Figure 6

Interactions of hypoxia and cytokines modulate induction of angiogenic activity by rheumatoid arthritis fibroblast-like synoviocytes. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were cultured in normoxia (21% oxygen), hypoxia (1% oxygen) or with 10 ng/ml IL-4 or TNFα for 24 hours. Alternatively the cells were co-stimulated with cytokines whilst cultured in hypoxia to mimic the environment in RA joints. Supernatants from these cultures were applied to wells of a 96-well plate containing growth factor-reduced matrigel and pre-seeded human microvascular endothelial cell (HMEC)-1 cells and left to form tubules for 4 to 6 hours. (a) A single picture was captured from the centre of each well with a camera (QICAM FAST; QImaging) attached to a microscope (CKX41; Olympus), with a representative example for each condition shown here. Using AngioSys Image Analysis software we analysed several parameters of angiogenesis: (b) percentage of field area covered by HMEC-1 tubules, (c) number of tubules, and (d) tubule junctions formed in the field area. Data expressed as the mean ± standard error of the mean of ≥3 independent experiments with supernatants from each condition assayed in triplicate wells. Samples analysed using one-way analysis of variance with Bonferroni's post-hoc test for multiple comparisons versus control (*P < 0.05, **P < 0.01, ***P < 0.001).