Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity

Background: Vascular endothelial growth factor (VEGF) requires heparin-like molecules for full activity. Results: VEGF, VEGF receptor-2, and neuropilin-1 complexes bind heparin synergistically. Neuropilin-1 enhances VEGF signaling and is dependent on heparan sulfate. Conclusion: Heparin influences VEGF receptor-1, VEGF receptor-2, and neuropilin-1 through distinct mechanisms and regulates VEGF-induced signaling. Significance: Heparin-like molecules with specific structural features might be used to selectively manipulate the VEGF system to regulate angiogenesis. Angiogenesis is a highly regulated process orchestrated by the VEGF system. Heparin/heparan sulfate proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors, yet the mechanisms of action have not been fully defined. In the present study, we characterized molecular interactions between receptors and co-receptors, using surface plasmon resonance and in vitro binding assays. Additionally, we demonstrate that these binding events are relevant to VEGF activity within endothelial cells. We defined interactions and structural requirements for heparin/HS interactions with VEGF receptor (VEGFR)-1, NRP-1, and VEGF(165) in complex with VEGFR-2 and NRP-1. We demonstrate that these structural requirements are distinct for each interaction. We further show that VEGF(165), VEGFR-2, and monomeric NRP-1 bind weakly to heparin alone yet show synergistic binding to heparin when presented together in various combinations. This synergistic binding appears to translate to alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increases VEGF binding and activation of VEGFR-2 and ERK1/2 in endothelial cells and that these effects require sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output of the system. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1.