Figure 1. IL-6 stimulates angiogenesis in the aortic ring assay.

A. Phase contrast images of aortic rings embedded in collagen I with the indicated concentrations of VEGF or human IL-6. Human and mouse IL-6 experiments were carried out with aortas isolated from wild-type C57BL/6 mice (8–12 weeks) and rat IL-6 experiments were carried out with aortas isolated from Wistar rats weighing 180-200g. B. Angiogenic sprouts were counted after a week in culture for mouse aortic rings or after 4 days in culture for the rat aortic rings following treatment with VEGF, hIL-6 or mIL-6 or rIL-6. Significant increases in microvessel sprouting are observed in VEGF and IL-6 treated rings (n=9 per group) compared with PBS-treated controls, however no difference in number of sprouts is observed between VEGF and IL-6 treated rings. The p value for medium vs VEGF (p<0.058) and medium vs VEGF (0.054) just failed to reach significance. Statistical analysis carried out using student T-test is shown as (**) p ≤ 0.01; (***) p ≤ 0.001. C. Length of sprouts was measured using ImageJ analysis. Mean length of sprouts (n=9 per group) shows no significant difference between the VEGF and IL-6 treated sprouts. D. 10nM of VEGFRi inhibited vessel sprouting in VEGF (10ng/ml) treated rings but not in rat IL-6 (10ng/ml) treated rings. Statistical analysis carried out using student T-test is shown as (**) p ≤ 0.01; (***) p ≤ 0.001 E. Aortic ring vessel stained for endothelial cells using BS1 lectin (green) and for IL-6Rα (red). F. Western blot analysis of protein extracted from mouse aortas treated with VEGF (30ng/ml) or mouse IL-6 (30ng/ml). Mouse IL-6 activated downstream pSTAT3 and VEGF induced downstream pERK levels.