Abstract
Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.
Highlights
Plays a central role in pathophysiological neovascularization in human disease [1, 2]
We recently reported that a specific bicyclic peptide based on the C-terminal NP binding domain of VEGF-A165 [37] is an antagonist of VEGF binding to NP-1 and inhibits the anti-chemorepulsive effect of VEGF-A165 in dorsal root ganglion (DRG) neuronal explants [38]
To elucidate the contribution of each subdomain to the binding and biological activity of VEGF-A165 mediated through neuropilin, we evaluated the biological effects of peptides corresponding to the subdomains in endothelial cells
Summary
Plays a central role in pathophysiological neovascularization in human disease [1, 2]. VEGF-A165 exerts its biological effects through high affinity binding to two tyrosine kinase receptors, Flt-1 (VEGFR1) and KDR (VEGFR2), which are expressed in most vascular endothelial cells [6]. Overexpression of NP-1 in mice results in increased capillary formation, vasodilatation, and malformation of the heart [34], whereas mice deficient in NP-1 exhibit defects in embryonic axonal patterning and an array of vascular abnormalities including defective development of large vessels and impaired neural and yolk sac vascularization [35]. Evaluation of EG3287 in endothelial cells demonstrates that VEGF-A165 binding to NP-1 is required for stable binding to KDR, full activation of KDR and downstream signaling and biological responses This antagonist should be a valuable tool for probing the biological role of NP-1 in diverse cell types, and will be useful for designing improved neuropilin antagonists
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