title: Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2.
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.
authors.affiliation: Department of Rheumatology, Translational Research Group, Dublin Academic Medical Centre, St Vincent's University Hospital, Dublin, Ireland.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.forename: Catherine
authors.initials: C
authors.lastname: Sweeney
authors.forename: Ceara
authors.initials: C
authors.lastname: Walsh
authors.forename: Peadar
authors.initials: P
authors.lastname: Rooney
authors.forename: Jennifer
authors.initials: J
authors.lastname: McCormick
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Veale
authors.forename: Ursula
authors.initials: U
authors.lastname: Fearon
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D042702:Angiopoietin-2 / Q000502:physiology*; D015535:Arthritis, Psoriatic / Q000503:physiopathology*; D001172:Arthritis, Rheumatoid / Q000503:physiopathology*; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000502:physiology*; D005260:Female; D006801:Humans; D015850:Interleukin-6 / Q000502:physiology; D016209:Interleukin-8 / Q000502:physiology; D008297:Male; D008875:Middle Aged; D009389:Neovascularization, Pathologic / Q000503:physiopathology*; D010003:Osteoarthritis / Q000503:physiopathology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051881:Receptor, Notch1 / Q000502:physiology*; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000502:physiology*; D015398:Signal Transduction; D013583:Synovial Membrane / Q000503:physiopathology*; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology*
chemical_list: D042702:Angiopoietin-2; C508606:CXCL8 protein, human; C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; C497761:NOTCH1 protein, human; C106075:NOTCH4 protein, human; D011518:Proto-Oncogene Proteins; D051881:Receptor, Notch1; D000074562:Receptor, Notch4; D051880:Receptors, Notch; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A