Angiogenesis RT² Profiler™ PCR Array data and expression analysis of human rheumatoid arthritis cells. PCR arrays were used with cDNA from rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) from five RA patients and incubated for either (a) 4 hours or (b) 24 hours in 1% oxygen (hypoxia) and (c) from three RA patients exposed to 1 mM dimethyloxalylglycine (DMOG) for 24 hours. Values are fold-change versus normoxia (21% oxygen) or dimethyl sulfoxide (DMSO) control and were analysed against housekeeping gene β-actin, 18S ribosomal RNA, hypoxanthine phosphoribosyltransferase 1 and 60S ribosomal protein L13a. Genes significantly increased or decreased by a factor ≥2 (P < 0.05, dotted line) in all patients are shown. Data are mean ± standard error of the mean and were analysed by paired t test of ΔCt values, comparing normoxia versus hypoxia (a and b) or DMSO control versus DMOG (c). (d) Western blotting demonstrates induction of hypoxia-inducible factor (HIF)-1α and HIF-2α in total protein lysates from RA FLS in response to 1% oxygen or 1 mM DMOG for 24 hours. An antibody against α-tubulin was used as a loading control. A lane irrelevant to the study was removed as indicated by the line to show lanes of interest adjacent to one another. (e) Freshly dissociated total human RA synovial membrane cells from four patients were analysed for expression of vascular endothelial growth factor (VEGF), leptin, ephrin A3 (EFNA3) and angiopoietin-like (ANGPTL)-4 using quantitative PCR. Changes in mRNA are expressed as fold-change relative to levels under 21% oxygen set as 1.0 (dotted line). *P < 0.05, **P < 0.01, ***P < 0.001. EFNB2, ephrin B2; FIGF, C-fos-induced growth factor (VEGF D); HAND2, Heart and neural crest derivatives expressed 2; HPSE, heparanase; ID3, inhibitor of DNA-binding 3, dominant negative helix-loop-helix protein; NRP2, neuropillin 2; PLAU, plasminogen activator urokinase.
Larsen et al. Arthritis Research & Therapy 2012 14:R180 doi:10.1186/ar3934