The binding of resveratrol to vascular endothelial growth factor (VEGF

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Bioactive Constituents, Metabolites, and Functions

The binding of resveratrol to vascular endothelial growth factor (VEGF) suppresses angiogenesis by inhibiting the receptor signalling WEIHUI HU, Ray R. Duan, Yiteng Xia, Qingping Xiong, Huaiyou WANG, Gallant Kar-Lun Chan, Siyue Liu, Tina T.X. Dong, Qiwei Qin, and Karl W.K. Tsim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05977 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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Journal of Agricultural and Food Chemistry

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The binding of resveratrol to vascular endothelial growth factor (VEGF)

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suppresses angiogenesis by inhibiting the receptor signalling

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Wei-Hui Hua,b , Ran Duana,b, Yi-Teng Xia a,b, Qing-Ping Xiongb,c, Huai-You Wanga,b, Gallant Kar-

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Lun Chana,b, Si-Yue Liub,#, Tina Ting-Xia Donga,b,d, Qi-Wei Qind, Karl Wah-Keung Tsim*,a,b,d

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aShenzhen

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Institute, Hi-Tech Park, Nanshan, Shenzhen, China

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bDivision

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and Technology, Clear Water Bay Road, Hong Kong, China

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cJiangsu

Key Laboratory of Edible and Medicinal Bioresources, HKUST Shenzhen Research

of Life Science and Center for Chinese Medicine, The Hong Kong University of Science

Key Laboratory of Regional Resource Exploitation and Medicinal Research, Huaiyin

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Institute of Technology, Jiangsu, China

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dJoint

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Conservation and Exploitation, College of Marine Sciences, South China Agricultural University,

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Guangzhou 510642, PR China

Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource

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*Corresponding Author: Prof. Karl W. K. Tsim, Division of Life Science, Center for Chinese

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Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong

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Kong, China Tel: +852- 2358 7332; fax: +852- 2358 1559; E-mail address: [email protected].

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# Present address: Saint Edward's School, Vero Beach, FL 32963, USA

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Abbreviations: HUVECs, human umbilical vein endothelial cells; VEGF, vascular endothelial

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growth factor; VEGFR1, vascular endothelial growth factor receptor 1; VEGFR2, vascular

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endothelial growth factor receptor 2; VEGFR3, vascular endothelial growth factor receptor 3; JNK,

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c-Jun N-terminal kinase; eNOS, endothelial nitric oxide synthase; Akt, protein kinase B; Erk,

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extracellular signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MTT, 3-

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(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DCFH-DA, 2’7’-Dichlorofluorescein

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diacetate


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Abstract:

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Resveratrol is a polyphenol commonly found in plants and food health products, such as grape and

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red wine; this was identified for its binding to vascular endothelial growth factor (VEGF) by using a

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HerboChips screening: the binding, therefore, resulted in alterations of VEGF binding to its receptor

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as well as pharmaceutical roles of VEGF in angiogenesis. Several lines of evidence gave support to

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the inhibitory activities of resveratrol in VEGF-triggered angiogenesis. In human umbilical vein

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endothelial cells (HUVECs), compared with VEGF-induced group, resveratrol, at a high

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concentration, suppressed VEGF-mediated endothelial cell proliferation, cell migration, cell invasion

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and tube formation by 80 ± 9.01%, 140 ± 3.78%, 110 ± 7.51% and 120 ± 10.26%, separately.

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Moreover, resveratrol inhibited the sub-intestinal vessel formation in zebrafish embryo. In signalling

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cascades, application of resveratrol in HUVECs reduced the VEGF-triggered VEGF receptor-2

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phosphorylation and JNK phosphorylation. Moreover, the VEGF-mediated phosphorylations of

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eNOS, Akt and Erk were obviously decreased by 3 ± 0.37 folds, 2 ± 0.27 folds and 6 ± 0.23 folds,

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separately, in the present of resveratrol at high concentration. Parallelly, the VEGF-induced ROS

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formation was significantly decreased by 50 ± 7.88% to 120 ± 14.82% under resveratrol treatment.

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Thus, our results provided support to anti-angiogenic roles of resveratrol, as well as its related

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signalling mechanisms, in attenuating the VEGF-mediated responses. The present results supported

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possible development of resveratrol that should be considered as a therapeutic agent in terms of

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prevention and clinical treatment of diseases related with angiogenesis.

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Keywords: Herbal medicine, resveratrol, angiogenesis, VEGF, VEGFR2



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Introduction

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Angiogenesis is a highly regulated process, during which the formation of new blood vessels is taken,

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based on pre-existing vessels. Intricate and highly regulated angiogenesis plays a significant role in

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wound healing, embryogenesis and formation of corpus luteum.1, 2 Angiogenesis can be divided into

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several responses that are temporally regulated, including endothelial proliferation, migration and

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differentiation, and protease induction. Synchronization of these complex events and coordination of

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this complicated procedure of angiogenesis are controlled by a series of activators and inhibitors, e.g.

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growth factors and cytokines, secreted by endothelial cells, matrix cells and tumour cells.3 Activation

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of different receptors by numerous ligands, including the placental growth factor (PlGF), acidic and

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basic fibroblast growth factors (aFGF and bFGF), angiopoietins (mostly Ang-1 and -2), platelet-

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derived growth factor (PDGF), hepatocyte growth factor (HGF), and platelet derived-endothelial cell

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growth factor (PD-ECGF) are required

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number of growth factors exerting effects on angiogenesis, vascular endothelial growth factor

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(VEGF) is the most significant signal protein that contributes to increase the survival and mitogenic

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capability of vascular endothelial cells.9

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The specific pharmaceutical effects of VEGF on endothelial cells are primarily mediated by reacting

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with two types of receptor tyrosine kinases (RTKs), vascular endothelial growth factor receptor

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(VEGFR-1 and VEGFR-2). Despite VEGF could be engaged with the two receptors, VEGFR-2 plays

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a major role in regulating most of biological functions of VEGF, including endothelial cell

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mitogenesis and cell permeability, thus triggering the pre-existing endothelial cell survival, cell

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proliferation, cell migration, as well as new tube formation.10-12 After VEGFR-2 activation, the

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phosphorylations of multiple downstream molecules, including ERK, JNK, PI3K, AKT, P70S6K and

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for a proper angiogenic process. In spite of an increasing


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p38MAPK, are subsequently activated contributing to proliferation, migration, and tube formation of

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endothelial cells.13

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Angiogenesis without control is closely related with diseases, including diabetes, solid tumors,

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rheumatoid atherosclerosis and arthritis. Particularly, the natural growth and metastasis of tumours

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have been shown to reply on tumour angiogenesis. Therefore, inhibitors on angiogenesis have been

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expected to be useful and meaningful in clinical treatment for those diseases correlated with

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angiogenesis, including tumours.14 Several inhibitors exerting effects on angiogenesis targeting at

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VEGF and its receptor signaling pathways have been qualified a “license” by the FDA (Food and

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Drug Administration). Bevacizumab (from Genentech), also named as Avastin, is one kind of

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monoclonal antibodies against human VEGF, which is initially certificated as an inhibitor of VEGF-

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VEGFR2 pathway.15 Other small molecules, named sunitinib (SU11248, from Pfizer)16 and sorafenib

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(BAY 43-9006, from Bayer),17 are also reacted as inhibitors for angiogenesis. Thus, the discovery of

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inhibitors attenuating the VEGF/VEGFR2 signaling pathway has shed a future in developing new

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drugs for treatment of angiogenesis-related diseases.

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Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is one of stilbenoids and belongs to phytoalexin

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category exiting in several plants. One of the major sources of resveratrol is from grapes, blueberries,

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raspberries, mulberries. The content of resveratrol in red wine is rich, which is presumed to be of

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much benefits for human health. Resveratrol is also a major constituent in Polygoni Cuspidati

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Rhizoma et Radix, especially in the root and rhizome of Polygonum cuspidatum Sieb. et Zucc.

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Polygoni Cuspidati Rhizoma et Radix is a traditional Chinese medicinal herbs, and which has been

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extensively used in Asia from ancient times for its excellent pharmaceutical activities in anti-oxidant

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effects, anti-bacterial effects and anti-inflammations.18 According to our previous investigations,

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Polygoni Cuspidati Rhizoma et Radix was found to exert inhibitory effects on endothelial cell 5 ACS Paragon Plus Environment


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migration, cell invasion and tube formation based, as well as to inhibit the formation of neo-vessel in

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zebra fish embryo19 The recruitment of resveratrol showed a hepatoprotective activity by interrupting

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signal transduction and expression of proteins related with cell cycle,20 exhibited activities of cancer

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chemo-prevention21 and possessed pharmaceutical effects of anti-platelet aggregation.22 In this study,

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we determined the pharmaceutical activities of resveratrol in angiogenesis, based on in vitro and in

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vivo models. Moreover, the inhibitory roles of resveratrol in angiogenesis were shown to be triggered

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by binding with VEGF and thereafter to suppress downstream signalling of VEGFR2.

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Materials and Methods

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Chemicals

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HPLC grade acetonitrile (ACN) and formic acid were from Merck (Darmstadt, Germany). Deionized

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water (18 MΩ cm-1) was supplied from a Millipore Milli-Q water system (Milford, MA). Other

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reagents were of analytical purity. The Grace Prevail C18 HPLC column and trans-resveratrol, a

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reference compound, was bought from Chengdu Institute of Biology. CAS (Chengdu, China), and its

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purity was over 98%, detected by HPLC-DAD. A stock solution of resveratrol at 100 mM was freshly

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prepared in dimethyl sulfoxide (DMSO).

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Cell cultures

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HUVECs were obtained from Lonza (San Diego, CA), and the cultures were performed for all cell-

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based experiments from 3 to 6 passages. HUVECs were cultured in EGM-2® BulletKit media strictly

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following illustrations (Lonza). Recombinant human VEGF (VEGF165) was obtained from R&D

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systems (Minneapolis, MN). Inhibitors, including LY294002, U0126 and L-Name, were bought from

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Sigma-Aldrich (St. Louis, MO). DCFH-DA was from Sigma-Aldrich. The following antibodies:

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phospho-eNOS (Ser1177), eNOS, phospho-Akt (Ser473), Akt, phospho-VEGFR2 (Tyr1175)

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(19A10), VEGFR2 (55B11), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK 6 ACS Paragon Plus Environment

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(Erk1/2), phospho-SAPK/JNK (Thr183/Tyr185) (81E11), SAPK/JNK were purchased from Cell

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Signalling Technology (Danvers, MA); GAPDH antibody was from Sigma-Aldrich.

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Analyses of resveratrol by HPLC and HerboChips

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The extract of Polygoni Cuspidati Rhizoma et Radix (the root and rhizome of P. cuspidatum) was

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kindly supplied by Yunnan Baiyao Group Tianzihong Pharmaceutical Co. Ltd. (Kunming, Yunnan,

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China) and further qualified by Yunnan Institute of Materia Medica (Kunming, Yunnan, China)

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following The Pharmacopoeia Commission of PR China 2015. In brief, 50 g Polygoni Cuspidati

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Rhizoma et Radix were smashed into powder and successively dissolving in 1,000 mL of 50%

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ethanol (1:20 w/v) for 72 hours as the extraction. All herbal extracts were dried in a lyophilizer, kept

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sealed in 4 °C until use.23 For resveratrol identification existing in herbal extract, 50 mg of powder at

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100 mg and 2 mL of 50% methanol were together added into a 15-mL centrifugal tube. The tube was

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centrifuged at 1,000 x g for 5 min after sonicated for 15 min. After centrifuge, supernatant was

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directly used for analysis. A solution of resveratrol, at a concentration of 1,000 mg/L, was made as

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the stocking solution. An Agilent 1200 HPLC series system, equipped with a degasser, an auto-

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sampler, a binary pump and a thermos-stated column compartment, was used for the chemical

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analysis. An Agilent, Grace VisionHT C18 column (4.6 x 250 mm, 5 μm) was used for

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chromatographic separation. Acetonitrile (as Solvent A) and 0.2% formic acid (as Solvent B) were

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used as mobile phase. The separation was performed at room temperature, and the flow rate was set at

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1.0 mL/min. The gradient elution applied here was set at below: Solvent A was gradually increased

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from 10% to 35% from 0 to 60 min and then increased up to 100% at 96 min. Before sample injection,

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0.45 μm Millipore syringe filter unit was used to filter herbal extract. Ten μL of filtered herbal extract

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and the standard solution of resveratrol, at a concentration of 1,000 mg/L, were separately performed

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the injection for HPLC chromatogram analysis; while the wavelength was set at 280 nm, together

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with a whole spectral scanning from 190 nm to 400 nm. The content of resveratrol in this herb was 7 ACS Paragon Plus Environment


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precisely quantified according to Chinese Pharmacopoeia. Each fraction was related with 1 min. The

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blank chips went through surfaces activation with the application of epoxy groups before dotted with

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herbal extract. Then, the previously collected herbal fractions were separately dotted on surface of

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activated chips in rows with application of an automatic arrayer (Biodot A101, Shuai Ran Precision,

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Taiwan) and then went through fixation.

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Screening of HerboChips

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Dialysis method was used to generate biotinylated protein, as described previously.19 Herbal extracts

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were reacted with the biotinylated protein probe according to standard screening protocol of

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HerboChips.19 In brief, biotinylated VEGF, at a weight of 20 ng, was added onto each cave and

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reacted for 60 min at 4 oC. Then, biotinylated protein unbound with chip was removed from chips

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rinsed for 4X with application of Tris-buffered saline with 0.1% Tween 20 solution (TBST solution).

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Streptavidin-Cy5TM (Invitrogen Life Technologies, Carlsbad, CA) was selected to hybridize with the

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chip. The hybridization reaction was taken 1 hour at 4 oC. Streptavidin-Cy5TM was applied to detect

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biotinylated protein, which was bound with herbal fractions. Lastly, Herbochips were scanned under

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a fluorophore microarray scanner (GenePix 4100A, Molecular Devices Corp., Sunnyvale, CA) to

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obtain the fluorescence intensity of streptavidin-Cy5TM. The fluorescence results were further

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quantified with application of Gene-Pix Pro 7 (ver. 7.1.16) software on a microarray scanner.

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Molecular docking

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The crystal structure of protein was obtained from Protein Data Bank (PDB), and the legend

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molecules, resveratrol (PubChem: 445154) and bevacizumab (PubChem: 24801581) were both

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obtained from NCBI-PubChem database. The structures of legend molecules were separately

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transferred into MOL2 mode for molecular docking analysis by using Chemoffice 2014 (Cambridge

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Soft, Cambridge, MA). The docking calculations were performed on the basis of DockingServer,

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which was an interface on the basis of websites to cope with all aspects of molecular docking with 8 ACS Paragon Plus Environment

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application of AutoDock tools. Molecular docking calculations were separately performed based on

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bevacizumab/resveratrol-VEGF protein model. By using AutoGrid program, affinity (grid) maps of

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40×40×40 Å grid points, corresponded to x, y, and z, and 0.375 Å spacing, were automatically shown,

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and box center was designed as followed: x: 0.38 Å, y: -2.98 Å and z: 20.51 Å.24 With this grid

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designed, most of binding modes were generated for the most favourable binding interaction, and

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energies could be approximately predicted.25 The binding actions were run with the application of the

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vina software, and the binding results could be shown with PyMOL molecular graphics system.

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Moreover, the interactions of resveratrol/or its analogues with other growth factors, including EGF,

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bFGF and PDGF, were analysed following similar auto-docking analysis.

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Immunoprecipitation assay

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Immunoprecipitation assay was performed to confirm the binding interaction between resveratrol and

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VEGF. Briefly, 100 µL 0.5 µM resveratrol solution, with VEGF protein or biotinylated VEGF, was

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incubated 1 hour at 4 oC. Then, 100 µL of PureProteome Streptavidin Magnetic Beads were added

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separately into VEGF solution and biotinylated VEGF solution. The interaction reaction was taken

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for 24 hours at 4 oC. Then, the tubes containing mixtures were placed into magnetic stand, allowing

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the beads to migrate to the magnet. Then, the solution was carefully removed without disturbing the

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beads. After three rounds of washing with PBS, acetonitrile (ACN) was applied to precipitate the

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VEGF/biotin-labelled VEGF complex. The supernatant was subjected to UPLC analysis to determine

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the amount of resveratrol, based on a Waters ACQUITY UPLC system (Waters, Milford, MA) with

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an Agilent, Grace VisionHT C18 column (4.6 x 250 mm, 5 μm). The mobile phases used here were

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determined as followed: A, 0.1% diluted aqueous formic acid; B, 0.1% formic acid in ACN, and the

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elution of gradient was determined at below: Solvent B was increased from 10% to 35% by 60 min

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and then increased to 100% by 96 min. The sample and column temperatures were at room

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temperature and 35 oC. Mass spectrometric detection was performed together with a UPLC and a 9 ACS Paragon Plus Environment


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SynaptTM quadrupole time-of-flight (Q-TOF) High-Definition Mass Spectrometer (Waters), equipped

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with an electrospray ionization source operating under positive ionization mode. After optimization,

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the mass spectrometric parameters were shown as follows: sample cone, 25 V; capillary voltage, 2.5

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kV; source temperature, 120 oC; extraction cone, 4.0 V; and desolvation temperature was set at 350

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oC.

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and 50 L/hour. Argon was here determined as a collision gas. A lock mass of leucine-enkephalin at a

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concentration of 200 pg/mL in 50% ACN-water solution (including 0.1% formic acid) was employed

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as an external reference to generate a [M-H]- ion in negative mode at m/z 226.8728 via a lock spray

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interface with flow rate set at 5 mL/min to acquire accurate mass during the analysis. The sample was

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scanned in full-scan mode from m/z 80 to 800 in 1 sec scan intervals.

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Surface plasmon resonance (SPR)

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The binding between resveratrol and VEGF protein was performed on a Biacore S200 (GE Life

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Sciences) with a GE series dextran-coated (CM5) sensor chip. Briefly, reaction temperature was set at

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25 oC, and HBS-T (150 mM NaCl, 10 mM Hepes, 0.05% polysorbate 20, 3.4 mM EDTA, pH 7.4)

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was selected as the running buffer. The sensor surface of chip used for capture was prepared by

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covalently immobilizing VEGF onto the chip surface according to EDC/NHS (1-Ethyl-3-[3-

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dimethylaminopropyl] carbodiimide hydrochloride)/N-hydroxysuccinimide) coupling chemistry.

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After surface activation, VEGF protein dissolved in coupling buffer (0.1 M acetate buffer, pH 4.5)

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was put onto the activated surface of chip, until the detected RU (resonance unit) signal of VEGF

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protein reached at around 6500 RU. To remove uncoupled protein, the chip with activated coupled

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surfaces were washed and reacted with 10 mM glycine-HCl of which pH was set at 1.5. Next, a series

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of concentrations of resveratrol were diluted with running buffer (3-fold dilutions from 100 μM to

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0.137 μM) and flown through chip surface with VEGF protein coupled. The interaction between

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VEGF protein and resveratrol was detected in real time. A reference channel with no protein

Nitrogen was selected as a desolvation, and a cone gas was, respectively, set at a flow rate of 600


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immobilized was served as a reference control. Data was analysed with the application of GE Biacore

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S200 control software.

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Cell viability assay

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The MTT assay was firstly performed to study the effects of resveratrol on HUVECs. Briefly, 5 × 103

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endothelial cells in 100 μL of medium were seeded on sterile 96-well plate. After incubation for 24

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hours, the medium in each well was changed with 100 μL of fresh medium, containing VEGF at 10

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ng/mL or a series of concentrations of resveratrol. Ten μL/well of MTT solution with concentration

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set at 5 mg/mL was added after drug treatment for 48 hours. After incubating at 37 oC for another 4

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hours, 150 μL of 100% dimethylsulfoxide (DMSO) was added into each well to dissolve the

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formazan salt following medium aspiration. The colour intensity of formazan solution was read under

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a microplate spectrophotometer and the wavelength was set at 570 nm. The LDH release was detected

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with application of a cytotoxicity detection kit plus (LDH) (Roche Diagnostics, Indianapolis, IN).

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The LDH content of each group was quantified following the below formula: cytotoxicity (%) =

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(experimental value − low control)/ (high control − low control) × 100.

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Migration assay

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A wound-healing assay was used to study cell migration in vitro.19,26 In brief, endothelial cells

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(HUVECs) were seeded on a sterile 12-well plate with the density set at 20×104/well. After

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incubation for 24 hours, a straight and single wound, located in the centre of each well, was

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performed to remove the attached endothelial cells: this wound was created with application of a

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sterile 200 μL plastic pipette tip. Then, each well was taken photos with application of a phase-

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contrast microscope (At0) after washed by pre-warmed PBS. Fresh medium containing VEGF, at a

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concentration of 10 ng/mL or different concentrations of resveratrol was added to certain wells

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separately, and the wells treated only fresh medium were regarded as a control. After drug treatment

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for 8 hours, three areas of endothelial cells were decided randomly and taken pictures (At8). With 11 ACS Paragon Plus Environment


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application of Tscratch software (CSE lab, Switzerland), the wound area of each well, before and

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after drug treatments, was measured. The recovery percentage of endothelial cells was calculated on

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the basis of the following formula: Recovery (%) = At0-At8/At0 × 100%.

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Cell invasion assay

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Endothelial cells were seeded on Transwell Boyden Chamber (8-μm pore; Corning Inc., Lowell, MA),

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in which matrigel was allowed to be polymerizd for 1 hour at 37 oC. Next, 100 μL of cell suspension

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in serum-free medium were plated on upper compartment with concentration set at 20 x 104 cells/mL.

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Certain wells contained a series of different concentrations of resveratrol or VEGF, at a concentration

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of 10 ng/mL. The lower chambers were added with 500 μL fresh medium only. After incubated for

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24 hours at 37 ˚C with drug, pre-warmed PBS was used to rinse of HUVECs failing to attach and

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grow; while invasive cells, succeeded into the lower surface, were undergoing dehydration firstly

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with pre-cold methanol and then stained with crystal violet, at a percentage of 0.01%. After staining,

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photos of endothelial cells invaded into lower surface were taken with application of a phase-contrast

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microscope. Images were further analysed by counting of cell number manually. The ratio of invasion

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was demonstrated as % of the control group.

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Tube formation assay

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Tube formation assay was performed based on previous descriptions.19,27 Briefly, Matrigel was used

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to pre-coat 12-well plate firstly, and matrigel polymerization was lasted for 1 hour at 37 ˚C. After

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matrigel polymerization, endothelial cells (HUVECs) containing a series of different concentrations

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of resveratrol or 10 ng/mL VEGF were plated onto each well of plates coated with matrigel with the

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cell density set at 20×104 cells/well. After incubated for 8 hours at 37 °C with drug, tube-like

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structures of HUVECs were photographed with a phase-contrast microscope. Images from different

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drug-treated groups were analysed by counting of the branching points manually, which were located

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in three fields in each well. The fields were determined randomly. 12 ACS Paragon Plus Environment

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Zebrafish angiogenesis assay

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To perform angiogenesis-related assay in vivo, wild-type zebrafish model was selected.19 Zebrafishes

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were fed in a system equipped with flow water and regular aeration, and maintained at a cycle of 10

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hours: 14 hours of dark/light, while the temperature was set at 28.5 oC. Zebrafish maintenance and

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experimental conduction were published strictly following the guidelines and requirements of Animal

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and Plant Care Facility of HKUST. All the experiments performed on animals were given official

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approval by the Animal Ethics Committee in HKUST. By natural spawning of mating matured fishes,

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embryos were obtained and gathered. Healthy embryos were chosen and dechlorinated manually.

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Dechlorinated embryos were assigned to each well of 12-well plate (8-10 embryos per well) and were

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fed with egg water containing a series of different concentrations of resveratrol and VEGF at 28.5 oC.

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The embryos, separately fed with egg water or DMSO, served as solvent and vehicle control. After

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drug treatment for 48 hours, treated embryos were checked with morphological characteristics and

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eyes for viability. After drug treatment, the embryos were fixed with paraformaldehyde (4%) at 4 oC

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for at least 20 hours. After fixation, embryos were rinsed separately by PBS with 0.1% Tween 20

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solution (PBST), 50% methanol and methanol, 5 min each. To dehydrated zebrafish embryos,

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embryos were maintained in methanol and stored at -20 oC refrigerator for 2 hours. To end the

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dehydration reaction, embryos were rinsed for 4 times, 5 min each, in PBST solution. For alkaline

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phosphatase-based vascular staining, embryos were then kept in buffer 9.5T for 15 min at room

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temperature. Buffer 9.5T was freshly prepared by 1M Tris-9.5, 1M MgCl2, deionized water and 20%

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Tween 20. Next, all embryos were stained with the application of newly prepared nitro-blue

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tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate

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Danvers, MA) under dark. To remove left NBT/BCIP, the stained embryos were rinsed continuously

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with PBST for 3 times, 5 min each. Finally, photos of stained vessels located in the embryo sub-

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intestinal were taken with the application of a stereomicroscope (Nikon AZ100), which was equipped

(NBT/BCIP)


(Cell

Signalling

Technology,


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with a digital camera (Olympus DP71). ImageJ software was used to quantify the area and branch of

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sub-intestinal vessels. The experiment was separately performed three times and the same number of

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fish embryos was used for each treatment group.

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Western blot analysis

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Western blot was performed to measure phosphorylations of eNOS (S1177), VEGFR2 (Tyr1175),

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p44/42 MAPK (Erk1/2) (Thr202/Tyr204), SAPK/JNK (Thr183/Tyr185) and Akt (S473). Endothelial

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cells were incubated in medium without serum for 60 min before drug treatment. After drug

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application, medium was aspirated and cultures were subsequently collected in freshly prepared low-

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salt lysis buffer (2% SDS, 10% glycerol, 200 mM 2-mercaptoethanol, 125 mM Tris-HCl, pH 6.8) and

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transferred into centrifuge tubes. Next, tubes containing cell lysates were heated at 95 °C for three

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times, 5 min each, and tubes were vortexed. By electrophoresis, the protein extracts were separated

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into targeted sizes of protein using 7% or 8% acrylamide gels, and the gels were transferred onto

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nitrocellulose membranes. The membranes representing different sizes of protein were obtained.

300

Then, the membranes were blocked with application of 5% milk solution, which was freshly made in

301

a Tris-buffered saline containing 0.1% Tween-20 solution (TBST), for 60 min at room temperature.

302

After blocking, different primary antibody solutions, targeted different kinds of proteins, were used to

303

immerse membranes at 4 °C. The antibody used was diluted at a ratio of 1:1,000. After primary

304

antibody incubation for 24 hours, TBST solution was used to rinse membranes for 4 times, 5 min

305

each, and then membranes were reacted with horseradish peroxidase-conjugated secondary anti-rabbit

306

antibody, which was diluted at a ratio of 1:2,000. After incubated with targeted secondary antibody

307

for 120 min at room temperature, TBST solution was used to rinse membranes for another 4 times, 5

308

min each. ECL (Invitrogen) was selected to visualize the reactive bands, and images of reactive bands

309

were taken by using the Chemidoc Imaging System (Bio-Rad; Hercules, CA). The band intensities of

310

control group and different groups with drug treatments, run on the same piece of gel and 14 ACS Paragon Plus Environment

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photographed under standardized ECL conditions, were further analysed and compared on the related

312

software, which were taken on the basis of a calibration plot from a parallel gel with one of the

313

samples diluted at different ratios. To quantify the western blot in phosphorylation, the band at 10

314

min from each group was compared with the corresponding control at 0 min.

315

Fluorescence intensity analysis

316

HUVEC cells were seeded on a sterile 12-well plate with cell density set at 20×104/well. After

317

incubation for 24 hours, fresh medium containing biotinylated VEGF, at 10 ng/mL or together with a

318

series of concentrations of resveratrol, was applied onto the cultures. The well, treated only fresh

319

medium, was regarded as a background control. After incubated for 24 hours at 37 °C with drug,

320

Streptavidin-Cy5TM was used to hybridize with the biotinylated VEGF. For 1 hour at 37 oC. After

321

washing, cultures were fixed in cold-ethanol, and photos were taken by a laser confocal fluorescent

322

microscopy. Relevant software was used for image analysis, quantifying the total green fluorescence

323

intensity of photos in each group.

324

Measurement of reactive oxygen species

325

The formation of ROS was measured with 2’7’-Dichlorofluorescein diacetate (DCF-DA, Sigma).

326

After incubated with a series of concentrations of resveratrol and VEGF (10 ng/mL) for 48 hours at

327

37 oC, endothelial cells were reacted with DCF-DA (100 μM) at 37 oC for 30 min. Then, pre-warmed

328

PBS was used to rinse cells for three times, 5 min each, to remove unreacted DCF-DA. Pictures were

329

taken with the application of a laser confocal fluorescent microscopy. Relevant software was used for

330

image analysis, quantifying the total green fluorescence intensity of photos in each group.

331

Statistics and other assays

332

Statistical analysis was performed by one way analysis of variance (ANOVA), following a

333

Bonferroni multiple comparisons test using the SPSS 16.0 software. Data were expressed as the mean

334

± standard error of the mean (SEM) and the statistical significance was set at p

The binding of resveratrol to vascular endothelial growth factor (VEGF

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