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. 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-1α interactions mediate hypoxia-induced angiogenesis and invasion in inflammatory arthritis.
|
Authors | Wei Gao, Catherine Sweeney, Mary Connolly, Aisling Kennedy, Chin Teck Ng, Jennifer McCormick, Douglas J Veale, Ursula Fearon |
Journal | Arthritis and rheumatism
(Arthritis Rheum)
Vol. 64
Issue 7
Pg. 2104-13
(Jul 2012)
ISSN: 1529-0131 [Electronic] United States |
PMID | 22275240
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
|
Copyright | Copyright © 2012 by the American College of Rheumatology. |
Chemical References |
- NOTCH1 protein, human
- Receptor, Notch1
- Vascular Endothelial Growth Factor A
- Matrix Metalloproteinase 2
- Matrix Metalloproteinase 9
|
Topics |
- Arthritis, Rheumatoid
(metabolism)
- Cell Movement
(physiology)
- Endothelial Cells
(metabolism)
- Female
- Humans
- Hypoxia
(metabolism)
- Knee Joint
(metabolism)
- Male
- Matrix Metalloproteinase 2
(metabolism)
- Matrix Metalloproteinase 9
(metabolism)
- Neovascularization, Pathologic
(metabolism)
- Receptor, Notch1
(metabolism)
- Synovial Membrane
(metabolism)
- Vascular Endothelial Growth Factor A
(metabolism)
|