Journal of Ethnopharmacology 151 (2014) 565–575

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Molecular mechanisms of angiogenesis effect of active sub-fraction from root of Rehmannia glutinosa by zebrafish sprout angiogenesis-guided fractionation Cheuk-Lun Liu a,b,c, Hin-Fai Kwok a,b, Ling Cheng a,b, Chun-Hay Ko a,b, Chun-Wai Wong a,b, Tina Wai Fong Ho a,b, Ping-Chung Leung a,b, Kwok-Pui Fung a,b,c, Clara Bik-San Lau a,b,n a

Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, Hong Kong State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, Hong Kong c School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, Hong Kong b

art ic l e i nf o

a b s t r a c t

Article history: Received 29 May 2013 Received in revised form 17 August 2013 Accepted 10 November 2013 Available online 16 November 2013

Ethnopharmacological importance: The root of Rehmannia glutinosa (Rehmanniae Radix (RR)) is clinically used as a wound-healing agent in traditional Chinese medicine. Angiogenesis acts crucially in the pathogenesis of chronic wound healing. The present study investigated the angiogenesis effect and its underlying mechanism of RR through zebrafish sprout angiogenesis guided-fractionation. Materials and methods: The in vivo angiogenesis effect was studied by analyzing the number of ectopic sprouts formed upon sub-intestinal vessel of transgenic TG(fli1:EGFP)y1/ þ(AB) zebrafish embryos by fluorescence microscopy. Quantitative real-time PCR gene expression of the zebrafish embryos was further performed using a panel of 30 angiogenesis-associated genes designed for zebrafish sprout angiogenesis. Classical in vitro angiogenesis assays using human microvascular endothelial cells (HMEC-1) was accompanied. Results: We demonstrated that among all RR sub-fractions tested, C1-1 treated-zebrafish embryos possessed the most potent angiogenesis activities (from 190 to 780 ng/ml, po0.001) in sprout formation in the zebrafish model. Quantitative gene expression of the treated embryos demonstrated significant up-regulation in MMP-9 (po0.05), ANGPT1 (po0.05), EGFR (po0.05), EPHB4 (po0.01), and significant down-regulation in Ephrin B2 (po0.05), Flt-1 (po0.05) and Ets-1 (po0.05). C1-1 treatment could also significantly (po0.001–0.05) stimulate HMEC-1 cell migration in scratch assay. Significant increase (po0.05) in mean tubule length was observed in the C1-1-treated HMEC-1 cells in the tubule formation assay. Conclusions: Our zebrafish sprout angiogenesis model-guided fractionation revealed that C1-1 possessed the most potent angiogenesis effect in RR. The design of the panel with 30 tailor-made angiogenesis-associated genes exhibited in zebrafish gene expression analysis showed that C1-1 could trigger differential expression of various angiogenesis-associated genes, such as VEGFR3 and MMP9, which played key role in angiogenesis. The pro-angiogenic activity of C1-1 was further confirmed in the translated study in motogenic and tubuleinducing effect using HMEC-1. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Bioassay-guided fractionation Rehmannia glutinosa zebrafish Angiogenesis PCR array

1. Introduction In chronic wound healing, such as diabetic wounds, impaired angiogenesis is one of the pivotal pathological phases resulted in the delay of wound repair (Chabbert-Buffet et al., 2003; Martin et al., 2003). Microangiopathy induced impaired angiogenesis could reduce the blood flow and oxygenation essential for normal wound healing (Martin et al., 2003). n Corresponding author at: The Chinese University of Hong Kong, Institute of Chinese Medicine, Room 305, Science Centre East Block, Shatin, New Territories, Hong Kong. Tel.: þ852 3943 6109; fax: þ 852 2603 5248. E-mail address: [email protected] (C.B.-S. Lau).

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.11.019

Pro-angiogenic agents could aid in enhancing angiogenesis, thus, improving the recovery of chronic wounds. Rehmanniae Radix (RR) is commonly used in traditional Chinese medicine to treat burns and eczema (Wagner et al., 2011). In ulcer healing, RR extract was found to facilitate gastric ulcer healing (Wang et al., 1999; Zhang et al., 2008). Our lab was one of the pioneering groups in investigating the effect of cutaneous wound healing of RR (Lau et al., 2007–2009). Previously we found that aqueous extract of RR alone (Lau et al., 2008), and in a 2-herb formula (Tam et al., 2011), could significantly improve wound healing in diabetic foot ulcer animal model. In particular, RR extract increased the VEGF expression for improved angiogenesis resulting in the promotion of wound healing (Lau et al., 2009). These studies suggested that the enhanced wound healing effects of RR aqueous

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extract in difficult wound model using diabetic rats could be due to the improved angiogenesis. To illustrate the complete effect of angiogenesis, in vivo angiogenesis models should be used. Among them, chick chorioallantoic membrane assay (CAM) (Li et al., 2010) is a simple and inexpensive method of which the angiogenesis effects are examined by counting the number of blood vessels formed. However, several shortcomings of CAM assay include the fact that since vascular network exist before the treatment, blood capillaries newly formed become difficult to observe. Also, the slides placed on the embryos could trigger immune response that masks the lately formed capillaries. Other in vivo angiogenesis models, such as rabbit corneal pocket (Ziche and Morbidelli, 2009) are rather invasive, required heavy surgical experimental manipulations, expensive and difficult to quantify the data (Jain et al., 1997), and therefore inefficient for drug screening. To tackle these setbacks, the use of a non-invasive real-time observation of transgenic zebrafish TG(fli1:EGFP)y1 with fluorescent vasculature (by the expression of enhanced green fluorescent protein marker in the endothelial cells (Lawson and Weinstein, 2002a)) for quantitative screening of the drug possessing angiogenesis effect could be a favorable choice (Hong et al., 2009; Lawson and Weinstein, 2002a; Raghunath et al., 2009; He et al., 2010; Tang et al., 2010; Liu et al., 2011). The quantification can be achieved by assessing the number of ectopic vessels present in the sub-intestinal vessel (SIV) region of zebrafish embryos to evaluate the degree of pro-angiogenesis effect of the testing agent (Raghunath et al., 2009). High-throughput screening of angiogenic sprouts using zebrafish embryos could provide a better acuity to the total effect of angiogenesis in vivo, compared with in vitro cell lines study alone (Stern and Zon, 2003; Raghunath et al., 2009). Through zebrafish sprout angiogenesis-guided fractionation, we previously reported that, norviburtinal was found to possess angiogenesis activities in RR (Liu et al., 2011); however, the compound might only played a minor part. Other active subfraction(s) or compound(s) in RR responsible for its angiogenesis effect are yet to be identified. In searching for these, bioassay-guided fractionation was further applied. This guided approach prevents overlooking minute but active compounds that are often missed in studies which only isolate major components. Indeed, most of the studies applying zebrafish embryos as the model for pro-angiogenic agents detecting through sprout(s)induction in sub-intestinal vein were mainly concentrated in the phenotype level (Hong et al., 2009; Raghunath et al., 2009; He et al., 2010; Liu et al., 2011). In the sense of focused PCR array, limited methodologies were developed for the in-depth study of the pro-angiogenesis effects of testing agent(s) followed by its ectopic vessels assessment upon SIV of zebrafish embryos. Here we further investigated the angiogenesis effect and its underlying mechanisms of RR through zebrafish sprout angiogenesis-guided fractionation. In addition, dedicating to the zebrafish sprout angiogenesis platform, a focused angiogenesis associated panel was designed to examine the pro-angiogenesis effect of the active sub-fraction(s) of RR. Using human microvascular endothelial cells, classical in vitro angiogenesis assays were also used to further translate and support our findings from zebrafish angiogenesis model.

Chinese University of Hong Kong, with voucher specimen number 2008–3200. Also, the sample was authenticated using Thin Layer Chromatography (TLC) with chemical reference marker catalpol, in accordance to the methodology suggested by the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission, 2010). As for extraction, the sliced raw herb of RR (20 kg) was successively extracted with distilled water ten times volume of weight under reflux twice, one-hour each. The water extracts were filtered and concentrated under reduced pressure using a vacuum rotary evaporator, and then freeze-dried, with yield of around 39% w/w and was kept in the desiccator until use.

2.2. Zebrafish culture The transgenic zebrafish line TG(fli1:EGFP)y1/ þ(AB) with endothelial cells expressing EGFP (enhanced Green Fluorescent Protein), was purchased from the Zebrafish International Resource Centre, University of Oregon, USA and cultured with reference to previous report (Lawson and Weinstein, 2002a; Hong et al., 2009). The zebrafish were maintained at 28 1C on a 14 h (light): 10 h (dark) photoperiod and were fed with brine shrimp and tropical fish food twice daily. The handling of zebrafish was under the animal license issued by the Hong Kong Special Administrative Region and was approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong (AEEC No. (08217) in DH/HA&P/8/2/1 Pt.4).

2.3. Collection of zebrafish embryos and herbal treatment protocol The zebrafish embryos were generated by natural pair-wise mating of 3 to 4 pairs of the TG(fli1:EGFP)y1/ þ(AB) zebrafish of 4 to 8 months old. Embryos were rinsed with 2 μg/ml of methylblue solution for disinfection and then cultured in the sterilized embryo medium (0.06 g/l Instant Oceans Salt, Aquarium Systems, USA). Healthy and regular embryos were selected at their 1–4 cell stage (Kimmel et al., 1995) and distributed into a 6-well microplate with 25–30 embryos per well depending on the assay. The medium in the wells was replaced by RR fractions in various concentrations (0.19–25 mg/ml). A maximum of 0.5%v/v DMSO was used as vehicle control group for those treatments of non-polar fractions which were dissolved in 0.5%v/v DMSO. After 24 h of treatment, the viability and gross morphological state of embryos were examined. The samples were prepared in sterilized Milli-Q water containing a maximum of 0.5%v/v DMSO. Treated embryos in individual wells were then incubated for 72 h to 96 h (Serbedzija et al., 1999). Embryos receiving DMSO (0.2%v/v) served as negative controls, depending on the solvents used in the tested samples. Vascular endothelial growth factor (VEGF) (Invitrogen, CA, USA) was used as positive control (Liu et al., 2011).

2.4. Screening of zebrafish embryos using fluorescence microscopy 2. Materials and methods 2.1. Plant material and extraction The root of Rehmannia glutinosa (RR) Libosch. (family Scrophulariaceae) was purchased from Henan province, Mainland China and was morphologically authenticated by a botanist. Small amount of the raw herb sample was deposited as voucher specimen in the museum of the Institute of Chinese Medicine, the

After 72 to 96 h post-fertilization (hpf) (Serbedzija et al., 1999), the embryos were examined using an Olympus IX71S8F-2 inverted microscope (Olympus, Tokyo, Japan) for the presence of the number of ectopic vessels in the sub-intestinal vessel (SIV) region as an indication of pro-angiogenesis effect (Raghunath et al., 2009). The mean sprout number was assessed by the sum of the number of sprouts present in each sample group over the total number of embryos in the sample group. Captured images were analyzed using Image J 1.38  (NIH, USA).

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Fig. 1. A flow chart showing the fractionation procedures of RR (modified from Liu et al. (2011)).

2.5. Bioassay-guided fractionation of RR The aqueous crude extract of RR was fractionated by sequential solvent partition using dichloromethane (DCM), ethyl acetate (EtOAc), and then n-butanol. The partitioned extracts were further concentrated to give fractions P1 (DCM, 13.7 g), P2 (EtOAc, 19.6 g), P3 (n-butanol, 320 g) and P4 (water, 7440 g) (Fig. 1). Based on our previous published work on biological activities of RR (Liu et al., 2011), fraction P1 was further fractionated by column chromatography, using a silica gel (Kieselgel 60, 230– 400 mesh, Merck KGaA, Darmstadt, Germany) column (31  4 cm), with initial mobile phase of a gradient of hexane–ethyl acetate (9:1 to 1:9), and then eluted with ethyl acetate, ethyl acetate– acetone (1:1), acetone, and finally methanol, to afford six subfractions (Fig. 1): C1 (0.41 g), C2 (1.67 g), C3 (1.31 g), C4 (2.42 g), C5 (2.78 g) and C6 (4.0 g) grouped according to the differences in their compositions indicated by TLC. Basing on the biological activities (see results later; also see Liu et al., 2011), sub-fraction C1 was further subjected to column chromatography using silica gel column eluted with hexane, hexane–ethyl acetate (9:1), ethyl acetate and finally methanol to give six fractions C1-1 (46.3 mg), C1-2 (58.3 mg), C1-3 (46.0 mg), C1-4 (39.9 mg), C1-5 (37.1 mg) and C1-6 (55.1 mg) grouped according to the differences in their compositions indicated using TLC.

2.6. Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) analysis of sub-fraction, C1-1 UHPLC chromatographic separation was conducted using Agilent 1290 Infinity UHPLC system (Santa Clara, CA, USA), equipped with binary solvent delivery system and an auto-sampler. A 100 mm  3.0 mm Zorbax Eclipse Plus C18 1.8 μm column (Agilent Technologies, Santa Clara, CA, USA) was utilized. The mobile phase composed of (A) 0.1% formic acid in water and (B) acetonitrile. The profile gradient was optimized as follows: flow rate, 0.3 ml/min: 0–1 min, 25% B; 1–6 min, 25–60% B; 6–36 min, 90% B. The injection volume was 15 ml and the column temperature was maintained at 40 1C. Mass spectrometry was conducted using Agilent 6530 AccurateMass QTOF mass spectrometer (Santa Clara, CA, USA), equipped with Jet Stream electrospray ionization (ESI) source. Source parameters were as follows: positive ion mode, capillary 3500 V, nebulizer 45 psi, drying gas 9 l/min, gas temperature 350 1C, skimmer voltages 65 V, octapoleRFpeak 750 V, fragmentator 180 V. Mass spectra were recorded across the range of m/z 50–2000 with accurate mass

measurement. The full-scan data were analyzed using software by Agilent MassHunter Workstation (Santa Clara, CA, USA).

2.7. Detection of zebrafish mRNA expression level by real-time PCR (RT-PCR) Total RNA from control zebrafish embryos, and C1-1 treated embryos were extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. Real-time PCR was performed with CFX96™ Real Time System and iScript one-step RT-PCR kit with SYBRs Green (Bio-Rad, Hercules, USA). β-actin was used as house-keeping gene. The primers used were listed in Table 1. The cycling conditions were 50 1C for 10 min, 95 1C for 5 min, then 50 cycles of 95 1C for 10 s and 60 1C for 30 s. The samples were performed in triplicate. Gene expression differences were analyzed using two-cycle threshold calculation.

2.8. Human microvascular endothelial cell (HMEC-1) culture and sample treatment protocol Human microvascular endothelial cells (HMEC-1, purchased from the American Type Culture Collection, Manassas, VA, USA) were maintained in MCDB 131 medium (Sigma, St. Louis, USA), supplemented with hydrocortisone (Sigma, St. Louis, USA), 125 ng/ml human epidermal growth factor (Sigma, St. Louis, USA), 10% v/v fetal bovine serum (FBS; GIBCO, USA) and 1% penicillin–streptomycin (PS; GIBCO, USA). All cells were maintained at 37 1C, in 5% CO2 humidified incubator.

2.9. HMEC-1 endothelial cell proliferation assay HMEC-1 cells were seeded at 15,000 cells per well in 96-well plate in the supplemented MCDB 131 medium. Cells were then starved in medium containing 0.5% v/v FBS for 24 h. HMEC-1 were exposed to different concentrations of C1-1 dissolved in final concentration of 0.5% v/v DMSO. The treatment lasted for 48 h at 37 1C. MTT (3-[4,5-dimethylthiazol-2-yl]  2,5- diphenyltetrazolium bromide; Sigma, USA) solution (5 mg/ml) in 1  PBS was added directly to the medium in each well, and the plate was then incubated at 37 1C for 4 h. The medium was then aspirated and replaced with DMSO, and the relative amount of viable cells was determined at optical density at 540 nm and expressed as the percentage of control samples without treatment.

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Table 1 List of genes and its real-time PCR primers used. Gene name

Forward (F) and reverse (R) primer sequences

Epidermal growth factor receptor (EGFR)

F-ACGCAGACGAGTATTTAGTGCCCA R-AGTTTCCAAAGCTGCTGTTCAGGC F-TTCAAATCGGACCTGCGAGAGTGT R-AGATCACAGATGATGTGGCCTGCT F-AACTGTGGCTGCACAGAGAGATGA R-TAGAGCACCTCAGCATGTCTGCAT F-TGTGCACAGAACGAGATGTTTGCG R-ACCAAACACACAAGTGGCACCATC F-ATACACACACTCAGACTCGCGCTT R-AGGAATTGGTGTTGCGCACAGAAG F-TGACTCGGGTTATTACCGCTGCTT R-TGGATGCTCTGGGTCTCGAACAAA F-AGTCAGTCGTCCTTCAACAGCCTT R-TAAACGTGCCCTTGGGTTTGTGTG F-TCTCACCTGGACAAAGCCTCCATT R-AAGCCATTCAGCTGACTTTCCAGC F-AGCTTTGACGATGACCGCAAATGG R-TCAGAATGCTCTAAACCCAGGGCA F-AACCACCGCAGACTATGACAAGGA R-GTGCTTCATTGCTGTTCCCGTCAA F-ATAAGCATGCGCTGAGGAAGAGGA R-AGCTGCAACAATCCAACTCCATGC F-CATGCAAAGGCCTTGATGGGAACT R-TTGATCTCCACAACTAGGCACGCT F-ACGGCTCTCTGCTTTACATCACCA R-TTCAGAGGGTTGAGCTCCCTGTTT F-TGGTCAAAGAGCCAGATTGCCCTA R-GTTTCCAGATAAGCAAGGCGGCAA F-ATGAGGGCATGCAGATACCTTCCA R-TTGACCACGGCATGTTTGAGCATC F-TCTTTCGCAAATCTGCAAGCCTGG R-TGGCACTGAAACAGGCACTTTAGC F-ACATGCAAGTGTGCACTGATGCTC R-TTCCGACATGCTGTCCTTGTCTGT F-TGAGCTACCTGAGCCAGAAACAGT R-TCTTCGCCACAAAGTTCTCTCCCA F-TGGGTCCTCACATCAACATCGTCA R-TGTTTCTGTGCAGGTAGTCCACCA F-TGGACACCAATGGCTACGATGTCT R-TGTCACCCTTACAAGCAGGAGGTT F-GCATCGACAGCATGAACAACTGCT R-ACAGGATGACGATGGCCATGAGAA F-GTCGGCCAAATGCTGATGGAGAAA R-TGTGTGTTGGGCTCCTGTGACATA F-AGGACAAACCGAAAGAGGCTGTGA R-GTTCTTGTGCCGGCCAATCATCTT F-GCAAGATAACGCGCAATTCGGAGA R-TGCATCTCTGTGTCATGAGCCTGT F-TCTGGGAACTCGCTTTGTCTCCAA R-TCTTCTGAACCCTGCAGCCATTCT F-AGGTGGTTCAATGGAAGGAGTGGA R-ATTTGACGTCCTGGTCTGGCTTGA F-ATTCCTCGTCTCCGCTGCTTGTTA R-TGTCGCTCAGCTCTGGATCTTTGT F-TCCCCTTGTTCACAATAACC R-TCTGTTGGCTTTGGGATTC

Hepatocyte growth factor receptor (Met) Insulin-like growth factor 1 (IGF1) Connective tissue growth factor (CTGF) Neuropilin 2 (NRP2) Vascular endothelial growth factor receptor 3 (VEGFR3) v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1) Hypoxia-inducible factor 1 alpha subunit (HIF1A) Matrix metallopeptidase 2 (MMP-2) Matrix metallopeptidase 9 (MMP-9) TIMP metallopeptidase inhibitor 2 (TIMP2) Plasminogen (PLG) Integrin alpha-V (ITGAV) Integrin β-3 (ITGβ3) cadherin-associated protein β (β-catenin) Platelet endothelial cell adhesion molecule (PECAM1) Angiopoietin 1 (ANGPT1) TEK tyrosine kinase (Tie-2) Platelet-derived growth factor receptor β (PDGFR-β) Type 2 cadherin 5, vascular endothelium (CDH5) Sphingosine-1-phosphate receptor 1 (S1PR1) Fibroblast growth factor 2 (FGF2) Fibroblast growth factor receptor 2 (FGFR2) Sonic hedgehog (Shh) Transforming growth factor β1 (TGFβ1) Ephrin-B2 (Ephrin B2) Ephrin type-B receptor 4 (EPHB4) β-actin

2.10. HMEC-1 scratch assay The migration of HMEC-1 cells was examined using the wound healing method (Liu et al., 2013). HMEC-1 (1.5  105 cells) were seeded into each well of a 24-well plate and incubated with complete medium at 37 1C and 5% CO2. Cells were then starved in medium with 0.5% v/v FBS for 24 h. HMEC-1 were scrapped horizontally and vertically with a P100 pipette tip (Eppendorf AG, Hamburg, Germany) and two views on the cross were photographed on each well attached to the microscope at 40  magnification. The medium was replaced with fresh medium in the absence or presence of C1-1. After 6 h incubation, the second

set of images was photographed. To determine the migration of HMEC-1, the images were analyzed using Tscratch software (Gebäck et al., 2009). Percentage of the closed area was measured and compared with the value obtained before treatment. An increase of the percentage of closed area (% control) indicated the migration of cells. 2.11. HMEC-1 tubule formation assay The effects of C1-1 on HMEC-1 differentiation and vascular formation were assessed by tubule formation on Matrigel (Merchan et al., 2003). HMEC-1 cells were seeded onto 96-well

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plates at 1.5  105 cells per well over 60 ml Matrigel (BD BioSciences, USA). Fresh media in the absence or presence of C1-1 were subsequently added. Tubular structures were photographed after 24 h. The total tubule length formation was measured for quantification of angiogenesis by an imaging software, Image-Pro Plus version 6.0 (Media Cybernetics, USA).

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endothelial cells were performed using Students' t-test or Mann– Whitney test. As for multiple group comparison of cell proliferation of HMEC-1, statistical significance was assessed by one-way ANOVA followed by post-hoc Dunnett's test. All statistical tests were performed at 5% level of acceptance (po0.05). Data were presented as means7standard deviation (S.D.) unless otherwise specified.

2.12. Statistical analysis Comparison of results of real-time PCR experiments of angiogenesis-associated genes for zebrafish embryos, cell migration and tubule length formation (% control) with HMEC-1

3. Results 3.1. Angiogenesis effects of bioassay-guided fractions of RR in zebrafish model Sub-fraction C1 was further subjected to fractionation to afford six sub-fractions, namely, C1-1 to C1-6 (Fig. 1). The mean sprout number of sub-fraction C1-1 treated embryos was 2.85 70.24, which was significantly higher (po 0.01) than that of the control (0.15 70.04). It possessed the most potent activities (from 190 to 780 ng/ml, p o0.001) in sprout formation in the zebrafish angiogenesis model among all those sub-fractions tested in RR (Figs. 2 and 3) (Liu et al., 2011). Other upstream sub-fractions and the crude extract of RR have already been studied in our previous work (Liu et al., 2011).

3.2. UHPLC/QTOF-MS analysis of sub-fraction, C1-1

Fig. 2. The angiogenesis effects of the six sub-fractions C1-1 to 6 of RR extract in TG (fli1:EGFP)y1/þ (AB) zebrafish embryos, in terms of mean sprout number. Data are presented as mean 7S.E.M.; n ¼14 to 92. np o 0.05, nnp o 0.01, nnnp o0.001 as compared with control. n represents the total number of zebrafish embryos used in sprout-evaluation.

Using UHPLC/QTOF-MS, the total ion chromatogram (TIC) of C1-1 was revealed by ESI ion source in positive mode (Fig. 4). The molecular weight of major peaks present in C1-1 were screened against our established in-house database for all the known compounds found in RR based on literature review. However, no match was found. This revealed that RR sub-fraction C1-1 might contain novel compounds which are yet to be structurally elucidated.

Fig. 3. The effect of C1-1 treatment on ectopic vessels formation upon sub-intestinal vessels of TG(fli1:EGFP)y1/þ (AB) zebrafish embryos. (A) Control: embryo treated with 0.2% DMSO with no sprout induced in sub-intestinal vessel (SIV). Sprouts induced in SIV of embryo treated with C1-1 at (B) 190 ng/ml, (C) 390 ng/ml, and (D) 780 ng/ml. Ectopic vessels (sprouts) formed in the SIV (magnification: 100  ) as positive indication of angiogenesis effect of testing agent; the arrow indicated sprouts.

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Fig. 4. Total ion chromatogram (TIC) of the active sub-fraction C1-1 of aqueous crude extract of RR by UHPLC/QTOF-MS in positive mode using ESI. Table 2 Angiogenesis-associated genes for zebrafish angiogenesis study in category. (See Table 1 for full names). Category

Gene names

Angiogenic and its associated growth factors and receptors Transcription factors Matrix degradation/ endothelial cell migration Cell adhesion molecules Tubule formation and morphogenesis/smooth muscle cell recruitment and differentiation Blood vessel maturation/formation of arteries and veins

EGFR, Met, IGF1, CTGF, NRP2, VEGFA, Flt-1, KDR, VEGFR3 Ets-1, HIF1A MMP-2, MMP-9, TIMP2, PLG ITGAV, ITGB3, β-catenin, PECAM1 ANGPT1, Tie-2, PDGFR-β, CDH5, S1PR1, FGF2, FGFR2, Shh, TGFβ1 Ephrin B2, EPHB4

Fig. 5. C1-1 induced mRNA expression of angiogenesis-associated genes in zebrafish embryos quantified using real-time PCR normalized against house-keeping gene, β-actin. Data are presented as mean of relative mRNA amount (fold of control)7 S.E.M. of three independent experiments. np o 0.05, nnpo 0.01, vs. control group.

3.3. Angiogenesis effect of C1-1 in angiogenesis-related mRNA expression level in zebrafish To further study the underlying in-depth mechanisms of active sub-fraction C1-1 in zebrafish embryo model, total RNA of the C1-1 treated embryos and the untreated control were subjected to quantitative real-time PCR with a selection of tailor-made angiogenesis-associated genes broadly categorized in Table 2 according to their functions and/or stages in angiogenesis. In brief, the target genes were chosen through the considerations of current thoughts in angiogenesis, zebrafish biology and its sub-intestinal vessel sprout formation (Lawson and Weinstein, 2002b; Muñoz-Chápuli et al., 2004; Oettgen, 2005; Dejana et al., 2007; Le Bras et al., 2010; Herbert and Stainier, 2011). Also, commercially available software and design, such as the collections in PCR arrays (SABiosciences, Qiagen GmbH, Hilden, Germany) of the angiogenesis arrays (for

human, mouse and rat), endothelial cell biology, extracellular matrix (ECM) and adhesion array, wound-healing array, and the interacting networks of between-gene-target by Gene Network Central (GNC) Pro (SABiosciences, Qiagen GmbH, Hilden, Germany) were also integrated in the selection process. Although some of those target genes listed in Table 2 might also be involved in multiple functions and/or overlapping stages, they were placed in their representative contributing groups. As for gene expression of 780 ng/ml C1-1 treated zebrafish embryo, significant up-regulation with more than 2-fold was found in epidermal growth factor receptor (EGFR) (p o0.05), angiopoietin 1 (po 0.05), matrix metallopeptidase 9 (MMP-9) (p o0.05) and ephrin type-B receptor 4 (EPHB4) (p o0.01) (Fig. 5). These are the genes concerning angiogenic growth factors and receptors, matrix degradation, tubule formation and vessel maturation in angiogenesis. On the other hand, significantly down-regulated genes demonstrated in the gene panel were ephrin-B2 (p o0.05), vascular endothelial growth factor receptor 1 (Flt-1) (p o0.05) and Ets-1 (p o0.05) upon C1-1 treatment (Fig. 5). These genes were associated with transcription factors, vessel differentiation and receptors for angiogenic growth factors. Meanwhile, trends of increase (while not statistically significant) were observed in integrin β3 (ITGβ3), hepatocyte growth factor receptor (Met), connective tissue growth factor (CTGF), VEGFA, platelet-derived growth factor receptor β (PDGFR-β), fibroblast growth factor receptor 2 (FGFR2) and VEGFR3 under C1-1 treatment (Fig. 5). These are the genes regarding cell adhesion, tubule morphogenesis and angiogenic growth factors and their receptors, 3.4. Effect of endothelial cell proliferation of C1-1 in HMEC-1 cells In Fig. 6A, no significant change was found in the endothelial cell proliferation when treated with concentrations of C1-1 at or below 31.25 mg/ml, indicating that these concentrations were nontoxic and probably did not affect the cellular activities of HMEC-1. However, 62.5 mg/ml of C1-1 was found cytotoxic (p o0.001) and

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Fig. 6. (A) Effect of C1-1 on HMEC-1 proliferation. The value from the baseline control group was set at 100%. Data are expressed as mean 7 S.D. from three individual experiments. #p o0.001 versus control. (B) Quantitative analysis of the C1-1-induced HMEC-1 cell migration. np o 0.05, nnpo 0.01 and nnnp o 0.001 for differences in wound closure (% control) from baseline cultures without treatment. Data are expressed as mean 7 S.D. from two individual experiments. (C) C1-1-induced HMEC-1 migration after 6 h. Images were captured at 0 h and different concentrations (3.9 and 15.6 mg/ml) of C1-1 were added to the wells. Another set of images were captured after 6 h incubation of C1-1.

hence this high concentration was not used in the angiogenesis assays in the study. 3.5. Cell migration effect of C1-1 in HMEC-1 cells Effects of C1-1 on HMEC-1 endothelial cell migration were assessed using the migration scratch assay. As shown in Fig. 6B and C, C1-1 (at 2.0 to 15.6 μg/ml) could significantly stimulate cell migration of HMEC-1 from 26.5 to 36.4% (p o0.001 to 0.05). Thus, C1-1 was found to significantly promote in vitro endothelial cells migration for angiogenesis. 3.6. Tubule formation of C1-1 in HMEC-1 cells Angiogenesis is a complex process involving endothelial cell proliferation and migration, leading to subsequent vascular structure formation (Arnaoutova et al., 2009). Tubule formation assay was used to assess the ability of endothelial cell differentiation in angiogenesis after treatment of C1-1. Our results demonstrated that when HMEC-1 were cultured on Matrigel without addition of

C1-1, HMEC-1 formed little and simple hollow tubule-like structure only (Fig. 7). Yet, after incubation of C1-1 for 24 h, a more complex and branched tubular structure was observed. In Fig. 7B, quantification revealed that 1.6 mg/ml of C1-1 could significantly enhance the tubule formation ability of HMEC-1 (p o0.05) on Matrigel.

4. Discussion In chronic wound healing such as diabetic wound, impaired angiogenesis is one of the pathological stages in delaying wound repair (Chabbert-Buffet et al., 2003; Martin et al., 2003). Proangiogenic agents could help to enhance the angiogenesis, thus, improves chronic wound healing. Our previous studies found that aqueous extract of RR (Lau et al., 2008) could significantly enhance wound healing in diabetic foot ulcer animal model with enhanced the VEGF expression observed for the improved angiogenesis (Lau et al., 2009).

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Fig. 7. Effect of tubule formation of C1-1-treated HMEC-1 on Matrigel. (A) Morphological changes of HMEC-1 differentiation in vascular network in three-dimensional matrigel after treatment of control and 1.6 mg/ml C1-1 at 24 h. (B) Quantitative analysis of the total tubule length in C1-1-treated HMEC-1 with the use of Image-Pro Plus software. Data are expressed as the tubule length (% control)7S.D. from three individual experiments.

Using zebrafish sprout angiogenesis-guided fractionation, we previously reported that, norviburtinal was found to possess angiogenesis effect in RR, however, in a rather moderate manner (Liu et al., 2011). The active component(s) in RR responsible for its angiogenesis effect is still not well understood. In searching for other active component(s), bioassay-guided fractionation was further applied. Here, the angiogenesis effects and its underlying mechanism of the further guided fractions of RR were studied using a transgenic zebrafish embryo angiogenesis model. Sub-fraction C1 (from the DCM partition of RR aqueous extract) was further subjected to fractionation to afford six sub-fractions, of which C1-1 with the mean sprout number of 2.85 70.24, was significantly higher (p o0.01) than that of the control (0.15 70.04). It possessed the most potent activities (from 190 to 780 ng/ml, p o0.001) in sprout formation in the zebrafish angiogenesis model among all other sub-fractions tested in RR (Fig. 2). Indeed, the potency of C1-1 active sub-fraction (0.78 μg/ml) was460-fold and 41200-fold higher than that of norviburtinal (50 μg/ml) and RR crude extract (1000 μg/ml) respectively, for the concentrations by which their maximum effects reached (Liu et al., 2011). More importantly, C1-1 (0.78 μg/ml) could induce around 3-sprout formed per embryo treated (by mean sprout number), whereas only around 1-sprout per embryo was induced (by mean sprout number) for both norviburtinal (50 μg/ml) and RR crude extract (1000 μg/ml) (Liu et al., 2011). Furthermore, based on the results from LCMS (Fig. 4), norviburtinal (along with other compounds in our RR chemical library) was not found in sub-fraction C1-1. Hence, there might be other novel compound(s) in C1-1, other than norviburtinal, which is responsible for the angiogenesis effect in RR. Currently, most of the studies applying zebrafish as a screening model for pro-angiogenic agents detecting through sprout(s)-

induction in sub-intestinal vein were mainly focused in phenotypic level (Hong et al., 2009; Raghunath et al., 2009; He et al., 2010; Liu et al., 2011). Here we designed a wider set of angiogenesisassociated genes to reveal a broader spectrum of actions in angiogenesis for their gene expression levels concerning the testing of pro-angiogenic agents. Selection of angiogenesis-associated genes was broadly categorized in Table 2 according to their functions and/or stages in angiogenesis. Target genes were chosen through reviewing the current thoughts in different stages, signaling, mechanism and the blood vessel morphogenesis concerned in angiogenesis, with special emphasis to the zebrafish biology and its sub-intestinal vessel sprout formation (Lawson and Weinstein, 2002b; MuñozChápuli et al., 2004; Oettgen, 2005 Dejana et al., 2007; Le Bras et al., 2010; Herbert and Stainier, 2011). As for the aspect of angiogenic and its associated growth factors and receptors (Lawson and Weinstein, 2002b; Muñoz-Chápuli et al., 2004; Oettgen, 2005; Herbert and Stainier, 2011), epidermal growth factor receptor (EGFR) was significantly up-regulated (p o0.05); while trends of up-regulations were shown in the 780 ng/ml C1-1-treated zebrafish embryo expression of hepatocyte growth factor receptor (Met), connective tissue growth factor (CTGF), VEGFA and VEGFR3 by quantitative real-time PCR analysis. On the other hand, significant down-regulation of vascular endothelial growth factor receptor 1 (Flt-1) (p o0.05) was found in C1-1 treatment (Fig. 5). Angiogenesis was demonstrated by up-regulated expression of these growth factor receptors, EGFR (Repertinger et al., 2004), hepatocyte growth factor receptor (Kaga et al., 2012), CTGF (HallGlenn et al., 2012), VEGFA (Herbert and Stainier, 2011) and VEGFR3 (Siekmann and Lawson, 2007; Tammela et al., 2008; Gore et al., 2011). In particular, VEGFR3 was required for positive regulation in

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Fig. 8. Proposed gene interaction of C1-1 in angiogenesis by zebrafish embryo and its motogenic and tubule inducing effect by human microvascular cell line.

angiogenesis (Tammela et al., 2008) and essential for endothelial cell sprouting in zebrafish and mice (Siekmann and Lawson, 2007; Tammela et al., 2008). Indeed, selective EGFR inhibitor was reported to demonstrate inhibition in EGF-induced endothelial cell migration and tubule formation; the inhibitor also blocked mice cornea neovascularization (Hirata et al., 2002). EGFR played crucial roles in angiogenesis. Regarding C1-1 treatment for genes responsible for matrix degradation in angiogenesis (Steinle et al., 2002; Moro et al., 2008; Santhekadur et al., 2012), matrix metallopeptidase 9 (MMP-9) was highly up-regulated by more than 2-fold (p o 0.05). In fact, MMP-9 up-regulation was shown being important in matrix degradation processes for angiogenesis (Santhekadur et al., 2012). Integrins and other cell adhesion molecules playing critical roles in angiogenesis for, in particular, cell migration and tubule formation, were also studied (Borges et al., 2000; Muñoz-Chápuli et al., 2004; Herbert and Stainier, 2011). A trend of increase for integrin β3 (ITGβ3) was observed under C1-1 treatment, which was reported to be crucial in angiogenesis for cell motility (Borges et al., 2000) (Fig. 5). Concerning transcription factors (Oettgen, 2005; Dejana et al., 2007; Seeger et al., 2009), Ets-1 was significantly down-regulated (p o0.05) upon C1-1 treatment (Fig. 5). The rationale for the down-regulation of Ets-1 could be induced by the fact that the activation of MMP-9 (Moro et al., 2008) and down-regulation of Flt-1 (Tchaikovski et al., 2008) could lead to down-regulation of ERK. This down-regulation of ERK might, in turn, trigger the down-regulation of Ets-1 (Chen et al., 2005). Although it has been reported that Ets-1 activation could induce angiogenesis (Hashiya et al., 2004), down-regulated Ets-1 was found to rescue

endothelial progenitor cells (EPC) reduction (Seeger et al., 2009), of which EPC showed importance in zebrafish hematopoiesis (Du et al., 2011), and improvement in vascular repair of zebrafish fin amputation model (Pozzoli et al., 2011). Hence, Ets-1 downregulation could be pro-angiogenic related. Tubule formation and morphogenesis, working closely with smooth muscle cell recruitment and differentiation, play vital roles in angiogenesis (Borges et al., 2000; Muñoz-Chápuli et al., 2004; Oettgen, 2005; Presta et al., 2005; Herbert and Stainier, 2011; HallGlenn et al., 2012). Angiopoietin 1 was highly up-regulated (p o0.05) with more than 2-fold. In addition, trends in upregulation of platelet-derived growth factor receptor β (PDGFR-β) and fibroblast growth factor receptor 2 (FGFR2) were observed (Fig. 5). The increase in expression of angiopoietin 1, PDGFR-β (Borges et al., 2000; Hall-Glenn et al., 2012), and FGFR2 (Presta et al., 2005) were found to be involved in angiogenesis, mainly for vessel stabilization. The later part of angiogenesis would be blood vessel maturation and the formation of arteries and veins (Lawson and Weinstein, 2002b; Muñoz-Chápuli et al., 2004; Oettgen, 2005; Carmeliet and Jain, 2011; Herbert and Stainier, 2011). Here we demonstrated a significant increase with more than 3-fold (p o0.001) in the ephrin type-B receptor 4 (EPHB4) expression after C1-1 treatment, of which this increase was reported to increase angiogenesis (Carmeliet and Jain, 2011). On the other hand, ephrin-B2 expression was significantly down-regulated (p o0.05) (Fig. 5). Decreased ephrin-B2 and increased VEGFR3 expression were shown in correlation with the knockdown of Flt-1 (Krueger et al., 2011); such knockdown triggered augmented angiogenic behavior, induced ectopic sprouting, resulting in the increase blood flow by the increased vessel developed in zebrafish

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(Krueger et al., 2011). Their results showed a high similarity of what we observed in the significant decrease of ephrin-B2 (p o0.05) and Flt-1 (po 0.05) (Fig. 5) and trend of increase in VEGFR3 expression in our C1-1 treated zebrafish embryo. Besides, EPHB4 and VEGFR3 are the vein specific markers in zebrafish and mice (Lawson and Weinstein, 2002b; Dejana et al., 2007). The up-regulation of EPHB4 and VEGFR3 supported our observation of which elevated ectopic sprouts from the subintestinal vein was significantly formed in C1-1 treated (from 190 to 780 ng/ml, p o0.001, Fig. 5) zebrafish embryos. Additionally, the interacting network for C1-1 induced angiogenesis in zebrafish was further complicated by reported interaction among these angiogenic-associated genes (Fig. 8). Hall-Glenn et al. (2012) reported the CTGF-induced PDGF-B expression in endothelial cells. Borges et al. (2000) observed that enhanced cell migration by PDGF in cell transfected with integrin beta 3 extracellular domain, and association of PDGFR-beta and integrin beta 3 extracellular domain was further found. Steinle et al. (2002) revealed that EPHB4 activated MMP9 expression that might contribute to angiogenesis. Thus, C1-1 treated zebrafish embryo angiogenic-associated gene expression demonstrated pro-angiogenic potency in various aspects of angiogenesis over a complex network (Fig. 8). With the prominent results of C1-1 triggered in both the phenotypic and molecular gene expression level of angiogenesis in zebrafish embryo model, we further translated the study to a human endothelial cell line. Indeed, majority of endothelial cells (EC) in human body belongs to microvasculature (Bouïs et al., 2001), HMEC-1, carries a majority of traits to that of primary microvascular endothelial cell, has been widely used for endothelial research (Lidington et al., 1999; Bouïs et al., 2001). Furthermore, the dermal origin of HMEC-1 further makes it favourable for the study of wound healing (Lidington et al., 1999). Thus, the human microvascular endothelial cell (HMEC-1) was used for in vitro angiogenesis assays of EC proliferation, migration and tubule formation. Endothelial cell migration is a vital step in angiogenesis, C1-1 treatment (2.0 to 15.6 μg/ml) could significantly (po 0.001 to 0.05) stimulate HMEC-1 cell migration by scratch assay (Fig. 6B and C). Another process of angiogenesis is endothelial cell proliferation; however, C1-1 did not induce any increase in HMEC-1 cell proliferation by MTT proliferation studies (as shown in Fig. 6A). Morphological differentiation of endothelial cells is one of the pivotal stages in angiogenesis, involving cell adhesion, cell migration, secretion of proteases, and tubule formation (Arnaoutova et al., 2009). A time-saving, yet, quantifiable in vitro angiogenesis assay including the above critical stages in differentiation is the tubule formation assay upon basement membrane such as matrigel. Significant increase (po 0.05) in mean tubule length (% control) was observed in the C1-1-treated HMEC-1 cells with matrigel complex in our tubule formation assay. This suggested that C1-1 could stimulate tubular formation in vitro in HMEC-1 cells, of which revealing its importance in later stages of angiogenesis for the formation of vascular structure. Taken together, zebrafish sprout-inducing model-guided fractionation suggested that C1-1 possessed the most potent angiogenesis effect in RR. To study the in-depth angiogenic effect induced by C1-1, a panel with 30 tailor-made angiogenesisassociated genes was designed. From gene expression analysis, C1-1 triggered up-regulation of various angiogenesis-associated genes, such as VEGFR3 and MMP9, which played crucial role in angiogenesis. The pro-angiogenic activity was also succeeded in the translated study in motogenic and tubule inducing effect using in vitro human microvascular cell line (Fig. 8). Apart from norviburtinal, sub-fraction C1-1 possessed active pro-angiogenesis effect of RR. However, large scale extraction from RR could aid to further characterize the active component(s) in C1-1.

5. Conclusion Our zebrafish sprout angiogenesis model guided-fractionation demonstrated that sub-fraction C1-1 possessed the most potent angiogenesis effect in RR. In-depth angiogenic effects induced by C1-1 were further studied with the design of a panel of 30 tailormade angiogenesis-associated genes. Zebrafish gene expression analyses showed that C1-1 could trigger up/down-regulation of various angiogenesis-associated genes, such as VEGFR3 and MMP9, which played crucial role in angiogenesis. The proangiogenic activity of C1-1 was also confirmed in the translated study in motogenic and tubule-inducing effect using in vitro human dermal microvascular cell line. Other active angiogenesis component(s) of RR are yet to be found.

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