AUTHOR=Huang Sheng-Teng , Chang Cheng-Chieh , Pang Jong-Hwei S. , Huang Hung-Sen , Chou Shen-Chieh , Kao Ming-Ching , You Huey-Ling 

TITLE=Drynaria fortunei Promoted Angiogenesis Associated With Modified MMP-2/TIMP-2 Balance and Activation of VEGF Ligand/Receptors Expression

JOURNAL=Frontiers in Pharmacology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2018.00979

DOI=10.3389/fphar.2018.00979

ISSN=1663-9812

ABSTRACT=<p><bold>Background and Purpose:</bold><italic>Drynaria fortunei</italic> J. Sm (<italic>D. fortunei</italic>), known as Gu-Sui-Bu, is used in traditional Chinese medicine to treat common injuries, including bone fractures and bruising. The specific functional mechanisms of the angiogenic and endothelial cell migration properties of <italic>D. fortunei</italic> are currently unclear. Thus, the purpose of this study is to validate the potential angiogenic and cellular migration properties and related mechanisms by <italic>D. fortunei</italic> both <italic>in vivo</italic> and <italic>in vitro</italic>.</p><p><bold>Experimental Approach:</bold> The present study investigates, both <italic>in vivo</italic> and <italic>in vitro</italic>, the wound healing effects of <italic>D. fortunei</italic> as associated with angiogenesis, specifically by the modulation of matrix metalloproteinases (MMPs) and upregulation of vascular endothelial growth factor (VEGF) ligand/receptors. In order to determine the potential <underline>angiogenic</underline> effects of <italic>D. fortunei</italic>, <italic>in vivo</italic> neovascularization of chick chorioallantoic membranes (CAMs) assay, and directed <italic>in vivo</italic> angiogenesis assay (DIVVA) were performed, while <italic>in vitro</italic> scratch wound healing, migration, and matrix-induced tube formation assays were performed by using human umbilical vascular endothelial cells (HUVECs). Furthermore, we used qPCR to analyze the gene expressions and Western blot to observe protein expressions of MMP-2, MMP-14, TIMP-2, RECK, and VEGF/VEGFRs.</p><p><bold>Results:</bold> This study identified five major compounds from the water extract of <italic>D. fortunei</italic>: protocatechuic acid, caffeic acid 4-<italic>O</italic>-β-<sc>D</sc>-glucopyranoside, 5,7-dihydroxychromone-7-<italic>O</italic>-rutinoside, neoeriocitrin, and naringin. <italic>D. fortunei</italic> was confirmed to activate <italic>in vivo</italic> angiogenesis by CAM and DIVVA assays. <italic>D. fortunei</italic> further exhibited <italic>in vitro</italic> angiogenic effects associated with cell migration, as demonstrated by the tube formation assay, transwell migration assay, and scratch wound healing assay. The extracellular MMP-2 activity was found to be dose-dependently augmented both <italic>in vitro</italic> and <italic>in vivo</italic> by <italic>D. fortunei</italic>. The mRNA and protein expressions of MMP-2, and MMP-14 were increased; while the tissue inhibitor metalloproteinase-2 (TIMP-2), and reversion-inducing cysteine-rich protein with kazal motifs (RECK) were both decreased. Furthermore, <italic>D. fortunei</italic> activated the gene and protein expressions of VEGF-A, -B, and VEGFR-2, -3.</p><p><bold>Conclusion:</bold><italic>D. fortunei</italic> increased MMP-2 activity, thereby stimulating angiogenesis and cell migration, both <italic>in vivo</italic> and <italic>in vitro</italic>, as a result of MMP-2 and TIMP-2 balance modulation and the activation of VEGF/VEGFRs expression.</p>