title: Basal Flt1 tyrosine kinase activity is a positive regulator of endothelial survival and vascularization during zebrafish embryogenesis.
abstract: BACKGROUND
  The role of Kdr (VEGFR-2/Flk-1) in vascular formation has been well described, but the role of Flt1 (VEGFR-1) is not well studied and is generally considered as a decoy receptor for trapping VEGF.
  
  
  METHODS
  The effects of VEGFR1/2 kinase inhibitor (VRI) and calycosin on Flt1 tyrosine kinase (TK) activity were evaluated by molecular docking, enzymatic inhibition assay, protein co-immunoprecipitation and siRNA gene knock-down analysis in HUVECs. Toxicities of the chemicals were examined using HUVECs viability. Their effects on angiogenesis and vessel formation were furthered studied in HUVECs in vitro and Tg(fli-1:EGFP) zebrafish in vivo. The gene and protein expression of VEGF and VEGF receptors were investigated by quantitative RT-PCR and Western blot.
  
  
  RESULTS
  VRI strongly inhibited physiological functions of both VEGF receptors and suppressed endothelial cell survival. This resulted in blood vessel loss in zebrafish embryos. Interestingly, calycosin co-treatment impeded VRI-induced blood vessel loss. Docking and kinase inhibition assay revealed that calycosin competed with VRI for the tyrosine kinase domain of Flt1 without affecting ATP binding. On the contrary, calycosin did not affect the interaction between VRI and Kdr-TK. Consistent with these results, calycosin counteracted the inhibition of Flt1-TK and PI3K phosphorylation induced by VRI in HUVECs. Further studies in vitro and in vivo showed that the minimizing effect of calycosin on VRI-mediated endothelial cytotoxicity was blocked by wortmannin (a PI3K inhibitor). The impeding effect of calycosin on VRI-induced blood vessel loss was absent in zebrafish embryos injected with Flt1 MO.
  
  
  CONCLUSIONS
  Flt1-tyrosine kinase (TK) activity contributed significantly in endothelial cells survival and vascular development during embryo angiogenesis in zebrafish by engaging PI3K/Akt pathway.
  
  
  CONCLUSIONS
  The roles of Flt1 activity in endothelial cell survival in physiological vascular formation may have been previously under-appreciated.
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao, China.
authors.forename: Shang
authors.initials: S
authors.lastname: Li
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.
authors.forename: Xue Lin
authors.initials: XL
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao, China.
authors.forename: Yuan Ye
authors.initials: YY
authors.lastname: Dang
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.
authors.forename: Yiu Wa
authors.initials: YW
authors.lastname: Kwan
authors.affiliation: State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China.
authors.forename: Shun Wan
authors.initials: SW
authors.lastname: Chan
authors.affiliation: Department of Pharmacology and Pharmacy, Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: George Pak Heng
authors.initials: GP
authors.lastname: Leung
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao, China. Electronic address: simonlee@umac.mo.
authors.forename: Simon Ming-Yuen
authors.initials: SM
authors.lastname: Lee
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao, China. Electronic address: maghoi@umac.mo.
authors.forename: Maggie Pui Man
authors.initials: MP
authors.lastname: Hoi
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000196:embryology*; D047108:Embryonic Development / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000196:embryology*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D000077191:Wortmannin; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*
chemical_list: D000730:Androstadienes; D007529:Isoflavones; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D040281:Vascular Endothelial Growth Factor Receptor-1; D000077191:Wortmannin; D029961:Zebrafish Proteins; C121707:7,3'-dihydroxy-4'-methoxyisoflavone; C583603:FLT1 protein, zebrafish
keywords: Calycosin; Endothelial cell; Flt1; Molecular docking; VEGF receptor inhibitor; Zebrafish