Apremilast interferes with the TGFß1-induced transition of human skin fibroblasts into profibrotic myofibroblasts: in vitro study.

OBJECTIVES: Fibroblast-to-myofibroblast transition and extracellular matrix overproduction represent progressive events in chronic inflammatory and fibrotic diseases, in which TGFß1 is one of the key mediators. Phosphodiesterase 4 (PDE4) acts as a proinflammatory enzyme through the degradation of cyclic adenosine monophosphate and it is overexpressed in skin fibroblasts. The study investigated how apremilast (a PDE4 inhibitor) interferes with the intracellular signalling pathways responsible for the TGFß1-induced fibroblast-to-myofibroblast transition and profibrotic extracellular matrix protein synthesis. METHODS: Cultured human skin fibroblasts were stimulated with TGFß1 (10 ng/ml) alone or combined with apremilast (1 and 10 µM) for 4, 16 and 24 h. Other aliquots of the same cells were previously stimulated with TGFß1 and then treated with apremilast (1 and 10 µM) for 4, 16 and 24 h, always under stimulation with TGFß1. Gene and protein expression of αSMA, type I collagen (COL1) and fibronectin were evaluated, together with the activation of small mothers against decapentaplegic 2 and 3 (Smad2/3) and extracellular signal-regulated kinase (Erk1/2) proteins. RESULTS: Apremilast reduced the TGFß1-induced increase in αSMA, COL1 and fibronectin gene expression at 4 and 16 h, and protein synthesis at 24 h of treatment in cultured fibroblasts, even for cells already differentiated into myofibroblasts by way of a previous stimulation with TGFß1. Apremilast inhibited the TGFß1-induced Smad2/3 and Erk1/2 phosphorylation at 15 and 30 min. CONCLUSION: Apremilast seems to inhibit in vitro the fibroblast-to-myofibroblast transition and the profibrotic activity induced by TGFß1 in cultured human skin fibroblasts by downregulating Smad2/3 and Erk1/2 intracellular signalling pathways.

Altered expression of RXFP1 receptor contributes to the inefficacy of relaxin-based anti-fibrotic treatments in systemic sclerosis.

OBJECTIVES: Relaxin is a potent anti-fibrotic hormone that has been tested to ameliorate fibrosis in systemic sclerosis (SSc), but with controversial results. The aim of the study is to sequence relaxin receptor gene RXFP1 and to assess its mRNA expression and protein levels in the skin of SSc patients and healthy subjects. METHODS: Fibroblasts were isolated from unaffected/affected skin samples of (n=16) limited-cutaneous-SSc-(LcSSc) and from affected ones of (n=4) diffuse-cutaneous-SSc-(DcSSc) patients. Fibroblasts from healthy subjects were used as controls. Sequencing of exonic target regions of interest for RXFP1 gene was performed, coupled with mRNA transcript variant analysis. RXFP1 mRNA and protein levels were assessed by quantitative-real-time-PCR-(qRT-PCR) and by immunocytochemistry-(ICC). Alpha-smooth-muscle-actin-(α-SMA) synthesis induced by transforming-growth-factor-beta-1-(TGF-ß1) stimulation was investigated in all fibroblasts with and without pre-treatment with serelaxin (a recombinant form of human relaxin-2 targeting the receptor RXFP1). RESULTS: Sequencing of RXFP1 gene showed no relevant mutations in all fibroblast populations. The analysis of mRNA transcripts revealed the presence of 13 different mRNA isoforms of RXFP1 (7 coding and 6 non-coding) upregulated in LcSSc/DcSSc-affected samples and not in LcSSc-unaffected and in healthy ones. On the contrary, ICC demonstrated the absence of RXFP1 in LcSSc/DcSSc-affected fibroblasts and the presence in LcSSc-unaffected and in healthy ones. To prove these findings, serelaxin pre-incubation was unable to counteract TGF-ß1-driven upregulation of α-SMA in LcSSc/DcSSc-affected fibroblasts only, but not in LcSSc-unaffected and healthy ones. CONCLUSIONS: The absence/altered expression of relaxin receptor RXFP1 in the affected fibroblasts of SSc patients could explain the inefficacy of relaxin-based anti-fibrotic treatments in the disease.

Sarcopenia in systemic sclerosis: the impact of nutritional, clinical, and laboratory features.

We evaluated the presence of sarcopenia in a population of systemic sclerosis (SSc) patients, with respect to nutritional, clinical, and laboratory features. A total of 62 patients who met the ACR/EULAR 2013 classification criteria were enrolled. Sarcopenia was defined according to the Relative Skeletal Mass Index (RSMI) and hand grip strength (HGS). Body composition was assessed with the calculation of the Body Mass Index (BMI), lean body mass (LBM) and fat mass (FM). Malnutrition was evaluated according to the ESPEN criteria. Clinical evaluation included nailfold capillaroscopy and skin evaluation by modified Rodnan Skin Score (mRSS), pulmonary function tests (PFT) with diffusing capacity for carbon monoxide adjusted for hemoglobin (DLCO), high-resolution computed tomography (HR-CT) of the lungs, echocardiography and high-resolution manometry (HRM) for esophageal involvement. Laboratory evaluation included erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), hemoglobin, creatinine, creatine kinase (CK), transaminases, lipid profile, glycemia, albumin, and vitamin-D. Antinuclear antibodies (ANA) and extractable nuclear antigens (ENA) were also assessed. Considering RSMI, the prevalence of sarcopenia is 42%. In this case, age, malnutrition, disease duration, mRSS, capillaroscopy score, esophageal involvement, ESR, and ANA titer are higher in the sarcopenic group, while DLCO and LBM are lower. Considering HGS, the prevalence of sarcopenia is 55%. Age, disease duration, malnutrition, FM, mRSS, capillaroscopy score, esophageal involvement, ESR, and ENA positivity are higher in the sarcopenic group, while DLCO is lower. By using both RSMI and HGS to assess sarcopenia in SSc, the results of this study demonstrated that this condition correlates with different nutritional, clinical, and biochemical parameters associated with the worsening of the disease.

Dual endothelin receptor antagonists contrast the effects induced by endothelin-1 on cultured human microvascular endothelial cells.

OBJECTIVES: To evaluate the ability of dual endothelin (ET) receptor antagonists (ETA/ETB -ETA/BRAs) to contrast the ET-1-induced effects on cultured human microvascular endothelial cells (HMVECs). METHODS: Some cultured HMVECs were untreated, or treated with ET-1 (100nM) or transforming growth factor ß1 (TGFß1, 10ng/mL) alone for 6 days, in order to induce the endothelial-to-mesenchymal transition (EndoMT). Other cultured HMVECs were pre-treated for 1hr with ETA/BRAs bosentan (10µM) or macitentan (1µM, 10µM) before the stimulation with ET-1 for 6 days. At the end of treatments, a mechanical injury was induced to cultured HMVECs (by scratching the cell monolayer with a sterile tip), and then the cell ability to re-fill the damaged area was determined after 24hrs. EndoMT phenotype markers and monocyte chemoattractant protein-1 (MCP-1) were evaluated by qRT-PCR and Western blotting. Statistical analysis was performed using Mann-Whitney-U non-parametric test. RESULTS: Both ET-1 and TGFß1 induced EndoMT and the MCP-1 over-expression in cultured HMVECs, as well as reduced the process of endothelial cell damage repair. Pre-treatment with ETA/BRAs let cultured HMVECs to significantly restore the in vitro damage of the cell monolayer and antagonised the EndoMT process as well as the MCP-1 over-expression (range p<0.05 - p<0.001). Conversely, untreated or TGFß1-treated HMVECs were found unaffected by the ETA/BRAs treatments. CONCLUSIONS: The treatment with dual ETA/BRAs seems to partially restore the altered cell function induced by ET-1 in cultured endothelial cells, and might justify their therapeutic efficiency in clinical conditions characterised by increased concentrations of ET-1.

Pulmonary hypertension: a correct diagnosis for a suitable therapy in scleroderma patients.

Systemic sclerosis (SSc) is a heterogeneous disorder characterised by dysfunction of the endothelium and dysregulation of fibroblasts, resulting in excessive production of collagen, and abnormalities of the immune system. Progressive fibrosis of the skin and internal organs is a pathologic hallmark of the disease, resulting in major organ damage and failure. Pulmonary hypertension (PH) is frequent in patients with SSc and, pulmonary arterial hypertension (PAH) represents one of the main causes of death. PH is not a specific disease, but a haemodynamic condition characterized by a mean pulmonary pressure ≥25mmHg. In SSc, because of the great variability in clinical manifestation, it is possible to identify pulmonary hypertension due to left heart disease, PH due to respiratory disease or pulmonary arterial hypertension. The knowledge of PH and the right diagnosis are crucial to assess the most appropriate therapeutic strategy. In this article, the new classification criteria of PH have been examined taking into account the SSc clinical evolution and focusing on the different underlying pathogenetic mechanisms.

Serum levels, tissue expression and cellular secretion of macrophage migration inhibitory factor in limited and diffuse systemic sclerosis.

OBJECTIVES: To investigate serum levels, tissue/cellular expression of macrophage migration inhibitory factor (MIF) in patients with limited (lSSc) and diffuse (dSSc) systemic sclerosis. METHODS: 10 lSSc-patients, 10 dSSc-patients and 10 controls were enrolled. MIF serum levels were assayed by ELISA. MIF and its receptors CD74/CD44 were evaluated by immunohistochemistry on skin biopsies from patients with dSSc, lSSc (affected and not-affected skin) and controls. MIF levels were assessed (ELISA) in supernatants of healthy dermal microvascular endothelial cells (MVECs) and in control (CTR), non-affected SSc (NA) and affected (SSc) fibroblasts treated for 48 h with 10% control serum and 10% SSc-serum. MIF supernatant (ELISA) and mRNA (quantitative real-time PCR) levels were determined in SSc dermal fibroblasts and in control dermal fibroblasts untreated or stimulated at 6 h-24 h-48 h with bleomycin (50 mU/ml). RESULTS: Serum MIF was significantly higher in dSSc (18.7±4.1 ng/ml, p<0.001) and in lSSc (10.4±4.4 ng/ml, p<0.001) patients respect to controls (2.6±1.4 ng/ml). Enhanced MIF immunoreactivity was found in keratinocytes, fibroblasts, endothelium, sebaceous/sweat glands from lSSc/dSSc affected skin. Faint MIF immunoreactivity was found in control skin and not-affected skin of lSSc patients. No differences were found in CD74/CD44 receptors' analysis among control and dSSc/lSSc affected and non-affected skin. MVECs and fibroblasts (CTR, NA and SSc) produced significantly more MIF, when stimulated with SSc serum respect to control-serum (p<0.001). Finally, MIF mRNA levels significantly increased at 6h (p<0.001) and decreased at 48 h (p<0.001) in control fibroblasts treated with bleomycin compared to control untreated. Simultaneously, MIF supernatant protein levels increased after 48 h (p<0.01) in bleomycin-treated fibroblasts respect to untreated ones. CONCLUSIONS: These results suggest that MIF could be implicated in the pathogenesis of SSc, probably acting as protective factor against the SSc stressful conditions.

Proteomics of human primary osteoarthritic chondrocytes exposed to extremely low-frequency electromagnetic fields (ELF EMFs) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF).

Osteoarthritis (OA) is the most frequent joint disease, characterized by degradation of extracellular matrix and alterations in chondrocyte metabolism. Some authors reported that electromagnetic fields (EMFs) can positively interfere with patients affected by OA, even though the nature of the interaction is still debated. Human primary osteoarthritic chondrocytes isolated from the femoral heads of OA-patients undergoing to total hip replacement, were cultured in vitro and exposed 30 min/day for two weeks to extremely-low-frequency electromagnetic field (ELF) with fixed frequency (100 Hz) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF) with variable frequencies, intensities and waveforms. Sham-exposed (S.E.) cells served as control group. Cell viability was measured at days 2, 7 and 14. After two weeks, cell lysates were processed using a proteomic approach. Chondrocyte exposed to ELF and TAMMEF system demonstrated different viability compared to untreated chondrocytes (S.E.). Proteome analysis of 2D-Electrophoresis and protein identification by mass spectrometry showed different expression of proteins derived from nucleus, cytoplasm and organelles. Function analysis of the identified proteins showed changes in related-proteins metabolism (glyceraldeyde-3-phosphate-dehydrogenase), stress response (Mn-superoxide-dismutase, heat-shock proteins), cytoskeletal regulation (actin), proteinase inhibition (cystatin-B) and inflammation regulatory functions (S100-A10, S100-A11) among the experimental groups (ELF, TAMMEF and S.E.). In conclusion, EMFs do not cause damage to chondrocytes, besides stimulate safely OA-chondrocytes and are responsible of different protein expression among the three groups. Furthermore, protein analysis of OA-chondrocytes treated with ELF and the new TAMMEF systems could be useful to clarify the pathogenetic mechanisms of OA by identifying biomarkers of the disease.

Human osteoarthritic chondrocytes exposed to extremely low-frequency electromagnetic fields (ELF) and therapeutic application of musically modulated electromagnetic fields (TAMMEF) systems: a comparative study.

Osteoarthritis (OA) is the most common joint disease, characterized by matrix degradation and changes in chondrocyte morphology and metabolism. Literature reported that electromagnetic fields (EMFs) can produce benefits in OA patients, even if EMFs mechanism of action is debated. Human osteoarthritic chondrocytes isolated from femoral heads were cultured in vitro in bidimensional (2-D) flasks and in three-dimensional (3-D) alginate beads to mimic closely cartilage environment in vivo. Cells were exposed 30 min/day for 2 weeks to extremely low-frequency electromagnetic field (ELF) with fixed frequency (100 Hz) and to therapeutic application of musically modulated electromagnetic field (TAMMEF) with variable frequencies, intensities, and waveforms. Cell viability was measured at days 7 and 14, while healthy-cell density, heavily vacuolized (hv) cell density, and cluster density were measured by light microscopy only for 3-D cultures after treatments. Cell morphology was observed for 2-D and 3-D cultures by transmission electron microscopy (TEM). Chondrocyte exposure to TAMMEF enhances cell viability at days 7 and 14 compared to ELF. Light microscopy analysis showed that TAMMEF enhances healthy-cell density, reduces hv-cell density and clustering, compared to ELF. Furthermore, TEM analysis showed different morphology for 2-D (fibroblast-like) and 3-D (rounded shape) cultures, confirming light microscopy results. In conclusion, EMFs are effective and safe for OA chondrocytes. TAMMEF can positively interfere with OA chondrocytes representing an innovative non-pharmacological approach to treat OA.

High-throughput 3D microvessel-on-a-chip model to study defective angiogenesis in systemic sclerosis.

In early systemic sclerosis (Scleroderma, SSc), the vasculature is impaired. Although the exact etiology of endothelial cell damage in SSc remains unclear, it is hypothesized that endothelial to mesenchymal transition (EndoMT) plays a key role. To perform physiologically relevant angiogenic studies, we set out to develop an angiogenesis-on-a-chip platform that is suitable for assessing disease parameters that are relevant to SSc and other vasculopathies. In the model, we substituted Fetal Bovine Serum (FBS) with Human Serum without impairing the stability of the culture. We showed that 3D microvessels and angiogenic factor-induced sprouts exposed to key pro-inflammatory and pro-fibrotic cytokines (TNFα and TGFß) undergo structural alterations consisting of destructive vasculopathy (loss of small vessels). We also showed that these detrimental effects can be prevented by compound-mediated inhibition of TGFß-ALK5 signaling or addition of a TNFα neutralizing antibody to the 3D cultures. This demonstrates that our in vitro model is suitable for compound testing and identification of new drugs that can protect from microvascular destabilization or regression in disease-mimicking conditions. To support this, we demonstrated that sera obtained from SSc patients can exert an anti-angiogenic effect on the 3D vessel model, opening the doors to screening for potential SSc drugs, enabling direct patient translatability and personalization of drug treatment.

Antibodies against specific extractable nuclear antigens (ENAs) as diagnostic and prognostic tools and inducers of a profibrotic phenotype in cultured human skin fibroblasts: are they functional?

BACKGROUND: The importance of systemic sclerosis (SSc) autoantibodies for diagnosis has become recognized by their incorporation into the 2013 ACR/EULAR classification criteria. Clear prognostic and phenotypic associations with cutaneous subtype and internal organ involvement have been also described. However, little is known about the potential of autoantibodies to exert a direct pathogenic role in SSc. The aim of the study is to assess the pathogenic capacity of anti-DNA-topoisomerase I (anti-Topo-I) and anti-centromeric protein B (anti-Cenp-B) autoantibodies to induce pro-fibrotic markers in dermal fibroblasts. METHODS: Dermal fibroblasts were isolated from unaffected and affected skin samples of (n = 10) limited cutaneous SSc (LcSSc) patients, from affected skin samples of diffuse cutaneous (DcSSc) patients (n = 10) and from healthy subjects (n = 20). Fibroblasts were stimulated with anti-Topo-I, anti-Cenp-B IgGs, and control IgGs in ratios 1:100 and 1:200 for 24 h. Cells were also incubated with 10% SSc anti-Topo-I+ and anti-Cenp-B+ whole serum and with 10% control serum for 24 h. Viability was assessed by MTT test, while apoptosis was assessed by flow cytometry. Activation of pro-fibrotic genes ACTA2, COL1A1, and TAGLN was evaluated by quantitative real-time PCR (qPCR), while the respective protein levels alpha-smooth-muscle actin (α-SMA), type-I-collagen (Col-I), and transgelin (SM22) were assessed by immunocytochemistry (ICC). RESULTS: MTT showed that anti-Cenp-B/anti-Topo-I IgGs and anti-Cenp-B+/anti-Topo-I+ sera reduced viability (in a dilution-dependent manner for IgGs) for all the fibroblast populations. Apoptosis is induced in unaffected LcSSc and control fibroblasts, while affected LcSSc/DcSSc fibroblasts showed apoptosis resistance. Basal mRNA (ACTA2, COL1A1, and TAGLN) and protein (α-SMA, Col-1, and SM22) levels were higher in affected LcSSc/DcSSc fibroblasts compared to LcSSc unaffected and to control ones. Stimulation with anti-Cenp-B/anti-Topo-I IgGs and with anti-Cenp-B+/anti-Topo-I+ sera showed a better induction in unaffected LcSSc and control fibroblasts. However, a statistically significant increase of all pro-fibrotic markers is reported also in affected LcSSc/DcSSc fibroblasts upon stimulation with both IgGs and sera. CONCLUSIONS: This study suggests a pathogenic role of SSc-specific autoantibodies to directly induce pro-fibrotic activation in human dermal fibroblasts. Therefore, besides the diagnostic and prognostic use of those autoantibodies, these data might further justify the importance of immunosuppressive drugs in the early stages of the autoimmune disease, including SSc.

MicroRNA-34a and MicroRNA-181a Mediate Visfatin-Induced Apoptosis and Oxidative Stress via NF-κB Pathway in Human Osteoarthritic Chondrocytes.

Current evidence suggests a complex interaction between adipokines and microRNA (miRNA) in osteoarthritis (OA) pathogenesis. The present study explored the role of miR-34a and miR-181a in regulating apoptosis and oxidative stress induced by visfatin in human OA chondrocytes. Chondrocytes were transfected with miR-34a and miR-181a inhibitors and stimulated with visfatin for 24 h, in the presence of nuclear factor (NF)-κB inhibitor (BAY-11-7082, 2 h pre-incubation). Apoptosis and reactive oxygen species (ROS) production were detected by cytometry, miRNA, antioxidant enzymes, nuclear factor erythroid (NRF)2 and B-cell lymphoma (BCL)2 expressions by quantitative real time polymerase chain reaction (real time PCR) and western blot. P50 NF-κB subunit was measured by immunofluorescence. Visfatin significantly induced apoptosis and superoxide anion production, increased miR-34a, miR-181a, superoxide dismutase (SOD)-2, catalase (CAT), NRF2 and decreased BCL2 gene and protein expression in OA chondrocytes. All the visfatin-caused effects were suppressed by using miR-34a and miR-181a inhibitors. Pre-incubation with BAY-11-7082 counteracted visfatin-induced expression of miRNA, BCL2, SOD-2, CAT and NRF2. Inhibition of miR-34a and miR-181a significantly reduced the activation of p50 NF-κB. Visfatin confirms its ability to induce apoptosis and oxidative stress in human OA chondrocytes; these effects appeared mediated by miR-34a and miR-181a via NF-κB pathway. We highlight the relevance of visfatin as potential therapeutic target for OA treatment.

Pulmonary arterial hypertension in systemic sclerosis: Diagnosis and treatment according to the European Society of Cardiology and European Respiratory Society 2015 guidelines.

Scleroderma (systemic sclerosis) is an autoimmune connective tissue disease which presents endothelial dysfunction and fibroblast dysregulation, resulting in vascular and fibrotic disorders. Pulmonary hypertension is frequent in patients with systemic sclerosis: the natural evolution of the disease can induce the development of different forms of pulmonary hypertension, representing one of the main causes of death. Among the different forms of pulmonary hypertension in systemic sclerosis, pulmonary arterial hypertension is the most frequent one (rate of occurrence is estimated between 7% and 12%). This pulmonary vascular complication should be treated with a combination of drugs that is able to counteract endothelial dysfunction, antagonizing the endothelin-1 system and replacing prostaglandin I2 and nitric oxide activity. A correct diagnosis is mandatory, because it is possible only for pulmonary arterial hypertension to use specific drugs that are able to control the symptomatic condition and the evolution of the disease. According to the most recent guidelines, for the patients with systemic sclerosis, also without pulmonary hypertension symptoms, echocardiography screening for the detection of pulmonary hypertension is recommended. Pulmonary arterial hypertension screening programs in systemic sclerosis patients is able to identify milder forms of the disease, allowing earlier management and better long-term outcome.

Effects of selexipag and its active metabolite in contrasting the profibrotic myofibroblast activity in cultured scleroderma skin fibroblasts.

BACKGROUND: Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). Selexipag is a prostacyclin receptor agonist synthesized for the treatment of pulmonary arterial hypertension. The study investigated the possibility for selexipag and its active metabolite (ACT-333679) to downregulate the profibrotic activity in primary cultures of SSc fibroblasts/myofibroblasts and the fibrogenic signaling molecules involved. METHODS: Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 µM to 0.3 µM) or ACT-333679 (from 10 µM to 0.1 µM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679-treated cells by western botting. RESULTS: Selexipag and ACT-333679 significantly reduced protein synthesis and gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, whereas FN was significantly downregulated at the protein level. Interestingly, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts. CONCLUSIONS: Selexipag and mainly its active metabolite ACT-333679 were found for the first time to potentially interfere with the profibrotic activity of cultured SSc fibroblasts/myofibroblasts at least in vitro, possibly through the downregulation of fibrogenic Erk1/2 and Akt signaling molecules.

Activation of the Wnt Pathway by Small Peptides: Rational Design, Synthesis and Biological Evaluation.

A computational analysis of the X-ray structure of the low-density lipoprotein receptor-related protein 6 (LRP6) with the Dickkopf-1 (DKK1) C-terminal fragment has allowed us to rationally design a small set of decapeptides. These compounds behave as agonists of the canonical Wnt pathway in the micromolar range when tested on a dual luciferase Wnt functional assay in glioblastoma cells. Two of the oligopeptides showed a lack of cytotoxicity in human primary osteoblasts isolated from sponge bone tissue (femoral heads or knees of elderly patients). According to the mechanism of action, the studies revealed a dose- and time-dependent increase in the viability of human osteoblasts. These results may indicate a potential therapeutic application of this class of compounds in the treatment of bone diseases related to aging, such as osteoporosis.

Histopathological findings in systemic sclerosis-related myopathy: fibrosis and microangiopathy with lack of cellular inflammation.

OBJECTIVES: The objective of this study was to identify specific histopathological features of skeletal muscle involvement in systemic sclerosis (SSc) patients. METHODS: A total of 35 out of 112 SSc-patients (32%, including 81% female and 68% diffuse scleroderma) presenting clinical, biological and electromyographic (EMG) features of muscle weakness, were included. Patients underwent vastus lateralis biopsy, assessed for individual pathologic features including fibrosis [type I collagen (Coll-I), transforming growth factor ß (TGF-ß)], microangiopathy [cluster of differentiation 31 (CD31), pro-angiogenic vascular endothelial growth factor A (VEGF-A), anti-angiogenic VEGF-A165b], immune/ inflammatory response [CD4, CD8, CD20, human leucocyte antigens ABC (HLA-ABC)], and membranolytic attack complex (MAC). SSc biopsies were compared with biopsies of (n = 35) idiopathic inflammatory myopathies (IIMs) and to (n = 35) noninflammatory myopathies (NIMs). Ultrastructural abnormalities of SSc myopathy were also analyzed by transmission electron microscopy (TEM). RESULTS: Fibrosis in SSc myopathy (81%) is higher compared with IIM (32%, p < 0.05) and with NIM (18%, p < 0.05). Vascular involvement is dominant in SSc muscle (92%), and in IIM (78%) compared with NIM (21%, p < 0.05). In particular, CD31 shows loss of endomysial vessels in SSc myopathy compared with IIM (p < 0.05) and with NIM (p < 0.01). VEGF-A is downregulated in SSc myopathy compared with IIM (p < 0.05) and NIM (p < 0.05). Conversely, VEGF-A165b is upregulated in SSc myopathy. The SSc immune/inflammatory response suggested humoral process with majority (85%) HLA-ABC fibral neoexpression and complement deposits on endomysial capillaries MAC, compared with IIM (p < 0.05), characterized by CD4+/CD8+/B-cell infiltrate, and NIM (p < 0.05). TEM analysis showed SSc vascular alterations consisting of thickening and lamination of basement membrane and endothelial cell 'swelling' coupled to endomysial/perimysial fibrosis. CONCLUSIONS: Fibrosis, microangiopathy and humoral immunity are predominant in SSc myopathy, even if it is difficult to identify specific histopathological hallmarks of muscle involvement in SSc, since they could be present also in other (IIM/NIM) myopathies.

Proteomic Investigation of Dermal Fibroblasts Isolated from Affected and Unaffected Skin Samples from Patients with Limited Cutaneous Systemic Sclerosis: 2 Distinct Entities?

OBJECTIVE: To identify using proteomic analysis the proteins of altered abundance in the affected and unaffected limited cutaneous systemic sclerosis (lcSSc) skin fibroblasts. METHODS: Excision biopsies (3 mm) were obtained from the affected and unaffected skin of 5 patients with lcSSc. Dermal fibroblasts were isolated enzymatically. Two-dimensional gel electrophoresis was used to separate and define proteins in affected and unaffected fibroblast lysates. Proteins of altered abundance were identified by mass spectrometry. Differences among skin samples were confirmed also by immunohistochemistry (IHC) and by quantitative real-time PCR (qRT-PCR) for type I collagen (Col-1) and vimentin (VIM). RESULTS: Proteomic analysis revealed different expressions of proteins involved in cytoskeleton organization (27%), extracellular matrix remodeling (11%), response to oxidative stress (22%), energy metabolism (19%), protein metabolism (5%), cellular homeostasis (5%), signal transduction (3%), and protein transcription, synthesis, and turnover (8%). IHC analysis showed that SSc-affected epidermis is thickened and the dermis is strongly reactive to Col-1 and VIM (typical markers of activated myofibroblasts) compared to SSc-unaffected skin, whose stainings are comparable to those of control healthy skin. Overexpression of Col-1 and VIM mRNA levels in affected lcSSc fibroblasts compared to unaffected lcSSc ones was confirmed by qRT-PCR. CONCLUSION: Consistent with previous studies, these findings are important for 2 reasons: first, because they reveal the opposite behavior of dermal fibroblasts in the unaffected and affected skin areas of the same patient with lcSSc; second, because they demonstrate the histological/histochemical similarities between unaffected skin from patients with lcSSc and healthy control skin.

Erythema nodosum associated with Staphylococcus xylosus septicemia.

Staphylococcus xylosus is a coagulase-negative staphylococcus. It is a commensal bacterium associated with skin and mucous membranes and occasionally it can cause human infections. We report the first case of erythema nodosum developed in a young woman with S. xylosus septicemia and specific serum antibody response.

Bosentan and macitentan prevent the endothelial-to-mesenchymal transition (EndoMT) in systemic sclerosis: in vitro study.

BACKGROUND: Systemic sclerosis (SSc) is characterized by early vascular abnormalities and subsequent fibroblast activation to myofibroblasts, leading to fibrosis. Recently, endothelial-to-mesenchymal transition (EndoMT), a complex biological process in which endothelial cells lose their specific markers and acquire a mesenchymal or myofibroblastic phenotype, has been reported in SSc. In the present study, we evaluated the ability of endothelin-1 (ET-1) dual receptor antagonists bosentan (BOS) and macitentan (MAC) to antagonize EndoMT in vitro. METHODS: Ten women with limited SSc were enrolled. They underwent double skin biopsy (affected and nonaffected skin). Fibroblasts and microvascular endothelial cells (MVECs) were isolated from biopsies. We performed mono- or coculture of MVECs (isolated from nonaffected skin) with fibroblasts (isolated from affected skin and stimulated with ET-1 and transforming growth factor beta [TGF-ß]). In cocultures, the MVEC layer was left undisturbed or was preincubated with BOS or MAC. After 48 h of coculture, MVECs were analyzed for their tube formation ability and for messenger RNA and protein expression of different vascular (CD31, vascular endothelial growth factor-A [VEGF-A], VEGF-A165b) and profibrotic (alpha-smooth muscle actin [α-SMA], collagen type I [Col I], TGF-ß) molecules. RESULTS: After 48 h, MVECs showed a reduced tube formation ability when cocultured with SSc fibroblasts. CD31 and VEGF-A resulted in downregulation, while VEGF-A165b, the antiangiogenic isoform, resulted in upregulation. At the same time, mesenchymal markers α-SMA, Col I, and TGF-ß resulted in overexpression in MVECs. Tube formation ability was restored when MVECs were preincubated with BOS or MAC, also reducing the expression of mesenchymal markers and restoring CD31 expression and the imbalance between VEGF-A and VEGF-A165b. CONCLUSIONS: With this innovative EndoMT in vitro model realized by coculturing nonaffected MVECs with affected SSc fibroblasts, we show that the presence of a myofibroblast phenotype in the fibroblast layer, coupled with an ET-1-TGF-ß synergic effect, is responsible for EndoMT. BOS and MAC seem able to antagonize this phenomenon in vitro, confirming previous evidence of endothelium-derived fibrosis in SSc and possible pharmacological interference.