Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice
Fig 3
Inhibition of endothelial cell proliferation, invasion and migration by CaD.
HUVECs were plated in 96-well plates, allowed to attach overnight, and then cultured for 24 h-48 h with CaD in the presence of VEGF165 (25 ng/ml)-CaD mixture (A) at the indicated concentration. The proliferation was measured as described in the Materials and Methods section. (B) Serum-starved HUVECs were allowed to migrate through trans-well membranes towards a vehicle, VEGF165 (25 ng/ml) and/or CaD at the indicated concentration for 24 h. Cells that had migrated to the underside of the membrane were processed for calcein-AM staining as described in the materials and methods section. (C) A monolayer of HUVECs was scratched and fresh medium containing vehicle, VEGF and/or CaD was then added. After 14 h, migration distance of HUVECs was quantified. Original magnification, 40x. (D) HUVECs were incubated with vehicle, VEGF165 (25 ng/ml) and CaD for 15 min. Cells were stained with phalloidin-Alexa Fluor 488 (left images) and DAPI (middle images). Merged view is represented in the right images. Original magnification, 40x. Scale bars represent 5 μm. The results shown are the means ± SD of four independent experiments conducted in triplicate. *P<0.05, **P<0.01, versus VEGF165-treated HUVECs.