Targeting non-canonical nuclear factor-κB signalling attenuates neovascularization in a novel 3D model of rheumatoid arthritis synovial angiogenesis

Objective. Angiogenesis is crucial in RA disease progression. Lymphotoxin beta receptor (LT beta R)-induced activation of the non-canonical nuclear factor-kappa B (NF-kappa B) pathway via NF-kappa B-inducing kinase (NIK) has been implicated in this process. Consequently, inhibition of this pathway may hold therapeutic potential in RA. We describe a novel three-dimensional (3D) model of synovial angiogenesis incorporating endothelial cells (ECs), RA fibroblast-like synoviocytes (RAFLSs) and RA synovial fluid (RASF) to further investigate the contributions of NF-kappa B in this process. Methods. Spheroids consisting of RAFLSs and ECs were stimulated with RASF, the LT beta R ligands LT beta and LIGHT, or growth factor bFGF and VEGF, followed by quantification of EC sprouting using confocal microscopy and digital image analysis. Next, the effects of anginex, NIK-targeting siRNA (siNIK), LT beta R-Ig fusion protein (baminercept) and a novel pharmacological NIK inhibitor were investigated. Results. RASF significantly promoted sprout formation, which was blocked by the established angiogenesis inhibitor anginex (P < 0.05). LT beta and LIGHT induced significant sprouting (P < 0.05), as did bFGF/ VEGF (P < 0.01). siNIK pre-treatment of ECs led to reductions in LT beta R-induced vessel formation (P < 0.05). LT beta R -Ig not only blocked LT beta-or LIGHT-induced sprouting, but also RASF-induced sprouting (P < 0.05). The NIK inhibitor blocked angiogenesis induced by LT beta, LIGHT, growth factors (P< 0.05) and RASF (P < 0.01). Conclusion. We present a novel 3D model of synovial angiogenesis incorporating RAFLSs, ECs and RASF that mimics the in vivo situation. Using this system, we demonstrate that non-canonical NF-kB signalling promotes neovascularization and show that this model is useful for dissecting relative contributions of signalling pathways in specific cell types to angiogenic responses and for testing pharmacological inhibitors of angiogenesis.