Search

OBJECTIVE To study the potential role of semaphorins in the pathogenesis of rheumatoid arthritis (RA). METHODS Microarray experiments were performed on Affymetrix GeneChip Human Exon 1.0 ST arrays in RA endothelial cells (ECs) and control ECs derived from circulating progenitors. Expression of class 3 and class 4 semaphorins and their receptors in the serum of RA patients and healthy controls was assessed by immunohistochemical analysis in synovial tissue and by enzyme-linked immunosorbent assay. RESULTS Microarray analysis revealed differential expression of class 3 and class 4 semaphorins and their receptors in RA ECs. Semaphorin 4A (SEMA4A), plexin D1, and neuropilin 1 messenger RNA (mRNA) levels were markedly increased in RA ECs by 1.75-, 2.21-, and 1.68-fold, respectively. Stimulation with tumor necrosis factor (TNF) led to a 2-fold increase in SEMA4A mRNA levels in RA ECs, and deficient SEMA4A expression modified RA EC angiogenic properties. Class 3 and class 4 semaphorins as well as their receptors were overexpressed in RA synovial tissue. A respective 1.30-fold increase and 1.54-fold increase in SEMA4A and SEMA3E, as well as a 24% decrease in SEMA3A, was observed in the serum of RA patients. Serum levels of SEMA4A, SEMA4D, and SEMA3A correlated with levels of inflammation and proangiogenic markers. In 2 independent cohorts of patients with low disease activity or with RA in remission, the presence of SEMA4A identified patients with residual disease activity. CONCLUSION Gene expression profiling of ECs identified class 3 and class 4 semaphorins as potential biomarkers and therapeutic candidates in RA, with confirmed overexpression in ECs, synovial vessels, and serum, and correlation with validated markers of inflammation and angiogenesis. Thus, semaphorins might be novel and appealing EC-derived inflammatory and proangiogenic targets in RA.

ObjectivesTo decipher the phenotype of endothelial cells (ECs) derived from circulating progenitors issued from patients with rheumatoid arthritis (RA).MethodsRA and control ECs were compared according to their proliferative capacities, apoptotic profile, response to tumour necrosis factor (TNF)-α stimulation and angiogenic properties. Microarray experiments were performed to identify gene candidates relevant to pathological angiogenesis. Identified candidates were detected by RT-PCR and western blot analysis in ECs and by immunohistochemistry in the synovium. Their functional relevance was then evaluated in vitro after gene invalidation by small interfering RNA and adenoviral gene overexpression, and in vivo in the mouse model of methyl-bovine serum albumin-(mBSA)-induced arthritis.ResultsRA ECs displayed higher proliferation rate, greater sensitisation to TNF-α and enhanced in vitro and in vivo angiogenic capacities. Microarray analyses identified the NAD-dependent protein deacetylase sirtuin-1 (SIRT1) as a relevant gene candidate. Decreased SIRT1 expression was detected in RA ECs and synovial vessels. Deficient endothelial SIRT1 expression promoted a proliferative, proapoptotic and activated state of ECs through the acetylation of p53 and p65, and lead the development of proangiogenic capacities through the upregulation of the matricellular protein cysteine-rich angiogenic protein-61. Conditional deletion of SIRT1 in ECs delayed the resolution of experimental methyl-bovine serum albumin-(mBSA)-induced arthritis. Conversely, SIRT1 activation reversed the pathological phenotype of RA ECs and alleviates signs of experimental mBSA-induced arthritis.ConclusionsThese results support a role of SIRT1 in RA and may have therapeutic implications, since targeting angiogenesis, and especially SIRT1, might be used as a complementary therapeutic approach in RA.

ObjectiveTo evaluate the antifibrotic effects of the pan-peroxisome proliferator-activated receptor (PPAR) agonist IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH).MethodsIVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterised by non-specific interstitial pneumonia and severe vascular remodelling of pulmonary arteries leading to PH. Mice received two doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks.ResultsIVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis in both mouse models compared with mice receiving 30 mg/kg of IVA337 or vehicle. Histological score was markedly reduced by 61% in the bleomycin model and by 50% in Fra-2 transgenic mice, and total lung hydroxyproline concentrations decreased by 28% and 48%, respectively, as compared with vehicle-treated mice. IVA337 at 100 mg/kg also significantly decreased levels of fibrogenic markers in lesional lungs of both mouse models. In addition, IVA337 substantially alleviated PH in Fra-2 transgenic mice by improving haemodynamic measurements and vascular remodelling. In primary human lung fibroblasts, IVA337 inhibited in a dose-dependent manner fibroblast to myofibroblasts transition induced by TGF-β and fibroblast proliferation mediated by PDGF.ConclusionWe demonstrate that treatment with 100 mg/kg IVA337 prevents lung fibrosis in two complementary animal models and substantially attenuates PH in the Fra-2 mouse model. These findings confirm that the pan-PPAR agonist IVA337 is an appealing therapeutic candidate for these cardiopulmonary involvements.

Background Synovial neoangiogenesis is an early and crucial event to promote the development of the hyperplasic proliferative pathologic synovium in rheumatoid arthritis (RA). A recent microarray analysis of Endothelial cells (ECs) issued from patients with RA and matched controls has revealed a differential expression of semaphorin family in RA ECs, which is known to contribute to angiogenesis and autoimmunity/inflammation. Objectives Our aim was to study the potential implication of semaphorins in RA pathogenesis. Methods mRNA levels of class 3 and 4 Semaphorins (SEMA) SEMA3A, SEMA3E, SEMA4A and SEMA4E, as well as their receptors PlexinD1 (PLXND1) and Neuropillin-1 (NRP1), were measured by real-time quantitative PCR in RA (n = 15) and control (n = 15) ECs. Protein expression of these 4 semaphorins and their receptors was evaluated in ECs of 10 RA patients and controls by western blot, and in the synovial tissue of 10 RA patients and 5 controls by immunohistochemistry and immunofluorescence. Serum concentrations of these 4 semaphorins were measured by sandwich ELISA in a cohort of 130 patients with RA (85% women, mean age: 58 ± 12 years and mean disease duration: 11 ± 12 years) and 30 age- (56 ± 10 years) and sex- (82% women) matched controls. Results SEMA4A, SEMA3E and their receptors PLXND1 and NRP1 mRNA (Real time quantitative PCR) and protein (western blot analyses) levels were found to be markedly increased in RA ECs compared to control ECs. The expression of SEMA4A, SEMA3A, SEMA3E, PLXND1 and NRP1 was strikingly increased in the synovial tissue of patients with RA. Confocal microscopy with double labeling for CD31 confirmed the prominent endothelial expression of these class 3 and 4 semaphorins and their receptors. SEMA3A serum levels were significantly lower in RA compared to controls (13.93±5.69 vs. 18.29±6.69 ng/mL, P Conclusion Gene expression profiling of ECs identified semaphorins as potential biomarkers and therapeutic candidates in RA. Class 3 and 4 semaphorins are overexpressed in ECs, synovial vessels and the serum of patients with RA. They also correlated with validated markers of inflammation and angiogenesis. Thus, semaphorins might be novel and appealing EC-derived inflammatory and proangiogenic targets in RA. Disclosure of Interests Jerome Avouac Grant/research support from: research grant from Pfizer, Sonia Pezet: None declared, Eloise Vandebeuque: None declared, Yannick Allanore Grant/research support from: Inventiva, F Hoffman La-Roche, Sanofi, BMS, Pfizer, Consultant for: Actelion, Bayer, BMS, Boehringer, Roche, Sanofi

Background Angiogenesis through the recruitment and activation of endothelial cells (ECs) is a crucial event to promote the development of the hyperplasic proliferative synovium in rheumatoid arthritis (RA). Objectives Our aim was to decipher the phenotype of ECs derived from circulating progenitors issued from patients with RA. Methods Proliferation capacities between RA and control ECs was compared using the xCELLigence™ RTCA System. rh-TNFα-induced EC activation was analyzed by adhesion cell expression, VEGF synthesis and stress fiber formation. Angiogenic properties of ECs were assessed in vitro by tube formation on Matrigel and migration capacities through VEGF stimulation in modified Boyden chambers, and in vivo in a mouse model of tumoral neovascularization. Microarray experiments were then performed on Affymetrix GeneChip® Human Exon 1.0 ST Arrays in ECs issued from 18 RA patients compared to 11 age and sex-matched healthy controls to identify gene candidates relevant to pathological angiogenesis. Expression of identified candidates was assessed by RT-PCR and western blots in ECs and by immunohistochemistry in the synovial tissue of RA patients and controls. Their functional importance was then evaluated in vitro after gene invalidation by siRNA and gene overexpression via adenovirus, and in vivo in the mouse model of methyl-BSA-induced arthritis. Results EPC-derived ECs issued from RA patients displayed higher proliferation rate, greater sensitization to TNFα, with increased VEGF production, ICAM/VCAM expression, and more prominent stress fiber formation, as well as enhanced angiogenic capacities, characterized by accelerated tube formation and increased migration capacities through VEGF stimulation, compared co control ECs. The subcutaneous transplantation of murine colon carcinoma (CT-26) cells with RA ECs in CB17-SCID mice markedly amplified tumor growth and intra-tumoral neovessel density, compared to the transplantation of control ECs. Supervised microarray analyses identified the NAD+-dependent protein deacetylase sirtuin-1 (SIRT1) as a relevant gene candidate. A strikingly decreased SIRT1 gene/protein expression and enzyme activity was detected in RA ECs. A markedly decreased SIRT1 expression was also observed in RA synovium, and more particularly in blood vessels. Invalidation of SIRT1 with specific siRNA in control ECs was associated with a proliferative and activated profile upon TNFα stimulation, trough the acetylation of p53 and p65, and with the proangiogenic capacities through the upregulation of the matricellular protein CYR61. Conditional deletion of SIRT1 in ECs trough a Cre-LoxP recombination system increased angiogenesis and worsened signs of arthritis and in experimental models of tumor angiogenesis and methyl-BSA-induced arthritis. Conversely, overexpression of SIRT1 via adenovirus in RA ECs reversed this activated and proangiogenic phenotype, and activation of SIRT1 with resveratrol alleviated signs of experimental methyl-BSA-induced arthritis. Conclusion SIRT1 expression is reduced in synovial blood vessels of patients with RA. SIRT1 invalidation in control ECs reproduces the proliferative, activated and proangiogenic profile of RA ECs, and these effects were reversed by SIRT1 overexpression in RA ECs. These results support the implication of SIRT1 in RA synovial neoangiogenesis and may have direct therapeutic implications, since targeting angiogenesis, and especially SIRT1, might be used as a complementary therapeutic approach in RA. Acknowledgement This work was supported by the French Society of Rheumatology (SFR), Arthritis R&D and a research grand from Pfizer (“Bourse Passerelle”). Disclosure of Interests Agathe Leblond: None declared, Sonia Pezet: None declared, Anne Cauvet: None declared, Claudine Casas: None declared, Julie Pires Da Silva: None declared, Roxane Herve: None declared, Luca Semerano Grant/research support from: pfizer, Speakers bureau: pfizer, roche, msd, bms, Christophe Lemaire: None declared, Yannick Allanore Grant/research support from: Inventiva, F Hoffman La-Roche, Sanofi, BMS, Pfizer, Consultant for: Actelion, Bayer, BMS, Boehringer, Roche, Sanofi, Jerome Avouac Grant/research support from: research grant from Pfizer

Systemic sclerosis primarily affects women. This sex bias raises the question on the role female hormones could play in the development of fibrosis, which is largely unknown. Our aim was to evaluate the effects of estrogens in the development of experimental dermal fibrosis, in the mouse models of bleomycin-induced dermal fibrosis and tight skin (Tsk-1) mice, and on the activation of dermal fibroblasts by transforming growth factor-β (TGF-β). Estrogen inhibition, obtained through gene inactivation for the estrogen receptor-αknockout or treatment with tamoxifen, exacerbated skin fibrosis in the bleomycin model and in the Tsk-1 mice. In the dermal fibroblasts, treatment with 17-β-estradiol significantly decreased the stimulatory effects of TGF-β on collagen synthesis and myofibroblast differentiation, decreased the activation of canonical TGF-β signaling, and markedly reduced the expression of the TGF-β target genes. Tamoxifen reversed the inhibitory effects of estrogens by restoring Smad2/3 phosphorylation and TGF-β-induced collagen synthesis. Our results demonstrate a beneficial effect of estrogens in dermal fibrosis. Estrogens reduce the TGF-β–dependent activation of dermal fibroblasts, and estrogen inhibition leads to a more severe experimental dermal fibrosis. These findings are consistent with the prominent development of systemic sclerosis in postmenopausal women and the greater severity of the disease in men.

Background The pathogenesis of systemic sclerosis (SSc) involves a distinctive triad of autoimmune, vascular and inflammatory alterations resulting in fibrosis. Evidence suggests that peroxisome proliferator-activated receptors (PPARs) play an important role in SSc-related fibrosis and their agonists may become effective therapeutic targets. Objective To determine the expression of PPARs in human fibrotic skin and investigate the effects of IVA337, a pan PPAR agonist, in in vitro and in vivo models of fibrosis. Methods The antifibrotic effects of IVA337 were studied using a bleomycin-induced mouse model of dermal fibrosis. The in vivo effect of IVA337 on wound closure and inflammation were studied using an excisional model of wound healing. Results Low levels of PPARα and PPARγ were detected in the skin of patients with SSc compared with controls. In mice, IVA337 was associated with decreased extracellular matrix (ECM) deposition and reduced expression of phosphorylated SMAD2/3—intracellular effector of transforming growth factor (TGF)-β1. Although the antifibrotic effect of pan PPAR was similar to that of PPARγ agonist alone, a significant downregulation of several markers of inflammation was associated with IVA337. Despite its anti-inflammatory and antifibrotic properties, IVA337 did not interfere with wound closure. In vitro effects of IVA337 included attenuation of transcription of ECM genes and alteration of canonical and non-canonical TGF-β signalling pathways. Conclusions These findings indicate that simultaneous activation of all three PPAR isoforms exerts a dampening effect on inflammation and fibrosis, making IVA337 a potentially effective therapeutic candidate in the treatment of fibrotic diseases including SSc.

Background We aimed to investigate the efficacy of abatacept in preclinical mouse models of digestive involvement, pulmonary fibrosis, and related pulmonary hypertension (PH), mimicking internal organ involvement in systemic sclerosis (SSc). Methods Abatacept has been evaluated in the chronic graft-versus-host disease (cGvHD) mouse model (abatacept 1 mg/mL for 6 weeks), characterized by liver and intestinal fibrosis and in the Fra-2 mouse model (1 mg/mL or 10 mg/mL for 4 weeks), characterized by interstitial lung disease (ILD) and pulmonary vascular remodeling leading to PH. Results In the cGvHD model, abatacept significantly decreased liver transaminase levels and markedly improved colon inflammation. In the Fra-2 model, abatacept alleviated ILD, with a significant reduction in lung density on chest microcomputed tomography (CT), fibrosis histological score, and lung biochemical markers. Moreover, abatacept reversed PH in Fra-2 mice by improving vessel remodeling and related cardiac hemodynamic impairment. Abatacept significantly reduced fibrogenic marker levels, T-cell proliferation, and M1/M2 macrophage infiltration in lesional lungs of Fra-2 mice. Conclusion Abatacept improves digestive involvement, prevents lung fibrosis, and attenuates PH. These findings suggest that abatacept might be an appealing therapeutic approach beyond skin fibrosis for organ involvement in SSc. Electronic supplementary material The online version of this article (10.1186/s13075-018-1694-9) contains supplementary material, which is available to authorized users.

Background Recent accumulating evidences indicate a crucial role of macrophage lineage in the pathogenesis of fibrotic diseases including systemic sclerosis (SSc). CD163 is a surface marker expressed by M2 macrophages that accumulate during the healing phase of acute inflammation. It is actively released from the plasma membrane in response to certain inflammatory stimuli and enters the circulation in its soluble form (sCD163). Objectives In this study, we aimed to evaluate the performance of serum and urinary sCD163 concentrations as possible biomarker in SSc. Methods Urine and serum samples were obtained from SSc patients, fulfilling the 2013 ACR/EULAR classification criteria for SSc, and age- and sex-matched controls. Serum and urinary sCD163 concentrations were measured by commercially available ELISA kit (R and D systems) and evaluated for their significance as potential biomarkers. Statistical analysis was carried out using Mann-Whitney U test and the relationship between parameters was statistically examined by Spearman’s rank test. Results Two hundred and three SSc patients were included, 163 (80%) were female, with a mean ±standard deviation (SD) age of 59±13 years and a mean ±SD disease duration of 12±9 years. Eighty-one (41%) patients had diffuse cutaneous SSc and mean ±SD mRSS was 6.6±7.7. Lung fibrosis on imaging was observed in 33% of the patients, 7% had pulmonary arterial hypertension, 44% had history of digital ulcers and 41% were taking immunosuppressive therapy. Control group consisted of 47 age- and sex-matched patients with non-inflammatory diseases, being osteoporosis for the very large majority. Serum sCD163 levels were significantly higher in SSc patients compared with controls (mean ±SD: 529±251 vs 385.1±153 ng/ml; p Conclusions To our knowledge this is the first evaluation of both serum and urinary sCD163 levels in SSc. Our results show a significant difference for sera values that should be prioritised for further studies as compared to urinary concentrations conversely to what has been described in lupus. Our results further support that the M2 macrophages/CD163 signalling system may play a role in the pathogenesis of SSc. However, further studies are required to address the exact role of CD163 in the pathogenesis of SSc and to determine whether it could help in the risk-stratification of the patients in this heterogeneous disease. Disclosure of Interest None declared

Background Cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) is an immunoregulatory membrane receptor resulting in the down-regulation of T-cell responses. A previous report showed that abatacept (CTLA4-Ig) prevented and induced regression of inflammation-driven dermal fibrosis in two different mouse models of systemic sclerosis (SSc). 1 Objectives We aimed to assess the effects of abatacept in two complementary mouse models reflecting digestive involvement, lung fibrosis and pulmonary hypertension (PH), mimicking severe SSc organ damage. Methods Abatacept was given in the chronic graft-versus-host disease (cGvHD) mouse model, characterised by digestive involvement, and in the Fra-2 mouse model which is characterised by non-specific interstitial pneumonia and pulmonary vascular remodelling leading to PH. Mice were treated by intraperitoneal injections of abatacept (1 mg/mL in the cGvHD model for 6 weeks; 1 mg/mL or 10 mg/mL in the Fra-2 mouse model for 4 weeks) or human IgG1 (100 mg) used as a negative control. Results In the cGvHD model, treatment of allogeneically mice with abatacept led to a significant reduction of alanine aminotransferase (24%, p=0.014) and aspartate aminotransferase levels (61%, p When assessed by chest micro-CT imaging, Fra-2 mice treated with abatacept displayed a 12% decrease in lung density (10 mg/ml, p=0.037) as well as an increase in functional residual capacity as compared to IgG1-treated mice (16% for 1 mg/ml, p=0.001% and 14% for 10 mg/ml, p=0.005). Consistent with these results, abatacept 10 mg/L decreased histological fibrosis score (Ashcroft score) as well as hydroxyproline content by 79% (p=0.009) and 31% (p=0.044) respectively, as compared to IgG1-treated mice. Treatment with abatacept 10 mg/mL markedly reduced protein levels in the lesional lungs of Fra-2 mice of the fibrogenic markers MCP1 by 79% (p=0.043) and osteopontin by 87% (p=0.039). Levels of TGF-b were also reduced with abatacept (61% for 1 mg/mL, p=0.037% and 69% for 10 mg/mL, p=0.013). Further, abatacept decreased M1 and M2 macrophages infiltration as well as T-cell proliferation in the lesional lungs of Fra-2 mice. Upon treatment with abatacept a reduction of right ventricular systolic pressure (28.1±1.5 mmHg vs 36.0±5.1 mmHg, p=0.037 for 10 mg/mL) and right ventricular hypertrophy (0.29±0.01 vs 0.33±0.010, p=0.037 and 29±0.01% vs 0.33±0.01% for 10 mg/mL, p=0.037) was observed compared to IgG1-treated mice. Consistent with these findings, abatacept 10 mg/mL was associated with significant decrease in percent medial wall thickness and numbers of muscularized distal pulmonary arteries. Conclusions We demonstrate that treatment with abatacept improves digestive involvement, prevents lung fibrosis and attenuates PH in SSc pre-clinical mice models. These findings suggest that abatacept might be an appealing therapeutic approach for severe internal organ involvement in SSc beyond its already demonstrated effects on skin fibrosis. Reference [1] Ponsoye M, et al. Ann Rheum Dis2016Dec;75(12):2142–9. Disclosure of Interest None declared

Background Neoangiogenesis is a crucial event to promote the development of the hyperplasic proliferative pathologic synovium in Rheumatoid arthritis (RA). Ultrasound (US) is sensitive for detection of power Doppler (PD) vascularisation. Objectives To explore the associations between a set of complementary circulating angiogenic markers reflecting different angiogenic processes and a comprehensive US assessment in patients with RA. Methods Serum levels of eight angiogenic markers (Vascular Endothelial Growth Factor (VEGF), Placenta Growth Factor (PlGF), Tie-2, Angiopoietin-1, soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), Interleukin-8 (IL-8, CXCL8), CYR61 (CCN1) and Angiostatin), reflecting endothelial cell activation, proliferation, survival, growth and migration, as well as vessel maturation and stabilisation, were measured by quantitative ELISAs in a total of 125 patients with RA, who were all systematically assessed in parallel by PDUS, performed on 32 joints. Results Synovitis was detected in 84 patients with RA (67.2%). Among these patients, 53 patients (42.4%) had positive Doppler signal, including 31 with moderate to marked hyperemia. Serum levels of soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1) (808±293 ng/mL vs. 697±240 ng/mL, p=0.022) and Tie-2 (16.2±7.5 ng/mL vs. 13.8±4.9 ng/mL, p=0.038), were more likely to be increased in patients with synovial hyperemia detected on at least one joint (Power Doppler grade ≥1). sVCAM-1, Tie-2 and Angiostatin concentrations gradually increased together with the grade of the semiquantitative PDUS scale (figure 1A-C) and concentrations of these three markers were markedly increased in patients with moderate to marked hyperemia (Power Doppler grade 2 and 3). Levels of sVCAM-1 (r=0.20, p=0.028), Tie-2 (r=0.28, p=0.001), and Angiostatin (r=0.25, p=0.006) correlated with a global arthritis sum score, defined by the sum of the semiquantitative PDUS scores for all joints examined. Among the 81 patients with a DAS28-CRP≤3.2, 22 patients had synovial hyperemia detected on at least one joint (Power Doppler grade 1 in 13 patients, grade 2 in 6 patients and grade 3 in 3 patients). Patients with synovial hyperemia on at least one joint were more likely to have significantly increased levels of PlGF (18.9±11.2 pg/mL vs. 13.1±9.5 pg/mL, p=0.022) and Tie-2 (15.7±5.8 ng/mL vs. 12.6±3.4 ng/mL, p=0.004) than patients with absence of synovial hyperemia. Conclusions Serum levels of the angiogenic markers Tie-2, sVCAM-1 and Angiostatin were strongly associated with synovial vascularisation and inflammation assessed by PDUS among patients with established RA. Moreover, Tie-2 and PlGF were associated with persistent disease activity in RA patients in mow disease activity. These findings suggest that it may possible to find surrogate serum angiogenic biomarkers of active synovitis that might replace PDUS examination, in case of further confirmation of their pertinence. Disclosure of Interest A. Leblond: None declared, S. Pezet: None declared, A. P. Trouvin: None declared, M. Elhai: None declared, V. Gonzalez: None declared, Y. Allanore Grant/research support from: Pfizer, J. Avouac: None declared

Objective Interstitial lung disease (ILD ) in systemic sclerosis (SS c) runs a highly variable course, and prediction tools are highly desired. The aim of this study was to assess the diagnostic and prognostic performance of 4 candidate serum biomarkers for SS c‐associated ILD . Methods Serum samples from a combined cohort of SS c patients (from Paris, France and Oslo, Norway; n = 427) were analyzed by enzyme‐linked immunosorbent assay for concentrations of lung epithelial–derived surfactant protein D (SP ‐D), Krebs von den Lungen 6 glycoprotein (KL ‐6), CCL 18, and OX 40 ligand (OX 40L). Lung fibrosis was measured by high‐resolution computed tomography and pulmonary function tests. Associations of these candidate biomarkers with baseline disease involvement and prediction of disease progression over time (mean ± SD follow‐up 3.2 ± 4.4 years) were investigated. Results In SS c patients at baseline, serum levels of KL ‐6 correlated with the forced vital capacity (FVC ) (r = −0.317, P < 0.001), diffusing capacity for carbon monoxide (r = −0.335, P < 0.001), and extent of lung fibrosis (r = 0.551, P < 0.001). In multivariate analyses, serum levels of KL ‐6 and SP ‐D, but not CCL 18 and OX 40L, were associated with lung fibrosis (odds ratio [OR ] 2.41, 95% confidence interval [95% CI ] 1.43–4.07 [P = 0.001] and OR 3.15, 95% CI 1.81–5.48 [P < 0.001], respectively). In SS c patients with ILD at baseline, longitudinal, multivariate analyses showed that CCL 18 serum levels were an independent predictor of a >10% decrease in the FVC (hazard ratio [HR ] 2.90, 95% CI 1.25–6.73; P = 0.014) and de novo development of extensive disease (HR 3.71, 95% CI 1.02–13.52; P = 0.048). Matrix‐based logistic regression models for the diagnosis and prognosis of SS c‐associated ILD were constructed, and these models discriminated 3 groups of risk (mild, moderate, or high) for the diagnosis or worsening of lung fibrosis according to the serum levels of SP ‐D (for diagnosis) and serum levels of CCL 18 (for progression of disease). Conclusion These results show that SP ‐D is a relevant diagnostic biomarker for SS c‐associated ILD , whereas KL ‐6 could be used to assess the severity of lung fibrosis. CCL 18 appears to be a potential predictive marker for progression of ILD in SS c.

Objective. To evaluate the performance of serum and urinary sCD163 concentrations as possible biomarker in systemic sclerosis (SSc). Methods. Urine and serum samples were obtained from SSc patients and age- and sex-matched controls. Serum and urinary sCD163 concentrations were measured by commercially available ELISA kit. SSc patients were assessed following international guidelines. Cross-sectional analyses were performed. Results. Two hundred and three SSc patients were included. The control group consisted of 47 age- and sex-matched patients having noninflammatory diseases, mainly osteoporosis. Serum sCD163 levels were significantly higher in SSc patients compared with controls (mean ± SD: 529 ± 251 versus 385 ± 153 ng/mL; p<0.001). Urinary sCD163 concentrations were higher in SSc patients than controls, but this did not reach significance (236 ± 498 versus 176 ± 173 ng/mg uCr; p=0.580). The sCD163 concentrations were not associated with clinical, laboratory, and instrumental characteristics of SSc patients. Conclusion. To our knowledge, this is the first evaluation of both serum and urinary sCD163 levels in SSc. Our results show a significant difference for sera values that should be prioritized for further studies as compared to urinary measurements. Our results further support that the M2 macrophages/CD163 signaling system may play a role in the pathogenesis of SSc, although we could not identify a subset of SSc patients with higher concentrations.

Background Neoangiogenesis is a crucial event to promote the development of the hyperplasic proliferative pathologic synovium in Rheumatoid arthritis (RA). Ultrasound (US) is sensitive for detection of power Doppler (PD) vascularization. Objective To explore the associations between a set of complementary circulating angiogenic markers and a comprehensive US assessment in patients with RA. Patients and methods Serum levels of eight angiogenic markers were measured by quantitative ELISAs in a total of 125 patients with RA, who were all systematically assessed in parallel by PDUS, performed on 32 joints. Results Serum levels of soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1) and Tie-2 were more likely to be increased in patients with synovial hyperemia detected on at least one joint (Power Doppler grade ≥1). sVCAM-1, Tie-2 and Angiostatin concentrations gradually increased together with the grade of the semiquantitative PDUS scale and concentrations of these three markers were markedly increased in patients with moderate to marked hyperemia (Power Doppler grade 2 and 3). Levels of sVCAM-1, Tie-2, and Angiostatin correlated with a global arthritis sum score, defined by the sum of the semiquantitative PDUS scores for all joints examined. Levels of Tie-2 and Placenta Growth Factor (PlGF) were associated with PDUS features indicating residual disease activity. Conclusion Our results support the relevance of measuring serum levels of vascular markers to evaluate the intensity and extent of synovial vascularization. Angiogenic markers, and particularly Tie-2, could be a valuable surrogate of active synovitis and their place in relation to PDUS in clinical practice deserve further investigation.

Background Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors known to modulate fibrosis. The pan-PPAR agonist IVA337 recently demonstrated efficacy in prevention and treatment of experimental skin fibrosis (1). Objectives Our objective was to evaluate the antifibrotic effects of IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH). Methods IVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterized by non-specific interstitial pneumonia and severe vascular remodeling of pulmonary arteries leading to PH. Mice received 2 doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks. Results Both 30 mg/kg and 100 mg/kg doses of IVA337 were well tolerated in all mouse models. IVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis induced by bleomycin compared to mice receiving 30 mg/kg of IVA337 or vehicle. Indeed, IVA337 (100 mg/kg) strongly reduced by 61% and 28% tissue density on histological measurements and total lung hydroxyproline concentrations, respectively, as compared to vehicle. IVA337 at 100 mg/kg also significantly decreased col1, col3 and fibronectin in lesional lungs. Similarly, Fra-2 transgenic mice treated with 100 mg/kg of IVA337 displayed reduced lung density (20% vs. vehicle) and significant increase of functional residual capacity (30% vs. vehicle) when assessed by chest micro-CT imaging. These results were emphasized by a 50% reduction of the Ascroft fibrosis score (Figure 1A) and by a 48% reduction of hydroxyproline concentrations upon IVA337 (100 mg/kg) compared to vehicle treated mice. Successful targeting of the TGF-b signaling axis was observed in both mouse models upon treatment with 100 mg/kg of IVA337. 100 mg/kg IVA337 also significantly reduced T cell and B cell infiltration in lesional lungs of Fra-2 transgenic mice. Regarding vessel remodeling and related pulmonary hypertension, treatment with 100 mg/kg of IVA337 led to a substantial attenuation of right ventricular systolic pressure and right ventricular hypertrophy compared to mice receiving the vehicle (Figure 1B and 1C). Furthermore, IVA337 given at 100 mg/kg markedly reduced medial wall thickness (Figure 1D) and the number of muscularized distal pulmonary arteries. In vitro in primary human lung fibroblasts, IVA337 inhibited in a dose-dependent manner TGFβ-mediated fibroblasts to myofibroblasts transition and PDGF-mediated proliferation. Conclusions We demonstrate that treatment with 100 mg/kg IVA337 prevents lung fibrosis in two complementary animal models and substantially attenuates PH in the Fra-2 mouse model. These findings confirm that the pan-PPAR agonist IVA337 is an appealing therapeutic candidate for systemic sclerosis both, for skin and key cardiovascular complications. References Ruzehaji et al, Ann Rheum Dis 2016;75(12):2175–2183. Disclosure of Interest J. Avouac: None declared, I. Konstantinova Employee of: INVENTIVA, C. Guignabert: None declared, J. Sadoine: None declared, G. Thomas: None declared, S. Pezet: None declared, A. Cauvet: None declared, L. Tu: None declared, J. M. Luccarini Employee of: INVENTIVA, J. L. Junien Consultant for: INVENTIVA, P. Broqua Employee of: INVENTIVA, Y. Allanore Grant/research support from: Inventiva

To elucidate the role of gene candidates involved in pulmonary hypertension (PH) associated with systemic sclerosis (SSc).Gene candidates were identified through microarray experiments performed on Affymetrix GeneChip Human Exon 1.0 ST arrays in endothelial progenitor cell (EPC)-derived endothelial cells (ECs) obtained from patients with SSc-associated PH, patients with SSc without PH, and healthy control subjects. Expression of identified gene candidates was assessed by quantitative sandwich enzyme-linked immunosorbent assay in the serum, and by immunohistochemistry in lesional lung tissue. The functional importance of the identified gene candidates was then evaluated in fos-related antigen 2-transgenic (Fra-2-Tg) mice that spontaneously develop SSc-like features associated with an intense pulmonary vascular remodeling.Microarray experiments revealed that the matrix metalloproteinase 10 (MMP-10) gene was the top up-regulated gene in SSc-associated PH EPC-derived ECs. Circulating serum proMMP10 concentrations were markedly increased in patients with SSc-associated PH compared to SSc patients without PH and healthy controls. Consistent with these observations, a strong MMP10 staining of the thickened wall of distal pulmonary arteries was found both in the lungs of patients with SSc-associated PH and in the lungs of Fra-2-Tg mice. Daily treatment of Fra-2-Tg mice with neutralizing anti-MMP10 antibodies did not significantly affect the development and severity of pulmonary fibrosis, but did reverse established PH and markedly reduced pulmonary vascular remodeling by reducing cell proliferation, cell survival, and the platelet-derived growth factor signaling axis.Gene expression profiling of EPC-derived ECs identified MMP10 as a novel candidate gene in SSc-associated PH. MMP10 is overexpressed in the serum and pulmonary arteries of patients with SSc-associated PH, and its blockade alleviates PH in the Fra-2-Tg mouse model. MMP10 appears to be a prospective treatment target for this devastating disorder.

Treatment for fibrosis represents a critical unmet need, because fibrosis is the leading cause of death in industrialized countries, and there is no effective therapy to counteract the fibrotic process. The development of fibrosis relates to the interplay between vessel injury, immune cell activation, and fibroblast stimulation, which can occur in various tissues. Immunotherapies have provided a breakthrough in the treatment of immune diseases. The glycoprotein OX40-OX40 ligand (OX40L) axis offers the advantage of a targeted approach to costimulatory signals with limited impact on the whole immune response. Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. OX40L is overexpressed in the fibrotic skin and serum of patients with SSc, particularly in patients with diffuse cutaneous forms. Soluble OX40L was identified as a promising serum biomarker to predict the worsening of lung and skin fibrosis, highlighting the role of this pathway in fibrosis. In vivo, OX40L blockade prevents inflammation-driven skin, lung, and vessel fibrosis and induces the regression of established dermal fibrosis in different complementary mouse models. OX40L exerts potent profibrotic effects by promoting the infiltration of inflammatory cells into lesional tissues and therefore the release of proinflammatory mediators, thereafter leading to fibroblast activation.

Objective Activated T cells are the main component of the inflammatory skin infiltrates that characterise systemic sclerosis (SSc). Our aim was to investigate the efficacy of abatacept, which tempers T-cell activation, in reducing skin fibrosis in complementary mouse models of SSc. Methods The antifibrotic properties of abatacept were evaluated in the mouse models of bleomycin-induced dermal fibrosis and sclerodermatous chronic graft-versus-host disease, reflecting early and inflammatory stages of SSc. Thereafter, we studied the efficacy of abatacept in tight skin (Tsk-1) mice, an inflammation-independent mouse model of skin fibrosis. Results Abatacept efficiently prevented bleomycin-induced skin fibrosis and was also effective in the treatment of established fibrosis. In this model, abatacept decreased total and activated T-cell, B-cell and monocyte infiltration in the lesional skin. Abatacept did not protect CB17-SCID mice from the development of bleomycin-induced dermal fibrosis, which supports that T cells are necessary to drive the antifibrotic effects of abatacept. Upon bleomycin injections, skin interleukin (IL) 6 and IL-10 levels were significantly reduced upon abatacept treatment. Moreover, treatment with abatacept ameliorated fibrosis in the chronic graft-versus-host disease model, but demonstrated no efficacy in Tsk-1 mice. The tolerance of abatacept was excellent in the three mouse models. Conclusions Using complementary models, we demonstrate that inhibition of T-cell activation by abatacept can prevent and induce the regression of inflammation-driven dermal fibrosis. Translation to human disease is now required, and targeting early and inflammatory stages of SSc sounds the most appropriate for positioning abatacept in SSc.