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
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
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DOI：
10.1074/jbc.m414581200
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发表时间：
2005-09-09
影响因子：
4.8
通讯作者：
Kimata, K
中科院分区：
文献类型：
--
作者：
Ashikari- Hada, S;Habuchi, H;Kimata, K
血管内皮生长因子（VEGF）是一类具有强大血管生成活性的糖蛋白。我们以前报道过肝素对VEGF有亲和力（165），VEGF的主要亚型，而2- o -去硫肝素和6- o -去硫肝素有弱但显著的亲和力(ashikarii - hada， S., Habuchi， H., Kariya， Y., Itoh， N., Reddi, a.h .，和Kimata， K. (2004) J. Biol。化学，279,12346-12354)。在这项研究中，我们首先检测了肝素和改性肝素（完全去硫的n -硫酸肝素、2- o -去硫的肝素和6- o -去硫的肝素）对VEGF（165）依赖性的有丝分裂活性和人脐静脉内皮细胞I型胶原凝胶的管形成的影响。肝素增强了VEGF活性，但改性肝素没有增强VEGF活性，这表明肝素/硫酸肝素中己醛酸的2- o -硫酸盐基团和n -磺酰基氨基的6- o -硫酸盐基团对VEGF活性都是必需的。然后，我们检查了VEGF受体（VEGFR）的激活，以了解其机制。我们有几个新的发现；1)肝素使VEGF（165）诱导的VEGFR-2磷酸化增强1.7倍；2)肝素酶/肝素酶处理使细胞表面硫酸肝素耗损，而氯酸钠处理优先还原细胞表面HS的三硫酸双糖单位，导致这种磷酸化减少，表明肝素样结构域参与了VEGFR-2的磷酸化；3)没有外显子7编码区的VEGF(121)，不具有与肝素结合的能力，没有表现出这些作用。因此，硫酸肝素/肝素的肝素样结构域可能通过外显子7编码区与VEGF（165）和VEGFR-2形成复合物，从而增强VEGF（165）依赖的信号传导。
Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF(165), 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 VEGF(165)-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 VEGF(165) 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 VEGF(165)-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) VEGF(121), 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 VEGF(165) and VEGFR-2 via the exon 7-encoded region, thereby enhancing VEGF(165)-dependent signaling.