title: Targeting non-canonical nuclear factor-κB signalling attenuates neovascularization in a novel 3D model of rheumatoid arthritis synovial angiogenesis.
abstract: 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.
  
  
  
  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.
  
  
  
  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).
  
  
  
  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.
authors.affiliation: Amsterdam Rheumatology and immunology Center.|Department of Clinical Immunology & Rheumatology.|Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Chrissta X
authors.initials: CX
authors.lastname: Maracle
authors.affiliation: AstraZeneca R&D, Gothenburg, Sweden.
authors.forename: Paulina
authors.initials: P
authors.lastname: Kucharzewska
authors.affiliation: Amsterdam Rheumatology and immunology Center.|Department of Clinical Immunology & Rheumatology.|Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Boy
authors.initials: B
authors.lastname: Helder
authors.affiliation: Amsterdam Rheumatology and immunology Center.|Department of Clinical Immunology & Rheumatology.|Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Corine
authors.initials: C
authors.lastname: van der Horst
authors.affiliation: MD Anderson Cancer Centre, University of Texas, Houston, TX, USA.
authors.forename: Pedro
authors.initials: P
authors.lastname: Correa de Sampaio
authors.affiliation: Amsterdam Rheumatology and immunology Center.|Department of Clinical Immunology & Rheumatology.|Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Ae-Ri
authors.initials: AR
authors.lastname: Noort
authors.affiliation: Amsterdam Rheumatology and immunology Center.|Department of Clinical Immunology & Rheumatology.|Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Katinka
authors.initials: K
authors.lastname: van Zoest
authors.affiliation: Angiogenesis Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Arjan W
authors.initials: AW
authors.lastname: Griffioen
authors.affiliation: AstraZeneca R&D, Gothenburg, Sweden.
authors.forename: Henric
authors.initials: H
authors.lastname: Olsson
authors.affiliation: Amsterdam Rheumatology and immunology Center s.w.tas@amc.uva.nl.|Department of Clinical Immunology & Rheumatology.|Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Sander W
authors.initials: SW
authors.lastname: Tas
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D006801:Humans; D053259:Lymphotoxin beta Receptor; D053308:Lymphotoxin-beta / Q000494:pharmacology; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism*; D009389:Neovascularization, Pathologic / Q000378:metabolism* / Q000473:pathology; D010455:Peptides / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D015398:Signal Transduction; D013582:Synovial Fluid; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology; D000070918:Synoviocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14 / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D000097782:NF-kappaB-Inducing Kinase
chemical_list: D005346:Fibroblast Growth Factors; D053259:Lymphotoxin beta Receptor; D053308:Lymphotoxin-beta; D016328:NF-kappa B; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14; D042461:Vascular Endothelial Growth Factor A; D000097782:NF-kappaB-Inducing Kinase; C558923:anginex peptide; C000599771:baminercept
keywords: NF-κB signalling; NIK; angiogenesis; rheumatoid arthritis