INTRODUCTION: Hypoxia and T-helper cell 1 (Th1) cytokine-driven inflammation are 
key features of rheumatoid arthritis (RA) and contribute to disease pathogenesis 
by promoting angiogenesis. The objective of our study was to characterise the 
angiogenic gene signature of RA fibroblast-like synoviocytes (FLS) in response 
to hypoxia, as well as Th1 and T-helper cell 2 (Th2) cytokines, and in 
particular to dissect out effects of combined hypoxia and cytokines on hypoxia 
inducible transcription factors (HIFs) and angiogenesis.
METHODS: Human angiogenesis PCR arrays were used to screen cDNA from RA FLS 
exposed to hypoxia (1% oxygen) or dimethyloxalylglycine, which stabilises HIFs. 
The involvement of HIF isoforms in generating the angiogenic signature of RA FLS 
stimulated with hypoxia and/or cytokines was investigated using a DNA-binding 
assay and RNA interference. The angiogenic potential of conditioned media from 
hypoxia-treated and/or cytokine-treated RA FLS was measured using an in vitro 
endothelial-based assay.
RESULTS: Expression of 12 angiogenic genes was significantly altered in RA FLS 
exposed to hypoxia, and seven of these were changed by dimethyloxalylglycine, 
including ephrin A3 (EFNA3), vascular endothelial growth factor (VEGF), 
adipokines angiopoietin-like (ANGPTL)-4 and leptin. These four proangiogenic 
genes were dependent on HIF-1 in hypoxia to various degrees: EFNA3 >ANGPTL-4 
>VEGF >leptin. The Th1 cytokines TNFα and IL-1β induced HIF-1 but not HIF-2 
transcription as well as activity, and this effect was additive with hypoxia. In 
contrast, Th2 cytokines had no effect on HIFs. IL-1β synergised with hypoxia to 
upregulate EFNA3 and VEGF in a HIF-1-dependent fashion but, despite strongly 
inducing HIF-1, TNFα suppressed adipokine expression and had minimal effect on 
EFNA3. Supernatants from RA FLS subjected to hypoxia and TNFα induced fewer 
endothelial tubules than those from FLS subjected to TNFα or hypoxia alone, 
despite high VEGF protein levels. The Th2 cytokine IL-4 strongly induced 
ANGPTL-4 and angiogenesis by normoxic FLS and synergised with hypoxia to induce 
further proangiogenic activity.
CONCLUSION: The present work demonstrates that Th1 cytokines in combination with 
hypoxia are not sufficient to induce angiogenic activity by RA FLS despite HIF-1 
activation and VEGF production. In contrast, Th2 cytokines induce angiogenic 
activity in normoxia and hypoxia, despite their inability to activate HIFs, 
highlighting the complex relationships between hypoxia, angiogenesis and 
inflammation in RA.