Notch-1 mediates hypoxia-induced angiogenesis in rheumatoid arthritis

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 1a (HIF-1a) under normoxic and hypoxic conditions (13%) 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 ?-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-1a 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-1a expression, suggesting that Notch-1/HIF-1a signaling is bidirectional. Finally, 3% hypoxiainduced angiogenesis, endothelial cell migration, endothelial cell invasion, and proMMP-2 and proMMP-9 activities were inhibited by Notch-1 siRNA and/or the ?-secretase inhibitor DAPT. Conclusion 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-1a interactions mediate hypoxia-induced angiogenesis and invasion in inflammatory arthritis.