogeneic cell type with an activated phenotype in patients with systemic sclerosis (SSc). CD14(+) monocytes have the potential to differentiate into extra-cellular matrix (ECM) producing cells, possibly participating in fibrogenesis.. To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts.. CD14(+) cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment.. GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses.. SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties.

Topics: Actins; Adult; Aged, 80 and over; Case-Control Studies; Cell Differentiation; Cell Shape; Cells, Cultured; Collagen Type I; Endothelin-1; Female; Gels; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-4; Lipopolysaccharide Receptors; Male; Middle Aged; Monocytes; Myofibroblasts; Phenotype; Scleroderma, Systemic; Up-Regulation

Endothelin-1 (ET-1) has been implicated in the development of pulmonary fibrosis, based on its capacity in vitro to promote extracellular matrix (ECM) production and contraction, and on studies showing elevated expression of ET-1 and its receptors in patients with pulmonary fibrosis. However, the in vivo fibrogenic effect of ET-1 is not well characterized. We used the adenoviral-mediated gene transfer of ET-1 to overexpress ET-1 transiently in murine lungs by intratracheal administration. An increased expression of ET-1 for 3 to 10 days after injection resulted in a moderate but reversible fibrotic response, peaking on Day 14 after infection and characterized by the deposition of ECM components, myofibroblast formation, and a significant inflammatory infiltrate, mainly in the peribronchiolar/perivascular region. Adenoviral-mediated ET-1 overexpression activated focal adhesion kinase (FAK) both in vitro, using primary murine lung fibroblasts, and in vivo, intratracheally administered in the lungs of mice. The inhibition of FAK with the compound PF-562,271 prevented ET-1-mediated collagen deposition and myofibroblast formation, thereby preventing the development of lung fibrosis. In conclusion, we demonstrate that the overexpression of ET-1 directly in the lungs of mice can initiate a fibrogenic response characterized by increased ECM deposition and myofibroblast formation, and that this effect of ET-1 can be prevented by inhibition of FAK. Our data suggest that the ET-1/FAK axis may contribute importantly to the pathogenesis of fibrotic disorders, and highlight FAK as a potential therapeutic target in these devastating diseases.

Topics: Adenoviridae; Animals; B-Lymphocytes; Cell Differentiation; Cells, Cultured; Endothelin-1; Enzyme Activation; Focal Adhesion Protein-Tyrosine Kinases; Genetic Vectors; Humans; Indoles; Lung; Mice; Mice, Inbred C57BL; Myofibroblasts; Phosphorylation; Protein Processing, Post-Translational; Pulmonary Fibrosis; Recombinant Proteins; Scleroderma, Systemic; Sulfonamides; T-Lymphocytes

The objective of the study was to investigate the role of endothelin-1 in the pathogenesis of scleroderma renal crisis in patients with systemic sclerosis. We used immunohistochemical analysis with anti-endothelin-1 and anti-von Willebrand factor antibodies in comparing kidney biopsies from patients with systemic sclerosis and scleroderma renal crisis (n = 14); from normal kidneys (n = 5); and from patients with typical hemolytic uremic syndrome and thrombotic microangiopathy (n = 5), antiphospholipid syndrome (n = 6), diabetic nephropathy (n = 5), minimal change disease with cyclosporine toxicity (n = 5), or nephroangiosclerosis (n = 5). Kidney biopsies from all systemic sclerosis patients presented specific lesions: glomerular lesions with thickened capillary walls (n = 6, 42.8%), mesangiolysis (n = 3, 21.4%), fibrin thrombi (n = 3, 21.4%), hypertrophy of juxtaglomerular apparatus (n = 5, 35.7%), arteriolar lesions showing mucinous intimal thickening and lumen mucoid occlusions (n = 13, 92.8%), proliferation of intimal cells (ie, "onion-skin" lesions; n = 13, 92.8%), fibrinoid necrosis (n = 3, 21.4%), and fibrin thrombosis (n = 4, 28.6%). Chronic lesions in large arteries showed modifications such as fibrous intimal thickening (n = 13, 92.8%). The pattern of endothelial staining for endothelin-1 in both glomeruli and arteriolar lesions appears to be specific for scleroderma renal crisis. Glomerular endothelin-1 staining without arteriolar staining was seen in hemolytic uremic syndrome; and isolated arteriolar staining (without glomerular staining) was seen in a number of conditions including antiphospholipid nephropathy, cyclosporine toxicity, and diabetic nephropathy. Endothelin-1 is overexpressed in glomeruli and arterioles of patients with scleroderma renal crisis, which suggests that endothelin-1 might be a therapeutic target in this condition.

Topics: Acute Kidney Injury; Adult; Aged; Antiphospholipid Syndrome; Endothelin-1; Female; Hemolytic-Uremic Syndrome; Humans; Kidney; Male; Middle Aged; Scleroderma, Systemic; von Willebrand Factor

In patients with systemic sclerosis (SSc), the relationship between innate immune activation, represented by increased expression of interferon (IFN)-regulated genes, and vascular injury/activation, manifest by increased endothelin-1 (ET-1), endothelin converting enzyme-1 (ECE1) and intercellular adhesion molecule-1, is uncertain.. To investigate the potential roles of innate immune ligands in both these pathogenic pathways.. The effect of known Toll-like receptor (TLR) ligands was tested in vitro on dermal microvascular and pulmonary arterial endothelial cells, and on dermal fibroblasts cultured from healthy controls and patients with SSc. To test the effect of double-stranded RNA (dsRNA) on vascular activation/injury in vivo, polyinosinic/polycytidylic acid (poly(I:C)) was administered continuously over 7 days by subcutaneous osmotic pump.. dsRNA/poly(I:C), but not other TLR ligands, highly stimulated ET-1 protein and mRNA (EDN1), as well as intercellular adhesion molecule-1 (ICAM-1) and IFN-regulated MX2, by endothelial cells and dermal fibroblasts. Poly(I:C) induced EDN1, ECE1, and ICAM-1 mRNA expression in poly(I:C) treated skin. Poly(I:C)-induced EDN1, ECE1 and MX2 was not blocked in mice with the type I IFN receptor deleted. However, poly(I:C)-induced EDN1 and ECE1, but not poly(I:C)-induced ICAM-1 expression was blocked in mice with the TLR3 signalling protein TRIF/TICAM-1 deleted.. Together these data show that the dsRNA can regulate genes associated with vascular activation, as seen in SSc, that type I IFNs do not mediate these effects, and that EDN1 and ECE1 but not ICAM-1 activation is mediated by TLR3.

Topics: Animals; Cells, Cultured; Endothelial Cells; Endothelin-1; Endothelium, Vascular; Fibroblasts; Gene Expression Regulation; GTP-Binding Proteins; Humans; Immunity, Innate; Ligands; Mice; Mice, Inbred C57BL; Myxovirus Resistance Proteins; Poly I-C; Pulmonary Artery; RNA, Double-Stranded; RNA, Messenger; Scleroderma, Systemic; Skin; Toll-Like Receptor 3

Systemic sclerosis (SSc) is a rare multisystem chronic disease and its etiology is still unknown. To obtain and generate reasonable treatment methods, new mediators or targets are needed. Urotensin-II (U-II) is predominantly a vasoactive peptide with fibrotic and prothrombotic features. Like endothelin-1 (ET-1), U-II could play an important role in SSc pathogenesis given its properties of convenient one-to-one SSc pathogenetic pathways. A consecutive group of 55 patients diagnosed with SSc and 30 healthy controls were included in the study. Patients and healthy controls were evaluated for clinical and laboratory manifestations, specific organ involvement, autoantibodies, and activity scores specific for SSc. In addition, plasma ET-1 and plasma levels of U-II-like immunoreactivity of both groups were compared. ET-1 level significantly increased in the SSc group in contrast to the healthy controls (6.38 ± 1.39 and 0.99 ± 0.27 pg/ml; p = 0.006). U-II was also significantly elevated in patients, and the plasma levels of U-II-like immunoreactivity were positively correlated with ET-1 (8.19 ± 1.74 and 1.02 ± 0.19 pg/ml; p = 0.003 and p = 0.0001; r = 0.887). For reasonable treatment of SSc, we need to focus on new targets such as ET-1 and U-II. This study hypothesized that these mediators could have a role in SSc pathogenesis, and U-II antagonist might be a potential alternative therapy for these patients.

Topics: Case-Control Studies; Endothelin-1; Female; Humans; Male; Middle Aged; Scleroderma, Systemic; Severity of Illness Index; Urotensins

Systemic sclerosis (SSc) is a disorder of systemic and dermal fibrosis of uncertain etiology. Recently, we found that SSc epidermis is abnormal, taking on an activated phenotype observed during wound healing and tissue repair. As epithelial-fibroblast interactions are important during wound repair and in fibrosis in general, we investigated further the phenotype of the SSc epidermis, and tested whether the SSc epidermis provides a pro-fibrotic stimulus to fibroblasts. In this study we show that in SSc epidermis keratinocyte maturation is delayed, and wound-associated keratins 6 and 16 are induced, in both involved and clinically uninvolved skin. Phosphorylation array analysis revealed induction of stress-induced mitogen-activated protein kinase signaling and mesenchymal feedback through hepatocyte growth factor/c-Met in SSc epidermis. SSc epidermal cells maintained with normal fibroblasts in three-dimensional co-culture were found to stimulate fibroblasts, leading to contractility and connective tissue growth factor expression. These effects depend on elevation of IL-1alpha by the epidermal cells and induction of endothelin-1 and transforming growth factor-beta in fibroblasts. Antagonism of endogenous IL-1alpha using IL-1 receptor antagonist blocked gel contraction by SSc epidermis. We propose that in SSc, epidermal cells are in a persistently activated state and are able to promote dermal fibrosis. These findings are important because biologic therapies could target epithelial-fibroblast interactions in the disease.

Topics: Biopsy; Cell Communication; Cells, Cultured; Coculture Techniques; Connective Tissue Growth Factor; Endothelin-1; Epidermis; Epithelial Cells; Fibroblasts; Fibrosis; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1alpha; Keratin-16; Keratin-6; Phosphorylation; Proto-Oncogene Proteins c-met; Scleroderma, Systemic; Signal Transduction; Stress, Physiological; Transforming Growth Factor beta

Systemic Sclerosis (SSc) is a multisystem connective tissue disease characterized by fibrosis of the skin and internal organs and extensive vasculopathy. We report a case of a 41 year-old white woman with a 10 year-history of limited scleroderma, who developed the rare combination of Scleroderma Renal Crisis (SRC) and Systemic Sclerosis related Pulmonary Arterial Hypertension (SScPAH) in the same time. Although the patient received the proposed antihypertensive treatment, the renal function did not recover, and she initiated on renal replacement therapy. SRC and SScPAH are two aspects of SSc vasculopathy characterized by endothelial dysfunction mediated by endothelin-1 and other vasoactive hormones. Further new studies with therapies directed towards the underlying mechanisms of SRC (i.e. endothelin-receptor antagonists), which are proven helpful in SScPAH, should take place to establish new therapeutic options and improve prognosis of these patients, for which our therapeutic armamentarium is currently poor.

Topics: Acute Kidney Injury; Adult; Endothelin-1; Endothelium, Vascular; Familial Primary Pulmonary Hypertension; Female; Humans; Hypertension, Pulmonary; Renal Replacement Therapy; Scleroderma, Systemic

Pulmonary arterial hypertension (PAH) is the major complication of systemic sclerosis (SSc) and the main cause of morbi-mortality. It is important to find predictors for this vascular problem. The objective of this study was to determine the serum levels of different biomarkers in patients with SSc and secondary PAH and to compare them with those of healthy control subjects to define their potential role as predictors of PAH. Cross-section study in which 20 patients with SSc were included. PAH was diagnosed by echocardiogram. The optical densities of endoglin (Eng), endothelin-1 (ET-1), platelet-derived growth factor (PDGF), tumoral necrosis factor alpha (TNF-alpha), Transforming growth factor beta 2 (TGF-beta2) and Interleukin 8 (IL-8) were measured in 20 patients with SSc and 20 healthy controls matched by sex. The differences found between the group of patients with PAH and the control group were (mean or median and range): ET-1 (0.20; 0.10-0.35 vs. 0.16; 0.10-0.24; P = 0.0276), IL-8 (195.7; 45.5-504 vs. 118.9; 23-299.5; P = 0.0364), TNF-alpha (0.70; 0.50-0.96 vs. 0.48; 0.38-0.65; P = 1 x 10(-8)) and Eng (0.95; 0.57-1.72 vs. 0.75; 0.57-0.89; P = 0.0028). A correlation was found between the progression of the disease and the development of Raynaud's phenomenon (Rho: 0.67 and P = 0.0011), ET-1 and Eng (Rho: 0.53 and P = 0.0196), and between IL-8 and Eng (Rho: 0.68 and P = 0.0019). In conclusions, the elevation of the serum levels of Eng and ET-1 could represent a useful tool as PAH biomarkers. Nevertheless, the diagnostic value of these markers needs to be determined by prospective studies.

Topics: Adult; Age of Onset; Aged; Antigens, CD; Biomarkers; Case-Control Studies; Cross-Sectional Studies; Echocardiography; Endoglin; Endothelin-1; Female; Humans; Hypertension, Pulmonary; Male; Receptors, Cell Surface; Scleroderma, Systemic

Topics: Administration, Oral; Adult; Antihypertensive Agents; Bosentan; Cytokines; Endothelin-1; Female; Follow-Up Studies; Humans; Hypertension, Pulmonary; Interleukin-12; Male; Middle Aged; Receptors, Endothelin; Scleroderma, Systemic; Sulfonamides

We evaluated endothelin (ET)-1 plasma levels and some clinical measures in patients with primary Raynaud's phenomenon (PRP), and in patients with systemic sclerosis (SSc) and secondary RP (SRP), in the latter according to their different nailfold videocapillaroscopy (NVC) patterns of microangiopathy (early, active, and late).. Ninety-nine patients with SSc, 49 with PRP, and 45 control subjects were studied. NVC was performed in all patients to distinguish the pattern of microvascular damage, and the morphological alterations were scored by a semiquantitative rating scale. ET-1 plasma levels were evaluated in all individuals by ELISA.. ET-1 plasma levels were significantly higher (p=0.001) in patients with both PRP and SRP, compared to controls. A significant positive correlation (p=0.03) was found between ET-1 plasma levels and SRP duration, but not between ET-1 plasma levels and PRP duration. Significant correlations were observed in patients with SSc between ET-1 plasma levels and clinical measures (e.g., digital ulcers), as well as the score value of single NVC measures, such as the number of capillaries, "ramified" capillaries, and enlarged capillaries (p<0.05). Finally, the highest ET-1 plasma levels were found in patients with SSc showing the late pattern of microangiopathy when compared to the early pattern (p=0.03) and to controls (p=0.003).. Highest ET-1 plasma levels were detected in the more advanced stage of the SSc microangiopathy, namely the late NVC pattern, characterized by capillary loss and increased tissue fibrosis; this might support the involvement of ET-1 in the progression of the microvascular/fibrotic SSc damage.

Topics: Adolescent; Adult; Aged; Capillaries; Endothelin-1; Female; Humans; Male; Microscopic Angioscopy; Middle Aged; Raynaud Disease; Scleroderma, Systemic; Skin; Young Adult

Systemic sclerosis is a connective tissue disorder with unclear aetiology and pathogenesis. However, there is evidence that microvascular changes belong to the early symptoms of the disease. These are associated with increased serum levels of markers of endothelium activation, such as adhesion molecules and growth factors. The stable prostacyclin analogue iloprost is licensed for vascular symptoms (Raynaud's phenomenon) and was recently shown to exert short-term effects on these markers. In this study, serum samples (n = 13) from patients with systemic sclerosis were examined for serum levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1, E-selectin, endothelin-1 and vascular endothelial growth factor over 6 months after iloprost infusions in order to detect possible long-term effects. Iloprost significantly reduced initially elevated levels of these markers, partly until the end of the observation period (E-selectin, VCAM-1, endothelin-1). These effects provide serological evidence for the benefits of iloprost infusions that are seen clinically in patients with systemic sclerosis.

Topics: Adult; Aged; Biomarkers; E-Selectin; Endothelin-1; Female; Humans; Iloprost; Infusions, Intravenous; Intercellular Adhesion Molecule-1; Middle Aged; Scleroderma, Systemic; Statistics, Nonparametric; Time Factors; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A; Vasodilator Agents

Impaired endothelium-dependent vasodilator function may contribute to vascular damage in patients with systemic sclerosis (SSc). This study was undertaken to investigate whether increased activity of the endothelin 1 (ET-1) system plays a role in the occurrence of endothelial dysfunction in patients with SSc.. In 12 patients with SSc (6 with diffuse cutaneous SSc [dcSSc] and 6 with limited cutaneous SSc [lcSSc]), forearm blood flow responses to graded doses of acetylcholine (ACh) and sodium nitroprusside (SNP) given intraarterially were assessed by plethysmography, during infusion of saline and following selective blockade of ETA receptors with BQ-123 (10 nmoles/minute).. During saline infusion, the vasodilator response to ACh was blunted in patients with SSc as compared with that in healthy controls (P<0.001), whereas the response to SNP was not different between groups (P=0.27). The vasodilator effect of ETA receptor antagonism was higher in patients than in controls (P<0.001), indicating enhanced ET-1-mediated vasoconstriction in SSc. In patients, ETA receptor blockade resulted in a potentiation of the vasodilator response to ACh (P<0.001 versus saline), but did not affect the response to SNP (P=0.31). Notably, both the vasodilator effect of ETA receptor antagonism and the improvement in the responsiveness to ACh following BQ-123 infusion were higher in patients with dcSSc than in those with lcSSc (P<0.01).. ET-1-dependent vasoconstrictor tone is increased predominantly in the subgroup of SSc patients with dcSSc, in whom acute blockade of ETA receptors was able to improve impaired endothelium-dependent vasodilator function. Our results suggest novel vasculoprotective effects of ETA receptor antagonism and support further exploration of strategies that target the ET-1 pathway in SSc.

Topics: Acetylcholine; Adult; Antihypertensive Agents; Case-Control Studies; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Female; Humans; Infusions, Intra-Arterial; Male; Middle Aged; Nitroprusside; Peptides, Cyclic; Plethysmography; Receptor, Endothelin A; Regional Blood Flow; Scleroderma, Systemic; Vasodilation; Vasodilator Agents

Pulmonary hypertension in systemic sclerosis appears in up to 35% of patients, is characterized by increased pulmonary artery resistance, and carries a poorer prognosis than in patients with other causes of pulmonary hypertension. The recommended therapy for stage III disease, based on clinical trials and by the Israeli Ministry of Health for 2006, includes bosentan (Tracleer), an endothelin-1 antagonist. This is a case report of a patient with severe pulmonary hypertension secondary to systemic sclerosis where therapy with prostacyclins was contraindicated due to the development of congestive heart failure and severe atherosclerosis. The patient responded well to bosentan monotherapy for 4 years until his death.

Topics: Antihypertensive Agents; Bosentan; Endothelin-1; Fatal Outcome; Humans; Hypertension, Pulmonary; Male; Middle Aged; Prognosis; Scleroderma, Systemic; Sulfonamides

Systemic sclerosis (SSc) is characterized by both vasculopathy and autoimmunity. The interplay between these pathogenetic links requires further exploration. The aim was to assess the interrelationship of endothelin-1 (ET-1), a vasoconstrictor peptide, whose levels are usually elevated in the plasma of the patients with SSc and the function of monocyte-derived dendritic cells (MDDCs), which serve as organizers of the immune response. MDDCs were grown from 5 patients with SSc and severe Raynaud's phenomenon and 5 healthy volunteers. The cells were further stimulated by synthetic ET-1, lipopolysaccharide (LPS) or both. The production of endogenous ET-1, TNFalpha and IL-12 was assessed by RT-PCR and/or ELISA. The plasma levels of ET-1 were significantly higher in patients with SSc compared to healthy controls (p = 0.0005). The production of ET-1 by MDDCs was negligible in all examined conditions, while the release of TNFalpha and IL-12 was stimulated by LPS but not by ET-1. The in vitro concentration of the exogenous ET-1, where added, was comparable to the plasma levels of ET-1 in patients with SSc. High plasma levels of ET-1 are characteristic for the patients with SSc and severe Raynaud's phenomenon. An in vitro model with concentrations of ET-1 comparable to those in the plasma of SSc patients has been elaborated. The examined function of MDDCs from SSc patients and healthy volunteers did not differ under these conditions and was not dependent on the presence of ET-1.

Topics: Adult; Dendritic Cells; Endothelin-1; Female; Humans; Interleukin-12; Male; Middle Aged; Monocytes; Peptides; Raynaud Disease; Scleroderma, Systemic; Tumor Necrosis Factor-alpha

Systemic sclerosis (SSc) is a systemic inflammatory disorder, resulting in severe vascular dysfunction. The endothelin (ET) system has vasoconstrictor and profibrotic properties and has been shown to be activated in SSc. ET antagonists are currently used in SSc-related pulmonary arterial hypertension, but the endothelial impact of ET antagonists remains less known in SSc. We thus assessed the effects of the dual ET(A)-ET(B) antagonist, bosentan, on endothelial dysfunction in a murine model of SSc, the heterozygous tight-skin mice 1 (TSK1(+)). Six-week-old TSK1(+) were either untreated or treated for 6 weeks with bosentan (100 mg/kg/day), and compared with controls. Endothelial function was evaluated in isolated mesenteric resistance arteries, using a small vessel myograph. TSK1(+) displayed endothelial dysfunction, as shown by a decreased response of mesenteric arteries to acetylcholine, especially in the presence of L-nitro-arginine methyl ester (L-NAME), corresponding to NO-independent, prostaglandin-mediated relaxation. The NO-independent relaxation was partially restored in bosentan-treated TSK1(+), and this was abolished by a cyclo-oxygenase inhibitor. Therefore, the murine model of SSc, TSK1(+) exhibits severe endothelial dysfunction of peripheral resistance arteries. The ET antagonist bosentan prevents endothelial alterations, suggesting a major role of ET in the adverse vascular effects of SSc.

Topics: Acetylcholine; Animals; Bosentan; Cyclooxygenase Inhibitors; Diclofenac; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Enzyme Inhibitors; Mesenteric Arteries; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Prostaglandins; Receptors, Endothelin; Scleroderma, Systemic; Sulfonamides; Vasodilation; Vasodilator Agents

To study the expression of adhesion molecules in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and the effects of therapy with the endothelin-1 (ET-1) receptor antagonist, bosentan.. In all, 35 patients with SSc and 25 healthy donors (HD) were selected for this study. Of 35 patients, 10 had isolated PAH assessed by Doppler echocardiography and treated with bosentan. Peripheral blood (PB) lymphocytes were isolated by density gradient centrifugation, and the expression of lymphocyte function-associated antigen-1 (LFA-1), very late antigen-4 (VLA-4) and L-selectin on CD3 T cells was assessed by double immunofluorescence and flow-cytometry. As endothelial activation markers, serum soluble P-selectin, platelet/endothelial cell adhesion molecule (PECAM)-1, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1 and von Willebrand factor (vWF) antigen were assessed by ELISA. In patients with SSc-PAH, T cell subsets and soluble endothelial markers were assessed at baseline and after 6 and 12 months of bosentan therapy.. In patients with SSc-PAH, serum soluble ICAM-1, VCAM-1, P-selectin and PECAM-1 levels were higher than in HD at baseline and fell to normal values after 12 months of bosentan therapy. CD3-LFA1 T cells were significantly higher in PAH-SSc at baseline than in HD or SSc and significantly decreased after therapy. CD3-L-selectin T cells were significantly lower in SSc-PAH at baseline than in HD or SSc and rose to normal levels after bosentan therapy.. This study confirms that endothelial activation occurs in SSc, and suggests that changes in the T cell/endothelium interplay take place in SSc-associated PAH. Bosentan seems to be able to hamper these changes and restore T cell functions in these patients.

Topics: Adult; Aged; Analysis of Variance; Antibodies, Antinuclear; Antihypertensive Agents; Autoantibodies; Bosentan; Case-Control Studies; CD3 Complex; Cell Adhesion Molecules; Centromere; Endothelin-1; Female; Humans; Hypertension, Pulmonary; Integrin alpha4beta1; L-Selectin; Lymphocyte Function-Associated Antigen-1; Male; Middle Aged; Scleroderma, Systemic; Sulfonamides; T-Lymphocytes

Growth of fibroblasts from bronchoalveolar lavage fluid (BALF) in patients with systemic sclerosis (SSc) has previously been described. The purpose of the present study was to characterise fibroblasts from BALF and bronchial biopsies from SSc patients with alveolitis and from controls, to analyse fibroblast proliferation, migration, stress fibres and proteoglycan production. BALF and bronchial biopsies were collected from 10 patients with SSc and alveolitis and from 15 controls. Outgrowth of fibroblasts was observed from the BALF of four patients, particularly in those with a markedly increased percentage of eosinophils in BALF, but not in any member of the control group. Increased levels of granulocyte-macrophage colony-stimulating factor, correlating with the percentage of eosinophils in BALF, were found in patients when compared with controls. Fibroblasts from BALF showed an elongated, mobile phenotype and increased proteoglycan production compared to the corresponding biopsy fibroblasts. In conclusion, outgrowth of fibroblasts with an altered phenotype is reported from bronchoalveolar lavage fluid in systemic sclerosis patients with alveolitis and an increased percentage of eosinophils in the bronchoalveolar lavage fluid. These findings indicate a possible role for eosinophil-fibroblast interaction in pulmonary fibrosis in systemic sclerosis.

Topics: Adult; Aged; Biopsy; Bronchi; Bronchoalveolar Lavage Fluid; Cell Division; Cell Movement; Endothelin-1; Female; Fibroblasts; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Lung Volume Measurements; Male; Middle Aged; Proteoglycans; Pulmonary Fibrosis; Scleroderma, Systemic; Stress Fibers

According to current knowledge, endothelin (ET)-1 plays an important role in the pathogenesis of systemic sclerosis (SSc). We assessed ET plasma levels in SSc patients according to the clinical presentation and the presence of complications such as pulmonary arterial hypertension (PAH).. Sixty-three consecutive patients with SSc were included. The control group included 17 healthy patients. ET plasma level was determined for all patients. Pulmonary function test and pulmonary high resolution computed tomography were performed in 44 patients and echocardiography in 51 patients, to screen for PAH, always confirmed by a right heart catheterization.. ET plasmatic levels were higher in SSc patients than in healthy group subjects but the difference was not significant (3.72+/-1.13 vs 3.40+/-0.71 pmol/l, p=0.27). ET plasmatic levels were significantly higher in patients with PAH than in patients without PAH (4.28+/-0.65 vs 3.62+/-1.07 pmol/l, p=0.04) and in patients with anticentromere antibodies (3.96+/-1.11 vs 3.19+/-1.12 pmol/l, p=0.03). There was a positive linear correlation between ET plasmatic levels and systolic pulmonary arterial pressure (r=0.34, p=0.013). The best cut-off value for ET plasmatic level to discriminate patients affected by PAH was determined by ROC curve method: 4.1 pmol/l (sensibility 85.7%, specificity 66%).. ET plasmatic levels were higher in SSc patients affected by PAH and patients with anticentromere antibodies. There was a positive linear correlation between ET plasmatic levels and systolic pulmonary arterial pressure. Assessment of ET plasmatic levels for detection and monitoring of pulmonary hypertension during SSc is warranted in larger prospective studies.

Topics: Aged; Biomarkers; Cardiac Catheterization; Echocardiography; Endothelin-1; Female; Humans; Male; Middle Aged; Radiography; Reference Values; ROC Curve; Scleroderma, Localized; Scleroderma, Systemic

Fibrosis is excessive scarring caused by the accumulation of extracellular matrix proteins and is a common end pathway in many chronic diseases. Endothelin-1 is a possible contributor to the persistent fibrotic phenotype of fibroblasts isolated from fibrotic lesions. In this report we used a specific dual endothelin A/B receptor antagonist, bosentan, to determine the role of endogenous endothelin signaling in maintaining the profibrotic phenotype of lung fibroblasts from scleroderma patients. Bosentan treatment of lung fibroblasts cultured from normal individuals and individuals with scleroderma was assessed using Affymetrix genome-wide expression profiling, real-time polymerase chain reaction and Western blot analysis and revealed that approximately one-third of the transcripts elevated greater than two-fold in fibrotic fibroblasts were reduced by Bosentan treatment. Genes whose overexpression in fibrotic fibroblasts that were dependent on endogenous endothelin signaling included the matrix or matrix-associated genes type I collagen, fibronectin and CCN2. The elevated adhesive property of fibrotic fibroblasts was also reduced by endothelin receptor antagonism. Basal expression of collagen, fibronectin and CCN2 and adhesion to matrix was not affected. Thus endogenous endothelin signaling contributes to the fibrotic phenotype of fibrotic fibroblasts, suggesting that antagonizing endothelin receptors may be of benefit in combating fibrotic disease.

Topics: Biopsy; Bosentan; Cell Adhesion; Cells, Cultured; Collagen Type I; Connective Tissue Growth Factor; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Fibroblasts; Fibrosis; Gene Expression Profiling; Gene Expression Regulation; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Lung; Receptor, Endothelin A; Receptor, Endothelin B; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Sulfonamides

Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibrosis of the skin and internal organs. Although the esophagus is the most frequently affected part of the gastrointestinal tract, all other segments can be involved. The present study was undertaken to evaluate the fibrotic process and the expression of fibrogenic cytokines in the gastric wall of SSc patients with gastroesophageal involvement.. Full-thickness surgical and endoscopic gastric biopsy samples were obtained from 14 SSc patients and 10 controls. Tissue sections were either stained with Masson's trichrome or by immunohistochemistry and analyzed for the expression of types I, III, and IV collagen, alpha-smooth muscle actin (alpha-SMA), transforming growth factor beta (TGFbeta), connective tissue growth factor (CTGF), and endothelin 1 (ET-1).. In the gastric wall of SSc patients, Masson's trichrome staining and immunohistochemistry for types I and III collagen revealed a high amount of collagen in the lamina propria that increased toward the muscularis mucosae. In addition, muscle layers showed features of atrophy, with wide areas of focal fibrosis surrounding smooth muscle cells. Type IV collagen was present around glands and small vessels, suggesting a thickening of the basal lamina. The expression of the fibrogenic cytokines TGFbeta and CTGF, ET-1, and the myofibroblast marker alpha-SMA was stronger in SSc patients than in controls.. A pronounced deposition of collagen, the presence of myofibroblasts, and increased expression of several profibrotic factors are important hallmarks in the stomach of patients with SSc. The fibrotic involvement of the gastric wall may account for muscle atrophy leading to stomach hypomotility in SSc.

Topics: Actins; Aged; Biopsy; Case-Control Studies; Collagen Type I; Collagen Type III; Collagen Type IV; Connective Tissue Growth Factor; Cytokines; Endothelin-1; Female; Fibrosis; Gastroscopy; Gene Expression; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Scleroderma, Systemic; Severity of Illness Index; Stomach; Transforming Growth Factor beta

To describe the effect of bosentan and its dual inhibition of endothelin-1 ETA and ETB receptors on digital ulcers in patients with systemic sclerosis (SSc).. Patients receiving bosentan for SSc-related digital ulcers were identified in eight centers, and their characteristics and follow-up were recorded.. Nine (six with diffuse and three with limited cutaneous forms of SSc) patients (median age: 54 years) had received bosentan for digital ulcers. Complete healing occurred in seven (median time to improvement: 4 weeks). Another experienced a significant decrease in the number of ulcers (from 22 to 5) in 8 weeks, while one had no improvement. After a median follow-up of 24.3 months, only one recurrence was observed. Raynaud phenomenon improved in all but one patient.. These data suggest that some patients may benefit from bosentan to treat digital ulcers. The short time to healing in these patients with rather chronic ulcers argues strongly in favor of its use. These results also strengthen the evidence that endothelin-1 plays an important role in the vascular manifestations of SSc.. Bosentan can be effective in the treatment of digital ulcers in some SSc patients with SSc, probably especially those involving substantial ischemia. Bosentan is not a first-line drug in this indication yet and must be carefully used by specialists in SSc. Forthcoming results from the international RAPIDS-2 study should clarify the indications for bosentan in the treatment of SSc-related digital ulcers.

Topics: Adult; Aged; Antibodies, Antinuclear; Bosentan; Endothelin-1; Female; Fingers; Follow-Up Studies; Humans; Ischemia; Male; Middle Aged; Raynaud Disease; Scleroderma, Systemic; Skin Ulcer; Sulfonamides; Time Factors; Treatment Outcome

Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis. One of several complications of SSc, pulmonary arterial hypertension (PAH) can be refractory to treatment, both novel and established. In the present study we investigated the ratio of circulating nitric oxide to endothelin-1 in patients with both SSc and PAH, and determined whether polymorphisms in NOS2 (the nitric oxide synthase 2 gene) are associated with susceptibility to PAH. Endothelin-1 in plasma and nitric oxide metabolites (nitrate and nitrite) in serum were measured. The nitric oxide/endothelin-1 ratio was significantly lower in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. We confirmed the presence of two single nucleotide polymorphisms at positions -1,026 and -277 and a pentanucleotide repeat (CCTTT) at -2.5 kilobases. There were significant differences in single nucleotide polymorphisms between patients with SSc who had PAH and those who did not, and between patients with both SSc and PAH and healthy control individuals. The CCTTT repeat was significantly shorter in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. Transcriptional activity were analyzed using the luciferase reporter assay. The transcriptional activity of NOS2 was much greater in fibroblasts transfected by a vector with a long allele of the CCTTT repeat than in those transfected by a vector with a short allele. Polymorphisms in the NOS2 gene are associated with transcriptional activity of the NOS2 gene and with susceptibility to SSc-related PAH.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Endothelin-1; Female; Genetic Predisposition to Disease; Haplotypes; Humans; Hypertension, Pulmonary; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type II; Osmolar Concentration; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Scleroderma, Systemic; Tandem Repeat Sequences; Transcription, Genetic

HLA-B35 is associated with an increased risk for developing isolated pulmonary hypertension (iPHT) in systemic sclerosis, but the mechanisms underlying this association have not been fully elucidated yet. Endothelin-1 (ET-1) is the main pathogenetic molecule implied in the development of iPHT; therefore, we sought to determine if ECV304 cells transfected with the HLA-B35 allele produce increased amounts of ET-1 after incubation with physiological concentrations of interleukin-1 beta (IL-1beta). ECV304 cells transfected with HLA-B*3501 and HLA-B*0801 polymorphic alpha chain or with pIRESneo2 were incubated with 100 U/ml of IL-1beta for 6, 12, 24, 36 and 48 h. ET-1 levels were determined using EIA kit (CAYMAN Chemical, Ann Arbor, MI) in supernatants from different cell cultures; the relative expression of the preproendothelin-1 (PPET-1) gene was also determined by reverse transcription-polymerase chain reaction. Cells expressing the HLA-B35 allele showed significantly increased levels of ET-1 at all the selected times compared with controls or HLA-B8-transfected cells. The relative expression of the PPET-1 gene was also increased in a proportionally direct manner. The HLA-B35 allele influences the production of ET-1 in HLA-B35-transfected ECV304 cells by promoting the expression of its precursor, PPET-1. Our results provide an explanation for the epidemiological association existing between iPHT and HLA-B35.

Topics: Alleles; Cell Line; Endothelial Cells; Endothelin-1; HLA-B Antigens; HLA-B35 Antigen; HLA-B8 Antigen; Humans; Hypertension, Pulmonary; Interleukin-1; RNA, Messenger; Scleroderma, Systemic; Transfection; Up-Regulation

Systemic sclerosis (SSc) is a chronic, multisystemic, autoimmune disease characterised by vascular changes and varying degrees of fibrosis of the skin and visceral organs. Organ systemic involvement in SSc is associated with an altered function of endothelial cells, perivascular infiltrating mononuclear cells and interstitial fibrosis. To evaluate the relationship between systemic manifestations and immunological markers of endothelial cell activation, serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) were determined by an enzyme-linked immunosorbent assay in 31 SSc patients and in 30 healthy controls. In comparison with the control group, higher serum concentrations of sVCAM-1, sE-selectin, VEGF and ET-1 were detected in SSc patients (in all cases p<0.001). Elevated concentrations of sVCAM-1 (p<0.05), sE-selectin (p<0.05), VEGF (p<0.05) and ET-1 (p<0.01) dominated in the serum of SSc patients with organ systemic involvement compared to those without systemic manifestation of the disease. These results suggest that the serum levels of sVCAM-1, sE-selectin, VEGF and ET-1 may reflect the extent of internal organ involvement in SSc patients and point to a pathogenic role of these molecules in systemic manifestation of the disease.

Topics: Biomarkers; E-Selectin; Endothelin-1; Female; Humans; Male; Middle Aged; Predictive Value of Tests; Scleroderma, Systemic; Severity of Illness Index; Solubility; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A

We studied circulating levels of endothelin-1, catecholamines and nitric oxide after a mental arithmetic test in 14 patients with early ischemic lesions of the extremities due to systemic sclerosis and slightly impaired peripheral vascular flow. The test induced an increase (P<0.01) in blood pressure, heart rate, endothelin-1 and catecholamine levels, whereas it did not change the low basal levels of nitric oxide. In healthy subjects (n=20) the test significantly (P<0.01) decreased endothelin-1 without affecting nitric oxide. The low basal levels of nitric oxide and the high plasma concentration of endothelin-1 after psychological stress cannot be explained by an impaired release from the limited ischemic lesions alone. This suggests a diffuse microvascular derangement that aggravates the course of peripheral microvascular ischemic lesions.

Topics: Adult; Aged; Blood Pressure; Endothelin-1; Female; Heart Rate; Humans; Ischemia; Male; Middle Aged; Nitric Oxide; Psychological Tests; Scleroderma, Systemic; Stress, Psychological

The objectives of the project were the following: (1) to establish a group of patients with a confirmed diagnosis of systemic sclerosis (Ssc), (2) to perform a detailed entrance examination of each patient, (3) to determine concentrations of potential activity markers, and (4) to make a comprehensive examination of each patient 1 year after inclusion into the study. A total of 49 patients were examined, 36 with a limited form of SSc, 9 with diffuse SSc, and 4 with other forms of SSc. We determined plasma or serum levels of the N-terminal propeptide of procollagen type III (NPIIIP), interleukin-6 (IL-6), soluble receptor for interleukin-2 (sIL-2r), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), von Willebrand factor antigen (vWFAg), and big endothelin-1 (BET-1) using commercial kits, and urinary excretion of pyridinoline (PYR) and deoxypyridinoline (D-PYR) using high-performance liquid chromatography. Correlations of these markers with selected clinical data were calculated. The mean levels of all potential activity markers were increased compared with normal values, but differences were not significant. The levels of NPIIIP, D-PYR, and IL-6 were normal. The measured values after 1 year did not differ from the entry values. At entry, NPIIIP concentrations correlated with the finger-to-palm distance, and D-PYR corresponded with findings on a simplified health assessment questionnaire (FQ). IL-6 levels correlated with the leukocyte count, sIL-2r with the FQ, and ET-1 with the diffuse lung capacity for carbon monoxide. In general, we found only a few clinical correlates of potential activity markers. Our data confirmed the correlations of collagen metabolism markers with skin involvement and FQ, as was reported previously. Larger studies in this field are needed.

Topics: Adult; Aged; Biomarkers; Collagen; Endothelin-1; Female; Humans; Intercellular Adhesion Molecule-1; Interleukin-6; Male; Middle Aged; Receptors, Interleukin-2; Scleroderma, Systemic; Vascular Cell Adhesion Molecule-1

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce alpha-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of alpha-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins alpha-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis.

Topics: Cell Membrane; Cells, Cultured; Collagen; Contractile Proteins; Endothelin A Receptor Antagonists; Endothelin-1; Fibroblasts; Fibrosis; Humans; Lung; Phenotype; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; rac GTP-Binding Proteins; Receptor, Endothelin A; Scleroderma, Systemic; Signal Transduction

Topics: Antihypertensive Agents; Bosentan; Endothelin Receptor Antagonists; Endothelin-1; Humans; Hypertension, Pulmonary; Lung; Randomized Controlled Trials as Topic; Scleroderma, Systemic; Sulfonamides; Treatment Outcome

This study examines endothelin-induced modulation of extracellular matrix synthesis and remodeling by fibroblasts, and its potential role in the pathogenesis of systemic sclerosis (scleroderma). Endothelin-1 promoted fibroblast synthesis of collagen types I and III, but not fibronectin, by a mechanism dependent upon both ETA and ETB receptors. Conversely, endothelin-1 inhibited both protein expression of matrix metalloproteinase 1 and zymographic activity exclusively via ETA receptors. A dual regulatory role for endothelin-1 in transcriptional regulation was suggested by the ability of endothelin-1 to enhance steady-state levels of collagen mRNA and activate the proalpha2(I) collagen (Col1a2) promoter, but in contrast to reduce matrix metalloproteinase 1 transcript expression and suppress transcription of a human matrix metalloproteinase 1 promoter reporter construct in transient transfection assays. Although endothelin-1 significantly enhanced remodeling of three-dimensional collagen lattices populated by normal fibroblasts, this was not observed for lattices populated by systemic sclerosis fibroblasts. Promotion of matrix remodeling was dependent upon ETA receptor expression and was blocked by specific inhibitors of tyrosine kinases or protein kinase C. Reverse transcriptase polymerase chain reaction, S1 nuclease, and functional cell surface binding studies showed that normal and systemic sclerosis fibroblasts express both ETA and ETB receptors (predominantly ETA), but that ETA receptor mRNA levels and ETA binding sites on fibroblasts cultured from systemic sclerosis skin biopsies are reduced by almost 50%. Endothelin-1 is thus able to induce a fibrogenic phenotype in normal fibroblasts that is similar to that of lesional systemic sclerosis fibroblasts. Moreover, reduced responsiveness to exogenous endothelin-1 in systemic sclerosis suggests that downstream pathways may have already been activated in vivo. These data further implicate dysregulated endothelin-receptor pathways in fibroblasts in the pathogenesis of connective tissue fibrosis.

Topics: Cells, Cultured; Collagen; Connective Tissue; Endothelin-1; Extracellular Matrix; Fibroblasts; Gene Expression; Humans; Matrix Metalloproteinase 1; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Reference Values; RNA, Messenger; Scleroderma, Systemic; Transcription, Genetic

To evaluate the relative roles of endothelium and platelets in the pathogenesis of primary RP and RP secondary to SSc.. Endothelial derived ET-1, t-PA, PAI-1, and platelet derived beta-TG, PDGF, TGF-beta were measured in 36 patients with primary RP, 14 patients with RP secondary to SSc and 30 age and sex matched controls.. A significative increase of ET-1, t-PA, PAI-1, TGF-beta, and beta-TG were the most relevant changes in patients with RP secondary to SSc with respect to the controls. Less relevant increases of t-PA, PAI-1, PDGF, and beta-TG levels were observed in patients with primary RP vs controls.. These data seem to confirm the involvement of endothelial cells and platelets in the pathogenesis of RP, with mild changes in primary RP and more relevant changes in RP secondary to SSc.

Topics: Adult; beta-Thromboglobulin; Biomarkers; Endothelin-1; Endothelium; Female; Humans; Male; Middle Aged; Plasminogen Activator Inhibitor 1; Platelet Activation; Platelet-Derived Growth Factor; Raynaud Disease; Scleroderma, Systemic; Tissue Plasminogen Activator; Transforming Growth Factor beta

Topics: Adrenomedullin; Adult; Aged; Case-Control Studies; Endothelin-1; Female; Humans; Hypertension, Pulmonary; Male; Middle Aged; Peptides; Scleroderma, Systemic

The precise mechanism of diffuse pigmentation in systemic sclerosis (SSc) is still unknown. We suspected the participation of endothelin-1 (ET-1), which is produced by keratinocytes, in the hyperpigmentation in SSc. The aims of this study are to demonstrate the hyperproductivity of ET-1 from epidermal cells in SSc by in situ hybridization histochemistry, and to show a correlation between the hyperproductivity of ET-1 in keratinocytes and skin hyperpigmentation.. In situ hybridization histochemistry was performed on nine SSc specimens (five cases of diffuse scleroderma (dSSc), four cases of acrosclerosis (lSSc)), and compared with four normal control specimens. We counted the grains on 10 x 10 microm(2) of epidermis and microvessels in each histology and examined the degree of skin pigmentation using the skin reflectance factor (Y).. In the specimens of the SSc patients, the number of grains on the epidermis was remarkably higher than those of the control specimens (P < 0.01). We found a close correlation between the number of grains and the skin reflectance factor in dSSc patients (P = 0.02). Correlations were not identified between serum ET-1 and skin pigmentation and between serum ET-1 and the frequency of grains on the epidermis. As for grains on microvessels, lSSc patients showed a greater frequency than dSSc patients.. The findings of this study suggest that an increase in the ET-1 productivity of keratinocytes is experienced in SSc patients, especially in dSSc patients. The results suggest a strong correlation between the ET-1 productivity of keratinocytes and skin pigmentation in severe cases of SSc. We conclude from these results that keratinocyte-derived ET-1 plays an important role in the pathogenesis of the hyperpigmentation of the skin in SSc patients.

Topics: Adult; Aged; Endothelin-1; Epidermis; Female; Humans; In Situ Hybridization; Keratinocytes; Male; Middle Aged; Scleroderma, Systemic; Skin Pigmentation

Scleroderma renal crisis (SRC) developed in two patients with systemic sclerosis (SSc) and they died from respiratory failure. Findings on autopsy revealed congestion and oedema in both lungs and intimal thickening of the small renal arteries in both patients. Immunohistological investigations showed positive staining of anti-human endothelin (ET)-1 in the media of the small renal arteries and ET-B receptor in the medial smooth muscle of the small renal arteries. This observation suggests an important pathophysiological role of ET-1 in the development of SRC in some patients with SSc.

Topics: Acute Kidney Injury; Aged; Endothelin-1; Fatal Outcome; Female; Humans; Immunohistochemistry; Kidney; Middle Aged; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Renal Artery; Scleroderma, Systemic

We investigated the potential modulation of cell surface intercellular adhesion molecule-1 (ICAM-1) expression and function by ET-1 in fibroblasts grown from skin biopsies of scleroderma (SSc) patients compared with healthy controls. Surface ICAM-1 expression was quantified by cell-bound ELISA and by FACS analysis. ICAM-1 function was investigated by measuring cell adhesion, and we studied ICAM-1 gene expression using RT-PCR. Fibroblast ET-1 binding sites were measured using 125I-labeled ET-1, and the modulation of ICAM-1 function by ET-1 was determined by measuring the binding of human U937 cells to fibroblasts in the presence of a mixed ETA/B receptor antagonist (bosentan) or a neutralizing anti-ICAM-1 antibody. ICAM-1 expression was significantly higher in SSc fibroblasts compared with normal controls. ET-1 increased ICAM-1 on both normal and SSc fibroblasts to comparable levels. RT-PCR demonstrated that ICAM-1 mRNA was upregulated by ET-1, and results from binding studies showed fibroblasts exposed to ET-1 supported more U937 cells than controls, a process that could be inhibited by bosentan and ICAM-1 neutralizing antibody. Autoradiography showed ET-1 receptors on both normal and SSc fibroblasts. Our findings indicate that SSc fibroblasts express intrinsically elevated levels of surface ICAM-1 and message. ET-1 can induce normal fibroblasts to express some SSc phenotypes and may function as a potent proinflammatory mediator, similar to cytokines, and therefore may also have immunoregulatory functions for immune cells infiltrating and binding to connective tissues.

Topics: Autoradiography; Cell Adhesion; Cells, Cultured; Endothelin-1; Fibroblasts; Gene Expression Regulation; Humans; Intercellular Adhesion Molecule-1; Polymerase Chain Reaction; Scleroderma, Systemic

Endothelin-1 (ET-1) has some relation to the pathogenesis of systemic sclerosis (SSc) and Raynaud's phenomenon. This study was performed to determine the localization of ET-1 in patients with SSc.. The localization of ET-1 on the specimen by skin biopsies from nine patients with SSc, was observed with immunoelectron microscopic techniques.. High-density deposits existed on the ribosomes and on the rough endoplasmic reticulum in the endothelial cells of microvessels of the upper dermis. The same findings were also seen in the fibroblasts of the dermis, but not found in the skin of normal controls. The level of deposits in the endothelial cells and dermal fibroblasts seemed to have a positive correlation with the serum levels of ET-1 of patients with SSc.. From these results, it can be seen that ET-1 is produced much more from the endothelial cells and fibroblasts of the dermis in the skin of SSc patients than from the normal controls. It is suspected that ET-1 is one of the pathogenetic factors of SSc.

Topics: Biopsy; Case-Control Studies; Endothelin-1; Endothelium, Vascular; Female; Fibroblasts; Humans; Immunoenzyme Techniques; Male; Microscopy, Immunoelectron; Middle Aged; Scleroderma, Systemic; Skin

We evaluated 30 consecutive patients and 48 age- and sex-matched controls to explore the possibility of a pathogenic contribution by plasma endothelin-1 in the cardiac expression of systemic sclerosis. Venous plasma endothelin-1 was measured by radio-immunoassay and left ventricular function by echocardiography. The patient group had elevated plasma endothelin-1 (2.6 +/- 0.2 vs. 1.8 +/- 0.1 pmol/1, P < 0.001), but endothelin-1 was not related to age, heart rate, blood pressure, total peripheral resistance, disease duration or systemic sclerosis score. Endothelin-1 was related to left ventricular hypertrophy in terms of septal thickness (r = 0.33, P < 0.01) and left ventricular mass index (r = 0.32, P < 0.01). Plasma endothelin-1 was further related to measures indicating reduced left ventricular filling; left atrial emptying index (r = -0.50, P < 0.0005), the first third filling fraction (r = -0.31, P < 0.05) and the time velocity integral of Doppler early/late filling velocity (r = -0.40, P < 0.001). Furthermore, circulating endothelin-1 was related to impaired left ventricular contractility as estimated by pre-ejection period/left ventricular ejection time (r = 0.32, P < 0.01) and end-systolic wall stress/volume index (r = -0.30, P < 0.05). We conclude that plasma endothelin-1 is elevated in relation to the degree of left ventricular hypertrophy, diastolic dysfunction and impaired contractility in systemic sclerosis. It may be of pathogenic importance to the cardiac involvement in systemic sclerosis which is not mediated via an increase in systemic blood pressure. It is not yet clear whether our findings are exclusive to systemic sclerosis patients or represent a generalized phenomenon in patients with impaired left ventricular function.

Topics: Adult; Aged; Atrial Natriuretic Factor; Cardiomyopathies; Case-Control Studies; Echocardiography; Endothelin-1; Female; Humans; Hypertrophy, Left Ventricular; Male; Middle Aged; Multivariate Analysis; Scleroderma, Systemic; Ventricular Dysfunction, Left

In addition to their vasoactive action, endothelins are potent peptides in the regulation of both cell proliferation and the turnover of extracellular matrix. Using immunohistochemical, autoradiographic, and molecular analyses, we have studied the localization and expression of endothelin-1 and endothelin A (ETA) and B (ETB) receptors in scleroderma-associated fibrotic lung disease. Increased ET-1 immunoreactivity was found in sclerotic tissue compared with control and was associated with the vasculature, pulmonary interstitium, and bronchial and alveolar epithelium. Microautoradiographic analysis after 125I-labeled ET-1 binding showed a two- to threefold increase in the expression of total ET-1 receptors in scleroderma lung tissue localized to the alveolar epithelium and the pulmonary interstitium which was composed of mainly fibroblastic cells with macrophages and some microvessels. RNAse protection assay revealed significantly reduced ETA receptor and slightly raised ETB message levels in systemic sclerosis lung. Surface expression of functional ET receptors was examined by targeted receptor blocking using mixed and receptor-subtype-selective ligands. A consistent decrease in ETA receptor binding sites was noted primarily within the interstitium and vasculature, in contrast to a slight increase in ETB receptors. Elevated ET-1 and the cell-specific pattern of endothelin receptor expression suggest that the endothelins may represent important mediators that influence the pathology of scleroderma-associated lung disease and other fibrotic conditions.

Topics: Adult; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Endothelin-1; Female; Gene Expression Regulation; HLA-DR Antigens; Humans; Immunohistochemistry; Lung; Middle Aged; Pulmonary Fibrosis; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; Scleroderma, Systemic

Endothelin-1 (ET-1), a potent fibroblast/smooth muscle cells mitogen, has been implicated in the pathogenesis of systemic sclerosis lung disease (SSc). Since monocytes and macrophages are thought to be activated in SSc, we hypothesized that alveolar macrophages (AM) and their precursors blood monocytes from patients with SSc produced more ET-1 than cells from healthy subjects. ET-1 and big ET-1 concentrations were measured in plasma, in bronchoalveolar lavage (BAL) fluids and in cell culture supernatants from monocytes and alveolar macrophages derived from 13 patients with definite SSc with lung involvement and from 10 control subjects. Plasma and BAL fluid ET-1 and big ET-1 levels were similar in both controls and patients with SSc. ET-1 and big ET-1 concentrations in unstimulated alveolar macrophage supernatants were similar in both groups. In contrast, LPS-stimulated alveolar macrophages from patients with SSc secreted higher amounts of ET-1 and big ET-1 than control subjects. ET-1 and big ET-1 concentrations in monocyte supernatants (either LPS-stimulated or not) were not different in patients and controls. These results show that AM from patients with SSc are hyperresponsive to LPS in vitro in terms of ET-1 and big ET-1 production and suggest that AM could participate in vivo in the overproduction of this potentially profibrotic mediator in patients with SSc.

Topics: Adolescent; Adult; Aged; Bronchoalveolar Lavage Fluid; Cells, Cultured; Endothelin-1; Endothelins; Female; Humans; Lipopolysaccharides; Lung Diseases, Interstitial; Macrophages, Alveolar; Male; Middle Aged; Monocytes; Prospective Studies; Protein Precursors; Scleroderma, Systemic

Endothelial cell injury in blood vessels of small arteries and capillaries is considered the primary event in the pathogenesis of systemic sclerosis (SSc). Because endothelin-1 (ET-1) is mainly released in the site of endothelial cell damage, thereby inducing a potent vasoconstriction, it was our intention to study ET-1 release in a group of SSc patients during a cold pressor test (CPT). Twelve SSc patients and a control group of 10 healthy subjects underwent CPT. Blood samples for ET-1 assay were collected at 90 and 180 seconds of exposure to cold stress. Heart rate and blood pressure were recorded at the same intervals. A capillaroscopic examination was performed in both groups before and after CPT. We observed significantly higher levels of plasma ET-1 in SSc patients compared with those of the control group at baseline (P=0.007) and at 90 (P=0.006) and 180 seconds (P=0.03) of CPT. During the test, the capillaroscopic examination showed a dramatic worsening of the vascular picture that was parallel to the increase in ET-1 plasma levels. This suggests that higher ET-1 plasma levels can have a part in the acute vascular reactivity of SSc patients undergoing CPT.

Topics: Aged; Case-Control Studies; Cold Temperature; Endothelin-1; Female; Humans; Male; Middle Aged; Scleroderma, Systemic

Forty-three patients with systemic scleroderma (SSc), 10 with non-SSc (6 cases of systemic lupus erythematosus and 4 cases of dermatomyositis), 14 cases of mild- or non-sclerotic type of scleroderma with capillaroscopic abnormalities of nailfolds (SSD; scleroderma spectrum disorders) and 10 healthy volunteers (HC) were subjected to examination of plasma levels of endothelin-1 (ET-1). The sex ratios (male/female) in the patients with SSc, non-SSc and HC were 7:36, 4:6 and 0:10, and the ranges of their ages were 22-74, 19-78 and 33-62 years old, respectively. The plasma levels of ET-1 in SSD, SSc (Barnett I;15), SSc (Barnett II;16), SSc (Barnett III;12 cases), non-SSc and HC were 1.67 +/- 0.37 2.04 +/- 0.58 2.04 +/- 0.68 1.85 +/- 041 191 +/- 0.7 and 1.31 +/- 0.34 pg/ml, respectively, confirming previous results from other laboratories. The plasma levels of ET-1 statistically differ between each collagen disease (SSD, SSc and non-SSc) and HC using Student's t-test (P < 0.05). Although a statistically significant difference was obtained in the plasma levels of ET-1 between the SSc group (6 cases) and HC (6 cases) measured at 06:00, 12:00, 18:00 and 24:00 h, there was no significant circadian variation of plasma levels of ET-1 at these times in both the SSc group and HC. The present study revealed that (1) the ET-1 level in HC showed no circadian fluctuation, and remained at a low level (0.8-1.6 pg/ml). (2) When compared to HC, ET-1 in blood plasma of patients with SSc was elevated (0.3-3 pg/ml) throughout the day and night (P < 0.05). (3) ET-1 tended to increase more at midnight (24:00 h) in the SSc group without PSL treatment, though no statistical significance was obtained. (4) TAT showed a significant increase at noon (12:00 h) suggesting coagulation activity in patients with SSc, but PlC did not show a significant increase compared to HC. In conclusion, the observed increase of vasoconstrictive ET-1 in the patients with SSc throughout the day and night may make maintenance of peripheral blood flow more difficult, may have some biological origin and should be further investigated.

Topics: Adult; Aged; Blood Coagulation; Circadian Rhythm; Endothelin-1; Female; Fibrinolysis; Humans; Male; Middle Aged; Reproducibility of Results; Scleroderma, Systemic; Vasoconstriction

Topics: Adult; Cold Temperature; Endothelin-1; Female; Humans; Male; Middle Aged; Raynaud Disease; Scleroderma, Systemic