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Objective During rheumatoid arthritis (RA), the angiogenic processes, occurring with pannus-formation, may be a therapeutic target. JAK/STAT-pathway may play a role and the aim of this work was to investigate the inhibiting role of a JAK-inhibitor, tofacitinib, on the angiogenic mechanisms occurring during RA. Methods After ethical approval, JAK-1, JAK-3, STAT-1, STAT-3 and VEGF expression was evaluated on RA-synovial-tissues. In vitro, endothelial cells (ECs), stimulated with 20 ng/ml of VEGF and/or 1 μM of tofacitinib, were assessed for tube formation, migration and proliferation, by Matrigel, Boyden chamber assay and ki67 gene-expression. In vivo, 32 mice received collagen (collagen-induced arthritis (CIA)) and 32 mice PBS (control). At day 19, CIA and controls mice were divided: 16 mice receiving vehicle and 16 mice receiving tofacitinib. At day 35, the arthritis score, the thickness of paw joints and the serum levels of VEGF and Ang-2 were evaluated. Results The expression of JAK-1, JAK-3, STAT-1, STAT-3 and VEGF in synovial tissue of RA-patients were significantly higher than healthy controls. In vitro, tofacitinib inhibited the ECs ability to form vessels, to proliferate and to migrate. In vivo, administration of tofacitinib prevented the increase of the arthritis score, the paw thickness, the synovial vessels and VEGF and Ang-2 serum-accumulation, when compared to CIA without tofacitinib. Conclusions We explored the anti-angiogenic role of tofacitinib, reporting its ability to inhibit in vitro the angiogenic mechanisms of ECs and in vivo the formation of new synovial vessels, occurring in CIA model. These findings suggest that the therapeutic effect of tofacitinib during RA may be also related to its anti-angiogenic activity.

Background:During rheumatoid arthritis (RA), a chronic autoimmune disease, the loop existing between inflammation and angiogenesis, characterised by new vessels formation associated with the increased recruitment of inflammatory cells, via the abnormal neo-angiogenesis in the synovial tissues, is considered an early important pathogenic mechanism.Tofacitinib, a potent and selective JAK inhibitor, showed a good profile of safety and efficacy in RA patients, slowing the radiographic progression of the disease. In the last years, many works confirmed that some pro-angiogenic genes are targets of STATs family, and among them, vascular endothelial growth factors (VEGF), a potent pro-angiogenic molecule, may promote the new vessels formation via JAK/STAT pathways.Objectives:The aim of this work was to investigate the inhibiting role of tofacitinib, on the angiogenic mechanisms occurring during experimental model of arthritis.Methods:Healthy control (HC) ECs were stimulated with VEGF and/or tofacitinib and assessed for tube formation and migration, by matrigel and Boyden chamber assay. Furthermore, after ethical approval the experimental model of arthritis was obtained, stimulating 32 mice with collagen (CIA) and 32 mice with PBS (control). At day-19, CIA and controls mice were divided in 16 mice receiving vehicle and 16 mice receiving tofacitinib. At day-35, the mice were scarified and the thickness of paw joints, the synovial vessels and the serum levels of VEGF and Ang-2 were evaluatedResults:In vitro, after tofacitinib-treatment, HC-ECs lose their ability to form vessels and to migrate.In vivo, tofacitinib significantly prevented the increase of paw thickness induced by the collagen administration and reduced the vessel density in synovial tissues of joints, when compared to CIA that did not received tofacitinib. Furthermore, the serum levels of VEGF and Ang-2 were higher in CIA mice, than in control mice. The administration of tofacitinib was able to prevent the VEGF and Ang-2 accumulation in CIA mice.Conclusion:During the last decade, the biological analogies between solid tumors and synovial pannus, and the encouraging results of anti-angiogenic treatments in oncology, lead to increasing interest for angiogenesis as a possible therapeutic target in RA. The present study demonstrated the anti-angiogenic efficacy of tofacitinib, opening a new perspective application for this molecule and improving our therapeutic skill to control the clinical evolution of RA.References:[1]Leblond A et al. Autoimmun Rev 2017;16:594-601.[2]Fleischmann R et al. N Engl J Med 2012;367:495-507.[3]Marrelli A, Autoimmun Rev 2011;10:595-8.Disclosure of Interests:Paola Cipriani Grant/research support from: Actelion, Pfizer, Speakers bureau: Actelion, Pfizer, Paola Di Benedetto Grant/research support from: Paola Di Benedetto received grant from Dompè outside this work., Piero Ruscitti Grant/research support from: Piero Ruscitti received grant from Pfizer outside this work., Speakers bureau: Piero Ruscitti received speaker honoraria BMS, MSD, Ely Lilly, SOBI outside this work, Onorina Berardicurti: None declared, Vasiliki Liakouli Grant/research support from: Vasiliki Liakouli received grant from Pfizer outside this work., Speakers bureau: Vasiliki Liakouli received speaker honoraria from Sanofi Genzyme outside this work., Francesco Carubbi Speakers bureau: Francesco Carubbi received speaker honoraria from Abbvie and Celgene outside this work., Noemi Panzera: None declared, Nicolò Grazia: None declared, Mauro Di Vito Nolfi: None declared, Barbara Di Francesco: None declared, Antonio Maurizi: None declared, Nadia Rucci: None declared, Anna Teti: None declared, Francesca Zazzeroni: None declared, Edoardo Alesse: None declared, Roberto Giacomelli Grant/research support from: Roberto Giacomelli received research grant from Pfizer.This study was supported by an unconditioned Research grant from Pfizer., Speakers bureau: Roberto Giacomelli received speaker honoraria from Abbvie, Roche, Actelion, BMS, MSD, Ely Lilly, SOBI and Pfizer outside this work.

Ferritin is an iron-binding molecule, which comprises 24 subunits, heavy (FeH) and light (FeL) subunits, suggested to have a pathogenic role by the ‘hyperferritinemic syndrome’. In this work, we tested (1) FeH and FeL in bone marrow (BM) and sera in patients with macrophage activation syndrome (MAS); (2) pro-inflammatory effects of ferritin, FeL, and FeH on macrophages; (3) ability of FeH-stimulated macrophages to stimulate the proliferation of peripheral blood mononuclear cells (PBMCs); (4) production of mature IL-1β and IL-12p70 in extracellular compartments of FeH-stimulated macrophages. Immunofluorescence analysis and liquid chromatography mass spectrometry (LC–MS/MS) based proteomics were performed to identify FeL and FeH in BM and sera, respectively, in the same patients. Macrophages were stimulated with ferritin, FeH, and FeL to assess pro-inflammatory effects by RT-PCR and western blot. The proliferation of co-cultured PBMCs with FeH-stimulated macrophages was tested. Immunofluorescence showed an increased FeH expression in BMs, whereas LC–MS/MS identified that FeL was mainly represented in sera. FeH induced a significant increase of gene expressions of IL-1β, IL-6, IL-12, and TNF-α, more marked with FeH, which also stimulated NLRP3. FeH-stimulated macrophages enhanced the proliferation of PBMCs. The ELISA assays showed that mature form of IL-1β and IL-12p70 were increased, in extracellular compartments of FeH-stimulated macrophages. Our results showed FeH in BM biopsies of MAS patients, whereas, LC–MS/MS identified FeL in the sera. FeH showed pro-inflammatory effects on macrophages, stimulated NLRP3, and increased PBMCs proliferation.