title: Calycosin promotes angiogenesis involving estrogen receptor and mitogen-activated protein kinase (MAPK) signaling pathway in zebrafish and HUVEC.
abstract: BACKGROUND
  Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo.
  
  
  METHODS
  Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 microM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 microM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 microM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting.
  
  
  CONCLUSIONS
  Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERalpha and ERbeta. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways.
authors.affiliation: Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Jing Yan
authors.initials: JY
authors.lastname: Tang
authors.forename: Shang
authors.initials: S
authors.lastname: Li
authors.forename: Zhen Hua
authors.initials: ZH
authors.lastname: Li
authors.forename: Zai Jun
authors.initials: ZJ
authors.lastname: Zhang
authors.forename: Guang
authors.initials: G
authors.lastname: Hu
authors.forename: Lorita Chi Veng
authors.initials: LC
authors.lastname: Cheang
authors.forename: Deepa
authors.initials: D
authors.lastname: Alex
authors.forename: Maggie Pui Man
authors.initials: MP
authors.lastname: Hoi
authors.forename: Yiu Wa
authors.initials: YW
authors.lastname: Kwan
authors.forename: Shun Wan
authors.initials: SW
authors.lastname: Chan
authors.forename: George Pak Heng
authors.initials: GP
authors.lastname: Leung
authors.forename: Simon Ming Yuen
authors.initials: SM
authors.lastname: Lee
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007529:Isoflavones / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000235:genetics / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology*; D015027:Zebrafish
chemical_list: D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D007529:Isoflavones; D011960:Receptors, Estrogen; C121707:7,3'-dihydroxy-4'-methoxyisoflavone; D020928:Mitogen-Activated Protein Kinases