Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Brescia, Italy.
Arterioscler Thromb Vasc Biol. 2011 Dec;31(12):e116-27. doi: 10.1161/ATVBAHA.111.235184. Epub 2011 Sep 15.
Heparan sulfate proteoglycans (HSPGs) modulate the interaction of proangiogenic heparin-binding vascular endothelial growth factors (VEGFs) with signaling VEGF receptor-2 (VEGFR2) and neuropilin coreceptors in endothelial cells (ECs). The bone morphogenic protein antagonist gremlin is a proangiogenic ligand of VEGFR2, distinct from canonical VEGFs. Here we investigated the role of HSPGs in VEGFR2 interaction, signaling, and proangiogenic capacity of gremlin in ECs.
Surface plasmon resonance demonstrated that gremlin binds heparin and heparan sulfate, but not other glycosaminoglycans, via N-, 2-O, and 6-O-sulfated groups of the polysaccharide. Accordingly, gremlin binds HSPGs of the EC surface and extracellular matrix. Gremlin/HSPG interaction is prevented by free heparin and heparan sulfate digestion or undersulfation following EC treatment with heparinase II or sodium chlorate. However, at variance with canonical heparin-binding VEGFs, gremlin does not interact with neuropilin-1 coreceptor. On the other hand, HSPGs mediate VEGFR2 engagement and autophosphorylation, extracellular signaling-regulated kinase(1/2) and p38 mitogen-activated protein kinase activation, and consequent proangiogenic responses of ECs to gremlin. On this basis, we evaluated the gremlin-antagonist activity of a panel of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. The results demonstrate that the highly N,O-sulfated derivative K5-N,OS(H) binds gremlin with high potency, thus inhibiting VEGFR2 interaction and angiogenic activity in vitro and in vivo.
HSPGs act as functional gremlin coreceptors in ECs, affecting its productive interaction with VEGFR2 and angiogenic activity. This has allowed the identification of the biotechnological K5-N,OS(H) as a novel angiostatic gremlin antagonist.
硫酸乙酰肝素蛋白聚糖 (HSPGs) 调节促血管生成肝素结合血管内皮生长因子 (VEGFs) 与内皮细胞 (ECs) 中信号 VEGFR2 和神经纤毛核心受体的相互作用。骨形态发生蛋白拮抗剂 Gremlin 是 VEGFR2 的一种促血管生成配体，与经典 VEGFs 不同。在这里，我们研究了 HSPGs 在 VEGFR2 相互作用、信号转导以及 Gremlin 在 ECs 中的促血管生成能力中的作用。
表面等离子体共振表明，Gremlin 通过多糖的 N-、2-O 和 6-O-硫酸化基团与肝素和硫酸乙酰肝素结合，但不与其他糖胺聚糖结合。因此，Gremlin 结合 EC 表面和细胞外基质中的 HSPGs。游离肝素和肝素酶 II 或氯化钠处理后，肝素和硫酸乙酰肝素消化或硫酸化不足可防止 Gremlin/HSPG 相互作用。然而，与经典的肝素结合 VEGFs 不同，Gremlin 不与神经纤毛-1 核心受体相互作用。另一方面，HSPGs 介导 VEGFR2 结合和自身磷酸化、细胞外信号调节激酶 (1/2) 和 p38 丝裂原活化蛋白激酶的激活，以及随后 EC 对 Gremlin 的促血管生成反应。在此基础上，我们评估了一系列化学硫酸化的大肠杆菌 K5 多糖衍生物对 Gremlin 的拮抗活性。结果表明，高度 N,O-硫酸化的衍生物 K5-N,OS(H) 与 Gremlin 具有高亲和力，从而抑制了体外和体内 VEGFR2 的相互作用和血管生成活性。
HSPGs 在 ECs 中作为功能性 Gremlin 辅助受体发挥作用，影响其与 VEGFR2 的有效相互作用和血管生成活性。这使得生物技术 K5-N,OS(H) 被鉴定为一种新型的血管生成 Gremlin 拮抗剂。
