Figure 2.

Hypoxia-induced hypoxia-inducible factor isoform dependence of angiogenic genes in human rheumatoid arthritis fibroblast-like synoviocytes. Hypoxia-induced hypoxia-inducible factor (HIF) isoform dependence of ephrin A3 (EFNA3), angiopoietin-like (ANGPTL)-4, leptin and vascular endothelial growth factor (VEGF) in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). RA FLS were transiently transfected with siRNA oligonucleotides complementary to hypoxia-inducible factor (HIF)-1α (siHIF-1α) or HIF-2α (siHIF-2α) or both simultaneously. An siRNA oligonucleotide complementary to siLuc was used as control. The cell cultures were subsequently exposed to 1% oxygen (hypoxia) for 24 hours. Total RNA was isolated and cDNA generated, and the mRNA level of (a) leptin and (c) ANGPTL-4, (e) VEGF and (g) EFNA3 was determined using quantitative PCR. Changes in mRNA expressed as fold-change relative to levels in the siLuc transfected normoxic controls set as 1.0 (dotted line). The secretion of (b) leptin, (d) ANGPTL-4 and (f) VEGF protein was measured using ELISA. Data expressed as mean ± standard error of the mean of ≥3 independent experiments with sample assayed in triplicate, and were analysed using one-way analysis of variance with Bonferroni's post-hoc test for multiple comparisons versus hypoxic siLuc transfected cells (*P < 0.05, **P < 0.01, ***P < 0.001).