title: Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis.
abstract: 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 Cgamma1. 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 Cgamma1. 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.
authors.affiliation: Department of Reproductive and Vascular Biology, Institute for Biomedical Research, The Medical School, University of Birmingham, Birmingham B15 2TT, UK. a.s.ahmed@bham.ac.uk
authors.forename: Shakil
authors.initials: S
authors.lastname: Ahmad
authors.forename: Peter W
authors.initials: PW
authors.lastname: Hewett
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Bahjat
authors.initials: B
authors.lastname: Al-Ani
authors.forename: Melissa
authors.initials: M
authors.lastname: Cudmore
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fujisawa
authors.forename: Jody J
authors.initials: JJ
authors.lastname: Haigh
authors.forename: Ferdinand
authors.initials: F
authors.lastname: le Noble
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Debabrata
authors.initials: D
authors.lastname: Mukhopadhyay
authors.forename: Asif
authors.initials: A
authors.lastname: Ahmed
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D018919:Neovascularization, Physiologic / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D014443:Tyrosine; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000502:physiology*
chemical_list: D009569:Nitric Oxide; D014443:Tyrosine; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1; D051057:Proto-Oncogene Proteins c-akt; D051966:Phospholipase C gamma