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(Circulation Research. 2006;99:715.)
© 2006 American Heart Association, Inc.

Molecular Medicine

Direct Evidence for Endothelial Vascular Endothelial Growth Factor Receptor-1 Function in Nitric Oxide–Mediated Angiogenesis

Shakil Ahmad^*, Peter W. Hewett^*, Ping Wang^*, Bahjat Al-Ani, Melissa Cudmore, Takeshi Fujisawa, Jody J. Haigh, Ferdinand le Noble, Ling Wang, Debabrata Mukhopadhyay^**, Asif Ahmed^**

From the Department of Reproductive and Vascular Biology (S.A., P.W.H., B.A.-A., M.C., T.F., A.A.), Institute for Biomedical Research, The Medical School, University of Birmingham, UK; Department of Biochemistry and Molecular Biology (P.W., L.W., D.M.), Mayo Clinic Foundation, Rochester, Minn; Department for Molecular Biomedical Research (J.J.H.), Flanders Interuniversity Institute of Biotechnology, Ghent University, Belgium; and Max Delbrueck Center for Molecular Medicine (F.l.N.), Berlin, Germany.

Correspondence to Prof Asif Ahmed, Department of Reproductive and Vascular Biology, Institute for Biomedical Research, The Medical School, University of Birmingham, Birmingham B15 2TT, UK. E-mail a.s.ahmed{at}bham.ac.uk

Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase C1. Consistent with these findings, the VEGFR-1–specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2–activated phospholipase C1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.

Key Words: angiogenesis • nitric oxide • eNOS • VEGF • VEGF receptors

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