Article Text

Extended report
Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2
  1. Wei Gao,
  2. Catherine Sweeney,
  3. Ceara Walsh,
  4. Peadar Rooney,
  5. Jennifer McCormick,
  6. Douglas J Veale,
  7. Ursula Fearon
  1. Department of Rheumatology, Translational Research Group, Dublin Academic Medical Centre, St Vincent's University Hospital, Dublin, Ireland
  1. Correspondence to Dr Ursula Fearon, Department of Rheumatology, Dublin Academic Medical Centre, St. Vincent's University Hospital, Dublin 4, Ireland; ursula.fearon{at}ucd.ie

Abstract

Objective Notch signalling pathways are critical for angiogenesis and endothelial cell (EC) fate; however the mechanisms regulating these processes in the inflamed joint remain to be elucidated. Here, we examine whether Notch signalling mediates vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2)-induced vascular function.

Methods Notch-1 intracellular domain (Notch-1 IC), Notch-4 IC, Delta-like-ligand 4, Hes-related transcriptional repressors-1 and 2 (Hrt-1, 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 an γ-secretase inhibitor N-(N-(3,5-Difluorophenacetyl-L-alanly))-S-phenylglycine-t-Butyl Ester (DAPT) in RA synovial explants or human microvascular EC. Interleukin (IL)-6 and IL-8 were measured by ELISA and MMP2 and 9 by gelatine zymography.

Results Notch-1 IC and Notch-4 IC protein expressions were demonstrated in RA and psoriatic arthritis synovial biopsies, with minimal expression observed in Osteoarthritis (OA). VEGF and Ang2 induced Notch-1 IC/ Notch-4 IC protein expression in synovial explant cultures and human microvascular EC levels were further potentiated by VEGF/Ang2 stimulation in combination. Notch-1, Delta-like-ligand 4, and Hrt-2 mRNA expression were significantly induced by VEGF and Ang2 alone and in combination. Furthermore VEGF/Ang2-induced EC invasion, angiogenesis and migration were inhibited by Notch-1 siRNA or DAPT. Conditioned media from VEGF/Ang2 stimulated RA synovial explants induced EC tube formation, an effect that was inhibited by DAPT. Finally, DAPT significantly decreased VEGF/Ang2 induced IL-6, IL-8, MMP2 and 9 expressions in RA synovial explants.

Conclusions Notch-1 mediates VEGF/Ang2-induced angiogenesis and EC invasion in inflammatory arthritis.

  • Inflammation
  • Rheumatoid Arthritis
  • Autoimmune Diseases

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