Abstract
BackgroundAngiogenesis 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.MethodsMicrovascular 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).ResultsAng2 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 angiogenenic 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).ConclusionTLR2 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.
Highlights
Rheumatoid Arthritis (RA) is a chronic progressive autoimmune disease characterised by proliferation of synovial membrane (SM), which leads to degradation of articular cartilage and subchondral bone
It has been shown that dominant negative forms of the essential TLR2 adapter molecules MyD88 and MAL/TIRAP ablate pro-inflammatory cytokine production in RA synoviocytes demonstrating that TLR2 and TLR4 signalling pathways are functional in these cells [10]
The aim of this study is to examine the effects of TLR2 activation on angiogenic processes using RA synovial explants and HMVEC cultures
Summary
Rheumatoid Arthritis (RA) is a chronic progressive autoimmune disease characterised by proliferation of synovial membrane (SM), which leads to degradation of articular cartilage and subchondral bone. RA synoviocytes manifest an abnormal phenotype characterised by increased proliferation, resistance to apoptosis and invasiveness of adjacent tissue. This leads to a self perpetuating and persistent infiltration of immune cells resulting in synoviocyte hyperplasia which transforms SM into an aggressive, tumour-like tissue -. 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
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