Figure 2
CuB inhibits angiogenesis in vitro and ex vivo.
(A) Wound healing assays were performed in the subconfluent HUVECs. Images of wound closure were captured at 0 h and 24 h after treatment. Data were expressed as percent wound closure compared to control. (B) HUVECs were seeded onto the upper surface of the matrigel-coated chambers with indicated concentrations of CuB for 24 h. Images of invaded cells were captured and total numbers of invaded cells were counted in 10 different microscopic fields. Each treatment was given in triplicates and data were plotted as percent invasion compared to control. (C) HUVECs were seeded in matrigel-precoated 96-well plates and treated with indicated concentrations of CuB for 6 h. Images of tube formation from each group was captured and total number of nodes and branches were calculated. Each treatment was given in triplicates and results were represented as average number of nodes and branches present in each treatment group. (D) The anti-angiogenic potential of CuB was determined by performing chorio-allantoic membrane (CAM) assay. Images x & y show the CAM membrane exposed after 8 days incubation of fertilized egg. Image z shows the effect of 25 nM CuB on the pre-existing vasculature after two days of exposure. The images are representative of three independent experiments. (E) The in vivo anti-angiogenic potential of CuB was determined by implanting matrigel plugs in the right flank of BALB/c mice and then by treating the mice with 0.1 mg/kg and 0.2 mg/kg b.w. doses of CuB. Imatinib at 60 mg/kg b.w. dose was used as positive control. Matrigel plugs from different animal groups were excised and photographed. H& E staining was performed to visualize the vascular formation. The arrows in the histopathological sections indicate the presence of endothelial cells. The images are representative of each group. In each case, results were obtained from three independent experiments, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.
