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Background:The new coronavirus 2 (SARS-CoV-2) disease 2019 (COVID-19) is a source of concern for the management of patients suffering from rheumatic and musculoskeletal diseases (RMDs) treated with immunomodulatory therapies (1).Objectives:We aimed to analyze the prevalence of SARS-CoV-2 infection in patients with RMDs living in Italy.Methods:During the first wave (March-May 2020) and during the second wave (October-December 2020) of COVID-19, we conducted a survey to investigate the incidence of SARS-CoV-2 infection in patients with RMDs followed at the Rheumatology Unit of the University of Campania, Italy. The demographic data, medication use, the frequency of respiratory symptoms and the incidence of COVID-19 confirmed by nasopharyngeal swab were collected with questionnaires administered by phone. The prevalence of COVID-19 of our cohort was compared to that of the general population (2).Results:During the first wave, we collected data from 900 patients with RMDs (Table 1): 320 patients with rheumatoid arthritis (RA), 295 patients with spondyloarthropathies (SpA), 283 patients with systemic lupus erythematosus (SLE), 2 patients with vasculitis. 546 (60%) were treated with bDMARD/tsDMARDs. Overall, a total of 11/900 (1%) cases were tested for COVID-19 due to compatible symptoms. 2 (0.2%) adult patients treated with bDMARDs were registered as swab test positive by PCR for COVID-19. 2 patients without confirmed COVID-19 developed pneumonia that required admission to hospital. No deaths occurred among the patients with confirmed COVID-19.During the second wave, data were collected from 470 patients who accepted to take part of the study (Table 1). 49 presented with symptoms that were compatible with COVID-19. 139 patients were tested whereas 30 patients (6%) had a swab confirmation of SARS-CoV-2 infection. Among them, 16 (53%) were treated with bDMARDs and a patient was treated with tofacitinib. we found no increase in COVID-19 prevalence in patients treated with bDMARD/tsDMARDs (p>0.05). A patient with SLE developed pneumonia that required admission to hospital and died.Lacking distinct prevalence data between first and second waves, we found no differences in total COVID-19 prevalence between general population living in Campania (215.752/5.802.000; 3.7%) and patients with RMDs (32/900; 3.5%). However, we had a significant increase in COVID-19 prevalence in our cohort during the second wave compared to the first. Nevertheless, no increase in mortality or hospitalization was recorded, confirming the safety of immunomodulatory therapies in patients with RMDs.Conclusion:In this cohort of patients with RMDs in a geographical region with a high prevalence of COVID-19, the risk of SARS-CoV-2 infection does not appear different from that observed in the general population.References:[1]Wang L., Wang Y., Ye D. Int J Antimicrob Agents. 2020:105948.[2]http://www.protezionecivile.gov.it/ (accessed 28.01.21)Table 1.Demographics and clinical characteristics of 900 patients with rheumatic diseases during the COVID-19 Pandemic.First waveSecond waveWomen, n 660 (73 %) 366(77%)Age, years, median (range)56 (54-57)53 (51-55)Rheumatoid Arthritis320 (35.5%)143 (30%)Spondyloarthritis295 (32%)110 (23%)Systemic Lupus Erythematosus283 (31%)217 (46 %)Vasculitis2 (0.2%)1 (0.2%)Prior ILD56 (6%)22 (4.6%)Smokers220 (24%)118 (25%)Hydroxychloroquine215 (23%)155 (32%)Steroids337 (37%)194 (41%)Prednisone equivalent dose, median (range)5 (0-75)5 (0-50)bDMARD/tsDMARDs546 (60%)247 (52%)csDMARDS387 (43%)185 (39%)Angiotensin-converting enzyme (ACE) inhibitors178 (19.8%)101 (21%)Angiotensin II receptor blockers(ARBs)153 (17%)61 (13%)Fever64 (7%)30 (6.3%)Cough83 (9%)36 (7%)Shortness of breath34 (3%)15 (3%)Sore throat32 (3%)11 (2.3%)Rhinorrhoea36 (3%)11 (2.3%)Headache5 (0.5%)2 (0.4%)Anosmia10 (1%)24 (5%)Myalgia2 (0.2%)1 (0.2%)Gastrointestinal symptoms24 (2.6%)3 (0.6%)Pneumonia2 (0.2%)2 (0.4%)Admission to hospital2 (0.2%)9 (1.9%)Swab confirmation of SARS-CoV-2 infection230Disclosure of Interests:None declared.

Background:Psoriatic arthritis (PsA) is a chronic inflammatory arthritis burdened by a series of metabolic comorbidities. Among them, obesity is very common in PsA, with a prevalence of 27%, as confirmed by a recent Spanish work (1). Obesity in PsA has been associated with higher disease activity and a worse effectiveness of biologic treatment in PsA. This has been certainly proven for anti-TNF-α as demonstrated by different studies reporting, in obese patients, a reduced treatment response and adherence. In particular, results coming from DAN-BIO and ICE-BIO registries, (2) point out that obesity is a risk factor for anti-TNF withdrawal due to poor response. Although a recent multi-centric, retrospective study in Spain has shown that obese subjects with psoriasis have a poor therapeutic response to secukinumab, (3) no data are currently available for secukinumab in PsA obese patients.Objectives:Our studies focused on the relationship between BMI and clinical response to secukinumab in PsA.Methods:We, retrospectively, analysed clinical data of 100 patients with PsA (57% female, median age 53 (49.2-55 years)) satisfying CASPAR criteria (4) for PsA, afferent to our clinic, who were treated with secukinumab. Patients were divided into 2 groups based on BMI (BMIResults:In the normal weight group 75% were female, median age was 50.5 (41-54.6), median BMI was 22 (20.2-23.3) and median DAPSA was 19.19 (15.6-24.2). The features of the overweight/obese patients were similar to the normal weight group (48% were female, median age 54 (50-59), median BMI 29 (27.4-30.1) and median DAPSA 21.2 (19-24.4)). Clinical response to therapy, evaluated as the achievement of low disease activity or remission according to DAPSA, was recorded 6 months after starting treatment. After 6 months of treatment, the variation of the DAPSA was inversely related to BMI: overweight/obese patients had in fact a better response to secukinumab compared to normal weight patients. By using a correlation coefficient (SPSS), to analyze the degree of association between BMI and DAPSA, we observed that BMI and DAPSA are inversely related in our PsA patients (p=0.05). Interestingly, analysis of serum levels of IL-17 in 20 obese patients compared to 20 non-obese patients, showed significantly higher serum levels of IL-17 in the former (Figure 1), indicating IL-17 as a key cytokine driving inflammation in PsA obese patients.Conclusion:These are the first data about clinical response to secukinumab in obese PsA patients. Our results support the relevance of IL-17 in driving systemic inflammation in obese PsA patients, also providing evidence that obese patients may have a better response to secukinumab compared to non-obese patients. Interestingly, this effect was notReferences:[1]Rubén Queiro, Lorenzo A, Tejón P et al. Obesity in psoriatic arthritis. Comparative prevalence and associated factors. Medicine 2019 Jul;98(28):e16400[2]Pil Højgaard, Glintborg B, Kristensen LE et al. The influence of obesity on response to tumour necrosis factor-a inhibitors in psoriatic arthritis:results from the DANBIO and ICEBIO registries. Rheumatology (Oxford). 2016 Dec;55(12):2191-2199[3]Jaime Notario, Deza G, Vilarrasa E et al. Treatment of patients with plaque psoriasis with secukinumab in a real-life setting: a 52-weeks, multicenter, retrospective study in Spain. Journ of Derm Treat 2019 Aug;30(5):424-429[4]Taylor W, Gladman D, Helliwell P et al. Classification criteria for psoriatic arthritis: development of new criteria from a large international study. Arthritis Rheum. 2006 Aug;54(8):2665-73.[5]Lluís Puig. Cardiometabolic Comorbidities in Psoriasis and Psoriatic Arthritis. Int J Mol Sci. 2017 Dec 25;19(1).Disclosure of Interests:Ilenia Pantano: None declared, DANIELA IACONO Speakers bureau: PFIZER, BRISTOL MAYERS SQUIBB, SANOFI, ENNIO GIULIO FAVALLI: None declared, GIUSEPPE SCALISE: None declared, Luisa Costa: None declared, Francesco Caso: None declared, Giuliana Guggino Grant/research support from: Pfizer, Celgene, Speakers bureau: Celgene, Sandoz, Pfizer, Raffaele Scarpa: None declared, francesco ciccia Grant/research support from: pfizer, novartis, roche, Consultant of: pfizer, novartis, lilly, abbvie, Speakers bureau: pfizer, novartis, lilly, abbvie

Background Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder associated with increased mortality (1), (standardized mortality ratio for all causes–SMR, being 1.47 in the last 50 years) (2). Objectives To investigate the mortality rate and predictive factors as assessed at admission in a cohort of RA patients followed at a South Italy tertiary centre. Methods RA patients admitted to the outpatient Clinic of a tertiary centre from 01/01/2008 to 31/12/2015 were investigated. Table 1 shows patients features (age, sex, disease duration, Simplified Disease Activity Index-SDAI index, Health Assessment Questionnaire-Disability Index-HAQ-DI, Rheumatoid Factor-RF and/or Anti-Citrullinated Cyclic Peptides Antibody-ACPA, concomitant comorbidity) as detected at baseline, and treatment during follow-up. RA patients mortality incidence rate (event/100person year) was calculated, baseline predictors of mortality were analyzed by univariate and multivariate cox-regression analysis. Results A total of 608 RA patients were included (497 women), median age 56.8 years, range 15–89.5. On December 31 2015, 589 out of patient were alive, 19 were known to have died and 7,3% had been lost to follow up. Mortality Incidence rate (MIR) in our cohort was 0.79 deaths/100 person-year, SMR with respect to Italian population was 0.79 (MIR in Italy 1/100 person-year). This feature might depend on the low prevalence of RF/ACPA positivity (69.13%) in our series. Cox regression analysis (univariate analysis) pointed out age at first visit (HR 1.14; p≤0.0001), diabetes (HR 4.395; p=0.004) and a HAQ value>1 (HR 6.02; p=0.02) as independent predictors of mortality. On the contrary, female sex resulted to have a protective role (HR 0.349, p=0.027). At multivariate analysis, the only baseline independent predictor of mortality was age at admission (HR 1.17, 95% CI 1.01–1.35, p=0.026). Conclusions The present study points out a South Italian RA SMR lower than that in general Italian population and significantly lower than that reported in RA patients from other countries (2). This result can depend on the lower percentage of RF/ACPA positive patients in our cohort in respect to general population. Epidemiological studies on the sierological profile of South Italian RA patients are needed. References Sokka T et al. ClinExpRheumatol. 2008;26(Suppl):S35–61. Dadoun S et al. Joint Bone Spine. 2013;80:29–33. Disclosure of Interest None declared

Background Cardiovascular (CV) morbidity and mortality are significantly greater in Systemic Lupus Erythematosus (SLE) patients than in the general population. Acetylsalicylic acid (ASA) is known to be associated with a decrease in the incidence of CV events in high-risk patients from general population [1]. Objectives We previously investigated ASA efficacy as primary prophylaxis in 167 SLE patients followed for 7 patient-years [2]. Here, we report data collected in 189 patients followed for 8,2 patient-years. Methods SLE patients consecutively admitted to a tertiary center, who, at admission, satisfied 1992 ACR and/or 2012 SLICC classification criteria for SLE and had not experienced any CV event, were enrolled into the study. Low-dose ASA (100 mg/day) was prescribed to each patient at first visit. Fourteen patients (7%) refused to take it. At each 6-monthly visit, ASA side effects, ASA discontinuation and occurred CV events were recorded. Results 189 consecutive SLE patients were followed-up for a median of 8 years (range 1–15). Among them, 164 regularly took the medication (ASA-treated) and 25 refused to take or discontinued it (non-ASA-treated patients). Six CV events occurred in the 164 ASA-treated (4.2/1000 person-years), 4 in the 25 non-ASA-treated patients (25.4/1000 person-years) (p=0.003). No relevant side effect related to ASA treatment was recorded. Conclusions Present data further support the role of ASA in the primary prophylaxis of CV events in SLE patients. References Vandvik PO, Lincoff AM, Gore JM, et al. Primary and secondary prevention of cardiovascular disease: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines.Chest. 2012;141 Iudici M, Fasano S, Gabriele Falcone L, et al. Low-dose Aspirin as Primary Prophylaxis for Cardiovascular Events in Systemic Lupus Erythematosus: A Long-Term Retrospective Cohort Study. Rheumatology (Oxford) Submitted for publication. Disclosure of Interest None declared

Background Sleep disturbances are frequently observed in systemic lupus erythematosus (SLE) patients, but the relationship with disease activity has not been unequivocally established. Objectives To assess the prevalence of and factors associated with sleep disorders in a cohort of SLE patients with persistent complete (no clinical activity, no serological activity and no treatment other than antimalarials) or clinical remission (only serological activity; stable immunosuppressive therapy with or without corticosteroids allowed) (1). Methods Consecutive adult SLE patients attending the outpatient clinic of the Second University of Naples were enrolled in a cross-sectional study. A complete clinical and laboratory assessment was carried out. Moreover, the Pittsburgh Sleep Quality Index (PSQI), Health Assessment Questionnaire – Disability Index (HAQ-DI), Short-Form 36 (SF-36), VAS pain, VAS fatigue, VAS global health, were administered. Results Ninety SLE patients (86 females) with a mean±SD age of 43±18 and a median disease duration of 17 years (range 2–38) were enrolled in the study. A poor sleep quality (i.e. PSQI score>5) was observed in 38 (42.2%) patients, with no difference between patients in complete remission (13/30) and those in clinical remission with or without GCs (25/51; p=0.651). PSQI score was found to be significantly correlated with physical component score of SF-36 (rho -0.467, 95% CI -0.632 to -0.262; p Conclusions Sleep disorders are frequent in SLE patients with persistent complete or clinical remission. An impaired mental and physical quality of life, disability, fatigue and pain represent the main features associated with a low quality of sleep. References Zen M, et al. Prolonged remission in Caucasian patients with SLE: prevalence and outcomes. Ann Rheum Dis 2015;74:2117–22 Disclosure of Interest None declared

Background Systemic Sclerosis (SSc) is characterized by vascular alteration with a progressive loss of capillaries resulting in insufficient blood flow and chronic peripheral hypoxia, associated to a failure of reparative angiogenesis, followed by progressive fibrosis of skin and internal organs[1]. An impaired production of angiogenic molecules is involved in this dysfunctional angiogenesis [2]. Tie2 is a trans-membrane receptor, exclusively expressed on the surface of endothelial cell (EC). Tie2 modulates the vessels quiescence, or alternatively, induces angiogenesis, via the interaction with its ligands, Ang1 and Ang2. Of note, activation of endothelial Tie2 signalling, by Ang1, which is produces by perivascular cells, enhances ECs barrier integrity and endothelial-pericyte interaction. On the contrary, Ang2, produced by ECs, normally acts as an Ang1 antagonist [3]. Objectives The aim of the present study was to assess if an impaired cross-talk between ECs and perivascular mesenchymal stem cells (MSCs), during SSc, may affect the normal interaction among Ang1, Ang2 and Tie2 thus contributing to the impaired angiogenesis. In this work, bone marrow derived MSCs were used as pericytes surrogate, considering that, perivascular cells share surface markers and differentiative ability with bone marrow MSCs, and MSCs express pericyte markers and cooperate with endothelial cells to form a vascular network, supporting the concept that pericytes are members of the adult multipotent MSCs family [4]. Methods We investigated Ang1, Ang2 and their receptor performing co-cultures with ECs and bone marrow MSCs obtained from patients and healthy controls (HC). After 48 hours, cells were sorted and analysed for molecular assays. Furthermore, we investigated, by ELISA assay, the proteins released in the supernatants. Finally, we silenced Ang-1 expression in HC-MSCs by Ang1-siRNA. Results At molecular level, SSc-MSCs, cultured alone, expressed lower amount of Ang1 when compared to HC-MSCs. After co-culture, a significant decreased of Ang1 mRNA levels was observed in the SSc-MSCs/SSc-ECs. On the contrary, SSc-ECs expressed higher levels of Ang2 and Tie2 in each co-culture condition, when compared to the expressions of cells cultured alone. The WB and ELISA assays mirrored the results observed in gene expression. HC-MSCs transfected with Ang1-siRNA lacked the ability to support the formation of tube like structure. Conclusions In this work we provided evidence that an imbalance of Ang1/Ang2 molecules and a decreased expression of their receptor, Tie2, during ECs-perivascular MSCs interplay, may modulate vessel stability, and vascular tube formation, thus contributing to the angiogenic alteration observed during SSc. References Cipriani P et al. Autoimmun Rev 2011,10:641-646. Cipriani P et al. Arthritis Res Ther 2013,16:442. Fukuhara S et al. Histol Histopathol 2010,25:387–396. Cipriani P et a. Angiogenesis 2013,16:595-607. Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is an autoimmune disorder characterized by microangiopathy and immunological abnormalities evolving into fibrosis. Endothelial damage occurs early in the disease. EGFL7, is a recently identified molecule expresses in vascular endothelial cells (ECs) and their mesodermal progenitors. EGFL7 controls blood vessel development by promoting ECs migration, proliferation, sprouting, adhesion (1,2). Its alteration results in abnormal vessel formation. Objectives We hypothesized that EGFL7 might play an important role in regulating SSc angiogenesis/vasculogenesis. Methods EGFL7 expression was investigated in the involved skin of 33 SSc patients, according to the different subsets (16 diffuse and 17 limited SSc) and stages (16 early and 17 late) of the disease and 27 healthy controls (HC), by using immunohistochemistry, immunofluorescence, RT-PCR analyses and Western Blot (WB). Furthermore, placenta biopsies from 4 healthy uncomplicated pregnancies were used as physiological angiogenesis controls. Microvascular endothelial cells (MVECs) and fibroblasts (FBs) both from patients and HC were also analyzed for EGFL7, by confocal laser scanning microscopy, RT-PCR analysis and WB. Results EGFL7 expression is clearly up-regulated in the placenta, that represents the physiological model of angiogenesis as observed by immunohistochemistry, immunofluorescence, RT-PCR and WB analyses, when compared to skin of HC and patients. Moreover, patients in the early stage and diffuse form display lesser expression of EGFL7 when compared to patients with limited subset, late stage, and HC, respectively. Cultured SSc-MVECs express a significantly higher amounts of EGFL7 mRNA in the early stage and lowest expression in the later stages of the disease, not mirroring the skin findings and suggesting a post transcriptional modification. Furthermore, cultured SSc-FBs express lower mRNA and protein levels of EGFL7. Interestingly, in cultured SSc and HC FBs only SSc myofibroblasts (a-SMA +) showed a strong immunopositivity for EGFL7, suggesting a role of this factor in the EndMT (endothelial-mesenchymal transition) transition. Conclusions We report the first observation of an abnormal expression of EGFL7 in the skin and in the myofibroblasts of the SSc patients, compared to controls which is generally considered a specific endothelial molecule. The alteration of EGFL7, seems to be involved in defective angiogenesis of SSc and in the modulation of EndMT, that leads to the fibroproliferative vasculopathy. References Campagnolo L et al. EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury. Am J Pathol. 2005 Jul;167(1):275-84 Schmidt M, et al. EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution. Development 2007 Aug;134(16):2913-23. Epub 2007 Jul 11. Disclosure of Interest None Declared

Background BM-MSCs are currently being studied to determine their potential role in the pathogenesis of autoimmune diseases. Experimental evidence have shown that they exhibit an anti-proliferative effect on T lymphocytes. In this setting BM-MSCs show a specific immunomodulatory function in inducing regulatory phenotype and function in T cells. T regulatory lymphocytes are a population of CD4+ CD25high Foxp3+ cells which play an important role in the induction of peripheral tolerance. SSc is a complex autoimmune disease, characterized by fibrosis of the skin and various internal organs. Our previous findings suggesting that BM-MSCs derived from SSc show a premature senescence, probably related to several pathologic stimuli which these cells encountered during their lifetimes. Objectives The aim of this work was to demonstrate that BM-MSCs are early senescent, assessing the activity of beta-galactosidase and cell cycle inhibitors (p53 and its downstream gene, p21) and investigate whether during SSc, BM-MSCs, although senescent, maintain their immunosuppressive properties, when co-cultured with T lymphocytes. Methods After ethical commitment approval MSCs were isolated from BM obtained from 10 SSc and 10 healthy controls (HC). At third passage, the cell cycle was analyzed by flow cytometry. The beta-galactosidase activity was measured by counting positive cells after staining with X-Gal. mRNA and proteins of p21 and p53 were measured by western blot and qRT-PCR. The treatment of the cells for 24h with doxorubicin was used to induce senescence. The immunomodulatory property of BM-MSCs was tested by co-culturing these cells with CD4+ T cells. Results Both HC and SSc BM-MSC showed stromal phenotype, and normal multidifferentiative ability. The study of cell cycle showed that about 80% of cells were in G0-G1 phase, without significant differences between SSc and HC. Interestingly, the SSc-MSCs showed an increase in beta-galactosidase compared to HC-MSC. After treatment with doxorubicin, the percentage of beta-galactosidase positive stained cells in SSc were higher with respect to HC, thus outlining that SSc cells show greater sensitivity to drug treatment. As far as the expression of p21 was concerned, intriguingly, we found a significant increase in SSc-MSCs compared with HC-MSCs. Doxorubicin treatment abolished p21 activation and, on the other hand, elicited p53 induction both in SSc and HC BM-MSCs. This regulation of p21/p53 machinery could be an attempt of BM-MSCs to bypass the doxorubicin induced. This protective mechanism seems lesser active in SSc cells. The above results were further confirmed by qRT-PCR. We found that senescent SSc MSC showed a preserved immunomodulatory activity maintaining the immunosuppressive effect and the ability to induce a regulatory phenotype on T as HC BM-MSCs. Conclusions SSc BM-MSC showed an increase of senescence biomarkers, suggesting premature aging, probably related to several pathologic stimuli. Although the early aging of SSc BM-MSCs, the finding that these cells showed a normal immunoregulatory activity, suggesting their potential to recover by activating a sort of adaptive mechanism. References Cipriani P et al. AR56(6). Landgraf K et al. Transpl Immunol 2011 Sep;25. Disclosure of Interest None Declared

Background B lymphocytes are key players in the development of chronic autoimmune sialoadenitis and ectopic lymphoneogenesis is a milestone in the natural history of Sjogren’s syndrome (SS) (1). Much effort has been spent to shed some light on the underlying mechanisms and to date several pathways have been identified. Recruitment and activation of B cells in target organs are orchestrated by a specific milieu of well characterized cytokines and chemokines (2). In this setting, targeted therapies aimed to delete autoreactive B cell clones or B-cell activating factors have been proposed. Recently, growing evidence about the positive effects of anti-CD20 monoclonal antibody rituximab (RTX) in SS pointed out a possible therapeutic application in these patients. However conclusive data about the effective role of RTX in glandular microenvironment are still lacking. Objectives Aim of the present study was to characterize the different histopathological and molecular features of salivary gland infiltrate following either conventional therapies or RTX in SS. Methods Eight females with SS underwent minor salivary gland (MSG) biopsy at the diagnosis and following either conventional therapies or RTX. Four females with sicca symptomatology but neither histologic nor sierologic features of SS acted as controls. Serial sections of 3μm thickness were cut and stained with hematoxylin and eosin for the assessment of several histologic scores according to literature (3). Immunofluoscence staining for anti-CD3, anti-CD20, anti-CD21 was performed to identify the grade of lymphoid organization and the presence of germinal center-like structures. Sections were counterstained with DAPI and analyzed with Olympus BX53 microscope. Part of MSG were processed for real time PCR and the following molecules, involved in inflammation and lymphoid organization, were tested: LTa, LTb, IL-1b, TNFa, BAFF, AID, CXCR4, CXCL12, CXCR5, CXCL13, CCR7, CCL19, CCL21. Results We observed that in SS-MSG the levels of proinflammatory cytokines and chemokines were increased with respect to control (all p Conclusions Our results confirm that RTX is currently the only therapeutic agent able to interfere with the perpetuation of salivary gland damage during SS. Therefore, selective targeting of pathogenic B cells appears to be a promising approach to restore normal glandular function. References Voulgarelis M et al, Nat Rev Rheumatol 2010;6:529-537. Youinou P et al, Arthritis Rheum 2010;62 (9):2605-2610. Christodoulou MI et al, Journal of Autoimmunity 2010;34:400-407. Disclosure of Interest None Declared

Background MVECs damage is a central event in the pathogenesis of SSc leading to capillaries rarefaction and consequent ischemia. VEGF is the best characterized angiogenic growth factor; its increase after ischemia is a timed controlled event, acting only in the first stages of new blood vessels formation when it regulates ECs sprouting and proliferation. During SSc, VEGF lacks this tight control and is strongly up-regulated over time both at local and systemic levels. Caveolae, specialized plasma membrane microdomains, abundant in ECs, and caveolin-1 (cav-1), their principal residual protein, compartmentalize VEGF-induced signaling toward angiogenesis. Objectives Here we aimed to prove whether caveolae/cav-1 act as platform for organizing and compartmentalizing VEGF/VEGFR2 signaling in MVECs during SSc. In fact an abnormal cav-1 expression in microvascular endothelium, may contribute to angiogenic defective response during the disease. Methods After ethical approval skin was collected from 10 patients with SSc and used for immunofluorescence (IF), qPCR and MVECs isolation. Normal skin was obtained from healthy donors (HC) who underwent surgery for trauma. Cav-1 detection and its co-localization with VEGFR-2 was perfomed on skin sections and MVECS isolated from SSc patients and compared to HC by IF, immunoprecipitation and Western Blot (WB). qPCR analysis was used to assess mRNA cav-1 expression level both in SSc skin and MVECs. To determine whether VEGF treatment could affect cav-1/VEGFR2 colocalization in MVECs during SSC, all the above was assessed after culturing these cells in the presence of VEGF. Results We found that VEGFR-2 colocalizes with cav-1 in blood vessels within SSc skin, as already observed in HC skin. However semi-quantitative analysis of IF cav-1 expression level revealed lower expression in SSc MVECS with respect to HC skin. This result was confirmed by qPCR, showing cav-1 was down-regulated in MVECs isolated from SSc patients when compared to HC cells. When we assessed cav-1/VEGFR.2 co-localization within MVECS, we observed that VEGFR-2 was localized in the caveolae directly bound by cav-1. This observation was further confirmed by cav-1 immunoprecipitation and WB for VEGFR-2 in SSc MVECs, without differences from HC. Interestingly, after culturing these cells in the presence of VEGF, we found a disruption of cav-1/VEGFR-2 co-localization both in HC and SSc MVECs, without cav-1 mRNA levels re-modulation Conclusions During SSc cav-1 expression down-regulation within MVECs, could possibly explain the dysfunctional, ineffective VEGF angiogenic signaling. This mechanism might maintain the redundancy of deregulated VEGF abundance in a vicious loop, strongly contributing to not compensative angiogenic response during SSc. References Del Galdo Fet al Curr Opin Rheumatol. 2008 Nov;20(6). Feng L et al J Cell Physiol. 2011 Aug 9 [Epub ahead of print]. Liao WX et al Mol Endocrinol. 2009 Sep;23(9). Disclosure of Interest None Declared