Background Angiogenesis plays a crucial role in the formation and maintenance of the pannus in inflammatory arthritis. The Notch signaling pathway is critical for vascular angiogenesis and endothelial cell (EC) fate; however the mechanisms involved in regulating these processes in the inflamed joint remain to be elucidated.
Objectives To examine if Notch signaling pathways mediate Vascular Endothelial Growth Factor (VEGF) and Angiopoietin 2 (Ang2)-induced vascular function in the inflamed joint.
Methods Whole tissue synovial biopsies obtained at arthroscopy and human microvascular endothelial cells (HMVEC) were utilized. Notch-1 IC (intracellular domain), Notch-4 IC, Notch ligand DLL-4, downstream target genes Hrt-1 and Hrt-2 mRNA and/or protein expression was measured by Real-time PCR and/or Western Blot. VEGF/Ang2 induced EC function was assessed using transwell invasion chambers, matrigel tube formation assays and wound repair scratch assays +/- Notch-1 siRNA or a γ-secretase inhibitor (DAPT). Pro-MMP-2/9 expression was measured by zymography.
Results Notch-1 IC and 4 IC protein expression were demonstrated in RA and PsA synovial explants with undetectable level in OA patients. VEGF and Ang2 induced Notch-1 IC and 4 IC protein expression in RA synovial explants cultures ex-vivo and HMVEC, levels of which were further potentiated following stimulation with VEGF and Ang2 in combination (p<0.05). mRNA expression of Notch-1, DLL-4 and Hrt-1 and Hrt-2 were significantly induced following stimulation with VEGF and Ang2 alone and in combination (p<0.05). VEGF-induced Notch-1 IC protein expression was completely blocked in the presence of Notch-1 siRNA (p<0.05). Furthermore VEGF/Ang2-induced EC invasion, tube formation, migration and pro-MMp-2/9 expression were inhibited in the presence of Notch-1 siRNA or DAPT (p<0.05).
Conclusions This study demonstrates Notch-1 IC and -4 IC expression in RA and PsA synovial explants. VEGF and Ang2 alone induce Notch-1 IC signaling pathways, with a synergistic increase in combination. VEGF/Ang2-indcued HMVEC function is inhibited in the presence of Notch-1 siRNA and DAPT. These results suggest that in inflammatory conditions, VEGF/Ang2-induced angiogenesis is mediated in part through Notch signaling pathways.
Disclosure of Interest W. Gao: None Declared, C. Sweeney: None Declared, C. Walsh: None Declared, P. Rooney: None Declared, J. McCormick: None Declared, U. Fearon: None Declared, D. Veale Grant/Research support from: Wyeth, GSK, Abbott, Opsona, Consultant for: Wyeth, GSK, Abbott, Pfizer, Schering Plough, Centocor, Speakers Bureau: Wyeth, GSK, Abbott, Pfizer, Schering Plough, Centocor, Mundipharma