F. Cannarile

Characterization of circulating endothelial microparticles and endothelial progenitor cells in primary Sjögren’s syndrome: new markers of chronic endothelial damage?

OBJECTIVE Chronic autoimmune diseases are associated with increased risk of cardiovascular death. Endothelial dysfunction represents the first stage of subclinical atherosclerosis and multiple factors contribute to endothelial injury. Among these, an altered balance between endothelial microparticle (EMP) release and endothelial progenitor cell (EPC) generation promotes endothelial dysfunction. The role of EMPs and EPCs in promoting endothelial damage in primary SS (pSS) has never been investigated. Our aim was to evaluate the role of EMPs and EPCs as markers of endothelial damage in pSS and their correlation with disease clinical and immunological features. METHODS Circulating EMPs (CD31(+)/CD42(-)), true EPCs (CD34(+)/KDR(+)/CD133(+)) and mature EPCs (CD34(+)/KDR(+)/CD133(-)) were quantified by FACS analysis in 34 pSS patients and 18 age- and sex-matched controls. Correlation between EMP and EPC levels and parameters of disease activity and damage, clinical features and markers of immunological dysfunction was performed. RESULTS Patients displayed higher EMP numbers with respect to healthy controls [HCs; mean 450 n/μl (S.D. 155) vs 231 (110), P < 0.0001]. EPC and mature EPC levels were higher in patients compared with HCs [mean 226 n/ml (S.D. 181) vs 69 (53), P < 0.001 and 166 (161) vs 36 (32), P < 0.0001, respectively). EMP levels directly correlated with disease duration from symptoms and diagnosis (ρ = 0.5, P < 0.01). Early EPCs inversely correlated with disease duration from symptoms (ρ = -0.5, P < 0.01) and diagnosis (ρ = -0.4, P < 0.05). CONCLUSION This is the first demonstration of chronic endothelial fragmentation characterizing pSS. The reparative potentiality of the endothelial layer appears to be preserved in the earliest stages of disease. During the course of the disease, progressive exhaustion of the precursor endothelial pool may be hypothesized, leading to defective vascular layer restoration and endothelial dysfunction.

Cardiovascular disease in systemic sclerosis.

Cardiovascular (CV) system involvement is a frequent complication of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). It still remains unclear if a premature atherosclerosis (ATS) occurs even in systemic sclerosis (SSc). Although microvascular disease is a hallmark of SSc, in the last few years a number of studies highlighted a higher prevalence of macrovascular disease in SSc patients in comparison to healthy individuals and these data have been correlated with a poorer prognosis. The mechanisms promoting ATS in SSc are not fully understood, but it is believed to be secondary to multi-system organ inflammation, endothelial wall damage and vasculopathy. Both traditional risk factors and endothelial dysfunction have been proposed to participate to the onset and progression of ATS in such patients. In particular, endothelial cell injury induced by anti-endothelial antibodies, ischemia/reperfusion damage, immune-mediated cytotoxicity represent the main causes of vascular injury together with an impaired vascular repair mechanism that determine a defective vasculogenesis. Aim of this review is to analyse both causes and clinical manifestations of macrovascular involvement and ATS in SSc.

T Regulatory and T Helper 17 Cells in Primary Sjögren’s Syndrome: Facts and Perspectives

Historically, primary Sjögrens syndrome (pSS) was thought to be a T helper (h) 1 driven disease due to the predominance of CD4+T lymphocytes and their products in target organs and peripheral blood of patients. In the last decades, the identification of a number of T cell subsets, including Th17, T regulatory (Treg), and follicular helper T cells, challenged this long-standing paradigm and prompted to identify their role in pSS pathogenesis. In addition the impact of abnormal proinflammatory cytokine production, such as IL-6, IL-17, IL-22, and IL-23, has also attracted considerable attention. However, although several studies have been carried out in experimental models and patients with pSS, many aspects concerning the role of Treg cells and IL-17/Th17 cell system in pSS pathogenesis are not fully elucidated. In particular, the role played by different IL-17-producing T cell subsets as well as the effects of pharmacological therapies on Treg/Th17 cell balance represents an intriguing issue. The aim of this review article is to provide an overview of current knowledge on Treg cells and IL-17-producing T cells in pSS pathogenesis. We believe that these insights into pSS pathogenesis may provide the basis for successful therapeutic intervention in this disease.

Central Hemodynamics and Arterial Stiffness in Systemic Sclerosis

Although microvascular disease is a hallmark of systemic sclerosis (SSc), a higher prevalence of macrovascular disease and a poorer related prognosis have been reported in SSc than in the general population. The simultaneous assessment of prognostically relevant functional properties of larger and smaller arteries, and their effects on central hemodynamics, has never been performed in SSc using the state-of-the-art techniques. Thirty-four women with SSc (aged 61±15 years, disease duration 17±12 years, and blood pressure 123/70±18/11 mm Hg) and 34 healthy women individually matched by age and mean arterial pressure underwent the determination of carotid-femoral (aortic) and carotid-radial (upper limb) pulse wave velocity (a direct measure of arterial stiffness), aortic augmentation (a measure of the contribution of reflected wave to central pulse pressure), and aortobrachial pulse pressure amplification (brachial/aortic pulse pressure) through applanation tonometry (SphygmoCor). Patients and controls did not differ by carotid-femoral or carotid-radial pulse wave velocity. Aortic augmentation index corrected for a heart rate of 75 bpm (AIx@75) was higher in women with SSc (30.9±16% versus 22.2±12%; P=0.012). Patients also had a lower aortobrachial amplification of pulse pressure (1.22±0.18 versus 1.33±0.25; P=0.041). SSc was an independent predictor of AIx@75 (direct) and pulse pressure amplification (inverse). Among patients, age, mean arterial pressure, and C-reactive protein independently predicted carotid-femoral pulse wave velocity. Age and mean arterial pressure were the only predictors of AIx@75. Women with SSc have increased aortic augmentation and decreased pulse pressure amplification (both measures of the contribution of reflected wave to central waveform) but no changes in aortic or upper limb arterial stiffness. Microvascular involvement occurs earlier than large artery stiffening in SSc.

SAT0621 Validation Study of Predictive Value of Capillaroscopic Skin Ulcer Risk Index (CSURI) in Scleroderma Patients Treated with Bosentan

Background CSURI is a capillaroscopic index, able to identify scleroderma (SSc) patients at high risk for new or non-healing digital ulcers (DU) in the next three months. CSURI has been validated, only in patients not treated with bosentan, in a large multicenter study in 2011. Objectives To evaluate the predictive value of CSURI in SSc patients assuming bosentan for the prevention of DU. Methods Seventy-six consecutive SSc patients treated with bosentan were enrolled in a multicenter study (F/M 4.4/1; mean age 56.4±13.6 years; diffuse/limited cutaneous subset 30/44). All patients undergone to NVC and CSURI was calculated according to published studies. At baseline all patients had a history of at least one DU in the last year, and 26 patients (30.3%) showed a current DU. At the time of the study 76.3% of patients were also treated with intravenous prostanoids, while no patients was assuming bosentan for pulmonary arterial hypertension. Results After 3 months from NVC 18/26 patients showed non healing ulcers and 18/76 patients developed new DU. Receiver operator characteristic curve, performed to analyze the prognostic accuracy of CSURI in regard to DU development, is reported in the figure. The area under the curve (AUC) was 0.69 (95% CI 0.57-0.79, p=0.0019) and the higher sensitivity and specificity were observed for a CSURI value of 2.5 (sensitivity 94.4; specificity 57.5; positive and negative likehood ratio 2.22 and 0.097, respectively). At the validated cut-off value of 2.96 sensitivity was 86.1%, specificity 60.0%, positive and negative likehood ratio 2.15 and 0.23, respectively, showing a lower negative predictive value. Conclusions In patients treated with bosentan, CSURI shows a lower positive predictive value if compared with SSc population observed in our previous validation study, while the negative predictive value can be considered acceptable. The cause of this different result is not evaluable by our study. SSc peripheral microangiopathy is sustained by a multifactorial process, only partially known, involving a complex cytokines and cells network. In this picture, bosentan could reduce the incidence of new DU, without significantly interfere with the parameters included in CSURI calculation. Some Authors observed a general improvement of NVC parameters in SSc patients treated with bosentan. These data are not in contrast with our study since CSURI calculation is obtained by considering the “worst” capillaroscopic image. Moreover, a longer period of observation as in the other studies should more significantly influence changes in NVC parameters. In our previous studies, CSURI showed a higher predictive value than history of DU in detecting SSc patients at risk for new lesions. Anyway in this study this role of primary prevention cannot be applicable. Moreover, a special attention could be pointed on patients with recurrent DU, regardless specific treatments. A combined approach based on clinical picture and CSURI could probably help in managing therapy of SSc patients with DU, but only prospective clinical trial could clarify the correct role of NVC in the evaluation and management of vasoactive treatments. Disclosure of Interest None declared

AB0592 Evaluation of Arterial Stiffness in A Cohort of Systemic Sclerosis Patients: A Case-Control Study

Background Systemic sclerosis (SSc) patients are characterized by premature cardiovascular (CV) mortality. In this setting, the disease represents an intriguing model to evaluate premature endothelial damage. Indeed, both macrovascular and microvascular damage characterizes subclinical atherosclerosis (ATS) in these patients (1). Traditional CV risk factors, SSc-related inflammatory and autoimmune mechanisms may contribute to accelerated ATS. Among indirect measures of subclinical ATS, aortic stiffness (AS), measured by carotid-femoral pulse wave velocity (cf-PWV), and augmentation index (Aix@75) are independent predictors of CV morbidity and mortality in several diseases. To date, studies evaluating subclinical ATS in SSc provided conflicting results. Moreover, factors influencing atherosclerosis in SSc have not been well defined. Objectives To evaluate cf-PWV and Aix@75 measures of AS in SSc patients and analyze relationship between disease-related features and AS. Methods Thirty-four consecutive female SSc patients (mean age 61±15 years) with mean disease duration of 17±12 years since Raynaud phenomenon and 34 healthy sex, age and arterial pressure (AP) matched controls (HC) were enrolled. Traditional CV risk factors, disease-specific clinical, metabolic and immunologic features were collected. Disease activity and severity were assessed by Valentini Disease Activity Index (VDAI) and Medsger Disease Severity Scale (MDSS), respectively. Cf-PWV was determined by means of applanation tonometry (SphygmoCor). AIx@75 was calculated as difference between the 2nd and 1st wave systolic peaks and standardised to 75 beats/min. The relationship between cf-PWV, AIx@75 and all variables was investigated with univariate (one-way ANOVA) and multivariate models. Results Mean blood AP was similar in patients and HC. About 1/3 patients were receiving immunosuppressive therapy, 33% iloprost, 41% calcium-chanel blockers, 9% beta-blockers, 9% angiotensin receptor blockers and 3% ACE-inhibitors. Mean VDAI was 1,37±1,32 and 15% of patients were characterized by high activity (score >3). Mean MDSS was 0,35±0,65 with 9% of patients who had a moderate-severe disease. Both absolute AIx (16,1±8 vs 11,5±7 mmHg; p=0,014) and AIx@75 (30,9±16 vs 22,2±12%; p=0,012) resulted significantly higher in SSc compared to HC, while cf-PWV was similar in the two populations. At multivariate analysis, age, mean AP and mean C-reactive protein resulted independent predictors of cf-PWV, while only age and mean AP of AIx@75. Of interest, limited SSc and anti-centromere positive patients were characterized by significantly higher AIx@75 in comparison to diffuse SSc and anti-Scl70 positive (p=0.045 and p=0.04, respectively). Conclusions Subclinical atherosclerotic damage in SSc seems to be mainly characterized by increased AIx, a measure of arterial stiffness that encompasses peripheral vascular reflectance and left ventricular ejection, thus reflecting a prevalent peripheral microvascular involvement. Moreover, several factors, including inflammatory state, may contribute to AS in these patients. Intriguing, the significant increase of AIx in limited SSc may suggest a more severe peripheral microvascular involvement in these patients. References Cannarile F et al. Ann Transl Med 2015;3:8. Disclosure of Interest None declared

AB0564 Disease Damage is Associated with Increased Aortic Stiffness in Systemic Lupus Erythematosus: A Cross-Sectional Study

Background Increasing evidence suggests that accelerated atherosclerosis accounts for increased morbidity and premature mortality for cardiovascular (CV) disease in systemic lupus erythematosus (SLE) patients (1). Both traditional CV risk factors and disease specific features, including disease activity and damage, have been demonstrated to significantly predict accelerated atherosclerosis. Among indirect measures of subclinical atherosclerosis, aortic stiffness (AS), measured by carotid-femoral pulse wave velocity (cf-PWV), is considered a strong independent risk factor for future CV morbidity and mortality in different clinical settings. Indeed, AS has been demonstrated to be increased in patients with inflammatory diseases with respect to controls (2). To date, few data suggest that SLE patients are characterized by a significantly increased AS with respect to controls. However, factors influencing AS in SLE have not been well defined. Objectives To evaluate cf-PWV as a measure of AS in SLE patients and analyze relationship between disease-related features and cf-PWV. Methods Forty consecutive SLE patients (90% females, mean age 45±12 years) with median disease duration of 12 years (IQR 5-19) were enrolled. Traditional CV risk factors, overt CV events, disease-specific clinical, metabolic and immunologic features were collected at enrollment. Disease activity and damage were assessed by the SLEDAI and SLICC indexes, respectively. Carotid-femoral PWV was determined by means of applanation tonometry (SphygmoCor). The relationship between cf-PWV and all variables was investigated with univariate (one-way ANOVA) and multivariate models. Results Average cf-PWV was 7.5±1.9 m/s mean. Mean blood pressure was 128/75±16/10 mmHg. Nine subjects (23%) were on anti-hypertensive treatment, 4 (10%) reported previous cardiovascular events, 17 (42%) subjects were treated with steroids, 29 (71%) with hydroxychloroquine and 15 (37%) with other immunosuppressants. Median SLEDAI and SLICC indexes were 0 (IQR 0-2) and 2 (IQR 1-3), respectively. Cf-PWV significantly increased across SLICC damage index categories (F=3.141, p<0.019). The association between cf-PVW and SLICC index persisted after adjustment for age, sex, mean arterial pressure, height, heart rate, disease duration, anti-hypertensive treatment, number of drugs for SLE therapy, C-reactive protein and previous cardiovascular events (p=0.031). Conclusions Disease damage appears to be a relevant factor influencing functional artery status in SLE. Present results support the need of adequate prevention of disease damage to slow the progression of subclinical atherosclerosis in SLE patients. References Bartoloni E et al; Expert Rev Clin Immunol 2007;3:531-41. Schillaci G, Bartoloni E et al; Ann Rheum Dis 2012;71:1151-6. Disclosure of Interest None declared

A3.1 Pathogenic role of IL-17 producing double negative (DN) T cells in primary sjögren’s syndrome

Background and Objectives We recently demonstrated that a CD3+ T cell population, that lacks both CD4 and CD8 molecules, defined as double negative (DN), is expanded in the peripheral blood of patients with long-standing primary Sjögren’s syndrome (pSS), produces IL-17, accumulates in minor salivary glands (MSGs) and is resistant to corticosteroids (CS) in vitro. Since IL-17 represents a key cytokine in the pathogenesis of pSS, we aimed to investigate glandular and peripheral DN T cells in early phases of the disease in order to verify a possible correlation with clinical parameters, MSGs histological patterns and possibly ectopic lymphoneogenesis. Materials and Methods Paired samples of peripheral blood mononuclear cells and MSGs from early pSS patients were evaluated by flow cytometry and immunofluorescence staining respectively to quantify DN T cells. Histological analysis to assess histological scores, B/T cell segregation and the presence of germinal center-like structures (GCs) was also performed. Ten patients with long-standing disease and 15 normal controls (NC) were also enrolled. Disease activity was assessed by the EULAR Sjögren’s syndrome disease activity index (ESSDAI) and patient reported dryness, fatigue, pain and global disease activity were recorded on a 100 millimeters (mm) visual analogic scale (VAS). Unstimulated salivary flow was also collected. Results In early stages of pSS, circulating DN T cells appeared not to be expanded as it occurs in patients with long-standing disease and were inversely correlated to circulating CD4+Th17 cells. The number of infiltrating DN T cells was associated with the extent of glandular involvement, defined with Tarpley biopsy score, and with the presence of GCs. Furthermore, dryness symptoms were directly correlated with glandular DN T cells and inversely correlated with circulating DN T cells. Circulating DN T cells displayed an in vivo activated phenotype, confirmed by the expression of CD25, CD69 and HLA-DR, which was not affected by the addition of CS to the culture, as it occurred for IL-17 expression. Conclusions Our findings suggest that DN T cells are in vivo activated Th17 cells involved in the pathogenic mechanisms leading to glandular dysfunction and damage in pSS. Furthermore, DN T cells may play a role in ectopic lymphoneogenesis development occurring during the disease.

SAT0030 Circulating Endothelial Microparticles and Endothelial Progenitors Cells in Primary SjÖGren's Syndrome: New Markers of Chronic Endothelial Damage?

Background Subclinical atherosclerosis can be considered a form of systemic involvement in patients with chronic inflammatory joint and connective tissue diseases. Multiple factors, including inflammatory and immune-mediated mechanisms, have been identified to promote atherosclerotic endothelial injury [1]. Recent studies focused on role played by the altered balance between endothelial microparticles (EMPs) release and endothelial progenitor cells (EPCs) generation in endothelial wall integrity disruption and subsequent endothelial dysfunction. EPCs represent a new marker of endothelial dysfunction cardiovascular (CV) disease patients and increased EMP levels have been demonstrated in diseases characterized by high inflammatory response, such as polymyalgia rheumatica [2]. There is evidence that primary Sjögrens syndrome (pSS) patients, free of previous CV manifestations, display signs of subclinical atherosclerosis, suggesting a greater CV risk [3,4]. However, exact pathogenic mechanisms involved in vascular damage in SS are still unclear. Increased levels of leukocyte and platelet MPs, reflecting systemic cell activation, have been demonstrated in SS. On the other hand, the role of EMPs and EPCs in SS has been never investigated. Objectives To evaluate endothelial injury degree in SS by EMP quantification and their repair potential by EPC and mature EPC measurement and to analyze possible correlation with disease-specific clinical and immunologic features. Methods 34 female pSS patients and 18 age- and sex-matched normal controls (NC) were enrolled. Number of circulating EMPs (CD31+/CD42–), EPCs (CD34+/KDR+/CD133+) and mature EPCs (CD34+/KDR+/CD133–) was quantified by FACS analysis. Parameters of disease activity and damage were measured by ESSDAI and SSDDI, respectively. Disease-related clinical features, laboratory markers of immunologic dysfunction and traditional CV risk factors were recorded. Results SS patients displayed higher EMP number with respect to NC (450±155 vs 231±110 n/μl mean ± SD; p<0.0001). Mean concentrations of EPCs as well as mature EPCs resulted significantly higher in patients in comparison to NC (226±181 vs 69±53 n/mL mean ± SD; p<0.001 and 166±161 vs 36±32 n/mL mean ± SD; p<0.0001, respectively). SS patients exhibited a significantly lower EPC/mature EPC ratio in comparison to NC (p<0.01). EMP levels directly correlated with disease duration from symptoms and diagnosis (rho=0.5; p<0.01). EPCs inversely correlated with disease duration from symptoms (rho= -0.5; p<0.01) and diagnosis (rho= -0.4; p<0.05). Conclusions Present data highlight for the first time a chronic persistent endothelial fragmentation characterizing pSS patients. Endothelial layer reparative potentiality appears to be preserved in the earliest stages of disease. Following disease course, a progressive exhaustion of the precursor endothelial pool may be hypothesized, leading to defective vascular layer restoration and endothelial dysfunction. Further investigations are needed to ascertain the role of EMPs and EPCs in promoting subclinical atherosclerosis in pSS. References Bartoloni E et al. Arthritis Care Res 2011;63:178 Pirro M et al. J Intern Med 2012;272:177 Vaudo G et al. Arthritis Rheum 2005;52:3890 Gerli R et al. Arthritis Care Res 2010;62:712 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2887

FRI0235 Static and Dynamic-Functional Trans-Cervical Ultrasonography of the Esophagus in Systemic Sclerosis Patients

Background Esophagus is the second internal organ to be affected in systemic sclerosis (SSc), resulting in gastro-oesophageal reflux, oesophagitis and, in more advanced stages, complete atony with megaesophagus. Trans-cervical ultrasonography (TCU) has been proposed as method to study proximal esophagus as it allows to view the five layers of the esophageal wall and permits a functional evaluation of the salivary bolus transit. Objectives To assess structural and functional alterations of the cervical esophagus in patients with SSc using TCU. Methods Thirty-nine patients with SSc and 39 healthy subjects underwent TCU of the esophagus (Esaote MyLab25 gold; 12 MHz linear probe) with patient in supine position with the neck extended and rotated to the right, using a posterior approach to the left thyroid gland. A functional evaluation was performed to study the transit of salivary bolus during swallowing and to detect possible salivary bolus reflux and its speed. In all subjects were recorded: age, sex, weight, height, BMI, symptoms of esophageal involvement (dysphagia, heartburn). In SSc patients were additionally recorded specific autoantibodies and videocapillaroscopic patterns, disease duration and organ involvement. Results Thickness of the muscle layer of esophagus was significantly lower in patients than in controls (1.55±0.04 mm vs 1.73±0.05 mm, P<0.05). There was no significant difference in thickness of the entire wall. A higher frequency of salivary reflux was detected by dynamic assessment in SSc patients (84.6% vs 38.4%, P<0.01). A “high speed” pattern (qualitative assessment) of bolus salivary reflux was found in 13 out of 39 patients (33%), but in none of control subjects. Quantitative assessment showed a significant difference in the speed of salivary reflux in patients compared to controls (4.8±0.5 cm/s vs 1.67±0.2 cm/s, P<0.01). There was a significant difference in wall and muscularis thickness among patients with SSc complicated by idiopathic pulmonary arterial hypertension (iPAH) compared to those not suffering from this complication (wall: 1.95±0.07 mm vs 2.4±0.11 mm, P<0.01; muscle: 1.24±0.06 mm vs 1.62±0.05 mm, P<0.01). Conclusions SSc patients have a significant reduction in the tunica muscularis thickness, probably related to an increase of collagen fibers in the lamina with consequent muscle atrophy. SSc patients compared to controls, have a high rate of cervical salivary reflux, likely related to altered distal esophageal peristalsis. Reduced esophageal wall and muscle layer thickness was found to be associated with the development of iPAH, confirming the already known relationship between pulmonary and esophageal involvement. According to our findings, esophagus TCU may represent a non-invasive and low-cost screening test to detect esophageal involvement in SSc. However, studies with larger series of patients and with direct comparison with instrumental methods validated for the study of esophagus in SSc, such as manometry, are needed. References Forbes A et al. Rheum 2008;48:iii36-iii39. Zhu SY et al. J Clin Ultrasound 2004;32:163-171. Denton CP et al. Eur respir Rev 2011;20:122, 270-276. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4984