title: Pro-angiogenic activity of notoginsenoside R1 in human umbilical vein endothelial cells in vitro and in a chemical-induced blood vessel loss model of zebrafish in vivo.
abstract: OBJECTIVE
  This study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish.
  
  
  METHODS
  The in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rg1 and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit II (XTT) assay. R1, Rg1 and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigelcoated wells was performed to evaluate the pro-angiogenic actions of R1. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-Nω-nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor II (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels.
  
  
  RESULTS
  R1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/Flk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemicallyinduced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs.
  
  
  CONCLUSIONS
  R1, similar to Rg1 and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Bin-Rui
authors.initials: BR
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.|School of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
authors.forename: Si-Jia
authors.initials: SJ
authors.lastname: Hong
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China. simonlee@umac.mo.
authors.forename: Simon Ming-Yuen
authors.initials: SM
authors.lastname: Lee
authors.affiliation: Laboratory of Cardiovascular Diseases, Xiyuan Hospital China Heart Institute of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, 100091, China. whongcong79@yahoo.com.
authors.forename: Wei-Hong
authors.initials: WH
authors.lastname: Cong
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Jian-Bo
authors.initials: JB
authors.lastname: Wan
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Zhe-Rui
authors.initials: ZR
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Qing-Wen
authors.initials: QW
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Yi-Tao
authors.initials: YT
authors.lastname: Wang
authors.affiliation: School of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
authors.forename: Zhi-Xiu
authors.initials: ZX
authors.lastname: Lin
mesh_terms: D000818:Animals; D001808:Blood Vessels / Q000473:pathology*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D003094:Collagen / Q000494:pharmacology; D004195:Disease Models, Animal; D004338:Drug Combinations; D036145:Ginsenosides / Q000737:chemistry / Q000494:pharmacology*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D007797:Laminin / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011509:Proteoglycans / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D015027:Zebrafish
chemical_list: D004338:Drug Combinations; D036145:Ginsenosides; D007797:Laminin; D047428:Protein Kinase Inhibitors; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D019869:Phosphatidylinositol 3-Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; C072936:notoginsenoside R1
keywords: angiogenesis; ginsenoside Re; ginsenoside Rg1; human umbilical vein endothelial cell; notoginsenoside R1; zebrafish