Brief Report: Association of Elevated Adipsin Levels With Pulmonary Arterial Hypertension in Systemic Sclerosis.

OBJECTIVE: Adipose tissues secrete adipokines, peptides with potent effects modulating fibrosis, inflammation, and vascular homeostasis. Dysregulated adipose tissue biology and adipokine balance have recently been implicated in systemic sclerosis (SSc). This study was undertaken to determine whether altered circulating adipokine levels correlate with SSc disease subsets or clinical manifestations. METHODS: Multiplex assays were used to measure circulating adipokine levels in 198 patients with SSc and 33 healthy controls. Data were evaluated for correlations between serum adipokine levels and demographic and clinical features, including pulmonary arterial hypertension (PAH). To assess the relevance of adipsin, an adipokine involved in complement pathway activation, in SSc, we analyzed publicly available genetic and transcriptomic data. RESULTS: Levels of adiponectin and adipsin differed significantly between controls and patients. Adipsin was significantly elevated in patients with limited cutaneous SSc (odds ratio [OR] 28.3 [95% confidence interval (95% CI) 7.0-113.8]; P < 0.0001), and its levels were associated with serum autoantibody status, pulmonary function and cardiovascular parameters, and PAH (OR 3.3 [95% CI 1.3-8.7]; P = 0.02). Elevated adipsin was more strongly associated with PAH than B-type natriuretic peptide was. Moreover, in SSc patients, adipsin gene single-nucleotide polymorphisms were associated with PAH. Transcriptome data set analysis demonstrated elevated adipsin expression in patients with SSc-related PAH. CONCLUSION: We identify adipsin as a novel adipose tissue-derived marker of SSc-related PAH. Circulating adipsin levels might serve as predictive biomarkers in SSc. Mechanistically, adipsin might represent a pathogenic link between adipocyte dysfunction and complement pathway activation and play an important role in the pathogenesis of SSc-related PAH.

Comparison of change in end tidal carbon dioxide after three minutes of step exercise between systemic sclerosis patients with and without pulmonary hypertension.

OBJECTIVES: Pulmonary hypertension (PH) is an important cause of morbidity and mortality in patients with SSc. The submaximal heart and pulmonary evaluation (step test) is a non-invasive, submaximal stress test that could be used to identify SSc patients with PH. Our aims were to determine whether change in end tidal carbon dioxide ([Formula: see text]) from rest to end-exercise, and the minute ventilation to carbon dioxide production ratio ([Formula: see text]), both as measured by the step test, differ between SSc patients with and without PH. We also examined differences in validated self-report questionnaires and potential PH biomarkers between SSc patients with and without PH. METHODS: We performed a cross-sectional study of 27 patients with limited or dcSSc who underwent a right heart catheterization within 24 months prior to study entry. The study visit consisted of questionnaire completion; history; physical examination; step test performance; and phlebotomy. [Formula: see text], [Formula: see text], self-report data and biomarkers were compared between patients with and without PH. RESULTS: SSc patients with PH had a statistically significantly lower median (interquartile range) [Formula: see text] than SSc patients without PH [-2.1 (-5.1 to 0.7) vs 1.2 (-0.7 to 5.4) mmHg, P = 0.035], and a statistically significantly higher median (interquartile range) [Formula: see text] [53.4 (39-64.1) vs 36.4 (31.9-41.1), P = 0.035]. There were no statistically significant differences in self-report data or biomarkers between groups. CONCLUSION: [Formula: see text] and [Formula: see text] as measured by the step test are statistically significantly different between SSc patients with and without PH. [Formula: see text] and [Formula: see text] may be useful screening tools for PH in the SSc population.

Platelets Induce Thymic Stromal Lymphopoietin Production by Endothelial Cells: Contribution to Fibrosis in Human Systemic Sclerosis.

OBJECTIVE: To investigate the relationship between vascular damage and fibrosis in systemic sclerosis (SSc) by testing the hypothesis that platelets contribute to skin fibrosis via the activation of human dermal microvascular endothelial cells (HDMECs) and subsequent production of profibrotic mediators. METHODS: A total of 203 SSc patients and 30 healthy donors were prospectively enrolled between 2012 and 2015 at the University Hospital of Bordeaux. Immunohistochemistry and immunofluorescence analyses were performed on skin biopsy sections from 18 SSc patients and 5 healthy donors. Serum thymic stromal lymphopoietin (TSLP) levels were measured by enzyme-linked immunosorbent assay in the entire cohort. HDMECs and fibroblasts were purified from biopsy sections. Extracellular matrix production by cultured fibroblasts was assessed by real-time quantitative polymerase chain reaction. RESULTS: Serum TSLP levels were significantly increased in SSc patients compared to healthy donors (P < 0.0001) and were associated with a higher frequency of vasculopathy (P = 0.02). The proportion of TSLP-positive dermal cells was increased in the skin of SSc patients compared with healthy donors (P < 0.0001) and was correlated with fibrosis (modified Rodnan skin thickness score) (r = 0.6146, P = 0.0001). In SSc dermis, TSLP was mainly expressed by CD31-positive endothelial cells. In vitro, activated platelets induced TSLP production by HDMECs in an interleukin-1ß-dependent manner. SSc fibroblasts responded differently according to their original TSLP environment. CONCLUSION: Taken together, these results identify HDMECs as contributors to TSLP production in SSc and suggest a potential mechanism by which platelets may profoundly affect the fibrotic process in SSc.

Enrichment of Scleroderma Vascular Disease-Associated Autoantigens in Endothelial Lineage Cells.

OBJECTIVE: Scleroderma patients with autoantibodies to CENPs and/or interferon-inducible protein 16 (IFI-16) are at increased risk of severe vascular complications. This study was undertaken to determine whether these autoantigens are enriched in cells of the vasculature. METHODS: Successive stages of embryoid bodies (EBs) as well as vascular progenitors were used to evaluate the expression of scleroderma autoantigens IFI-16 and CENP by immunoblotting. CD31 was included to mark early blood vessels. IFI-16 and CD31 expression were defined in paraffin-embedded skin sections from scleroderma patients and from healthy controls. IFI-16 expression was determined by flow cytometric analysis in circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells. RESULTS: Expression of CENP-A, IFI-16, and CD31 was enriched in EBs on days 10 and 12 of differentiation, and particularly in cultures enriched in vascular progenitors (IFI-16, CD31, and CENPs A and B). This pattern was distinct from that of comparator autoantigens. Immunohistochemical staining of paraffin-embedded skin sections showed enrichment of IFI-16 in CD31-positive vascular endothelial cells in biopsy specimens from scleroderma patients and normal controls. Flow cytometric analysis revealed IFI-16 expression in circulating hematopoietic progenitor cells but minimal expression in CECs. CONCLUSION: Our findings indicate that expression of the scleroderma autoantigens IFI-16 and CENPs, which are associated with severe vascular disease, is increased in vascular progenitors and mature endothelial cells. High level, lineage-enriched expression of autoantigens may explain the striking association between clinical phenotypes and the immune targeting of specific autoantigens.

Resolution of Skin Fibrosis by Neutralization of the Antifibrinolytic Function of Plasminogen Activator Inhibitor 1.

OBJECTIVE: Systemic sclerosis (SSc) is a fibrotic disease characterized by an obliterative vasculopathy with thrombosis and impairment of the coagulation-fibrinolysis balance. Plasminogen activator inhibitor 1 (PAI-1) is the major inhibitor of profibrinolytic plasminogen activators (PAs). This study was undertaken to evaluate the contribution of PAI-1 to SSc pathology in the skin. METHODS: PAI-1 was evaluated in skin from patients with diffuse SSc (dSSc) and those with limited SSc (lSSc) by immunohistochemistry. The contribution of PAI-1 to SSc pathology was tested in vivo in murine graft-versus-host disease (GVHD) and bleomycin models of progressive skin fibrosis and in vitro in dermal human microvascular endothelial cells (HMVECs) using a monoclonal antibody that selectively prevents the binding of PAI-1 to PA. RESULTS: Skin from patients with dSSc and those with lSSc showed increased PAI-1 levels in the epidermis and microvessel endothelium. PAI-1 neutralization in the GVHD model led to a dramatic, dose-dependent improvement in clinical skin score, concomitant with vasculopathy resolution, including a reduction in fibrinolysis regulators and vascular injury markers, as well as reduced inflammation. Resolution of vasculopathy and inflammation was associated with resolution of skin fibrosis, as assessed by reduction in collagen content and expression of key profibrotic mediators, including transforming growth factor ß1 and tissue inhibitor of metalloproteinases 1. Similar to the GVHD model, PAI-1 neutralization reduced dermal inflammation and fibrosis in the bleomycin model. PAI-1 neutralization stimulated plasmin-mediated metalloproteinase 1 activation in dermal HMVECs. CONCLUSION: Our findings indicate that neutralization of the antifibrinolytic function of PAI-1 resolves skin fibrosis by limiting the extent of initial vascular injury and connective tissue inflammation. These data suggest that PAI-1 represents an important checkpoint in disease pathology in human SSc.

Mast cells are a source of transforming growth factor ß in systemic sclerosis.

OBJECTIVE: To describe the cellular source of transforming growth factor ß (TGFß) in the dermis of patients with systemic sclerosis (SSc). METHODS: We performed electron microscopy (EM) with immunogold labeling on skin biopsy specimens from 7 patients with SSc and 3 healthy control subjects. For TGFß quantification, the numbers of gold particles per square micron were calculated. The origin of mast cells was confirmed and quantified by toluidine blue staining and light microscopy. Degranulation was assessed on toluidine blue-stained sections and on EM images. RESULTS: In all patients, active TGFß was observed uniquely in mast cell vesicles, some of which were released into the extracellular space. Patients with progressive SSc and a more recent onset of non-Raynaud's phenomenon symptoms had higher numbers of mast cells and gold particles per mast cell. Mast cells from healthy control subjects also contained active TGFß but, in contrast to SSc samples, showed a resting character with no or low-level degranulation and uniformly dense osmiophilic vesicles. CONCLUSION: Degranulation of skin mast cells can be an important mechanism of TGFß secretion in SSc.

Low circulating level of CD133+KDR+cells in patients with systemic sclerosis.

BACKGROUND: Results of previous studies on the level of circulating endothelial progenitor cells (EPCs), which are involved in vascular repair, in scleroderma (SSc) patients have been controversial. OBJECTIVES: To enumerate circulating EPC subsets and to examine their relation with endothelial dysfunction, biochemical markers of endothelial injury and vascular outcome in SSc patients. METHODS: Enumeration of circulating CD34+KDR+ and CD133+ KDR+EPCs was performed by flow cytometry. Endothelium-dependent vasodilation was evaluated by changes in flow-mediated dilation (FMD%) in the brachial artery. Serum level of vascular endothelial growth factor (VEGF) was measured by enzyme linked immunosorbent assay. RESULTS: SSc patients (n=52) were found to have significantly lower CD133+KDR+EPCs (3.0 vs. 7.0/µl, p<0.001) as well as FMD% (4.8% vs. 7.8%, p<0.001) compared with age and sex-matched controls (n=52). Among patients who had no concomitant cardiovascular risk factors (n=28), CD133+KDR+ EPC level was significantly lower than controls (3.8 vs. 7.3/µl, p=0.001) and correlated modestly with FMD% (r=0.29, p=0.03). Disease duration was the only determining factor identified for circulating CD133+KDR+ EPCs (p=0.03) by logistic regression analysis. Levels of serum VEGF (p=0.92) and KDR expression were not different between patients who had early and intermediate/late disease. Circulating CD34+KDR+ EPCs was not different between SSc patients and controls and did not correlate with any clinical or biochemical parameter. CONCLUSIONS: Lower circulating CD133 +KDR+ EPC subset was found in SSc patients and correlated with impaired endothelium-dependent vasodilation in patients without cardiovascular risk factors suggesting a potential role of deficient EPC recruitment contributing to endothelial dysfunction in this disease.

The tumour necrosis factor-related apoptosis-inducing ligand-osteoprotegerin system in limited systemic sclerosis: a new disease marker?

OBJECTIVE: To investigate the role of the TNF-related apoptosis-inducing ligand-osteoprotegerin (TRAIL-OPG) system in the pathogenesis of limited SSc (lSSc). METHODS: Circulating levels of TRAIL and of its soluble receptor OPG were measured by ELISA in serum samples obtained from 50 lSSc patients and 50 healthy controls. RESULTS: TRAIL serum levels in lSSc patients were similar to those of healthy controls, whereas the OPG serum levels were significantly increased (P < 0.0001). According to different subgroups of lSSc patients, TRAIL was not statistically different between each group and healthy controls; concerning OPG, the statistically different value was also maintained when comparing each single lSSc group with the whole control population. CONCLUSIONS: OPG serum levels, but not TRAIL, are elevated in lSSc patients. Since OPG binding to TRAIL inhibits TRAIL-TRAIL receptor interaction, the relative concentrations of these two molecules in the local micro-environment has to be considered. In this setting, OPG increase in lSSc patients may produce a detrimental effect by counteracting the vasoprotective activity of TRAIL. The TRAIL : OPG ratio and their relative levels of expression in lSSc patients should be taken into consideration as a possible novel marker of vascular damage.

Cartilage oligomeric matrix protein expression in systemic sclerosis reveals heterogeneity of dermal fibroblast responses to transforming growth factor beta.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) accumulates in systemic sclerosis (SSc) skin and is upregulated by transforming growth factor (TGF)beta. To further characterise the response to TGFbeta in SSc, we investigated TGFbeta1 and COMP expression and myofibroblast staining in SSc skin. METHODS: Skin biopsies from patients with diffuse cutaneous SSc (dSSc), limited cutaneous SSc (lSSc) and healthy controls were evaluated for COMP mRNA expression using real-time PCR. COMP, alpha-smooth muscle actin (SMA) and TGFbeta were assessed in skin sections and in cultured fibroblasts by immunohistochemistry. Clinical disease status was assessed by the modified Rodnan skin score (mRSS). RESULTS: Myofibroblasts expressing SMA and COMP were found coexpressed in many cells in dSSc dermis, but each also stained distinct cells in the dermis. Cultured SSc dermal fibroblasts also showed heterogeneity for COMP and SMA expression, with cells expressing SMA, COMP, both or neither. TGFbeta treatment increased COMP and SMA-expressing cells. COMP mRNA expression in lesional skin from patients with dSSc correlated with the mRSS and TGFbeta1 staining. CONCLUSION: These findings suggest that TGFbeta upregulation of COMP and/or SMA expression in subpopulations of fibroblasts contributes to different pathways of fibrosis and that multiple TGFbeta regulated genes may serve as biomarkers for the degree of SSc skin involvement.

Molecular subsets in the gene expression signatures of scleroderma skin.

BACKGROUND: Scleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production. METHODOLOGY AND FINDINGS: We analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc) with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of 'intrinsic' genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001) and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynaud's phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc. CONCLUSIONS AND SIGNIFICANCE: Genome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs in the skin of patients with scleroderma.

Relaxin receptors--new drug targets for multiple disease states.

Relaxin was discovered more than 75 years prior to the identification of the receptors that mediate its actions. There has been a slow emergence in understanding the role of relaxin, with it being denoted initially as a hormone of pregnancy due to its observed effects to relax pubic ligaments and soften the cervix of guinea pigs to facilitate parturition. However, many other physiological roles have been identified for relaxin, including cardiovascular and neuropeptide functions and an ability to induce the matrix metalloproteinases, so it is clear that relaxin is not exclusively a hormone of pregnancy but has a much wider role in vivo. The recent de-orphanisation of four receptors LGR7, LGR8, GPCR135 (SALPR) and GPCR142 (GPR100) that respond to and bind at least one of the three forms of relaxin identified to date, allows dissection of this system to determine the precise role of each receptor and enable the identification of new targets for treatment of numerous disease states. Relaxin has the potential to be useful for the treatment of scleroderma, fibrosis, in orthodontics and to facilitate embryo implantation in humans. Relaxin antagonists may act as contraceptives or prevent the development of breast cancer metastases. Recent research has added considerable knowledge to the signalling pathways activated by relaxin, which will aid our understanding of how relaxin produces its effects. The focus of this review is to bring together recent developments in the relaxin receptor field and to highlight their potential as drug targets.

Lack of increased expression of cell surface markers for circulating fibrocyte progenitors in limited scleroderma.

The aetiology and pathogenesis of scleroderma is incompletely understood. Recently, a cell called the fibrocyte has been shown to be derived from circulating monocytes with the ability to produce collagen. The aim of this study was to evaluate differences in the cell surface characteristics of circulating fibrocyte progenitors (monocytes) in patients with limited scleroderma compared to controls. A case-control study was performed in eight patients with limited scleroderma, which were matched with eight controls. Three-colour flow cytometry was used to assess the relative expression of cell surface markers. Statistical analysis then compared the relative expression between the two groups. In this preliminary study, there were no significant differences in the expression of circulating monocyte surface molecules involved with cell transformation, function, or migration presumed to give rise to fibrocytes, in a population of patients with limited scleroderma. Various explanations for the results are discussed.

Augmented production of transforming growth factor-beta by cultured peripheral blood mononuclear cells from patients with systemic sclerosis.

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

Parvoviral infection of endothelial cells and stromal fibroblasts: a possible pathogenetic role in scleroderma.

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disease (CTD) which differs from other CTDs by progressive irreversible fibrosis in lung, kidney, skin, and heart. It has a worse prognosis compared to several other CTDs. The pathogenesis may reflect a humorally mediated microangiopathy in concert with the overproduction of collagen triggered by immune-mediated cytokine production. Having previously demonstrated parvovirus B19 (B19) DNA in bone marrow and skin biopsies of SSc patients in the absence of B19 viremia, we sought to further elucidate a role for B19 in the pathogenesis of SSc. DESIGN: Twelve patients who fulfilled American College of Rheumatology criteria for a diagnosis of SSc were encountered. Ten were serologically screened for B19 infection. Solution phase polymerase chain reaction (PCR) for B19 DNA was performed on skin tissue from six patients, and in all biopsies, reverse transcriptase in situ PCR (RT in situ PCR) for B19 and tumor necrosis factor (TNF)-alpha mRNA was performed. B19 viral protein (VP2) expression was sought by immunohistochemistry and correlated to PCR findings and to light microscopy of hematoxylin and eosin-stained sections. Frozen tissue was also available on five of the patients. Two control groups were assessed for B19 and TNF expression comprising one with irrelevant primers and the other representing 18 cases of inflammatory skin lesions where the etiology was known and unrelated to B19 infection. In addition, frozen and paraffin-embedded tissues procured from skin lesions unrelated to B19 infection were assessed for B19 genome. In all cases, pretreatment with RNase was also performed to verify that any positive signal was indeed RNA based. RESULTS: Diffuse SSc was seen in seven patients, and limited disease in five. All patients had an antinuclear antibody--specifically, an antinucleolar, anticentromere, and/or anti-Scl 70 antibody. Eleven of the 12 had lung involvement, whereas eight patients had myocardial disease. Of 12 patients tested serologically, nine had B19-specific antibodies, which included immunoglobulin M (IgM)-specific antibodies in two cases. Solution phase PCR showed B19 DNA in the skin in three cases and in the bone marrow in three cases, including two in whom skin-based B19 DNA was observed. In all cases, RT in situ PCR demonstrated B19 and TNF-alpha mRNA in endothelia, fibroblasts, mast cells, and perivascular inflammatory cells. Immunohistochemistry to assess VP2 was either negative or equivocal. Immunofluorescent studies revealed prominent deposition of C5b-9 within the cutaneous vasculature from biopsies of all patients tested. The control samples were negative for B19 and TNF RNA and DNA. CONCLUSIONS: Parasitism of endothelia and fibroblasts by B19 with resultant enhanced TNF-alpha expression may be of pathogenetic importance in SSc even in the absence of demonstrable viremia. The vascular deposition of C5b-9 suggests a role for humoral immunity possibly induced by a state of endothelial neoantigenicity evoked by virally mediated cell injury. Treatment strategies include anti-viral therapy, including in the context of intravenous gamma-globulin and anti-TNF therapy.