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Systemic sclerosis (SSc, scleroderma) is a chronic, multisystem connective tissue disorder affecting the skin and various internal organs. Although the disease is characterized by a triad of widespread microangiopathy, fibrosis and autoimmunity, increasing evidence indicates that vascular damage is a primary event in the pathogenesis of SSc. The progressive vascular injury includes persistent endothelial cell activation/damage and apoptosis, intimal thickening, delamination, vessel narrowing and obliteration. These profound vascular changes lead to vascular tone dysfunction and reduced capillary blood flow, with consequent tissue ischemia and severe clinical manifestations, such as digital ulceration or amputation, pulmonary arterial hypertension and scleroderma renal crisis. The resulting tissue hypoxia induces complex cellular and molecular mechanisms in the attempt to recover endothelial cell function and tissue perfusion. Nevertheless, in SSc patients there is no evidence of significant angiogenesis and the disease evolves towards chronic tissue ischemia, with progressive and irreversible structural changes in multiple vascular beds culminating in the loss of capillaries. A severe imbalance between pro-angiogenic and angiostatic factors may also lead to impaired angiogenic response during SSc. Besides insufficient angiogenesis, defective vasculogenesis with altered numbers and functional defects of bone marrow-derived endothelial progenitor cells may contribute to the vascular pathogenesis of SSc. The purpose of this article is to review the contribution of recent studies to the understanding of the complex mechanisms of impaired vascular repair in SSc. Indeed, understanding the pathophysiology of SSc-associated vascular disease may be the key in dissecting the disease pathogenesis and developing novel therapies. Either angiogenic or vasculogenic mechanisms may potentially become in the future the target of therapeutic strategies to promote capillary regeneration in SSc.

Objective.To investigate the possible role ofFCGR2A519A>G andFCGR3A559A>C functional polymorphisms in the genetic predisposition to susceptibility to systemic sclerosis (SSc) or clinical phenotype.Methods.A total of 1566 patients with SSc and 2271 geographically matched controls were included in our study. We analyzed the genotype and allele frequencies of theFCGR2A519A>G andFCGR3A559A>C functional variants in 6 independent European cohorts of white patients with SSc, and white controls. The cohorts comprised 165 Dutch patients with SSc and 1326 controls, 236 Spanish patients with SSc and 257 controls, 267 German patients with SSc and 270 controls, 202 Swedish patients with SSc and 261 controls, 416 Italian patients with SSc and 157 controls, and additionally 280 English patients with SSc. Genotyping was performed using Taqman 5′ allelic discrimination assay. The study reached a 99% power to detect the effect of a polymorphism at an OR of 1.3.Results.NeitherFCGR2A519A>G norFCGR3A559A>C was significantly associated with susceptibility to SSc. We did not find an association with specific disease phenotypes, limited or diffuse cutaneous involvement, autoantibody profiles, or pulmonary involvement.Conclusion.Our study strongly suggests the lack of a role for theFCGR2A519A>G andFCGR3A559A>C polymorphisms in SSc susceptibility or clinical phenotype in 6 independent European cohorts.

Objective:Pregnant women with systemic sclerosis (SSc; scleroderma) have an increased risk of premature delivery and small full-term infants. During placental development, angiogenesis and vascular remodelling are essential for a successful pregnancy outcome. An analysis was made of the pathological changes and expression of angiogenic factors in SSc placentas.Methods:Placenta biopsies were obtained from three patients with SSc and four healthy uncomplicated pregnancies after delivery at 34–38 weeks of gestation. The sections were stained with Masson’s trichrome and phosphotungstic-acid-haematoxylin and immunostained for connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and receptors VEGFR-1 and VEGFR-2.Results:The pathological findings were signs of decidual vasculopathy, increased syncytiotrophoblast knotting, placental infarcts and villous hypoplasia. Severe and diffuse perivascular and stromal fibrosis of decidua and chorionic villi, and extensive deposition of fibrinoid material around decidual vessels and in intervillous spaces were observed. Strong CTGF expression in the vessel wall, decidual cells and fibroblasts and α-SMA+ myofibroblasts were found. VEGF and VEGFR-2 expression was stronger in SSc than in healthy placentas, while VEGFR-1 expression was similar to controls. PlGF immunopositivity was weaker in SSc.Conclusion:In SSc placentas, severe fibrosis and abnormal vascular remodelling were detected. This may result in reduced blood flow leading to deep sufferance of maternal placenta and possible premature delivery.

Objective Microparticles are small, membrane-coated vesicles that can serve as novel signaling structures between cells. The aim of the present study was to analyze the profile of microparticles in the blood of patients with systemic sclerosis (SSc; scleroderma) and healthy controls. Methods The study population consisted of 37 patients with SSc and 15 healthy subjects of comparable sex and age. Microparticles were isolated from plasma by high-speed differential centrifugation. Microparticles were stained with monoclonal antibodies against cell type–specific markers and were quantified by fluorescence-activated cell sorting analyses. Results The total number of microparticles was strongly increased in patients with SSc compared with healthy controls (mean ± SEM 88.0 ± 4.8 × 105 microparticles/ml plasma versus 42.3 ± 9.4 × 105 microparticles/ml plasma; P < 0.001). Similarly, significant increases were found for microparticles derived from platelets, endothelial cells, monocytes, and T cells, reflecting the activation of these cells in SSc. Platelets were the most common source of microparticles in the blood of patients with SSc (66.9 ± 5.2% of all microparticles) and healthy donors, followed by microparticles derived from endothelial cells (8.8 ± 0.9% in SSc patients). The modified Rodnan skin thickness score (MRSS) was inversely correlated with the total number of microparticles. Furthermore, patients with cutaneous ulcers showed a significantly lower total number of microparticles. In multivariate analysis, an additive model of age, C-reactive protein, MRSS, and subtype of disease accounted for 55% of the variability of the total microparticle count (r = 0.744). Conclusion The number of microparticles from different cellular sources is increased in the blood of SSc patients. Considering their role as important mediators of intercellular communication, microparticles could be a novel link between activated cellular compartments in the pathogenesis of SSc.

Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.

Systemic sclerosis (SSc) is characterised by a progressive microangiopathy that contributes significantly to the morbidity of patients with SSc. Besides insufficient angiogenesis, defective vasculogenesis with altered numbers of endothelial precursor cells (EPCs) might also contribute to the vascular pathogenesis of SSc. However, different protocols for isolation, enrichment, culture and quantification of EPCs are currently used, which complicate comparison and interpretation of the results from different studies.The aim of the European League Against Rheumatism Scleroderma Trials and Research (EUSTAR) group expert panel was to provide recommendations for standardisation of future research on EPCs. Consensus statements and recommendations were developed in a face to face meeting by an expert panel of the basic science working group of EUSTAR.The findings were: cardiovascular risk factors and medications such as statins should be described in detail. A detailed description of methods considering isolation, culture, enrichment and detection of EPCs should be given. For in vitro culture of EPCs, no protocol has been shown to be superior to another, but coating with laminin and type IV collagen would resemble most closely the situation in vivo. The endothelial phenotype should be confirmed in all in vitro cultures at the end of the culture period. We recommend using CD133, vascular endothelial growth factor type 2 receptor (VEGFR2) and CD34 in combination with a viability marker for quantification of EPCs in the blood. Finally, exact standard operating procedures for fluorescence-activated cell sorting (FACS) analysis are given that should be strictly followed.In summary, the EUSTAR recommendations will help to unify EPC research and allow better comparison between the results of different studies.

Objective:To evaluate the role of the single-nucleotide polymorphism (SNP) at position −670 in the FAS gene promoter (FAS−670G>A) in influencing the susceptibility, clinical features and severity of systemic sclerosis (SSc).Methods:350 white Italian SSc patients (259 with limited cutaneous SSc (lcSSc) and 91 with diffuse cutaneous SSc (dcSSc)) and 232 healthy individuals were studied. Patients were assessed for the presence of autoantibodies (anticentromere, anti-topoisomerase I (anti-Scl-70) antibodies), interstitial lung disease (ILD), pulmonary arterial hypertension and scleroderma renal crisis. FAS−670G>A SNP was genotyped by PCR restriction fragment length polymorphism assay. Serum levels of soluble FAS (sFAS) were analysed by ELISA.Results:A significant difference in FAS−670 genotype distribution was observed between SSc patients and healthy individuals (p = 0.001). The frequency of the FAS−670A allele was significantly greater in SSc than in controls (p = 0.001). No significant difference in genotype distribution and allele frequencies was observed between lcSSc and dcSSc, although a greater frequency of the FAS−670A allele was found in dcSSc. The FAS−670AA genotype significantly influenced the predisposition to SSc (OR 1.97, 95% CI 1.35 to 2.88, p = 0.001) and to both lcSSc (OR 1.84, 95% CI 1.23 to 2.75, p = 0.003) and dcSSc (OR 2.37, 95% CI 1.41 to 3.99, p = 0.001). FAS−670A allele frequency was greater, although not significantly, in anti-Scl-70 antibody-positive dcSSc and ILD dcSSc. sFAS was significantly higher in patients and controls carrying the FAS−670AA genotype compared with those carrying the FAS−670GG genotype (p = 0.003 in SSc, p = 0.004 in controls).Conclusion:The FAS−670A allele is significantly associated with susceptibility to SSc, suggesting a role for a genetic control of apoptosis in the pathogenesis of the disease.

Musculoskeletal involvement is more frequent than expected in patients with systemic sclerosis (SSc) and is a major cause of disability, even if the prognosis of the disease largely depends on visceral involvement. The most common clinical feature of musculoskeletal involvement is arthralgia; less frequent features are arthritis, flexion contractures, stiffness (affecting predominantly fingers, wrists and ankles), proximal muscle weakness (mainly of the shoulder and hip) and tendon sheath involvement. Tendon friction rubs are predictive of poor prognosis. If musculoskeletal involvement is suspected, serum creatinine phosphokinase, aldolase, lactate dehydrogenase, alkaline phosphate, rheumatoid factor and anticyclic citrullinated peptide autoantibodies should be checked routinely. Treatment for muscle involvement has not yet been considered adequately and, in the future, it is to be hoped that clinical trials will identify new drugs to control this aspect of SSc, which seriously compromises patients' quality of life.

Background:In systemic sclerosis (SSc) reduced capillary density decreases blood flow and leads to tissue ischaemia and fingertip ulcers. Nail fold videocapillaroscopy (NVC) is a diagnostic and follow-up parameter useful to evaluate the severity, activity and the stage of SSc microvascular damage. Autologous haemopoietic stem cell transplantation (HSCT) is a new treatment for patients with severe diffuse cutaneous systemic sclerosis (dcSSc) refractory to conventional therapies. We aimed to evaluate the improvement of microvasculature after HSCT using NVC.Methods:A total of 16 patients with severe dcSSc with a “late” videocapillaroscopy pattern underwent an immunesuppressive treatment: 6 were treated with HSCT and 10 with monthly pulse cyclophosphamide (CYC) 1 g for 6 months and then orally with 50 mg/day for further 6 months.NVC was performed before and after 3 months from the beginning of each treatment and then repeated every 3 months.Results:In all patients, before HSCT NVC showed large avascular areas and ramified capillaries and vascular architectural disorganisation (“late” pattern). At 3 months after HSCT, the NVC pattern changed from “late” into “active”, showing frequent giant capillaries (>6/mm) and haemorrhages, absence of avascular areas and angiogenesis phenomena; 1 year after HSCT, microvascular abnormalities were still in the “active” pattern. In patients treated with CYC, no NVC modifications were observed during 24 months of follow-up and the pattern always remained “late”.Conclusions:These results indicate that HSCT with a high dose CYC regimen may foster vascular remodelling, while CYC at lower doses and with a chronic regimen does not influence the microvasculature.

Objective T cell intracytoplasmic antigen 1 (TIA-1) and TIA-1–related protein (TIAR) are involved in posttranscriptional regulation of the expression of tumor necrosis factor α (TNFα) and other proteins. Given the pivotal role of TNFα in chronic inflammatory diseases, this study was undertaken to analyze sera from patients with systemic autoimmune diseases for the presence of autoantibodies to TIA proteins and to investigate the expression of these proteins in inflamed tissue. Methods The presence of autoantibodies to TIA proteins in sera from 385 patients with rheumatic diseases and healthy controls was determined by immunoblotting using recombinant antigens. Expression of TIA proteins in skin and kidney tissue was analyzed by immunohistochemistry. Serum levels of TNFα were measured by enzyme-linked immunosorbent assay. Results Autoantibodies to TIA-1 and/or TIAR were detected in 61% of patients with systemic lupus erythematosus (SLE), 42% of patients with systemic sclerosis (SSc), 15–31% of patients with other rheumatic diseases, and 6% of healthy controls. Compared with patients negative for anti-TIA antibody, anti-TIA antibody–positive SLE patients had higher disease activity (P = 0.01), elevated antibodies to double-stranded DNA (P = 0.0003), and increased serum TNFα levels (P = 0.018). In SLE patients, anti-TIAR antibodies were associated with lupus nephritis (P = 0.02), while in patients with SSc, anti–TIA-1 was associated with lung involvement (P = 0.02). Immunohistochemical analysis of skin and kidney tissue revealed aberrant expression of TIA proteins in skin lesions from SLE and SSc patients, as well as in glomerular cells from SLE patients. Conclusion Aberrant expression of TIA proteins in inflammatory tissue may lead to systemic autoantibody responses, particularly in SLE and SSc. Increased occurrence of anti-TIA autoantibodies in patients with severe organ involvement may point to a possible pathogenetic role.

Objectives: To determine the prevalence of anti-Ku antibodies in 625 patients with systemic sclerosis (SSc) from six European rheumatological centres and to evaluate their clinical and serological characteristics. Methods: Sera of 625 consecutive patients with either limited cutaneous or diffuse cutaneous SSc were tested for antibodies to Ku antigen together with other extractable nuclear antigens by counterimmunoelectrophoresis. A case–control design with calculation of bootstrap 95% confidence intervals derived from anti-Ku negative control patients was used to evaluate clinical associations of anti-Ku antibodies. Sera from anti-Ku positive patients with SSc and a control group were additionally tested by immunofluorescence on Hep-2 cell substrates and line immunoassay. Results: Anti-Ku antibodies were found in the sera of 14/625 (2.2%) patients with SSc. Of 14 anti-Ku positive patients with SSc, 10 had no other anti-extractable nuclear antigen (ENA) antibodies detected by counterimmunoelectrophoresis. Using a case–control study design, anti-Ku antibodies were significantly associated with musculoskeletal manifestations such as clinical markers of myositis, arthritis and joint contractures. In addition, a significant negative correlation of anti-Ku antibodies was found with vascular manifestation such as fingertip ulcers and teleangiectasias. There was a striking absence of anti-centromere antibodies as well as anti- polymyositis (PM)/scleroderma (Scl) antibodies in patients that were anti-Ku positive. As expected, anti-Scl70 and punctate nucleolar immunofluorescence patterns were present only in single cases. Conclusion: This is the largest cohort to date focusing on the prevalence of anti-Ku antibodies in patients with SSc. The case–control approach was able to demonstrate a clinically distinct subset of anti-Ku positive patients with SSc with only relative clinical differences in skeletal features. However, the notable exceptions were signs of myositis. This shows the importance of anti-Ku antibody detection for the prediction of this specific clinical subset.

Systemic sclerosis (SSc) is a multiorgan disease characterized by injury to vascular wall and extensive damage of the microvessels. The injury of the vascular wall is characterized by the formation of megacapillaries and avascular areas. The reduced capillary density leads to clinical manifestations such as digital ulcers. These lesions are extremely painful and lead to substantial functional disability. Management of digital ulcers includes non-pharmacologic and pharmacologic modalities. Despite the reduced blood flow and reduced partial oxygen pressure levels, there is paradoxically no evidence for a sufficient angiogenesis in the skin of patients with SSc. Angiogenesis is strongly disturbed in SSc, as demonstrated by Nailfold Video-Capillaroscopy changes, the damage of the vessels evolves progressively from early to late stages and is characterized by different morphological aspects. Almost all patients develop Raynaud's phenomenon which, together with structural vasculopathy, results in ulceration and critical digital ischemia. Many of the severe internal organ complications of SSc are vascular, including pulmonary arterial hypertension (PAH) and scleroderma renal crisis. Structural vascular damage occurs in many vascular beds and contribute to pulmonary, renal, cardiac and gastrointestinal complications. SSc has a high case-specific mortality due to organ-based complications including PAH, lung fibrosis, renal failure and involvement of the gastrointestinal tract.

In systemic sclerosis (SSc), the involvement of the interstitium or vascular system of the lung may lead to pulmonary arterial hypertension (PAH). PAH is often asymptomatic or oligosymptomatic in early SSc and, when it becomes symptomatic, pulmonary vascular system is already damaged. Exercise echocardiography (ex-echo), measuring pulmonary artery pressure (PAP) during exercise and allowing to differentiate physiologic from altered PAP responses, may identify subclinical PAH. Our aims were (a) to evaluate by ex-echo the change of PAP in patients with SSc without lung involvement; and (b) to correlate PAP during exercise (ex-PAP) values to clinical and biohumoral parameters of PAH. Twenty-seven patients with limited SSc (ISSc) without interstitial lung involvement were studied. Patients underwent rest and exercise two-dimensional and Doppler echocardiography by supine cycloergometer. Systolic PAP was calculated using the maximum systolic velocity of the tricuspid regurgitant jet at rest and during exercise values of systolic PAP exceeding 40 mmHg at ex-echo were considered as abnormal, and biohumoral markers potentially related to PAH were assessed. Eighteen of 27 SSc patients presented an ex-PAP > 40 mmHg, while in 9 of 27 patients ex-PAP values remained < 40 mmHg (48.8 +/- 4.5 mmHg versus 36.2 +/- 3.1 mmHg; P < 0.001). Other echocardiographic and ergometric parameters, clinical tests, and biohumoral markers were not different in the two groups. Ex-PAP significantly correlated with D-dimer (P = 0.0125; r2 = 0.2029). Ex-echo identifies a cluster of SSc patients with subclinical PAH that may develop PAH. This group should be followed up and may be considered for specific therapies to prevent disease evolution.

Increased evidence suggests an accelerated macrovascular disease in systemic sclerosis (SSc). Brachial artery flow-mediated vasodilation (FMD) and carotid intima-media thickness (IMT) are two indicators of subclinic cardiovascular disease and are frequently used as surrogate measures of subclinic atherosclerosis. The aim of this study was to evaluate macrovascular involvement in SSc. We studied 35 SSc patients (6 males and 29 females; 11 with diffuse and 24 with limited disease) and 20 healthy controls. Brachial artery FMD was assessed by method described by Celermajer in all patients and 13 control subjects. IMT was measured using high-resolution B-mode ultrasonography in patients and controls. Traditional risk factors for atherosclerosis (hypertension, dyslipidemia, and smoke) were also assessed. FMD was significantly impaired (3.41% +/- 4.56% versus 7.66% +/- 4.24%; P < 0.037) and IMT was significantly elevated compared with healthy controls (0.93 +/- 0.29 mm versus 0.77 +/- 0.13 mm; P < 0.005). FMD was not significantly different in SSc with increased IMT compared with those with normal IMT). No correlation was found between risk factors for atherosclerosis and the impairment of FMD or IMT in SSc patients. The impairment of endothelial function and structural changes of large vessels are evident in SSc, but do not seem associated with traditional risk factors for atherosclerosis. Prospective studies including also clinical outcomes are needed to assess the features and significance of macrovacular involvement in SSc.

Systemic sclerosis (SSc) is characterized by microvascular and macrovascular alterations. The D allele of the ACE I/D polymorphism is known to be associated with an increased incidence of atherosclerosis and has been recently proposed as associated with increased risk of SSc. This study evaluates the relationship between intima-media thickness (IMT), ankle-brachial pressure measurements (ABPI) and ACE I/D polymorphism in SSc patients.According to the presence of ACE D allele (analysed by PCR), 53 SSc patients (47 females and 6 males; median age was 60.4 +/- 10.68 yrs; range 40-75 yrs) were divided in carriers of the D allele (DD + ID) (n = 46) and carriers of the I allele (II) (n = 7). In these patients, IMT and ABPI [calculated as the posterior tibial artery pressure (mmHg) divided by the brachial pressure] were obtained. Forty-three healthy controls (40 women and 13 men; median age 56.3 +/- 10.23; range 40-70 yrs) of the same ethnicity were recruited.SSc patients had IMT significantly higher than controls (0.85 +/- 0.03 vs 0. 68 +/- 0.01; P0.03). No significant differences (P0.3) in ABPI values between patients (1.018 +/- 0.10) and controls (1.091 +/- 0.11) were found. SSc patients with ACE DD and ID genotype showed an IMT significantly greater (0.89 +/- 0.03) than those carrying the II genotype (0.61 +/- 0.01) (P0.04). ABPI was not different among ACE gene genotypes.Our findings confirm an increased prevalence of macrovascular disease in SSc patients and show that IMT is greater in patients carrying the ACE DD and ID genotype in comparison with II homozygotes. This suggests that, in SSc, the presence of ACE D allele may predispose to an involvement of the macrovascular system.

Introduction The vascular and nervous systems have several anatomic and molecular mechanism similarities. Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of class III semaphorin (Sema3) family, play a critical role in blood vessel guidance during physiological and pathological vascular development. Sema3E is a natural antiangiogenic molecule that causes filopodial retraction in endothelial cells, inhibiting cell adhesion by disrupting integrin-mediated adhesive structures. The aim of the present study was to investigate whether in systemic sclerosis (SSc) Plexin-D1/Sema3E axis could be involved in the dysregulation of vascular tone control and angiogenesis. Methods Sema3E levels were measured by quantitative colorimetric sandwich ELISA in serum samples from 48 SSc patients, 45 subjects with primary Raynaud's phenomenon (pRP) and 48 age-matched and sex-matched healthy controls. Immunofluorescence staining on skin sections from 14 SSc patients and 12 healthy subjects was performed to evaluate Sema3E and Plexin-D1 expression. Western blotting was used to assess Plexin-D1/Sema3E axis in human SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs, respectively) at basal condition and after stimulation with recombinant human vascular endothelial growth factor (VEGF), SSc and healthy sera. Capillary morphogenesis on Matrigel was performed on H-MVECs treated with healthy, pRP or SSc sera in the presence of Sema3E and Plexin-D1 soluble peptides. Results Serum Sema3E levels were significantly higher both in pRP subjects and SSc patients than in controls. In SSc, Sema3E levels were significantly increased in patients with early nailfold videocapillaroscopy (NVC) pattern compared to active/late patterns and pRP, and in patients without digital ulcers versus those with ulcers. In SSc skin, Sema3E expression was strongly increased in the microvascular endothelium. Cultured SSc-MVECs showed higher levels of phosphorylated Plexin-D1 and Sema3E expression than H-MVECs, and stimulation with SSc sera increased phosphorylated Plexin-D1 and Sema3E in H-MVECs. The addition of Sema3E-binding Plexin-D1 soluble peptide significantly attenuated the antiangiogenic effect of SSc sera on H-MVECs. Conclusions Our findings suggest that Plexin-D1/Sema3E axis is triggered in SSc endothelium and may have a role in the dysregulation of angiogenesis and vascular tone control by inducing neuro-vascular mechanism alterations clinically evident in particular in the early disease phases.

Objective To retrospectively analyse the features of calcinosis in a cohort of SSc patients. Methods Charts of SSc patients attending the Ulcer Unit of the Rheumatology Department, University of Florence and presenting a clinical suspicion of calcinosis were considered in the study. Data on clinical history, including recent skin changes, and clinical examination of all areas with suspected calcinosis, radiological imaging of the calcinotic area, demographics and SSc-related organ involvement and pain measured by a visual analogue scale were recorded. Results In 52 of 112 SSc patients, a total of 316 calcinoses were recorded and were divided into visible and palpable {154 [47.4%], clustered according to their macroscopic features as mousse [49 (31.8%)] and stone [: 105 (68.2%)]} and non-visible but palpable {: 162 [52.6%]: net [5 (3%)], plate [22 (13.8%)] and stone [135 (83.2%)]}. The X-ray-based classification of all calcinoses, both visible and non-visible, was as follows: stone, 289 (91.4%); net, 12 (3.8%) and plate, 15 (4.8%). Skin ulcers complicated 154 of 316 calcinoses (48.7%). Mousse calcinosis was associated with pulmonary arterial hypertension, the stone subset was suggestive of pulmonary involvement and justified further investigation and the net subset was the slowest to heal. Conclusion Our data indicate that calcinosis may be classified in SSc as mousse, stone, net and plate according to its clinical and X-ray features. This classification awaits validation for a possible use in clinical practice and to support early treatment and prevention of complications.

Objective: Qualitative capillaroscopy patterns in juvenile- and adult-onset systemic sclerosis (SSc) were studied in adulthood using data from the EULAR Scleroderma Trials and Research (EUSTAR) database. Methods: Data collected between June 2004 and April 2013 were examined with focus on capillaroscopy. In this retrospective exploratory study, series of patients with juvenile-onset SSc were matched with series of adult-onset SSc having the same gender and autoantibody profile. Results: 30 of 123 patients with juvenile-onset and 2108 of 7133 with adult-onset SSc had data on capillaroscopy. Juvenile-onset SSc showed scleroderma pattern more frequently than adult-onset SSc (93.3% and 88%). The OR was 2.44 and 95% Cl 0.57-10.41. An active scleroderma pattern was present in 58% of juvenile- and 61% of adult-onset SSc. The OR was 0.91 and 95% Cl 0.28-2.93. The late scleroderma pattern was present in 61% of juvenile- and 55.5% of adult-onset SSc. The OR was 1.06 and 95% Cl 0.34-3.56. Conclusion: This is the first exploratory study on the comparison of capillaroscopy between juvenile- and adult-onset SSc in adulthood. Juvenile-onset SSc had an increase prevalence of sderoderma pattern, but a similar distribution of the three patterns was suggested. Further studies are needed to define this issue. (C) 2015 Elsevier Inc. All rights reserved.

In systemic sclerosis (SSc), dermal capillaries are progressively lost with consequent chronic tissue hypoxia insufficiently compensated by angiogenesis. Clinical studies reported that intravenous cyclophosphamide (CYC) may improve SSc-related peripheral microvascular damage. Recently, we showed that CYC treatment may normalize SSc sera-induced abnormalities in endothelial cell-matrix interactions. Our objective was to evaluate in vitro the effects of sera from treatment-naive or CYC-treated SSc patients on dermal blood microvascular endothelial cell (dMVEC) angiogenesis, migration, proliferation and apoptosis. dMVECs were challenged with sera from 21 SSc patients, treatment-naive (n = 8) or under CYC treatment (n = 13), and 8 healthy controls. Capillary morphogenesis on Geltrex matrix was significantly reduced upon challenge with sera from naive SSc patients compared with healthy controls. When dMVECs were challenged with sera from CYC-treated SSc patients, their angiogenic capacity was comparable to that of cells treated with healthy sera. Wound healing capacity and chemotaxis in Boyden chamber were both significantly decreased in the presence either of naive or CYC-treated SSc sera compared with healthy sera. WST-1 assay revealed that cell proliferation was significantly decreased in dMVECs challenged with sera from naive SSc patients compared with healthy sera. Conversely, dMVEC proliferation was not impaired in the presence of sera from CYC-treated SSc patients. Accordingly, the percentage of TUNEL-positive apoptotic dMVECs was significantly higher in the presence of sera from naive SSc patients than healthy controls, while CYC-treated SSc sera did not induce dMVEC apoptosis. Levels of the angiostatic mediators endostatin, pentraxin 3, angiostatin and matrix metalloproteinase-12 were all significantly elevated in sera from naive SSc patients compared with sera from both healthy controls and CYC-treated SSc patients. In SSc, CYC treatment might boost angiogenesis and consequently improve peripheral microangiopathy through the normalization of the endothelial cell-matrix interactions, reduction of endothelial cell apoptosis and rebalance of dysregulated angiostatic factors.

Objectives In systemic sclerosis (SSc), vascular involvement is characterised by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) system disturbances. Neuropilin-1 (NRP1), a receptor for both class-3 semaphorins (Sema3s) and VEGF-A, is required for optimal VEGF-A/VEGFR-2 signalling. Here, we investigated the possible involvement of Sema3A/NRP1 axis in SSc. Methods Circulating Sema3A and soluble NRP1 (sNRP1) were measured in patients with SSc and controls. NRP1 and Sema3A expression in skin biopsies was evaluated by immunofluorescence and western blotting. NRP1 expression was assessed in SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs), and in SSc and control endothelial progenitor cell (EPC)-derived endothelial cells (ECs). The possible impact of transcription factor Friend leukaemia integration 1 (Fli1) deficiency on endothelial NRP1 expression was investigated by gene silencing. The binding of Fli1 to NRP1 gene promoter was evaluated using chromatin immunoprecipitation. Capillary morphogenesis was performed on Matrigel. Results Decreased sNRP1 levels in SSc were associated with active and late nailfold videocapillaroscopy patterns and digital ulcers. No difference in Sema3A was found between patients and controls. NRP1 was significantly decreased in SSc-MVECs both ex vivo and in vitro. NRP1 and Fli1 significantly decreased in H-MVECs challenged with SSc sera, while they were not different in SSc and control EPC-derived ECs. Fli1 occupied the NRP1 gene promoter and Fli1 gene silencing reduced NRP1 expression in H-MVECs. NRP1 gene silencing in H-MVECs resulted in a significantly impaired angiogenic capacity comparable to that of cells treated with SSc sera. Conclusion In SSc, NRP1 deficiency may be an additional factor in the perturbed VEGF-A/VEGFR-2 system contributing to peripheral microvasculopathy and defective angiogenesis.

The main pathologic hallmark of systemic sclerosis (SSc) is endothelial derangement; the pathologic alterations of the vessel wall in SSc are strikingly similar to the modification detected in the atherosclerotic lesions, and it is now evident that SSc is also characterized by accelerated macrovascular disease. Peptides related to angiotensin II, the final product of the renin-angiotensin system (RAS), play a role as regulators of endothelial cell function. Angiotensin-converting enzyme (ACE), the key enzyme in the RAS, is the predominant pathway of angiotensin II formation in blood and tissues. In intron 16 of the gene encoding for ACE an insertion/deletion (I/D) polymorphism, consisting of the presence or absence of a 287-base pair Alu sequence, has been identified. This polymorphism has been related to ACE enzyme levels, and data from experimental studies reported a functional role for this polymorphism in modulating the angiotensin II levels. We previously documented a high ACE D allele frequency in SSc patients and its role in increasing the risk of SSc, thus suggesting that the I/D polymorphism might be a useful genetic marker to identify SSc patients at risk to develop a severe vascular disease, frequently leading to gangrene. Moreover, our preliminary data, besides supporting the role of ACE I/D polymorphism as a predisposing factor to SSc, demonstrated its involvement in accelerated macrovascular disease by increasing the intima media thickness. Therefore, in SSc, not only endothelial dysfunction, but also vascular damage, linked to ACE I/D polymorphism, may significantly contribute to accelerated macrovascular disease, as the ACE D allele, by regulating both the production of angiotensin II and the degradation of bradykinin, contributes to mechanisms involved in the induction and maintenance of vessel wall modification.

Vascular disease is a hallmark of systemic sclerosis (SSc). It is present in every patient, being responsible both for the earliest clinical manifestations and the major life-threatening complications of the disease, and thus determining important morbidity and mortality. In SSc, progressive vascular injury leads to vascular tone dysfunction and reduced capillary blood flow, with consequent tissue ischemia and chronic hypoxia. These phenomena are often accompanied by abnormal levels of vascular factors. Microangiopathy in SSc may be easily assessed by nailfold videocapillaroscopy. The variety of derangements detected in the nailfold capillaries is accompanied by abnormal levels of different vascular mediators and appears to be the best evaluable predictor of the development of peripheral vascular complications, such as digital ulcers. The purpose of this review is to summarize in SSc the most relevant vascular biomarkers and the main associations between vascular biomarkers and capillaroscopic parameters and/or the presence of digital ulcers. Vascular biomarkers could become useful predictive factors of vascular damage in SSc, allowing an earlier management of vascular complications.

In systemic sclerosis (SSc), clinical evidence has shown that Bosentan may foster the regeneration of the peripheral microcirculatory network. The aim of this study was to verify in vitro the influence of Bosentan on the angiogenic performance of dermal microvascular endothelial cells (MVECs) and its possible capacity to counteract the antiangiogenic effects of SSc sera.Healthy dermal MVECs were challenged with Bosentan at different concentrations (0.1 μM, 1 μM, 10 μM) or with sera from patients with diffuse cutaneous SSc (n=8) and healthy subjects (n=8), alone or in combination with Bosentan (10 μM). Cell viability and chemoinvasion were determined by WST-1 and Boyden chamber assays, respectively. Angiogenesis was evaluated by capillary morphogenesis on Matrigel.Challenge of dermal MVECs with SSc sera induced a significant reduction in angiogenesis (p0.005 vs. basal condition; p0.001 vs. healthy sera). The addition of Bosentan could significantly restore angiogenesis in the presence of SSc sera (p0.01 vs. SSc sera alone). Healthy sera promoted cell viability which was, instead, significantly reduced with SSc sera (p0.005 vs. healthy sera). The addition of Bosentan to MVECs challenged with SSc sera significantly increased cell viability (p0.005 vs. SSc sera alone), reaching levels similar to MVECs treated with healthy sera. Co-incubation of MVECs with Bosentan and SSc sera significantly increased chemoinvasion (p0.005 vs. SSc sera alone) which was inhibited by SSc sera (0.001 vs. healthy sera).Bosentan effectively counteracts the antiangiogenic effects of SSc sera on dermal MVECs and fosters the restoration of a proangiogenic environment.

Objective Postnatal angiogenesis relies on a proper response of endothelial cells to angiogenic stimuli. In systemic sclerosis (SSc), endothelial cells are unresponsive to angiogenic factors. Since circumstantial and experimental evidence points to tissue kallikreins as powerful effectors of the angiogenic response, we undertook this study to investigate the kallikrein pattern of normal and SSc endothelial cells in order to identify differences that can account for defective angiogenesis. Methods Expression of 14 tissue kallikreins was studied by a microarray approach, by reverse transcription–polymerase chain reaction, and by Western blotting in endothelial cells isolated from the skin of clinically healthy subjects and SSc patients. Cell proliferation was quantified by direct cell counting. Invasion and capillary morphogenesis were evaluated in a Boyden chamber and in culture flasks layered with Matrigel. Cyclic nucleotide production was measured by enzyme immunoassay. MAP kinase and ERK activation were measured by Western blotting. Results Endothelial cells from SSc patients showed poor expression of kallikreins 9, 11, and 12 compared with endothelial cells from normal subjects. Antibodies against the relevant kallikreins on normal endothelial cells revealed that while kallikreins 9, 11, and 12 induced cell growth, only kallikrein 12 regulated invasion and capillary morphogenesis. Buffering of kallikrein 12 with antibodies resulted in the acquisition of an SSc-like pattern by normal cells in in vitro angiogenesis. Reduction of cAMP and cGMP production and of ERK phosphorylation upon administration of antikallikrein antibodies revealed that the activity of kallikreins 9, 11, and 12 was mediated by kinins. Conclusion Reduction of tissue kallikreins 9, 11, and 12 may be relevant to reduced angiogenesis in SSc patients.

In systemic sclerosis (SSc), Raynaud's phenomenon is the earliest clinical manifestation paralleled by nailfold capillaroscopic alterations that may occur months or even years before the onset of fibrosis.1 This evidence suggests a crucial role of microangiopathy characterised by a progressive loss of capillaries with the formation of avascular areas.1 ,2 Consequent chronic tissue hypoxia leads to skin ulcers and gangrene that heavily burden patients’ quality of life.1 ,2 Vascular endothelial growth factor-A (VEGF-A) is overexpressed in SSc skin, and increased circulating levels of VEGF-A correlate with the severity of nailfold capillary loss.2–4 It has been demonstrated that the VEGF-A primary transcript can be alternatively spliced in its terminal exon, producing two distinct mRNA splice variants that are translated to the proangiogenic VEGF165 and antiangiogenic VEGF165b isoforms.5 These two isoforms bind to the tyrosine kinase receptor VEGFR-2 with the same affinity, but binding of VEGF165b results in an insufficient tyrosine phosphorylation/activation of VEGFR-2 and incomplete or transient downstream signalling, which lead to an impaired angiogenic response.5 ,6 Recently, we have provided the first evidence that dermal expression and plasma levels of VEGF165b are raised in SSc patients, and VEGF165b overexpression prevents SSc dermal microvascular endothelial …

Neurogenic inflammation is caused by neuropeptides which are released by peripheral neurons and induce inflammatory signals. Two of the main neuropeptides involved are substance P (SP) and calcitonin gene related peptide (CGRP),1 both acting through several molecular mechanisms including activation of transient receptor potential vanilloid (TRPV) cation channels, particularly TRPV1 (also called vanilloid receptor type 1 or capsaicin (CAP) receptor).2 ,3 SP and CGRP have been found increased in synovial fluid from rheumatoid arthritis (RA) patients4 ,5 and may induce the production of interleukin (IL)-6 and IL-8 in RA synoviocytes,6 suggesting that neuropeptides may have a role in joint inflammation during RA. Interestingly, TRPV cation channels are expressed in various non-neuronal cell types and, recently, they have been found expressed in human synoviocytes.7 No previous study has investigated the possible link among SP, CGRP and TRPV channels in the inflammation process driven by RA synoviocytes. The aim of our work was to investigate whether SP, CGRP and CAP, a potent activator (agonist) of TRPV1, can increase IL-6 and IL-8 production by RA and healthy fibroblast-like synoviocytes, and whether neuropeptides may modulate TRPV1 expression in RA and healthy cells. Synoviocytes were obtained from knee-biopsy of three patients with active RA diagnosed according to the American College of Rheumatology 1987 revised criteria undergoing surgical joint replacement. Patients had not received any disease-modifying antirheumatic drug treatment. Healthy synoviocytes were collected from knee-biopsy of three subjects who underwent surgical intervention after accidental trauma. Synoviocytes (passage 2–4) were cultured at confluence, serum-starved for 24 h and incubated for …

Objective Defective angiogenesis, resulting in tissue ischemia, is particularly prominent in the diffuse form of systemic sclerosis (SSc). The present study was undertaken to identify possible differences between normal and SSc microvascular endothelial cells (MVECs) in the expression of the cell-associated urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) system, which is critical in the angiogenic process. Methods MVECs were isolated from the dermis of healthy individuals and from the dermis of patients with diffuse SSc. The uPA/uPAR system was examined at the protein and messenger RNA levels. Angiogenesis was assayed on Matrigel-coated porous filters and plates to evaluate cell proliferation, invasion, and capillary morphogenesis. Cleavage of uPAR and the activity of matrix metalloproteinase 12 (MMP-12) were evaluated by Western blotting. Results Compared with MVECs from healthy skin, MVECs from SSc patients showed higher expression of uPAR. However, in SSc MVECs, uPAR undergoes truncation between domain 1 and domain 2, as shown by flow cytometry, enzyme-linked immunosorbent assay, and Western blotting, a cleavage that is known to impair uPAR functions. These properties of SSc MVECs were associated with poor spontaneous and uPA-dependent invasion, proliferation, and capillary morphogenesis. The uPAR cleavage occurring in SSc MVECs was associated with overexpression of MMP-12. SSc MVEC–conditioned medium impaired uPA-dependent proliferation and invasion as well as capillary morphogenesis in normal MVECs in vitro. Both a general hydroxamate inhibitor of MMP activity and anti–MMP-12 antibodies restored this SSc MVEC–induced impaired functioning. Conclusion Overproduction of MMP-12 by SSc MVECs accounts for the cleavage of uPAR and the impairment of angiogenesis in vitro and may contribute to reduced angiogenesis in SSc patients.

The microvascular involvement in systemic sclerosis (SSc) is characterized by endothelial damage and smooth muscle cell migration in the intima. The vascular pathologic modifications in SSc are strikingly similar to those of atherosclerosis. SSc also is characterized by an accelerated macrovascular disease. The gene encoding for angiotensin-converting enzyme (ACE) is a 21-kb, 26-exon gene, localized on chromosome 17 (17q23). Polymorphic sites are an insertion/deletion (I/D) that consists of three genotypes: DD and II homozygotes, and ID heterozygote. ACE gene polymorphisms have been linked to vascular disorders (coronary artery disease, hypertension, cerebrovascular disease, hypertrophic cardiomyopathy, and diabetic or nondiabetic nephropathy). In particular, the possession of ACE D allele was associated with an increased risk of developing malignant vascular injury. ACE D allele frequency of the I/D polymorphism was associated with an increased risk of SSc, suggesting a genetic contribution to the disease. The discrepancy between the high prevalence of D allele and reduced ACE plasma levels in SSc demonstrate the lack of knowledge on the regulation and function of renin-angiotensin system in SSc.

Systemic sclerosis (SSc) is a challenge for the rheumatologist as it is easy to diagnose when it is evolved to skin fibrosis with obliterative vasculopathy and organ involvement but its diagnosis remains very difficult in the very early/early phase of the disease because the American College of Rheumatology (ACR) and LeRoy criteria, which are currently those largely used to classify SSc, were shown to be not sensitive enough to reach a very early or an early diagnosis of SSc.1–4 Therefore, the SSc diagnosis may be delayed for several years following the onset of Raynaud's phenomenon (RP) and even after the onset of the first non-RP symptom. This reality implies that the diagnosis, and consequently the therapy, are delayed until skin involvement and/or internal organ involvement are evident5–8 and, in too many cases, already irreversible.6 The fact that organ involvement may be present from the earliest stages of SSc corroborates the necessity of a very early or at least an early diagnosis of SSc to try to identify therapy which might achieve disease remission. Moreover, the rheumatologist is also facing today the problem to position the patient in the SSc evolution from its very early to the early and to the established phase. Previously, several attempts have been made to define the early phase of SSc7 and recently even the concept of very early SSc has been widely accepted and investigated.8 RP, despite its lack of specificity, has been proposed as a pivotal sign in a previous attempt to define criteria for the diagnosis of ‘early’ SSc9 and has been also considered as the main ‘sentinel’ sign10 for the identification of ‘very early …

Bosentan, a dual endothelin receptor antagonist, may reduce blood pressure by blocking the vasoconstrictor effect of endothelin-1. In systemic sclerosis (SSc) nailfold videocapillaroscopy (NVC); allows diagnostic and follow-up of microvascular damage. Distinct NVC patterns have been identified for the evaluation of severity of SSc microvascular damage. The objective of this study is to evaluate the modification of the microvasculature under Bosentan therapy in SSc patients with pulmonary arterial hypertension (PAH). Nine patients with PAH related to SSc in New York Heart Association classes III-IV were treated with Bosentan 125 mg twice a day. NVC optical probe videocapillaroscopy equipped with 100× and 200× contact lenses and connected to image analyse software was performed before and after 12 months of Bosentan therapy to evaluate the modification of microvasculature. Nine PAH SSc patients treated with Iloprost were used as controls. Before Bosentan therapy, seven patients showed at NVC severe loss of capillaries with large avascular areas and vascular architectural disorganisation which are typically "late" SSc pattern. After 12 months of Bosentan, NVC pattern changed in seven patients from "late" into "active" SSc pattern. The disappearance of avascular areas and capillary haemorrhages was the most striking result. Two patients had an "active" SSc pattern, not modified by Bosentan treatment. These data show that Bosentan may improve NVC pattern in SSC and the presence of new capillaries suggests that it may favour angiogenesis. Bosentan may improve and stabilise the microvasculature in long-term treatment modulating the structural modifications detected by NVC.

RP represents a common complaint in clinical practice, particularly among patients with rheumatic diseases. From its original description in the nineteenth century different definitions have been proposed. Today, this event is defined as primary RP when no disease is diagnosed while it is termed secondary RP when an underlying disease is disclosed. This classification satisfies the needs of the physician in practice. Clinical assessment by history and examination is the gold standard for making a diagnosis.

Aims Systemic sclerosis (SSc) may induce cardiac fibrosis and systo-diastolic dysfunction. Cardiovascular magnetic resonance (CMR) can detect replacement myocardial fibrosis with late gadolinium enhancement (LGE) and interstitial myocardial fibrosis with T1 mapping techniques. The aim of the study was to detect subclinical cardiac involvement with CMR in paucisymptomatic SSc patients with no previous history of myocardial disease, comparing it with skeletal muscle remodelling. Methods and results Thirty consecutive SSc patients (mean age: 51 ± 12 years, all women) and 10 healthy controls (mean age: 48 ± 15 years, all women) underwent clinical, biohumoral assessment, and CMR. Extracellular volume fraction (ECV) was calculated from pre- and post-contrast T1 values in the myocardium and skeletal muscle. Seventeen patients (57%) were asymptomatic, 13 (43%) paucisymptomatic (effort dyspnoea). All patients had normal biventricular volumes and systolic function, while LGE was present in seven patients (23%). Myocardial ECV was significantly increased in patients with SSc (30 ± 4%) than controls (28 ± 4%, P = 0.03), as was skeletal muscle ECV (23 ± 6% vs. 18 ± 4%, P < 0.01). Myocardial ECV did not differ between patients with and without LGE ( P = NS) and showed no significant correlations with clinical data, biventricular volumes, systolic, or diastolic function. Overall, myocardial ECV showed a significant correlation with skeletal muscle ECV ( R = 0.58, P < 0.001). Conclusion SSc is associated not only with myocardial replacement fibrosis, as detected by LGE, but also with interstitial remodelling of the myocardium and skeletal muscles, as detected by an increased ECV also in patients with normal biventricular function, with potential diagnostic, prognostic, and therapeutic clinical implications.

OBJECTIVE: The aim of this study was to evaluate the presence of digital lesions in very early diagnosis of SSc (VEDOSS) patients and its possible association with internal organ involvement. METHODS: One hundred and ten VEDOSS patients were investigated for the presence of digital ulcers (DUs), digital pitting scars, calcinosis, necrosis or gangrene, nailfold videocapillaroscopic abnormalities, disease-specific autoantibodies (ACA and anti-topo I) and internal organ involvement. RESULTS: Four patients reported a history of digital pitting scars, while 25 patients presented an active DU or reported a history of DUs. In particular, 16 patients presented with active DUs (14/16 also reporting a history of previous DUs), while the other 9 patients reported a history of DUs only. A statistically significant association between DUs and oesophageal manometry alteration was found in the whole DU population, as well as in the history of DU and the presence of active DU with/without a history of DU subgroups (P < 0.01, P = 0.01 and P < 0.05, respectively). DUs were observed in VEDOSS patients with internal organ involvement but not in those without organ involvement. CONCLUSION: DUs are already present in VEDOSS patients characterized by internal organ involvement, significantly correlating and associating with gastrointestinal involvement. DUs may be a sentinel sign for early organ involvement in VEDOSS patients.

Objective In addition to its chemotactic properties, recent evidence suggests that monocyte chemoattractant protein 1 (MCP-1) might participate in the fibrotic process by inducing the secretion of extracellular matrix (ECM) components. Since the factors that initiate the accumulation of inflammatory infiltrates and ECM deposits in systemic sclerosis (SSc) skin lesions are still unknown, this study was undertaken to examine the role of MCP-1 in SSc. Methods In situ hybridization and immunohistochemistry studies for MCP-1 were performed on skin biopsy specimens from patients with SSc and healthy controls. To identify possible stimulators of MCP-1 overexpression in SSc lesions, cultured dermal fibroblasts were incubated with recombinant platelet-derived growth factor (PDGF) and analyzed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. The chemotactic effects of SSc fibroblasts were examined using a modified Boyden chamber assay. To analyze the fibrotic potential of MCP-1, cultured dermal fibroblasts were incubated with recombinant MCP-1, and type I procollagen was measured by radioimmunoassay and real-time PCR. Results MCP-1 was expressed by fibroblasts, keratinocytes, and perivascular infiltrates throughout the skin, in involved as well as uninvolved skin areas, from 10 of 11 SSc patients, whereas no expression of MCP-1 was found in healthy controls. Stimulation with PDGF resulted in a significant increase in MCP-1 messenger RNA and protein, with differences between healthy control fibroblasts and fibroblasts from SSc patients. The chemotactic activity for peripheral blood mononuclear cells of SSc fibroblast supernatants increased in a time-dependent manner. Antibodies blocking MCP-1 decreased the chemotactic activity of SSc fibroblasts by a mean ± SD of 37 ± 12%. Despite an increase in type I collagen levels over time, no effect of recombinant MCP-1 on the synthesis of type I collagen was observed. Conclusion These data indicate that MCP-1 might contribute to the initiation of inflammatory infiltrates in SSc. Possible stimuli of MCP-1 in dermal SSc lesions include PDGF, which is known to be expressed in SSc. In contrast to previous findings in fibrotic lung diseases, no effect of MCP-1 on collagen synthesis was observed in SSc dermal fibroblasts in vitro.

Since activation of the immune system and a perivascular infiltrate of inflammatory cells are key features of SSc, immunosuppression has long been considered to be an anchor treatment. Non-selective immunosuppression remains central to the treatment of interstitial lung disease (ILD) and skin involvement, with CYC most widely used to obtain remission. The use of MTX as a first-line agent may be considered in the presence of skin involvement without ILD. More recently, MMF has shown encouraging results in observational studies, but still needs more formal evaluation to verify if it can be considered an alternative drug to CYC or a maintenance agent such as AZA. Rituximab has provided promising results in small open-label studies and other novel therapies targeting specific molecular and cellular targets are under evaluation. Patients with rapidly progressing diffuse cutaneous SSc should be evaluated for haematopoietic stem cell transplantation.

Introduction: Systemic sclerosis (SSc) is characterised by disturbed vessel morphology and an overproduction of vascular endothelial growth factor (VEGF). The VEGF gene located on chromosome 6p21.3 has several polymorphisms. Objective: To test the hypothesis that disturbed angiogenesis may be related to the genetic background of the VEGF gene. Materials and methods: EUSTAR centres included European Caucasian patients with SSc and matched controls with osteoarthritis. The VEGF gene was genotyped by polymerase chain reaction, followed by restriction enzyme analysis. The 634 C/T and 936 C/G mutations and an 18-base pair insertion/deletion at −2549 of the VEGF promoter region were tested. Results: 416 patients with SSc and 249 controls were included in the study population. Of the patients with SSc, 42% had a diffuse cutaneous subtype, 16% had increased pulmonary arterial pressure and 61% had decreased carbon monoxide diffusion capacity. The genotype frequencies in the patients with SSc and in controls were in Hardy–Weinberg equilibrium. The allele and genotype frequencies of the polymorphisms did not differ between patients with SSc and controls. No association was found between these polymorphisms and disease phenotypes. Conclusion: This study shows that there is no association between the three selected functional VEGF polymorphisms and SSc.

BackgroundThe oesophagus is the first gastrointestinal (GI) tract involved in systemic sclerosis (SSc), followed by the anorectum.ObjectiveEvaluation of oesophageal and anorectal involvement and their correlations in patients with very early diagnosis of SSc (VEDOSS).Patients and methods59 patients with VEDOSS, evaluated with oesophageal and anorectal manometry and investigated with lung function tests and chest HRCT. Demographic data, oesophageal and anorectal symptoms, Raynaud's phenomenon, autoantibodies, videocapillaroscopy patterns, puffy fingers and digital ulcers were recorded for all patients.ResultsIn 4 patients oesophageal manometry and in 17 patients anorectal manometry was not performed because of scarce tolerance. Oesophageal peristalsis was absent in 14 patients; its pressure and speed were significantly lower in 41 patients (p80% showed only an abnormal peristalsis (ConclusionsIn patients with VEDOSS, oesophageal and anorectal disorders are frequently detected, showing that very early SSc is characterised by GI involvement.

Stiff skin syndrome (SSS) is clinically characterized by stone-hard skin bound firmly to the underlying tissues, leading to a secondary limitation of joint mobility, often associated mild overlying hypertrichosis, and postural and thoracic wall abnormalities. First manifestations of SSS are usually observed between birth and early childhood with rock-hard skin that is most prominent in areas with abundant fascia such as on the buttocks and thighs, and the disease progresses until the skin of the entire body becomes fibrotic with subsequent growth retardation and joint contractures. SSS is a diagnosis of exclusion, with a distinctive clinical presentation without pathognomonic laboratory or pathological findings. The clinical differential diagnosis of stone-hard and thickened skin areas includes systemic sclerosis, scleroderma, eosinophilic fasciitis, and scleromyxedema. In SSS patients, an overexpression of ECM proteins was detected, whereas no inflammatory infiltrates or upregulation of pro-fibrotic cytokines were found. Data in the literature suggest that fibrosis in SSS might be independent from inflammation. SSS exhibits a spectrum of histopathologic findings and different disease stages might also account for controversial results.

Herewith we provide a critical digest of the recent literature on systemic sclerosis. The most relevant studies published over the last two years have been reviewed, with particular focus on the diagnosis, pathogenesis and treatment of the disease.

Objective The 1980 American College of Rheumatology (ACR) classification criteria for systemic sclerosis (SSc) lack sensitivity for early SSc and limited cutaneous SSc. The present work, by a joint committee of the ACR and the European League Against Rheumatism (EULAR), was undertaken for the purpose of developing new classification criteria for SSc. Methods Using consensus methods, 23 candidate items were arranged in a multicriteria additive point system with a threshold to classify cases as SSc. The classification system was reduced by clustering items and simplifying weights. The system was tested by 1) determining specificity and sensitivity in SSc cases and controls with scleroderma-like disorders, and 2) validating against the combined view of a group of experts on a set of cases with or without SSc. Results It was determined that skin thickening of the fingers extending proximal to the metacarpophalangeal joints is sufficient for the patient to be classified as having SSc; if that is not present, 7 additive items apply, with varying weights for each: skin thickening of the fingers, fingertip lesions, telangiectasia, abnormal nailfold capillaries, interstitial lung disease or pulmonary arterial hypertension, Raynaud's phenomenon, and SSc-related autoantibodies. Sensitivity and specificity in the validation sample were, respectively, 0.91 and 0.92 for the new classification criteria and 0.75 and 0.72 for the 1980 ACR classification criteria. All selected cases were classified in accordance with consensus-based expert opinion. All cases classified as SSc according to the 1980 ACR criteria were classified as SSc with the new criteria, and several additional cases were now considered to be SSc. Conclusion The ACR/EULAR classification criteria for SSc performed better than the 1980 ACR criteria for SSc and should allow for more patients to be classified correctly as having the disease. Copyright © 2013 by the American College of Rheumatology.

International audience; IntroductionMicrovascular damage and defective angiogenesis and vasculogenesis have a major role in the pathogenesis of systemic sclerosis (SSc). Epidermal growth factor-like domain 7 (EGFL7) is a proangiogenic molecule which is predominantly expressed and secreted by endothelial cells and their progenitors and controls vascular development and integrity. In this study, we investigated the possible involvement of EGFL7 in SSc.MethodsSerum EGFL7 levels from 60 patients with SSc and 35 age- and sex-matched healthy controls were examined by colorimetric sandwich enzyme-linked immunosorbent assay. The expression of EGFL7 in forearm skin biopsies (n = 16 SSc, n = 10 controls), cultured dermal microvascular endothelial cells (MVECs) (n = 3 SSc, n = 3 controls) and late-outgrowth peripheral blood endothelial progenitor cell (EPC)-derived endothelial cells (n = 15 SSc, n = 8 controls) was investigated by immunofluorescence and Western blotting.ResultsSerum EGFL7 levels were detectable in 68.6% of healthy controls and 45% of SSc cases (P < 0.05). Circulating levels of EGFL7 were significantly decreased in SSc patients compared with healthy controls (P = 0.01). Serum levels of EGFL7 were significantly lower in both limited cutaneous SSc and diffuse cutaneous SSc patients than in controls (P = 0.02 and P = 0.04, respectively). In SSc, decreased serum EGFL7 levels were significantly correlated with the severity of nailfold capillary abnormalities. Patients with the most severe capillary changes and digital ulcers had serum EGFL7 levels significantly lower than healthy controls, while the EGFL7 levels did not differ significantly between controls and SSc patients with less capillary damage and lack of digital ulcers. Endothelial EGFL7 expression was strongly downregulated or even almost completely undetectable in SSc-affected dermis compared with controls (P < 0.001). In cultured SSc dermal MVECs and late-outgrowth peripheral blood EPC-derived endothelial cells, EGFL7 was significantly downregulated compared with cells obtained from healthy subjects (P < 0.01 and P < 0.001, respectively).ConclusionsOur findings suggest that the loss of EGFL7 expression in endothelial cells and their progenitors might play a role in the development and progression of peripheral microvascular damage and the defective vascular repair process characteristic of SSc.

Objective The aims of this study were to obtain cross-sectional data on capillaroscopy in an international multi-center cohort of Systemic Sclerosis (SSc) and to investigate the frequency of the capillaroscopic patterns and their disease-phenotype associations. Methods Data collected between June 2004 and October 2011 in the EULAR Scleroderma Trials and Research (EUSTAR) registry were examined. Patients' profiles based on clinical and laboratory data were obtained by cluster analysis and the association between profiles and capillaroscopy was investigated by multinomial logistic regression. Results 62 of the 110 EUSTAR centers entered data on capillaroscopy in the EUSTAR database. 376 of the 2754 patients (13.65%) were classified as scleroderma pattern absent, but non-specific capillary abnormalities were noted in 55.48% of the cases. Four major patients' profiles were identified characterized by a progressive severity for skin involvement, as well as an increased number of systemic manifestations. The “early” and “active” scleroderma patterns were generally observed in patients with mild/moderate skin involvement and a low number of disease manifestations, while the “late” scleroderma pattern was found more frequently in the more severe forms of the disease. Conclusion These data indicate the importance of capillaroscopy in SSc management and that capillaroscopic patterns are directly related to the extent of organ involvement.

Systemic sclerosis (SSc) is an incurable chronic autoimmune disease associated with high morbidity and mortality. The current validated or proposed criteria are not appropriate to make a very early diagnosis of SSc. This implies that the diagnosis of SSc and, consequently, an appropriate therapy are delayed until the appearance of skin involvement and/or clinically detectable internal organ involvement when microvascular remodeling, tissue fibrosis, or atrophy are already irreversible. In a recent Delphi exercise, 4 signs/symptoms have been identified as necessary for the very early diagnosis of SSc: Raynaud's phenomenon (RP), puffy swollen digits turning into sclerodactily, antinuclear antibodies and specific SSc antibodies (anticentromere and antitopoisomerase‑I antibodies), and abnormal capillaroscopy with scleroderma pattern. Patients with very early SSc are the target of the recently launched the VEDOSS program, which has been designed to diagnose SSc very early and to examine whether this may change the disease prognosis. Although patients with RP, autoantibodies, and SSc capillaroscopic pattern could be easily followed up, there is still no agreement on the predictors that may allow us to identify patients who will develop an established disease.

Background Nailfold videocapillaroscopy (NVC) is a useful tool in systemic sclerosis (SSc) patients, useful not only for the diagnosis but also to predict the development of organ and vascular complications, such as the utility of the recently proposed semi-quantitative scoring for digital ulcers (DU) [1]. Objectives to prospectively evaluate the change of prevalence of NVC microvascular alterations and their possible correlations with changes in patients-reported outcomes (PROs) and clinical-reported outcomes (CROs) and prevalence of clinical characteristics related to DU in SSc patients. Methods SSc patients presenting at least one DU and attending the Rheumatology Wound Care Clinic of the Florence University Hospital or the London Royal Free Hospital were enrolled. Patients were assessed with PROs (HAQ-DI, UKFS, Cochin scale, VAS for pain, DU status and general disease status) and CROs (VAS for DU status) at baseline and after 16 weeks. DU number, appearance of new DU, presence of gangrene, infection, need for hospitalization or surgery procedures, painkillers use were recorded at each visit. NVC was performed at both visit and the presence of micro-haemorrhages, giant capillaries, irregularly enlarged capillaries, ramified capillaries, capillary disorganization and loss of capillaries was scored according to the previously proposed score (0 = no changes, 1 = from 0 to 33%; 2 = from 33% to 66%, 3 = more than 66%). Correlations were tested through Spearman test. Results twenty-five SSc patients were eligible for the study. No significant correlation was seen at baseline; when analysing changes between baseline and follow-up, a negative correlation was seen between irregularly enlarged capillaries and the majority of PROs; moreover, changes of number of DU showed negative correlation with changes of giant capillaries, possibly reflecting the evolution of microvascular damage. Conclusions NVC is useful tool to predicting DU development, but may not reflect short term change in DU status; therefore a longer-term evaluation may be necessary to detect NVC modifications paralleling DU changes in SSc patients. References Smith V., Pizzorni C., De Keyser F, et al. “Validation of the qualitative and semiquantitative assessment of the scleroderma spectrum patterns by nailfold videocapillaroscopy: preliminary results,” Arthritis and Rheumatism, vol. 60, pp. S164–S165, 2009. Disclosure of Interest None declared

Background SSc is characterized by diffuse microangiopathy leading digital ulcers (DU) in almost 50% of patients. The treatment of non-healing DU is often very difficult. It includes both systemic (prostanoids, bosentan) and local (wound care) therapies. In clinical practice, therapy of DU should be tailored mainly on clinical symptoms and according to specific clinician9s experience. At the moment, no guidelines for the use iloprost, a stable prostacyclin-analogue used for the management of DU in SSc, are not yet available. Objectives We report the therapeutical effects of our regimen for the use of iloprost in a cohort of SSc patients during a long-lasting follow-up period. Methods Fifty consecutive SSc patients recruited at two Rheumatology Units since January 2003, who received iloprost infusions for a time period >2 years were included in the analysis. Patients9 features were: males/females ratio 7/43; mean age at SSc diagnosis 43.5±12.7SD years; median disease duration at the beginning of therapy 2 years (range 0–18); mean follow-up with iloprost treatment 10±4.2SD years; diffuse SSc cutaneous subset in 13/50 (30%) patients; anti-Scl70 and anticentromere autoantibodies in 25 (50%), and 14 (28%) subjects, respectively. For all patients epidemiological, clinical, laboratory, and instrumental data were available for the study period. Iloprost schedule consisted in monthly i.v. infusion at 0.8–1 ng/kg body weight/min with average cumulative dosage per each session of 25 μg, according to patients9 tolerance. At the beginning of the treatment and in the presence of recent onset, severe and/or multiple DU at high risk of gangrene, patients were hospitalized for continuous infusion of iloprost (3 days, average 0.2 mg). Results 31/50 (62%) patients showed DU at the beginning of iloprost therapy: among them, 22 (71%) resolved during the follow-up, while the other 9 presented recurrent or chronic DU, despite the treatment. With regards the 19/50 patients without DU at baseline, only one developed skin lesions at the end of very long follow-up (8 years), when a general deterioration of clinical conditions, in particular severe pulmonary hypertension, lead to exitus. Considering the 31 SSc patients with DU at baseline, a diffuse skin subset was present in 3 out of 22 patients with healed DU, and in 5 out of 9 who did not (13.6% vs 55.5%, Fisher9s p=0.027); no other significant correlations were observed as regards visceral organ involvement and serological alterations. Finally, the concomitant administration of other vasoactive therapies seems to be scarcely relevant on the DU outcome. Conclusions Our data suggest that long term administration of iloprost seems to be effective for DU healing in SSc. Comparative studies with different treatment regimens are warranted to reach a consensus on standardized guidelines for the use of Iloprost for DU healing in SSc. Disclosure of Interest None declared

Background VEDOSS project relied on a specific set of inclusion criteria identifying patients in very early stage of systemic sclerosis (SSc) to be submitted to a tight follow up and unravel factors linked to the progression of the disease. The components of the ELF score (PIIINP, TIMP-1 and HA) have been shown to correlate with severity of skin and lung fibrosis in SSc. Objectives Our aim was to analyze the concentrations of ELF components in a subset of patients enrolled in the VEDOSS study and determine their potential diagnostic value. Methods Sera from 114 patients enrolled into the VEDOSS data base and 67 SSc controls fulfilling ACR/EULAR 2013 criteria (subsets: 33 diffuse; 34 limited) were obtained from 2 centres. Serum concentrations of ELF components were determined on Siemens Advia Centaur platform. Logistic regression analysis of the results was performed employing classification of SSc as state variable. Results Among the 114 patients enrolled from VEDOSS, 30 subjects had primary Raynaud9s phenomenon (PRP); 54 had Raynaud9s phenomenon (RP) and at least one of the following “red flags” (risk factors) for SSc: (Puffy fingers, ANA, SSc specific autoantibodies or SSc-specific capillaroscopic pattern) without fulfilling 2013 ACR/EULAR criteria (score 9) despite having no evidence of skin involvement nor interstitial lung disease at lung high resolution CT or pulmonary arterial hypertension, left ventricular systolic impairment or renal involvement. All serum biomarkers concentrations correlated with age (p Conclusions Our data indicate that the SSc score is a simple serum test based model that can be used in patients with RP to aid in the very early diagnosis of SSc. Furthermore, the identification of VEDOSS patients with a high SSc score test and already classified as SSc even in the absence of internal organ involvement can be used in intervention trials aimed to prevent further disease progression. Disclosure of Interest None declared

Background “Pet Therapy”, better defined as Animal-assisted Intervention (AAI), is based on the interaction between animal and human being and is a tool which may complement and support traditional therapies. It can be used on patients affected by different diseases, improving their quality of life from behavioral, physical and psychosocial point of view. Objectives The aim of our study was to evaluate the effect of AAI in improving quality of life and compliance to standard pharmacological treatment in a cohort of Systemic Sclerosis (SSc) patients. Methods 42 SSc patients attending at Scleroderma Unit and undergoing iloprost intravenous infusion were divided in three groups: 1) 14 SSc patients (mean age 60.4±8.6 yrs) submitted to 20 AAI sessions with a professional team (doctor, nurse, couple of animal-handler); 2)control (C1) 14 SSc patients (mean age 63.4±5.3 yrs) engaged in alternative social activity as create a cookbook; 3)control (C0) 14 SSc patients (mean age 62.3±6.8 yrs) without any alternative activity. All patients underwent psychological evaluation at baseline (t0) and at the end of project of AAI (t1); moreover the following test (italian standardized version) was performed at the beginning (s0) and at the end (s1) of each single session: General Anxiety State-Trait Anxiety Inventory (STAI-T), Beck Depression Inventory (BDI), Social Interaction Anxiety Scale (SIAS), Eysenck Personality Questionnaire-Revised (EPQ-R), the Social Phobia Scale (SPS), the Toronto Alexythymia Scale (TAS-20), the Thought Control Questionnaire (TCQ), the Visual Analog Scale (VAS), the State-anxiety (STAI-S). Results At the end of each session, AAI group showed a decrease of the anxiety state level while it was higher in the two control groups ( p s p s Conclusions In agreement with literature [1], our work showed that AAI might be a crucial tool to support the traditional therapies thanks to the creation of a different relationship between patient and doctor. This may allow a better compliance to the therapy in a chronic disease like SSc, where patient9s compliance is usually low. References Matuszek S. Animal-facilitated therapy in various patient populations: systematic literature review. Holist Nurs Pract. 2010 Jul-Aug;24(4):187–203. Disclosure of Interest None declared

Background Iloprost (ILO) is a synthetic analogue of prostacyclin PGI 2 with a potent vasodilator action, an anti-proliferative effect on smooth muscle cells and inhibition of platelet aggregation. ILO is employed intravenously for the control of Raynaud Phenomenon and digital ulcers. Up to now, no agreement has been reached on the regimen (dosage and frequency) to be used in SSc patients. Objectives To identify the most common side effects that are developed during ILO infusion in SSc. Methods 81 SSc patients, classified according to ACR EULAR criteria, derived from two Italian centers, were submitted to ILO infusion: patients were subclassified according to the edematous or fibrotic/atrophic phase of the disease. ILO was infused (6–8 hour infusion, dose ranging from 0.5 to 1.0 ng/kg/ min) (1) with a progressive increase of the dosage up to the achievement of patient9s tolerance: the infusion on pump was started at 5 ml/hour and increased every half an hour up to 20 ml/hour (a dose of 4 mcg/h which corresponds to 1ng/kg)according to patients tolerance and appearance of side effects. Results Out of 81 patients, 16 were in the edematous phase with puffy fingers (digital edema). In this cohort, 5 individuals (31.2%) developed diarrhea and 9 (56.2%) developed hypotension during the infusion. When the drug was withdrawn patients had an immediate amelioration but when ILO was restarted, they again developed diarrhea or hypotension that led to definitive interruption of the treatment. Moreover, 10/16 patients (62.5%) experienced significant and painful digital swelling which led to the tapering of the infusion dosage below 10 ml/hr (which corresponds to 0.5 ng/kg per min); out of 10 patients, only one could continue the treatment while the other 9 had to be withdrawn due to the maintenance of the intense digital pain due to vasodilation. Finally, 11 patients (68.7%) reported flushing and 7 (43.7%) headache, but they were always controlled with the reduction of the infusion below 10 ml/hr. Out of 81 patients, 65 were in the atrophic/ fibrotic phase: 10 patients (15.3%) developed diarrhea and 24 pts (37%) hypotension that led to temporary withdrawal: when ILO was restarted and kept below 10 ml /hr, no side effects were experienced. In 23 cases (35.3%) flushing and in 8 cases (12.3%) headache were experienced, but were well controlled with infusion reduction below 10 ml/hr. Conclusions ILO was well tolerated in SSc patients characterized by fibrotic phase, where side effects could be managed by reducing/modulating the infusion rate. In edematous patients, ILO infusion should be decided in case where the severity of Raynaud9s phenomenon or the presence of digital ulcers strongly suggests its use. References Wigley FM, et a Intravenous iloprost infusion in patients with Raynaud phenomenon secondary to systemic sclerosis. A multicenter, placebo-controlled, double-blind study Ann Intern Med. 1994;120:199–206 Disclosure of Interest None declared

Background In SSc, the management of DU is a problem in clinical practice, therefore their prevention is highly warranted. Sildenafil, iloprost, calcium channel blockers (CCB), ACE inhibitors (ACEi) and bosentan, alone or in combination, are used for DU prevention without unanimous agreement. Objectives DeSScipher (“to decipher the optimal management of SSc”) is an EC-funded FP7 research project that consists of five observational trials (OT) addressing different aspects of SSc. The aim of OT1 was to evaluate the efficacy of vasoactive and vasodilating drugs or a combination therapy for the management of DU in SSc. Methods Longitudinal data were collected for up to 24 months by 28 DeSScipher centres. In SSc patients, the efficacy of sildenafil, bosentan, sildenafil+bosentan, iloprost or CCB /ACEi for the prevention of new DU was evaluated. Clinical features, ulcer type and other drugs were also recorded. Time without new DU, proportion of patients without new DU, risk of developing new DU and the mean number of new DU per patient were evaluated at 6 months follow-up. Results In OT1, 1394 patients were enrolled. The history of DU was a significant risk factor for developing new DU (OR=3.146; (95%CI:1.19–8.31), p=0.021), therefore analysis was focused on secondary prevention (prevention of new DU in patients with DU history). 473/1394 (33.9%) had a history of DU and 268/473 (56.7%) had follow up data (58.2% limited and 41.8% diffuse SSc subset). 47/268 (17.5%) were on Bosentan, 33/268 (12.3%) on Sildenafil, 40/268 (14.9%) on Iloprost, 31/268 (11.6%) on Sildenafil and Bosentan combination and 117/268 (43.7%) on CCB/ACEi alone. In patients with history of DU in the last 24 weeks, the treatment with CCB/ACEi alone was associated with 7 fold increased risk of developing new DU compared to all other treatment arms (OR =7.313; 95%CI:1.248–42.85, p=0.027). Patients on concomitant immunosuppressive treatment had a trend towards faster DU recurrence. Conclusions History of DU in the past 24 weeks should prompt a more aggressive preventive strategy other than therapy with CCB/ACEi alone due to its lower efficacy to prevent DU recurrence compared to the other treatment options. Acknowledgement The DeSScipher project was funded by the European Community9s Framework Programme 7 (FP7-HEALTH-2012.2.4.4–2 - Observational trials in rare diseases) under grant agreement N° 305495. We acknowledge the contribution of the following EUSTAR centers: Ancona (member N°34), Assiut (168), Bad Bramstedt (187), Belgrade (55), Bucharest (100), Cluj-Napoca (16), Erlangen (106), Frankfurt (124), Hamburg (64), Iasi (162), Istanbul (21), Istanbul (133), Koln (44), Monserrato (142), Moscow (78), Moscow (190), Padua (31), Rome (94), Salford/Manchester (80), Tubingen (56), Wuppertal (192). Disclosure of Interest None declared