Notch‐1 mediates hypoxia‐induced angiogenesis in rheumatoid arthritis

W Gao, C Sweeney, M Connolly… - Arthritis & …, 2012 - Wiley Online Library
W Gao, C Sweeney, M Connolly, A Kennedy, CT Ng, J McCormick, DJ Veale, U Fearon
Arthritis & Rheumatism, 2012Wiley Online Library
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 …
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.
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