OP0291 ACTIVATION OF THE DEACETYLASE SIRTUIN-1 COUNTERACTS THE ACTIVATED AND PROANGIOGENIC PROFILE OF ENDOTHELIAL CELLS IN RHEUMATOID ARTHRITIS AND ALLEVIATES EXPERIMENTAL ARTHRITIS
Authors: Agathe Leblond, Sonia Pezet, Anne Cauvet, Claudine Casas, Julie Pires Da Silva, Roxane Hervé, Luca Semerano, Christophe Lemaire, Yannick Allanore, Jérôme Avouac
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> 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). <h3>Objectives</h3> Our aim was to decipher the phenotype of ECs derived from circulating progenitors issued from patients with RA. <h3>Methods</h3> 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 <i>in vitro</i> by tube formation on Matrigel and migration capacities through VEGF stimulation in modified Boyden chambers, and <i>in vivo</i> 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 <i>in vitro</i> after gene invalidation by siRNA and gene overexpression via adenovirus, and <i>in vivo</i> in the mouse model of methyl-BSA-induced arthritis. <h3>Results</h3> 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<sup>+</sup>-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. <h3>Conclusion</h3> 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. <h3>Acknowledgement</h3> This work was supported by the French&nbsp;Society of Rheumatology&nbsp;(SFR), Arthritis R&amp;D and a research grand from Pfizer ("Bourse Passerelle"). <h3>Disclosure of Interests</h3> Agathe Leblond: None declared, Sonia Pezet: None declared, Anne Cauvet: None declared, Claudine Casas: None declared, Julie Pires Da Silva: None declared, Roxane Hervé: 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, Jérôme Avouac Grant/research support from: research grant from Pfizer