A NOVEL 3D MODEL OF RHEUMATOID ARTHRITIS SYNOVIAL ANGIOGENESIS TO SCREEN NEW THERAPEUTIC COMPOUNDS

Background: Angiogenesis is a crucial mediator in rheumatoid arthritis (RA) pathogenesis and blocking this process is thought to have therapeutic potential in ameliorating the disease. Current in vitro models of angiogenesis focus solely on endothelial cells (EC), however, RA fibroblast like synoviocytes (FLS), as well as angiogenic mediators in synovial fluid (SF), are also important contributors to this process. Therefore, a model including both EC, FLS, and SF would be more representative of synovial angiogenesis, and could be used to screen new therapeutic compounds. Previous work demonstrates that the non-canonical NF-κB pathway, with its main regulator NF-κB inducing kinase (NIK), induces angiogenesis in EC. Yet, this remains to be studied in a co-culture model. Objectives: To generate a 3D model of RA synovial angiogenesis and to screen the effects of inhibition of non-canonical NF-κB signaling and other inhibitors of angiogenesis Methods: HUVEC and RA FLS were labeled with green or orange cell tracker dye, respectively, and incubated overnight to form spheroids. Subsequently, spheroids were harvested, plated in a collagen solution, and medium with growth factors or different concentrations of RASF was added. To activate non-canonical NF-κB signaling, lymphotoxinα1β2 (LT) or LIGHT was added. After 48 hours, spheroids were fixed and imaged through confocal microscopy. Cumulative EC sprout length was quantified using Leica QWin Plus software. As a proof of concept and to demonstrate NIK dependency of LT- or LIGHT-induced angiogenesis, EC were transfected with non-targeting or NIK-targeting siRNA before incorporation into the model and subsequent changes in sprout formation were measured. Results: Confocal analysis of the 3D model showed that spheroids formed sprouts containing true capillary-like structures. RASF induced sprout formation at all concentrations with a significant increase observed at 10% (p<0.05). LT and LIGHT also caused significant increases in cumulative sprout length and this LTβR-induced sprout formation was significantly decreased by siRNA-mediated knockdown of NIK in EC as compared to the non-targeting controls (p<0.05). Significant decreases in sprout formation were also observed in the presence of various established inhibitors of angiogenesis. Conclusions: The 3D model is an effective tool for studying RA angiogenesis. In this novel model, two key cellular mediators of this process are incorporated, EC and RA FLS, in addition to RASF, and true capillary-like structures are formed which can be easily quantified. Using this system, we have demonstrated that activation of the non-canonical NF-κB pathway induces angiogenesis in a NIK-dependent fashion. Therefore, targeting NIK may have therapeutic potential in reducing pathological angiogenesis and halting disease progression. Additional studies testing the efficacy of pharmacological NIK inhibitors are currently underway. We anticipate that this novel 3D model can be used to screen new pharmacological inhibitors of angiogenesis. Disclosure of Interest: None declared DOI: 10.1136/annrheumdis-2015-eular.4698Citation: Annals of the Rheumatic Diseases, volume 74, supplement 2, year 2015, page 654Session: Cytokines and inflammatory mediators (Poster Presentations )