Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

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 TNF alpha and IL-1 beta 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-1 beta synergised with hypoxia to upregulate EFNA3 and VEGF in a HIF-1-dependent fashion but, despite strongly inducing HIF-1, TNF alpha suppressed adipokine expression and had minimal effect on EFNA3. Supernatants from RA FLS subjected to hypoxia and TNF alpha induced fewer endothelial tubules than those from FLS subjected to TNF alpha 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.