Effect of dexrazoxane on angiogenesis in vitro. (A) Effect of Dexrazoxane on cell proliferation in endothelial cells. Cell proliferation was measured using the MTS assay 3 days after treatment of HDMEC (–▪–) and HUVEC (⧫) with Dexrazoxane. A one-way ANOVA showed that increasing the concentration of Dexrazoxane statistically significantly reduced cell proliferation (^***P-value <0.001) in both HUVECs and HDMECs. (B) Effect of Dexrazoxane on endothelial cell growth over 5 days. Cell numbers were counted on days 3, 4 and 5 after a single dose of 50 μM Dexrazoxane. The treatment groups were HUVEC control (▪), HUVEC treated with 50 μM Dexrazoxane (), HDMEC control () and HDMEC treated with 50 μM Dexrazoxane (□). Statistical significance of Dexrazoxane treatment vs control was determined using a t-test, indicated by ^*** (P-value <0.001) in the figure. Results show that Dexrazoxane treatment significantly reduced the number of cells. (C) Effect of increasing doses of dexrazoxane on the number of HUVEC in G2M phase of cell cycle. A one-way ANOVA shows that increasing the concentration of dexrazoxane statistically significantly increases the number of cells in G2-M phase with a P-value <0.001 (^***). (D) Effect of dexrazoxane on endothelial cell migration. The effect of 50 μM dexrazoxane on VEGF stimulated HUVEC migration was tested vs a control. Using a t-test, no statistical significance was found between the VEGF alone and VEGF+Dexrazoxane-treated migration of cells. However, in contrast, significant P-values of <0.001 (^***) were found comparing both of these to the untreated control cell migration. (E) Effect of dexrazoxane on the aortic ring assay. Aortic ring sprouting was scored on a scale of 0 (no sprouts), 1 (a few sprouts), 2 (intermediate sprouting), 3 (substantial sprouting), 4 (complete sprouting). The sprouting from each ring was assessed by two independent observers and the inter-observer variability was found to be <0.001. A 50-μM dose of Dexrazoxane had a statistically significant effect in reducing the sprouting index of the aortic ring (t-test, P-value <0.001).

Effect of dexrazoxane in vivo. Effect of dexrazoxane on angiogenesis in vivo, using the mouse sponge assay. (A) The number of vessels per unit area of sponge was counted after injection of carrier solution only, bFGF only and bFGF with 50 μM dexrazoxane. The top left panel shows a typical example of a sponge injected with bFGF alone, whereas the bottom left panel shows a typical example of a sponge injected with bFGF with dexrazoxane. (B) The number of vessels per unit area of sponge was counted after injection of 50 μM dexrazoxane into the tail vein on the day after injection of bFGF directly into the sponge. Both sponge assays produced a statistical significant difference between carrier solution control vs bFGF (P-value <0.01) and bFGF vs bFGF with dexrazoxane. No significant difference was found between carrier solution control vs bFGF with dexrazoxane.

Upregulation of thrombospondin-1 by dexrazoxane in vitro. (A) Western blotting of HUVEC whole cell lysate and medium after treatment with 50 μM Dexrazoxane for 2, 5, 9 and 24 h displayed an increase in THBS-1 protein expression over the time course. (B) This result was confirmed by ELISA using conditioned medium of cells treated with Dexrazoxane at different time points.

Downregulation of THBS-1 by siRNA. (A) Western blot showing the downregulation of THBS-1 in HUVEC at 24 and 48 h posttransfection with THBS-1 targeting siRNA. (B) Real-time PCR showing the downregulation (P-value <0.001) of THBS-1 in HUVEC, targeted by THBS-1 siRNA at 24 and 48 h posttransfection. (C and D) Real-time PCR showing no significant upregulation of the interferon response genes ISG 20 and OAS I in HUVEC targeted with THBS-1 siRNA (duplex 1) at 24 and 48 h posttransfection.

Effect of downregulation of THBS-1 on cell proliferation after treatment with dexrazoxane. (A) MTT assay showing cell proliferation 5 days posttransfection with THBS-1 siRNA and dose–response to 50 μM dexrazoxane given at 24-h intervals. The results show that the anti-proliferative effect of dexrazoxane is partially mediated by THBS-1 as its knockdown shows significant increase in cell proliferation (P-value <0.01 at 25 μM through to 100 μM) as compared with the untransfected and negative controls. (B) Real-time PCR showing statistically significant downregulation (P-value <0.01) of THBS-1 in HUVEC treated with THBS-1 siRNA at 5 days posttransfection. Although still significantly differentially expressed (knockeddown) at 5 days posttransfection, the percentage knockdown has decreased by 38% as compared with 24 h. (C) MTT assay showing cell proliferation 3–7 days posttransfection with THBS-1 siRNA at day 0 and again at day 3 with concurrent treatment with 50 μM dexrazoxane at 24-h intervals. (D) Real-time PCR showing downregulation of THBS-1 in HUVEC from 3 to 7 days with transfection with THBS-1 siRNA taking place at day 0 and again at day 3. The downregulation of THBS-1 was statistically significant at all time points, P-value <0.001. (E) Proportion of HUVEC in G2M phase of cell cycle 5 days posttransfection with THBS-1 siRNA and treatment with 50 μM dexrazoxane at 24-h intervals. Dexrazoxane treated cells alone vs the control showed statistical difference (P-value >0.01) in the number of cells in G2M phase. This effect was reversed with THBS-1 knockdown.