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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

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