title: Notch-1 mediates hypoxia-induced angiogenesis in rheumatoid arthritis.
abstract: OBJECTIVE
  To examine the effect of hypoxia on Notch-1 signaling pathway components and angiogenesis in inflammatory arthritis.
  
  
  METHODS
  The expression and regulation of Notch-1, its ligand delta-like protein 4 (DLL-4) and downstream signaling components (hairy-related transcription factor 1 [HRT-1], HRT-2), and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions (1-3%) were assessed in synovial tissue specimens from patients with inflammatory arthritis and controls and in human dermal microvascular endothelial cells (HDMECs) by immunohistology, dual immunofluorescence staining (Notch-1/factor VIII), Western blotting, and real-time polymerase chain reaction. In vivo synovial tissue oxygen levels (tissue PO2) were measured under direct visualization at arthroscopy. HDMEC activation under hypoxic conditions in the presence of Notch-1 small interfering RNA (siRNA), the γ-secretase inhibitor DAPT, or dimethyloxalylglycine (DMOG) was assessed by Matrigel tube formation assay, migration assay, invasion assay, and matrix metalloproteinase 2 (MMP-2)/MMP-9 zymography.
  
  
  RESULTS
  Expression of Notch-1, its ligand DLL-4, and HRT-1 was demonstrated in synovial tissue, with the strongest expression localized to perivascular/vascular regions. Localization of Notch-1 to synovial endothelium was confirmed by dual immunofluorescence staining. Notch-1 intracellular domain (NICD) expression was significantly higher in synovial tissue from patients with tissue PO2 of <20 mm Hg (<3% O2) than in those with tissue PO2 of >20 mm Hg (>3% O2). Exposure of HDMECs to 3% hypoxia induced HIF-1α and NICD protein expression and DLL-4, HRT-1, and HRT-2 messenger RNA expression. DMOG directly induced NICD expression, while Notch-1 siRNA inhibited hypoxia-induced HIF-1α expression, suggesting that Notch-1/HIF-1α signaling is bidirectional. Finally, 3% hypoxia-induced angiogenesis, endothelial cell migration, endothelial cell invasion, and proMMP-2 and proMMP-9 activities were inhibited by Notch-1 siRNA and/or the γ-secretase inhibitor DAPT.
  
  
  CONCLUSIONS
  Our findings indicate that Notch-1 is expressed in synovial tissue and that increased NICD expression is associated with low in vivo tissue PO2. Furthermore, Notch-1/HIF-1α interactions mediate hypoxia-induced angiogenesis and invasion in inflammatory arthritis.
authors.affiliation: Dublin Academic Medical Centre, St. Vincent's University Hospital, and University College Dublin, Dublin, Ireland.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.forename: Catherine
authors.initials: C
authors.lastname: Sweeney
authors.forename: Mary
authors.initials: M
authors.lastname: Connolly
authors.forename: Aisling
authors.initials: A
authors.lastname: Kennedy
authors.forename: Chin Teck
authors.initials: CT
authors.lastname: Ng
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: D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D007719:Knee Joint / Q000378:metabolism; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D051881:Receptor, Notch1 / Q000378:metabolism*; D013583:Synovial Membrane / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D042461:Vascular Endothelial Growth Factor A; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9