Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins.
Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF165, the major isoform of VEGF, whereas 2-O-desulfated heparin and 6-O-desulfated heparin have weak but significant affinity (Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., and Kimata, K. (2004) J. Biol. Chem. 279, 12346-12354). In this study, we first examined the effect of heparin and modified heparins (completely desulfated N-sulfated heparin, 2-O-desulfated heparin, and 6-O-desulfated heparin) on VEGF165-dependent mitogenic activity and tube formation on type I collagen gels of human umbilical vein endothelial cells. Both were enhanced by heparin, but not by modified heparins, suggesting that both the 2-O-sulfate group of hexuronic acid and the 6-O-sulfation group of N-sulfoglucosamine in heparin/heparan sulfate are necessary for VEGF165 activity. We then examined the activation of VEGF receptor (VEGFR) to understand the mechanism. We have made several new findings; 1) heparin yielded a 1.7-fold enhancement of VEGF165-induced phosphorylation of VEGFR-2; 2) depletion of cell surface heparan sulfate by heparinase/heparitinase treatment and preferential reduction of trisulfated disaccharide units of cell surface HS by sodium chlorate treatment resulted in the reduction of such phosphorylation, suggesting the involvement of a heparin-like domain in the phosphorylation of VEGFR-2; and 3) VEGF121, an isoform without the exon 7-encoded region, which has no capacity to bind to heparin, did not show these effects. It is therefore likely that a heparin-like domain of heparan sulfate/heparin forms a complex with VEGF165 and VEGFR-2 via the exon 7-encoded region, thereby enhancing VEGF165-dependent signaling.
Ashikari-Hada
Satoko
S
Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan.
Habuchi
Hiroko
H
Kariya
Yutaka
Y
Kimata
Koji
K
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D005091:Exons; D006493:Heparin / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D008934:Mitogens / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010766:Phosphorylation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D008934:Mitogens; D042461:Vascular Endothelial Growth Factor A; D006493:Heparin; D040301:Vascular Endothelial Growth Factor Receptor-2