title: Toll-like receptor 2 induced angiogenesis and invasion is mediated through the Tie2 signalling pathway in rheumatoid arthritis.
abstract: BACKGROUND
  Angiogenesis is a critical early event in inflammatory arthritis, facilitating leukocyte migration into the synovium resulting in invasion and destruction of articular cartilage and bone. This study investigates the effect of TLR2 on angiogenesis, EC adhesion and invasion using microvascular endothelial cells and RA whole tissue synovial explants ex-vivo.
  
  
  METHODS
  Microvascular endothelial cells (HMVEC) and RA synovial explants ex vivo were cultured with the TLR2 ligand, Pam3CSK4 (1 µg/ml). Angiopoietin 2 (Ang2), Tie2 and TLR2 expression in RA synovial tissue was assessed by immunohistology. HMVEC tube formation was assessed using Matrigel matrix assays. Ang2 was measured by ELISA. ICAM-1 cell surface expression was assessed by flow cytometry. Cell migration was assessed by wound repair scratch assays. ECM invasion, MMP-2 and -9 expression were assessed using transwell invasion chambers and zymography. To examine if the angiopoietin/Tie2 signalling pathway mediates TLR2 induced EC tube formation, invasion and migration assays were performed in the presence of a specific neutralising anti-Tie2mAb (10 ug/ml) and matched IgG isotype control Ab (10 ug/ml).
  
  
  RESULTS
  Ang2 and Tie2 were localised to RA synovial blood vessels, and TLR2 was localised to RA synovial blood vessels, sub-lining infiltrates and the lining layer. Pam3CSK4 significantly increased angiogenic tube formation (p<0.05), and upregulated Ang2 production in HMVEC (p<0.05) and RA synovial explants (p<0.05). Pam3CSK4 induced cell surface expression of ICAM-1, from basal level of 149±54 (MFI) to 617±103 (p<0.01). TLR-2 activation induced an 8.8±2.8 fold increase in cell invasion compared to control (p<0.05). Pam3CSK4 also induced HMVEC cell migration and induced MMP-2 and -9 from RA synovial explants. Neutralisation of the Ang2 receptor, Tie2 significantly inhibited Pam3CSK4-induced EC tube formation and invasion (p<0.05).
  
  
  CONCLUSIONS
  TLR2 activation promotes angiogenesis, cell adhesion and invasion, effects that are in part mediated through the Tie2 signalling pathway, key mechanisms involved in the pathogenesis of RA.
authors.affiliation: Department of Rheumatology, Dublin Academic Medical Centre and The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland.
authors.forename: Tajvur
authors.initials: T
authors.lastname: Saber
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Veale
authors.forename: Emese
authors.initials: E
authors.lastname: Balogh
authors.forename: Jennifer
authors.initials: J
authors.lastname: McCormick
authors.forename: Sinead
authors.initials: S
authors.lastname: NicAnUltaigh
authors.forename: Mary
authors.initials: M
authors.lastname: Connolly
authors.forename: Ursula
authors.initials: U
authors.lastname: Fearon
mesh_terms: D042702:Angiopoietin-2 / Q000378:metabolism; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000503:physiopathology*; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D055666:Lipopeptides / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D042787:Receptor, TIE-2 / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology; D046509:Tissue Culture Techniques; D051195:Toll-Like Receptor 2 / Q000819:agonists / Q000378:metabolism / Q000502:physiology*
chemical_list: D042702:Angiopoietin-2; D000906:Antibodies; D055666:Lipopeptides; C508075:Pam(3)CSK(4) peptide; D051195:Toll-Like Receptor 2; D018799:Intercellular Adhesion Molecule-1; D042787:Receptor, TIE-2; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9