Vascular Endothelial Growth Factor 165 (VEGF165) Activities Are Inhibited by Carboxymethyl Benzylamide Dextran That Competes for Heparin Binding to VEGF165and VEGF165·KDR Complexes*

We have previously shown that carboxymethyl dextran benzylamide (CMDB7), a heparin-like molecule, inhibits the growth of tumors xenografted in nude mice, angiogenesis, and metastasis by altering the binding of angiogenic growth factors, including platelet-derived growth factor, transforming growth factor β, and fibroblast growth factor 2, to their specific receptors. In this study, we explore the effect of CMDB7 on the most specific angiogenic growth factor, vascular endothelial growth factor 165 (VEGF165). We demonstrate here that CMDB7 inhibits the mitogenic effect of VEGF165on human umbilical vein endothelial cells (HUV-ECs) by preventing the VEGF165-induced VEGF receptor-2 (KDR) autophosphorylation and consequently a specific intracellular signaling. In competition experiments, the binding of 125I-VEGF165to HUV-ECs is inhibited by CMDB7 with an IC50of 2 µm. Accordingly, CMDB7 inhibits the cross-linking of125I-VEGF165to the surface of HUV-ECs, causing the disappearance of both labeled complexes, 170–180 and 240–250 kDa. We show that CMDB7 increases the electrophoretic mobility of VEGF165, thus evidencing formation of a stable complex with this factor. Moreover, CMDB7 reduces the125I-VEGF165binding to coated heparin-albumin and prevents a heparin-induced increase in iodinated VEGF165binding to soluble 125I-KDR-Fc chimera. Concerning KDR, CMDB7 has no effect on 125I-KDR-Fc electrophoretic migration and does not affect labeled KDR-Fc binding to coated heparin-albumin. In the presence of VEGF165,125I-KDR-Fc binding to heparin is enhanced, and under these conditions, CMDB7 interferes with KDR binding. These data indicate that CMDB7 effectively inhibits the VEGF165activities by interfering with heparin binding to VEGF165and VEGF165·KDR complexes but not by direct interactions with KDR.