Background/Purpose:

Pathological angiogenesis is a crucial part of disease progression in rheumatoid arthritis (RA) and is often considered the switch from acute to chronic inflammation. It is a complex process mediated by several different cell types in the inflamed synovium. However, many of the in vitromodels of angiogenesis focus solely on endothelial cells (EC), even though RA fibroblast-like synoviocytes (FLS) and immune cells also contribute to angiogenesis. Hence, a system that includes RA FLS as well as RA synovial fluid (SF) containing immune cell products such as cytokines, chemokines and growth factors, would be more representative of synovial angiogenesis. We have recently demonstrated that the non-canonical NF-kB pathway, with its main regulatory enzyme NF-kB inducing kinase (NIK), plays an important role in synovial angiogenesis. Therefore, we set out to investigate whether inhibition of this pathway may hold therapeutic potential in mitigating pathological angiogenesis associated with RA.

Objective:

Generate a representative 3D in vitromodel of RA synovial angiogenesis and screen the effects of targeting the non-canonical NF-κB pathway using siRNA or small molecule pharmacological inhibitors.

Methods:

Human primary EC were co-cultured with RA FLS after pre-incubation with cell tracker dyes and incubated overnight to form spheroids. Subsequently, spheroids were plated in collagen gel and stimulated with 10% RA SF, growth factors (GF) or well-known activators of non-canonical NF-kB signaling (lymphotoxin beta (LT) or LIGHT). To establish NIK dependency, EC were pre-transfected with NIK-targeting siRNA before incorporation into spheroids. Furthermore, several pharmacological inhibitors were screened for their ability to block sprout formation. Spheroids were imaged by confocal microscopy and quantified using Leica QWin Plus software.

Results:

We established a robust 3D model of angiogenesis containing both EC and RA FLS. LT, LIGHT, RA SF and GF stimulations led to significant increases in sprout formation as compared to basal conditions (p<0.05). The LT and LIGHT induced sprout formation proved to be NIK dependent as spheroids containing EC transfected with NIK targeting siRNA had significant reductions in vessel formation as compared to the non-targeting controls (p<0.05). Sprouting promoted by GF and RA SF was significantly blocked by the angiogenesis inhibitor Anginex (p<0.05). Finally, targeting of the non-canonical pathway using a small molecule pharmacological NIK inhibitor significantly reduced sprout formation caused by LT and LIGHT, and importantly also RA SF-induced sprout formation (p<0.05).

Conclusion:

We developed a novel 3D model that incorporates essential elements of synovial inflammation, namely EC, RA FLS and RA SF, which proves to be an effective tool for studying synovial angiogenesis. Using this system, we have further demonstrated a role for the non-canonical NF-kB pathway, and its central regulator NIK, in neovascularization associated with RA. Moreover, we have shown that the 3D model is useful for testing small molecule inhibitors of angiogenesis and found that targeting non-canonical NF-kB signaling is an effective method to block pathological angiogenesis.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeting-non-canonical-nf-kappa-b-signaling-inhibits-angiogenesis-in-a-novel-3d-model-of-rheumatoid-arthritis-synovial-angiogenesis/