POS0613 TOCILIZUMAB DECREASES ANGIOGENESIS IN RHEUMATOID ARTHRITIS THROUGH ITS REGULATORY EFFECT ON EMMPRIN/CD147
Authors: D. Zisman, M. Safieh, E. Simanovich, J. Feld, A. Kinarty, L. Zisman, T. Gazitt, A. Haddad, M. Elias, I. Rosner, L. Kaly, M. Amit Rahat
Journal: Annals of the Rheumatic Diseases (2021)

Background: Angiogenesis is an important contributor to the development of Rheumatoid arthritis (RA). Tocilizumab (TCZ), an anti-IL-6 receptor antibody, is an immunosuppressant used in the treatment of RA patients, but its effects on angiogenesis and the molecular mechanisms regulating new blood vessel formation are not fully elucidated. Objectives: To evaluate the concentrations of pro- and anti-angiogenic factors in serum samples of RA patients, before and after the initiation of TCZ treatment and to explore in an in vitro co-culture system the mechanisms of TCZ action. Methods: We evaluated the concentrations of EMMPRIN, VEGF, MMP-9, IL-6, NGAL, endostatin and thrombospondin-1 (Tsp-1) using commercial ELISA kits from 40 RA patients, before and 4 months after the initiation of TCZ treatment. The levels of secreted EMMPRIN, VEGF MMP-9 and Tsp-1 were measured in an in vitro co-culture system of HT1080 fibroblasts and U937 monocytes with and without addition of anti-EMMPRIN blocking antibody. In the tube formation assay serum samples and supernatnats from the co-cultures were added to endothelial layer. Images were obtained after 6 hours of incubation and the number of closed lumens were counted in two separate fields. In the wound assay, supernatants from the co-cultures, with or without the addition of the anti-EMMPRIN antibody were added to the endothelial layer after scratching. The scratch site area was measured immediately and compared to the area after 24 hours of incubation to assess the distance of cell migration. Results: Study population included 40 RA patients, 33 (82.5%) females, mean age of 57.5±11.1 years, disease duration of 7.7±5.6 years, and 53.9% positive for rheumatoid factor initiating treatment with TCZ. In this patient cohort, 25/40 (62.5%) patients were classified as “responders” according to EULAR criteria. Following 4 mounts of treatment, statistically significant reductions in the levels of EMMPRIN/CD147 (p=0.035), without significant changes in serum levels of MMP-9, VEGF, MMP-3 and MMP-7 and of the anti-angiogenetic factors Tsp-1 and endostatin were found. A statistically significant decrease in the ratio between the pro-angiogenic factor EMMPRIN and the anti-angiogenic factor Tsp-1 that was calculated for each patient 4 months after initiating TCZ was found(p=0.031). The decrease in angiogenesis was manifested by the reduced number of closed lumen tube-like structures formed by EaHy926 endothelial cell line after incubation with serum samples 4 months after initiation of TCZ, relative to the number of closed lumens formed prior to TCZ initiation (p=0.007). The ratio between EMMPRIN and Tsp-1 was significantly reduced in the responding patients versus non-responders (p=0.033), while the levels of VEGF, MMP-9, Tsp-1, and EMMPRIN were unchanged. In vitro, the accumulation of the pro-angiogenic factors EMMRPIN, VEGF and MMP-9 in the supernatants was increased in the co-culture, while the accumulation of the anti-angiogenic factor Tsp-1 was decreased. When EMMPRIN was neutralized with a blocking antibody, supernatants derived from these co-cultures exhibited reduced migration, proliferation, and tube-like structure formation in functional assays. Conclusion: Our findings suggest an important role for EMMPRIN in mediating pro-angiogenic signals in RA patients, with EMMPRIN/Tsp-1 ratio serving as a marker of angiogenesis in RA. When administered to RA patients, TCZ in turn, exerts an anti-angiogenic effect through its regulation of EMMRPIN/CD147 levels. Disclosure of Interests: None declared