Heparan sulfate proteoglycans mediate the angiogenic activity of the vascular endothelial growth factor receptor-2 agonist gremlin.

OBJECTIVE: 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.

METHODS AND RESULTS: 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.

CONCLUSIONS: 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.