Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice
Fig 6
Effect of CaD on VEGF165 binding to VEGFR-1/2, heparin and HS.
(A) bt-VEGF165 binding assay to immobilized VEGR-1/2 in the presence of increasing concentrations of CaD. Data are expressed as percentage of control (no CaD) ± SD in binding from four independent experiments; *p < 0.05 vs. no CaD. (B) bt-VEGF165 binding in the absence or presence of 1 μg/ml heparin or CaD (100 μM) to VEGFR-1 and VEGFR-2. Data are expressed as mean OD at 450 nm ± SD from four independent experiments; *p < 0.05 vs. no heparin or CaD (Ctrl). (C) Representative images were obtained by proximity ligation assay. Cells were fixed and incubated with antibodies to human VEGF (goat IgG) and to human VEGFR-2 (rabbit IgG) (upper panel) or to VEGF and heparin sulphate (mAb 10E4) (middle panel) or to VEGFR-2 and HS (lower panel) followed by proximity ligation assay reagents. Each red dot indicates a protein interaction. Nuclei are shown in blue. The antibody (Ab) control panel represents cells incubated with the corresponding IgG. (D) Quantified data presented as red dots (signal intensity)/cell. The error bar represents SD. n = 3 separate experiments *P<0.05, ***P<0.001 relative to that of VEGF alone. All images were taken with a Leica DMI3000 B microscopy, scale bar 100 μm and analyzed with NIH ImageJ software.