Background Angiogenesis plays a crucial role in the progression of rheumatoid arthritis (RA) and is considered a switch from acute to chronic inflammation. Previously, we have demonstrated that NF-κB inducing kinase (NIK) mediated activation of the non-canonical NF-κB pathway in endothelial cells (EC) induces angiogenesis. To further investigate the role of this pathway in RA angiogenesis, we developed an innovative 3D model that incorporates both EC and RA fibroblast-like synoviocytes (FLS) with the option to also add immune cells, thereby generating a model more representative of synovial angiogenesis.

Objective To develop a novel 3D in vitro model to study the interaction between RA FLS, EC and immune cells to further delineate the role of the non-canonical NF-κB pathway in pathological angiogenesis.

Methods In the traditional 2D model, RA FLS and HUVEC were co-cultured with or without the non-canonical NF-κB signalling inducing stimuli lymphotoxin α1β2 (LT) and LIGHT, or VEGF as a positive control. EC were visualised through immunohistochemical staining of CD31. In the novel 3D model, spheroids of HUVEC and RA FLS, pre-incubated with green or orange cell tracker dye, were plated in a collagen solution with medium containing LT, LIGHT or bFGF/VEGF. After 48 hours, spheroids were imaged through confocal microscopy. Cumulative EC sprout length and the number of sprouts was quantified using Leica QWin Plus software.

Results In the 2D model, LIGHT induced a significant increase in EC proliferation (p<0.05) and the LT-induced increase neared significance. Confocal analysis of the 3D model showed that spheroids containing HUVEC and RA FLS formed sprouts under all conditions. Importantly, both LT and LIGHT caused significant increases (p<0.05) in cumulative sprout length and total number of sprouts, which was comparable to that of bFGF/VEGF. Preliminary results indicate that siRNA-mediated targeting of NIK inhibits this process effectively.

Conclusion We demonstrate that induction of non-canonical NF-κB signalling by LT or LIGHT enhances angiogenic responses in EC in both models. However, the new 3D model allows visualization of actual blood vessel formation and incorporation of immune cells in future studies. Based on our findings, targeting NIK may be a novel therapeutic approach to block pathological angiogenesis in RA, thereby halting disease progression.