Background/objective Angiogenesis contributes to rheumatoid arthritis (RA) pathogenesis, however, many models focus solely on endothelial cells (EC). We developed a 3D-spheroid model including both EC and RA fibroblast-like-synoviocytes (FLS) to study angiogenesis associated with RA and to test efficacy of several inhibitors targeting this process. Previous work demonstrated a role for the non-canonical NF-κB pathway and its main regulator, NF-kB inducing kinase (NIK) in pathological angiogenesis. Here we aim to use the 3D model to further characterise its contribution to neovascularization.
Methods Human umbilical vein EC (HUVEC) were combined with RA FLS in spheroids and placed in a collagen gel. Spheroids were stimulated with RA synovial fluid (SF), growth factors (GF), LT or LIGHT. To establish NIK dependency in EC, EC were pre-transfected with a non-targeting or NIK-targeting siRNA before incorporation into spheroids. Pharmacological inhibitors were also added to determine their ability to abrogate EC sprouting induced by 10% RASF. Sprouting was imaged by confocal microscopy and quantified using the Leica QWin Plus software.
Results Spheroids formed sprouts under all conditions with significant increases observed upon stimulation with LT, LIGHT, 10% RA SF (p < 0.05) and growth factors (p < 0.01), as compared to basal levels. LT and LIGHT induced sprout formation was NIK dependent as spheroids containing HUVEC transfected with NIK targeting siRNA had reductions in vessel formation (p < 0.05) as compared to controls. Anginex blocked sprout formation induced not only by growth factors, but also by that induced by RA SF (p < 0.05). Inhibitors of the non-canonical NF-kB pathway were able to attenuate cumulative sprout growth promoted by LT, LIGHT and RA SF (p < 0.05).
Conclusion The 3D model is an effective tool for studying synovial angiogenesis in that it incorporates several elements essential to the process namely EC, RA FLS and immune cell factors found in RA SF. Using this system, we have further demonstrated a role for activation of the non-canonical NF-kB pathway, and its central regulator NIK, in neovascularization associated with RA. Moreover, we have shown this method to be useful for testing inhibitors of angiogenesis and found that targeting of non-canonical NF-kB signalling to be an effective method of blocking pathological angiogenesis.
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