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Antiangiogenic effects of AA-PMe on HUVECs in vitro and zebrafish in vivo

Authors Jing Y, Wang G, Xiao Q, Zhou Y, Wei Y, Gong Z

Received 22 November 2017

Accepted for publication 10 February 2018

Published 4 April 2018 Volume 2018:11 Pages 1871—1884


Checked for plagiarism Yes

Review by Single-blind

Peer reviewer comments 2

Editor who approved publication: Prof. Dr. Geoffrey Pietersz

Yue Jing,1,2,* Gang Wang,1,* Qi Xiao,1 Yachun Zhou,1 Yingjie Wei,3 Zhunan Gong1

1Center for New Drug Research and Development, College of Life Science, Nanjing Normal University, Nanjing, China; 2Central Laboratory of Stomatology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, China; 3Key Laboratory of Oral Drug Delivery System of Chinese Materia Medica of State Administration of Traditional Chinese Medicine, Jiangsu Branch of China Academy of Chinese Medical Science, Nanjing, China

*These authors contributed equally to this work

Abstract: Angiogenesis plays a vital role in many physiological and pathological processes and several diseases are connected with its dysregulation. Asiatic acid (AA) has demonstrated anticancer properties and we suspect this might be attributable to an effect on angiogenesis. A modified derivative of AA, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-L-proline methyl ester (AA-PMe), has improved efficacy over its parent compound, but its effect on blood vessel development remains unclear.
Methods: In this study, we investigated the antiangiogenic activity of AA and AA-PMe in zebrafish embryos and human umbilical vein endothelial cells (HUVECs). First of all, we treated HUVECs with increasing concentrations of AA-PMe or AA, with or without vascular endothelial growth factor (VEGF) present, and assessed cell viability, tube formation, and cell migration and invasion. Quantitative real-time polymerase chain reaction and Western blot analysis were later used to determine the role of vascular endothelial growth factor receptor 2 (VEGFR2)-mediated signaling in AA-PMe inhibition of angiogenesis. We extended these studies to follow angiogenesis using Tg(fli:EGFP) transgenic zebrafish embryos. For these experiments, embryos were treated with varying concentrations of AA-PMe or AA from 24 to 72 hours postfertilization prior to morphological observation, angiogenesis assessment, and endogenous alkaline phosphatase assay. VEGFR2 expression in whole embryos following AA-PMe treatment was also determined.
Results: We found AA-PMe decreased cell viability and inhibited migration and tube formation in a dose-dependent manner in HUVECs. Similarly, AA-PMe disrupted the formation of intersegmental vessels, the dorsal aorta, and the posterior cardinal vein in zebrafish embryos. Both in vitro and in vivo AA-PMe surpassed AA in its ability to block angiogenesis by suppressing VEGF-induced phosphorylation of VEGFR2 and disrupting downstream extracellular regulated protein kinase and AKT signaling.
For the first time, this study reveals that AA-PMe acts as a potent VEGFR2 kinase inhibitor and exerts powerful antiangiogenic activity, suggesting it to be a promising therapeutic candidate for further research.

Keywords: AA-PMe, angiogenesis inhibitor, zebrafish, HUVEC

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