Abstract

OBJECTIVE:

To examine the effect of hypoxia on Notch-1 signaling pathway components and angiogenesis in inflammatory arthritis.

METHODS:

Expression and regulation of Notch-1, its ligand DLL-4 and downstream signaling components (Hrt-1, Hrt-2) and HIF1α under normoxic and hypoxic conditions (1-3%) were assessed by immunohistology, dual-immunofluorescent staining (Notch-1/Factor VIII), Western blotting and Real-time PCR. In vivo synovial tissue oxygen levels (tpO(2) ) were measured under direct visualisation at arthroscopy. Microvascular endothelial cell (dHMVEC) activation under hypoxic conditions in the presence of Notch-1 siRNA, γ-secretase inhibitor (DAPT) or dimethyloxalylglycine (DMOG) was assessed by matrigel tube formation assay, migration assay, invasion assay and MMP2/9 zymography.

RESULTS:

Notch-1, its ligand DLL-4 and Hrt-1 expression were demonstrated in synovial tissue, with strongest expression localised to perivascular/vascular regions. Localization of Notch-1 to synovial endothelium was confirmed by dual-immunofluorescent staining. Notch-1 intracellular domain (IC) expression was significantly higher in synovial tissue from patients with tpO(2) <20mmHg (<3% O(2) ) compared to those with tpO(2) >20mmHg (>3% O(2) ). Exposure of dHMVEC to 3% hypoxia induced HIF1α and Notch-1 IC protein expression and DLL-4, Hrt-1 and Hrt-2 mRNA expression. DMOG directly induced Notch-1 IC expression, while siRNA Notch-1 inhibited hypoxia-induced HIF1α expression, suggesting that Notch-1/HIF1α signaling is bi-directional. Finally 3% hypoxia-induced angiogenesis, EC migration, EC invasion, and pro-MMP-2 and-9 activities were inhibited by Notch-1 siRNA and/or the γ-secretase inhibitor DAPT.

CONCLUSION:

Notch-1 is expressed in synovial tissue and increased Notch-1 IC expression is associated with low in vivo tpO(2) . Furthermore Notch-1/HIF1α interactions mediate hypoxia-induced angiogenesis and invasion in inflammatory arthritis.

Copyright © 2012 by the American College of Rheumatology.