Exogenous recombinant dimeric neuropilin-1 is sufficient to drive angiogenesis.
Neuropilin-1 (NRP-1) is present on the cell surface of endothelial cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between vascular endothelial growth factor-A(165) (VEGF-A(165)), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-γ, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A(165). This was evidenced by depleting the cell culture of exogenous VEGF-A(165), and using instead for routine culture VEGF-A(121), which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/endothelial co-culture model revealed that Fc rNRP-1 could induce endothelial cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A(165)-independent agonist of VEGFR-2 and stimulates angiogenesis in endothelial cells.
Department of Chemical and Structural Biology, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom.
Katarzyna A
KA
Uniewicz
Michael J
MJ
Cross
David G
DG
Fernig
D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D039942:Neuropilin-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000172:deficiency; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011799:Quinazolines / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D012995:Solubility; D042461:Vascular Endothelial Growth Factor A / Q000172:deficiency; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D011799:Quinazolines; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; C478783:ZM323881; C511613:vascular endothelial growth factor A (138-165); D039942:Neuropilin-1; D040301:Vascular Endothelial Growth Factor Receptor-2