title: Davallia bilabiata exhibits anti-angiogenic effect with modified MMP-2/TIMP-2 secretion and inhibited VEGF ligand/receptors expression in vascular endothelial cells.
abstract: BACKGROUND
  Davallia bilabiata Hosokawa (D. bilabiata), also called GuSuiBu, is popularly used as a substitute for Drynaria fortunei J. Sm for rheumatoid and degenerative arthritis in traditional Chinese medicine. Little is known about the underlying mechanisms of anti-angiogenesis responsible for arthritis in D. bilabiata which needs to be elucidated.
  
  
  OBJECTIVE
  The present study is intended to investigate the anti-angiogenic effect of D. bilabiata associated with the modulation of matrix metalloproteinases (MMPs) and down regulation of vascular endothelial growth factor (VEGF) ligand/receptors both in vivo and in vitro.
  
  
  METHODS
  We investigated the potential anti-angiogenic effect of D. bilabiata by the in vivo neovascularization of chick chorioallantoic membranes (CAM) assay, and the in vitro migration and matrix-induced tube formation assay using human umbilical vascular endothelial cells (HUVECs). The expressions of MMP-2, TIMP-2, RECK and VEGF/VEGFR were analyzed by real-time RT-PCR or Western blot method.
  
  
  RESULTS
  One major compound from water extract of D. bilabiata was identified as Epicatechin 3-O-β-D-allopyranoside. D. bilabiata was confirmed to inhibit in vivo angiogenesis by CAM assay. D. bilabiata also exhibited in vitro anti-angiogenic and anti-regrowth effects as demonstrated by tube formation assay, transwell migration assay and wound healing assay. The mRNA expressions of MMP-2, and MMP-14 were decreased. On the contrary, tissue inhibitor of metalloproteinase-2 (TIMP-2), reversion-inducing cysteine-rich protein with kazal motifs (RECK) were increased by D. bilabiata. The extracellular MMP-2 activity was found to be reduced both in vitro and in vivo by D. bilabiata as determined by gelatin zymography. Results from western blot analysis and ELISA further demonstrated the decrease of MMP-2 and increase of TIMP-2 secretion after D. bilabiata treatment. The gene expressions of VEGF-A, -B, -C, -D and VEGFR-1, -2, -3 were all inhibited by D. bilabiata.
  
  
  CONCLUSIONS
  We concluded that the anti-angiogenic effect of D. bilabiata was associated with the decreased MMP-2 activity mediated by the upregulation of TIMP-2 and RECK, and the suppression of VEGF/VEGFRs expression.
authors.affiliation: Department of Chinese Medicine and Mitochondrial Research Unit, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Chun-Ting
authors.initials: CT
authors.lastname: Liu
authors.affiliation: Department of Chinese Medicine and Mitochondrial Research Unit, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Kuo-Wei
authors.initials: KW
authors.lastname: Bi
authors.affiliation: Division of General Surgery, Ministry of Health and Welfare Pingtung Hospital, Pingtung, Taiwan.
authors.forename: Chao-Chun
authors.initials: CC
authors.lastname: Huang
authors.affiliation: Department of Chinese Medicine and Mitochondrial Research Unit, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Hsiao-Ting
authors.initials: HT
authors.lastname: Wu
authors.affiliation: Jen Li Biotech Company Ltd., Taiping District, Taichung 41143, Taiwan.
authors.forename: Hui-Ya
authors.initials: HY
authors.lastname: Ho
authors.affiliation: Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tao-Yuan,Taiwan; Department of Physical Medicine and Rehabilitation, Chang Gung Memorial Hospital, Taoyuan, Taiwan. Electronic address: jonghwei@mail.cgu.edu.tw.
authors.forename: Jong-Hwei
authors.initials: JH
authors.lastname: S Pang
authors.affiliation: Department of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan; School of Chinese Medicine, China Medical University, Taichung, Taiwan. Electronic address: sheng.teng@yahoo.com.
authors.forename: Sheng-Teng
authors.initials: ST
authors.lastname: Huang
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000187:drug effects / Q000502:physiology; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000235:genetics; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010936:Plant Extracts / Q000494:pharmacology*; D040262:Receptors, Vascular Endothelial Growth Factor / Q000235:genetics; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D064028:Tracheophyta; D042442:Vascular Endothelial Growth Factors / Q000235:genetics; D014945:Wound Healing / Q000187:drug effects
chemical_list: D020533:Angiogenesis Inhibitors; D058851:GPI-Linked Proteins; D010936:Plant Extracts; C115460:RECK protein, human; C560823:TIMP2 protein, human; D042442:Vascular Endothelial Growth Factors; D019716:Tissue Inhibitor of Metalloproteinase-2; D040262:Receptors, Vascular Endothelial Growth Factor; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14
keywords: Angiogenesis; Davallia bilabiata; Matrix metalloproteinases (MMPs); VEGFRs; VEGFs