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

Rheumatology (Oxford). 2017 Feb;56(2):294-302. doi: 10.1093/rheumatology/kew393. Epub 2016 Nov 17.


Objective: Angiogenesis is crucial in RA disease progression. Lymphotoxin β receptor (LTβR)-induced activation of the non-canonical nuclear factor-κB (NF-κB) pathway via NF-κB-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-κB in this process.

Methods: Spheroids consisting of RAFLSs and ECs were stimulated with RASF, the LTβR ligands LTβ 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), LTβR-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). LTβ and LIGHT induced significant sprouting (P < 0.05), as did bFGF/VEGF (P < 0.01). siNIK pre-treatment of ECs led to reductions in LTβR-induced vessel formation (P < 0.05). LTβR-Ig not only blocked LTβ- or LIGHT-induced sprouting, but also RASF-induced sprouting (P < 0.05). The NIK inhibitor blocked angiogenesis induced by LTβ, 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-κB 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.

Keywords: NF-κB signalling; NIK; angiogenesis; rheumatoid arthritis.

MeSH terms

  • Arthritis, Rheumatoid / metabolism
  • Arthritis, Rheumatoid / pathology
  • Cells, Cultured
  • Endothelial Cells / drug effects*
  • Endothelial Cells / metabolism
  • Endothelial Cells / pathology
  • Fibroblast Growth Factors / pharmacology
  • Humans
  • Lymphotoxin beta Receptor
  • Lymphotoxin-beta / pharmacology
  • Microscopy, Confocal
  • NF-kappa B / metabolism*
  • NF-kappaB-Inducing Kinase
  • Neovascularization, Pathologic / metabolism*
  • Neovascularization, Pathologic / pathology
  • Peptides / pharmacology
  • Protein Serine-Threonine Kinases / genetics
  • RNA, Small Interfering
  • Recombinant Fusion Proteins / pharmacology
  • Signal Transduction
  • Synovial Fluid
  • Synovial Membrane / metabolism
  • Synovial Membrane / pathology
  • Synoviocytes / drug effects*
  • Synoviocytes / metabolism
  • Synoviocytes / pathology
  • Tumor Necrosis Factor Ligand Superfamily Member 14 / pharmacology
  • Vascular Endothelial Growth Factor A / pharmacology


  • Lymphotoxin beta Receptor
  • Lymphotoxin-beta
  • NF-kappa B
  • Peptides
  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • Tumor Necrosis Factor Ligand Superfamily Member 14
  • Vascular Endothelial Growth Factor A
  • anginex peptide
  • Fibroblast Growth Factors
  • Protein Serine-Threonine Kinases
  • baminercept