http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.941882
ORIGINAL ARTICLE
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Chrysanthemum boreale flower floral water inhibits platelet-derived growth factor-stimulated migration and proliferation in vascular smooth muscle cells Do-Yoon Kim1*, Kyung-Jong Won2*, Mi-So Yoon1, Ho-Jin Yu1, Joo-Hoon Park1, Bokyung Kim2, and Hwan Myung Lee1 1
Department of Cosmetic Science, College of Natural Science, Hoseo University, Asan, Chungnam Prefecture, Republic of Korea and Department of Physiology, School of Medicine, Konkuk University, Chungju, Chungbuk Prefecture, Republic of Korea
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Abstract
Keywords
Context: Chrysanthemum boreale Makino (Compositae) (CBM) is a traditional medicine that has been used for the prevention or treatment of various disorders; it has various properties including antioxidation, anti-inflammation, and antitumor. Objective: The present study was designed to explore the in vitro effect of CBM flower floral water (CBMFF) on atherosclerosis-related responses in rat aortic smooth muscle cells (RASMCs). Materials and methods: CBMFF was extracted from CBM flower by steam distillation and analyzed using gas chromatography–mass spectrometry. The anti-atherosclerosis activity of CBMFF was tested by estimating platelet-derived growth factor (PDGF)-BB (10 ng/mL)-induced proliferation and migration levels and intracellular kinase pathways in RASMCs at CBMFF concentrations of 0.01–100 lM and analyzing ex vivo aortic ring assay. Results: Gas chromatography–mass spectrometry showed that the CBMFF contained a total of seven components. The CBMFF inhibits PDGF-BB-stimulated RASMC migration and proliferation (IC50: 0.010 lg/mL). Treatment of RASMCs with PDGF-BB induced PDGFR-b phosphorylation and increased the phosphorylations of MAPK p38 and ERK1/2. CBMFF addition prevented PDGF-BBinduced phosphorylation of these kinases (IC50: 008 and 0.018 lg/mL, for p38 MAPK and ERK1/ 2, respectively), as well as PDGFR-b (IC50: 0.046 lg/mL). Treatment with inhibitors of PDGFR, P38 MAPK, and ERK1/2 decreased PDGF-BB-increased migration and proliferation in RASMCs. Moreover, the CBMFF suppressed PDGF-BB-increased sprout outgrowth of aortic rings (IC50: 0.047 lg/mL). Discussion and conclusion: These results demonstrate that CBMFF may inhibit PDGF-BB-induced vascular migration and proliferation, most likely through inhibition of the PDGFR-b-mediated MAPK pathway; therefore, the CBMFF may be promising candidate for the development of herbal remedies for vascular disorders.
Anti-atherosclerosis, chemotherapy, natural products, vascular disease
Introduction Vascular smooth muscle cell (VSMC) migration and proliferation are important in the development progression of vascular neointima in atherosclerosis (Lee et al., 2012; Wang et al., 2012). These events were stimulated by various factors, including proinflammatory cytokines and peptide growth factors (Ross, 1999). Platelet-derived growth factors (PDGF)
*These authors contributed equally to this work. Correspondence: Hwan Myung Lee, Assistant Professor, Department of Cosmetic Science, College of Natural Science, Hoseo University, Asan-city, Chungnam Prefecture 336-795, Republic of Korea. Tel: +82 41 540 9551. Fax: +82 41 540 9538. E-mail: [email protected] Bokyung Kim, Professor, Department of Physiology, School of Medicine, Konkuk University, Chungju-city, Chungbuk Prefecture 380-701, Republic of Korea. Tel: +82 43 840 3726. Fax: +82 43 851 9329. E-mail: [email protected]
History Received 9 April 2014 Accepted 2 July 2014 Published online 21 October 2014
are a family of dimeric growth factors that are important to the regulation of VSMC proliferation and migration (Dammanahalli et al., 2012; Jiang et al., 2010; Won et al., 2008). The individual PDGF monomers may be one of four proteins: PDGF-A, B, C, or D (Fredriksson et al., 2004). In their dimeric form, PDGFs bind to and activate a PDGF receptor (PDGFR) that consists of two receptor tyrosine kinase subtypes, PDGFR-a and PDGFR-b, localized to the plasma membrane of cells. Once bound to a PDGF, PDGFRs activate diverse signaling molecules and regulatory proteins that contain Src homology 2-domains and thereby elicit various cellular responses like actin reorganization, proliferation, differentiation, or VSMC migration (Jiang et al., 2010; Won et al., 2011). Mitogen-activated protein kinases (MAPKs) are also signaling molecules important for regulating various cellular processes in cells, including proliferation, differentiation, and migration (Kim et al., 2009; Lee et al., 2008a,b).
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MAPK activation is induced by diverse stimuli, including growth factors, hormones, and cytokines (Lee et al., 2009). PDGFs can activate some MAPKs, including extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stressactivated protein kinase/c-Jun N-terminal kinase (Fukai et al., 1998). PDGF-induced activation of MAPK pathways is known to trigger VSMC migration and proliferation (Eguchi et al., 2001). Specifically, PDGFs stimulate the phosphorylation of p38 MAPK and ERK1/2 (Lee et al., 2007a), which are known to initiate migration or proliferation and growth, respectively, in VSMCs (Dubey et al., 2000; Gan et al., 2013). Chrysanthemum boreale Makino (Compositae) (CBM) is a perennial plant that is widely distributed in East Asia countries such as Korea, Japan, and China (Kim et al., 2010). CBM and other related species such as C. indicum Linne´ (Composite) and C. lavandulaefolium Makino (Fisch.) Mak. are traditional oriental medicines that have been used to treat a variety of disorders such as pneumonia, colitis, stomatitis, and carbuncle (Kim et al., 2003). CBM contains various pharmacologically active components, including flavonoids, sesquiterpene lactones, chrysanthemin, and essential oils (Kim et al., 2003). The components isolated from CBM exhibit diverse pharmacological activities including antibacterial, antitumor, anti-angiogenesis, anti-hypertension, and anti-inflammatory activities. CBM flowers (CBMF) are known to have antipyretic and anti-vertigo effects (Lee et al., 2003; Perry, 1980). Although some herbal medicines have been used for the prevention or treatment of cardiovascular diseases like atherosclerosis (Mashour et al., 1998), there are no reports of CBMF being used to treat vascular disorders. Since there are many reports about other herbal extracts inhibiting VSMC migration and proliferation that are key events in neointima formation (Choi et al., 2009; Won et al., 2009), we here explored whether CBMF floral water (CBMFF) could affect PDGF-BB-induced proliferation and migration of rat aortic smooth muscle cells (RASMCs).
Materials and methods Materials PDGF-BB was purchased from R&D Systems (Minneapolis, MN) and Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, phosphate buffered saline (PBS), trypsin-ethylenediamine tetraacetic acid (EDTA), and Hank’s balanced salt solution (HBSS) were from Hyclone (Logan, UT) or Invitrogen (Carlsbad, CA). Matrigel and type I collagen were purchased from BD Bioscience (Franklin Lakes, NJ), and collagenase was from Wako (Richmond, VA). Bovine serum albumin (BSA) and elastase were purchased from Sigma (St. Louis, MO) and the Diff-Quik stain kit was from Sysmex Corp (Kobe, Japan). AG1296, SB203580, and PD98059 were purchased from Tocris Bioscience (Bristol, UK). The antibodies used included anti-p38 MAPK, anti-phospho p38, anti-ERK1/2, anti-phospho ERK1/2, anti-PDGFR-b (Cell signaling, Beverly, MA), anti-phospho-tyrosine(4G10) (Upstate, Lake Placid, NY) and anti-b-actin (Sigma, St. Louis, MO) antibodies.
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Preparation of CBMFF Fresh CBMFs (20 kg) were collected from the farm of Hoseo University, Republic of Korea, in October 2010 and CBMFF was isolated by steam distillation for 2 h. In brief, the procedure comprises a passage of vapor from a boiler into a chamber holding plants, followed by condensation of the solute-containing steam by contact with cold water. The different densities of the oil and floral water (10 L) enabled the phases to effectively separate. The collected CBMFF were dried by freeze-drying, and the resulting extracts were dissolved in dimethyl sulfoxide (DMSO) and stored at 20 C. Analysis of CBMFF and compound identification CBMFF obtained by steam distillation of CBMF was dissolved in methanol and analyzed on a gas chromatograph (Agilent 6890N)-mass spectrometer (Aglient 5975i MS) (Agilent technologies, Palo Alto, CA) equipped with a DB5-MS column (30 m 250 lm, 0.25 lm film thickness; J&W Scientific, Folsom, CA). The samples were introduced by split mode at a split ratio of 1:10. The injector temperature was set at 280 C. The oven temperature was programmed as follows: isothermal at 50 C for 2 min, rising at 10 C/min to 300 C and held at this temperature for 10 min. Helium was used as the carrier gas at a rate of 1 mL/min. The interface temperature was 300 C. Effluent of the GC column was introduced directly into the source of the MS via a transfer line (230 C). Mass range was 50–800 m/z. Compounds were tentatively identified by comparison of mass spectra of each peak with those of an authentic sample in the Wiley7Nist05.L library. Cell isolation and culture All animal investigations were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996) and approved by the Animal Subjects Committee and by the Institutional Guidelines of Konkuk University, Korea. RASMCs were isolated from the aortas of male Sprague–Dawley (SD) rats (6 weeks old, 160–180 g, n ¼ 8) by collagenase and elastase treatment and were subsequently cultured in DMEM containing 10% FBS, 100 U/mL penicillin, 100 lg/mL streptomycin, and 200 mM L-glutamine. For all experiments, the RASMCs (used at passages 3–8) were grown to 70–80% confluence in DMEM and starved of FBS for 24 h. Immunoblotting After treatment with CBMFF and PDGF-BB, RASMCs were lysed with cold extraction buffer (20 mM HEPES [pH 7.5], 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, 2.5 mM 4-nitrophenylphosphate, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], and 1 complete proteinase inhibitor cocktail tablet [Roche, Indianapolis, IN]). Cell lysates were centrifuged at 17 000 g for 15 min at 4 C and the supernatants were collected as protein samples. Protein concentrations were determined using the protein assay reagents (Bio-Rad, Hercules, CA).
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DOI: 10.3109/13880209.2014.941882
Chrysanthemum boreale Makino in vascular smooth muscle cells
Protein homogenates were diluted 1:1 (v/v) with SDS loading buffer (40 mM Tris-HCl pH 6.8, 8 mM ethylene glycol tetraacetic acid, 4% 2-mercaptoethanol, 40% glycerol, 0.01% bromophenol blue, and 4% SDS) and boiled for 5 min. Samples (30–50 lg/lane) were separated on 12–14% polyacrylamide SDS gels and transferred electrophoretically to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membrane was blocked for 2 h at room temperature (RT) with PBS containing 0.05% Tween-20 and 5% fat-free dried milk and incubated overnight at 4 C with selective antibodies diluted 1000- to 2000-fold. Immune complexes were detected with horse radish peroxidase-conjugated antibodies (Amersham Pharmacia Biotech, Piscataway, NJ), followed by visualization on photographic film. Band intensities were quantified with Quantitation software package (Bio-Rad, Berkeley, CA). Immunoprecipitation To measure the activation of PDGFR, RASMCs were lysed in extraction buffer and lysates containing 200–300 lg of proteins were incubated with 4 lg/mL of anti-PDGFR-b antibody for 5 h at RT. The immunocomplex was precipitated by incubation with protein A-agarose beads (Roche, Branford, CT) at overnight at 4 C. Beads were subsequently washed with PBS containing 0.1% Tween-20, resuspended in SDS sample buffer, and boiled for 5 min. Protein samples collected were immunoblotted with anti-phospho-tyrosine (4G10) antibody as per the protocol described above. Migration assay Cell migration assays were performed in 48-well Boyden microchemotaxis chambers (Neuro Probe, Cabin John, MD). Polycarbonate membranes with 8 lm pores (Neuro Probe, Cabin John, MD) were coated with a 0.1 mg/mL solution of type I collagen from rat tail tendon (BD Bioscience, San Jose, CA) in distilled water and then dried for 60 min. RASMCs were harvested using trypsin–EDTA and resuspended in DMEM that contained 0.1% BSA. PDGF-BB, CBMFF, and test inhibitors were loaded in the bottom chamber and the membrane was laid over an aliquot of approximately 5 104 cells. The chamber was then incubated for 90 min at 37 C, fixed, and stained using a Diff-Quik staining kit (BD Bioscience, San Jose, CA). All samples used in the experiments contained 0.1% DMSO, which did not affect RASMC migration. The number of cells migrating through the membrane was determined by counting the cells in four randomly chosen regions of each well under a microscope at 400-fold magnification. Proliferation assay RASMC proliferation activity was measured via a 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay using a WelCountÔ cell viability assay kit (WelGENE, Korea). In brief, the cells were seeded at 2 103 cells per well in DMEM onto a 96-well plate coated with 0.1 mg/mL type I collagen. After incubating for 12 h, the DMEM was replaced with serum-free DMEM followed by incubation for an additional 6 h. After removing the medium, the remaining cells were treated with CBMFF, test inhibitors
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and PDGF-BB and incubated for 48 h. To measure proliferation, 20 lL of XTT solution was added to each well, followed by incubation for 4 h at 37 C in an atmosphere of 5% CO2. The plates were then thoroughly shaken, and the absorbance at 450 nm and 690 nm was measured using a microplate reader (Bio Tek, Seoul, Korea). Aortic ring assay Ex vivo migration and proliferation of the RASMCs were measured by an aortic ring assay using Matrigel according to a previously reported method with slight modifications (Nicocia & Ottinetti, 1990). The endothelium and adventitium of the aorta from four 5-week-old SD rats were removed enzymatically, and the vessels were cut into 1 mm rings. The rings were embedded onto 48-well plates coated with Matrigel, and then PDGF-BB and CBMFF suspended in FBS-free DMEM were simultaneously added. Following growth for 5 d, the rings were stained with Diff-Quik, photographed, and the length of the sprouts analyzed using Scion Image software (Institutes of Health, Houghton, MI). Statistical analysis Data are expressed as mean ± standard deviation (SD). Statistical evaluation of data was performed with Graphpad Prism version 5.0 (GraphPad Software, San Diego, CA) using a two-way analysis of variance (ANOVA). Values of p50.05 were considered statistically significant.
Results Identification of compounds in CBMFF To analyze the compounds of CBMFF in the absence of hydrophobic essential oil, we performed a GC-MS analysis and found that the floral water contained seven compounds (Table 1). Among them, 1,2-cis-1,5-trans-2,5-dihydroxy-4methyl-1-(10-hydroxy-1-isopropyl)cyclohex-3-ene (1.29%) was the highest content and all other compounds exhibited the following descending order: cis-4-hydrocy-2-methyl-5-(1hydrocy-1-isopropyl)-2-cyclohexen-1-one (1.29%), thymol (1.09%), methyl ester of ricinoleic acid (0.91%), eugenol (0.48%), cis-carveol (0.35%), and 2,4-pentadienenitrile (0.33%). CBMFF possessed not only bioactive compounds such as thymol and eugenol that have anticancer, antioxidant, antimicrobial, anti-inflammation, and vasorelaxant activities but also compounds with unknown activity. Effects of CBMFF on migration and proliferation in response to PDGF-BB The effect of CBMFF on RASMC migration and proliferation in response to PDGF-BB was assessed using a modified Boyden chamber and an XTT assay, respectively. At a concentration of 10 ng/mL, PDGF-BB induced RASMC migration (473.47 ± 21.97% of control, n ¼ 6; Figure 1). PDGF-BB-induced migration was suppressed by treatment with CBMFF (0.01–100 lg/mL) in a dose-dependent manner. On one hand, the maximum suppression was observed at a CBMFF concentration of 100 lg/mL (302.56 ± 20.62% of control, n ¼ 6). PDGF-BB treatment at 10 ng/mL also
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Table 1. Phytochemical components of the Chrysanthemum boreale Makino flower floral water. Compounds name (CAS no.) 2,4-Pentadienenitrile (1615-70-9) cis-Carveol (1197-06-4) Thymol (889-83-8)
RT
% of area
3.52 11.09 11.90
0.33 0.35 1.09
Activity Unknown Unknown Anti-cancer Anti-septic Anti-oxidant
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Anti-microbial
Eugenol (97-53-0)
12.77
0.48
Vasorelaxant Anti-inflammation Anti-cancer Anti-oxidation Anti-microbial
1,2-cis-1,5-trans-2,5-Dihydroxy-4-methyl-1-(10hydroxy-1-isopropyl)cyclohex-3-ene (87096-70-6) cis-4-Hydrocy-2-methyl-5-(1-hydroxy-1-isopropyl)-2cyclohexen-1-one (97762-02-2) Methyl ester of ricinoleic acid (141-24-2)
15.57
2.14
Vasorelaxant Unknown
15.80
1.29
Unknown
22.47
0.91
Unknown
References
Deb et al. (2011) and Hsu et al. (2011) Beena et al. (2013) Archana et al. (2009), Meeran and Prince (2012), and Satooka and Kubo (2011) Elissondo et al. (2008), Rivas et al. (2010), Wattanasatcha et al. (2012) Peixoto-Neves et al. (2010) Lee et al. (2007b) and Yeh et al. (2011) Manikandan et al. (2011), Pal et al. (2010), and Pisano et al. (2007) Devi et al. (2010), Ito et al. (2005), and Nagababu et al. (2010) Ali et al. (2005), Pei et al. (2009), and Shah et al. (2013) Damiani et al. (2003)
RT, retention time.
Figure 1. Effect of Chrysanthemum boreale Makino flower floral water on PDGF-BB-induced migration in RASMCs. RASMCs were treated with PDGF-BB (10 ng/mL) in the presence or absence of steam-distilled extract floral water of Chrysanthemum boreale Makino flower (CBMFF: 0.01–100 lg/mL) for 90 min. Cell migration was analyzed using a Boyden chamber assay. Cell migration in the quiescent state was considered as 100% (n ¼ 6). Results are represented as the mean ± SD. *p50.05 compared to the PDGF-BB-stimulated state by a two-way ANOVA.
elevated RASMC proliferation (232.03 ± 13.86% of control, n ¼ 6; Figure 2), and this response was suppressed by treatment with CBMFF in a dose-dependent fashion (0.01– 100 lg/mL). The maximum inhibition was observed at a CBMFF concentration of 100 lg/mL (118.58 ± 3.78% of control, n ¼ 6). On the other hand, CBMFF (0.01–100 lg/ mL) did not affect RASMC migration and proliferation at PDGF-BB-untreated states (Figures 1 and 2).
Figure 2. Effect of Chrysanthemum boreale Makino flower floral water on PDGF-BB stimulated proliferation in RASMCs. RASMCs were treated with or without steam-distilled floral water of Chrysanthemum boreale Makino flower (CBMFF: 0.01–100 lg/mL) and then stimulated by PDGF-BB (10 ng/mL) for 48 h. Cell proliferation was analyzed by performing the XTT assay. Cell proliferation in the quiescent state was expressed as 100% (n ¼ 6). Each value is expressed as the mean ± SD. *p50.05 compared to the PDGF-BB-stimulated state by a two-way ANOVA.
Effects of CBMFF on phosphorylation of PDGFR-b in RASMCs PDGFs normally bind to a cell surface protein tyrosine kinase receptor-like PDGFR-b, and this binding event leads to activation of the receptor and associated signaling proteins (Chen et al., 2006). Accordingly, to better understand the mechanism by which the CBMFF affects PDGF-BB activity, we examined the effect of the CBMFF on the activation of
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DOI: 10.3109/13880209.2014.941882
Chrysanthemum boreale Makino in vascular smooth muscle cells
PDGF-linked pathway proteins. As shown in Figure 3, treatment of RASMCs with PDGF-BB at 10 ng/mL for 10 min increased the level of phosphorylated PDGFR-b (369.83 ± 39.38% of control, n ¼ 4). Treatment with the CBMFF (0.01–100 lg/mL) dose dependently inhibited PDGF-BB-induced phosphorylation of PDGFR-b (n ¼ 4). This inhibitory response was significant starting at treatment with 0.01 lg/mL of CBMFF (265.69 ± 18.93% of control) and reached a maximum at 100 lg/mL (106.42 ± 11.70% of control). Moreover, we also tested the effects of PDGFR-b inhibition on PDGF-BB-induced migration or proliferation in RASMCs. Treatment with 10 lM of AG1296 suppressed PDGF-BB (10 ng/mL)-stimulated RASMC migration (97.46 ± 19.46% of control, n ¼ 8; Figure 3C) and RASMC proliferation (101.53 ± 10.27% of control, n ¼ 10; Figure 3D). Effects of the CBMFF on phosphorylation of PDGFR-b pathway kinases The activation of MAPKs is also known to be important for PDGF-stimulated VSMC migration and proliferation (Lee et al., 2007a). To investigate the mechanism of inhibitory effects of CBMFF on VSMC responses, we
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treated RASMCs with PDGF-BB and CBMFF. PDGF-BB (10 ng/mL) increased phosphorylation of p38 MAPK (351.32 ± 16.34% of control, n ¼ 4; Figure 4). The PDGF-BB-induced response was inhibited by CBMFF in a dose-dependent manner (0.01–100 lg/mL) (n ¼ 4) and reached a maximum by treatment with 100 lg/mL of CBMFF (101.42 ± 4.63% of control). Moreover, the elevated ERK1/2 phosphorylation in response to PDGF-BB (10 ng/ mL) was suppressed by the treatment with CBMFF in a dosedependent manner (0.01–100 lg/mL) (n ¼ 4; Figure 4), which was reached a maximum at a CBMFF concentration of 100 lg/mL (101.71 ± 5.77% of control; n ¼ 4). Notably, total protein levels of p38 MAPK and ERK1/2 were not affected by either the PDGF-BB or the CBMFF (Figure 4A). Moreover, to determine correlation between MPAKs, migration and proliferation in response to PDGF-BB in RASMCs, RASMCs were stimulated with PDGF-BB after treatment with inhibitors of MAPKs. As shown in Figure 4(D), PDGF-BB (10 ng/mL)-stimulated RASMC migration was inhibited by treatment with 30 lM of p38 MAPK inhibitor SB203580 (93.64 ± 10.11% of control, n ¼ 8) or 30 lM of ERK1/2 inhibitor PD98059 (99.58 ± 18.63% of control, n ¼ 8). PDGF-BB (10 ng/mL)-induced proliferation was also
Figure 3. Effect of Chrysanthemum boreale Makino flower floral water on PDGF-BB-induced phosphorylation of PDGF receptor-b. (A) RASMCs were incubated in the absence or presence of steam-distilled floral water of Chrysanthemum boreale Makino flower (CBMFF: 0.01–100 lg/mL) for 30 min, and then treated with PDGF-BB (10 ng/mL) for 10 min. The cell lysates were immunoprecipitated with anti-PDGFR-b antibody, and then immunoblotted with anti-phospho-tyrosine (4G10) antibody. The total expression of PDGFR-b was determined by immunoblotting with an antiPDGFR-b antibody. (B) A statistical graph was obtained from panel A. The ratio of phosphorylated/non-phosphorylated PDGFR-b in the basal state is expressed as 100% (n ¼ 4). (C) Effect of PDGFR inhibitor on PDGF-BB-induced RASMC migration. Cells were preincubated with PDGFR inhibitor AG1296 (10 lM) or steam-distilled extract floral water of Chrysanthemum boreale Makino flower (CBMFF; 100 lg/mL) and then they were stimulated with 10 ng/mL PDGF-BB from 90 min. (D) Effect of PDGFR inhibitor on PDGF-BB-induced RASMC proliferation. Cells were treated for 48 h with or without PDGF-BB (10 ng/mL), AG1296 (10 lM), or steam-distilled extract floral water of Chrysanthemum boreale Makino flower (CBMFF; 100 lg/mL). Cell migration (C) and proliferation (D) were examined using Boyden chamber assay and a XTT assay, respectively, as described in the Methods section. Migration and proliferation in the quiescent state are expressed as 100% (n ¼ 8), respectively. Two-way ANOVA, *p50.05 versus the PDGF-BB-stimulated state. IB, immunoblotting; IP, immunoprecipitation; p-PDGFR-b, phosphorylated PDGFR-b; Ab con, antibody control.
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Figure 4. Effects of Chrysanthemum boreale Makino flower floral water on phosphorylation of MAPKs in RASMCs. (A) After treatment without or with the steam-distilled floral water of Chrysanthemum boreale Makino flower (CBMFF: 0.01–100 lg/mL) for 30 min, RASMCs were stimulated with PDGF-BB (10 ng/mL) for 10 min. The cell lysates were immunoblotted with each MAPK antibody. The phosphorylation of p38 MAPK and ERK1/2 was detected using phospho-specific antibodies. The total expression of protein kinases and b-actin was measured using non-phospho-specific antibodies. (B) and (C) Statistical graphs of the phosphorylation levels of MAPKs were obtained from panel A. The basal levels of each phosphorylation are considered as 100% (n ¼ 4). (D) Effects of inhibitors of ERK1/2 and p38 MAPK on PDGF-BB-induced RASMC migration. Cells were incubated for 90 min with p38 MAPK inhibitor SB 203580 (30 lM), ERK1/2 inhibitor PD 98059 (30 lM), or steam-distilled extract floral water of C. boreale Makino flower (CBMFF; 100 lg/mL), and were then stimulated with 10 ng/mL PDGF-BB. Cell migration was examined using the Boyden chamber assay as described in the Methods section. (E) Effects of kinase inhibitors on the PDGF-BB-stimulated RASMC proliferation. Cells were treated with or without PDGF-BB (10 ng/mL), SB 203580 (30 lM), ERK1/2 inhibitor PD 98059 (30 lM), or steam-distilled extract floral water of C. boreale Makino flower (CBMFF; 100 lg/mL), for 48 h. Cell proliferation was tested using a XTT assay. Migration (D) and proliferation (E) in the quiescent state are expressed as 100% (n ¼ 8) Results are represented as the mean ± SD. Two-way ANOVA, *p50.05 compared with the PDGF-BBstimulated state. p-p38 MAPK, phosphorylated p38 MAPK; p-ERK1/2 MAPK, phosphorylated ERK1/2.
DOI: 10.3109/13880209.2014.941882
Chrysanthemum boreale Makino in vascular smooth muscle cells
suppressed by treatment with p38 MAPK inhibitor SB203580 at a concentration of 30 lM (103.52 ± 9.25% of control, n ¼ 8) or ERK1/2 inhibitor PD98059 at a concentration of 30 lM in RASMCs (91.97 ± 12.31% of control, n ¼ 8) (Figure 4E).
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Influence of CBMFF on PDGF-BB-stimulated sprout outgrowth of aortic rings To explore whether the CBMFF affects vascularization ex vivo, we treated aortic rings with CBMFF and analyzed sprout outgrowth using an aortic ring assay. Treatment of aortic rings with 10 ng/mL of PDGF-BB increased sprout outgrowth (358.03 ± 23.01% of control, n ¼ 6; Figure 5). This PDGF-BB-elevated aortic sprout outgrowth was inhibited by CBMFF in a dose-dependent manner (0.1–100 lg/mL) and this effect revealed maximum at a 100 lg/mL concentration of CBMFF (110.26 ± 15.40% of control, n ¼ 6). However, the vehicle in which CBMFF was suspended did not affect aortic sprout outgrowth.
Discussion In the present study, we have evaluated whether CBMFF, especially isolated by a steam-distilled extraction method, can affect RASMC migration and proliferation. We found that the CBMFF inhibited the migration and proliferation in response to PDGF-BB. Furthermore, to verify these in vitro findings, we performed ex vivo aortic ring assay that tests aortic sprout formation. The aortic ring assay revealed that CBMFF attenuated PDGF-BB-induced aortic sprout growth. Migration and proliferation of VSMC participated in the development of atherosclerosis (Lee et al., 2012; Wang et al., 2012). These findings collectively indicate that CBMFF inhibits VSMC migration and proliferation and
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may regulate vascular disorders such as atherosclerosis. CBMFF is known to have various biological properties including anticancer, inhibition of angiogenesis, and antiinflammation (Kim et al., 2010; Lee et al., 2003; Perry, 1980). Flower extract of Chrysanthemum indicum Linne´, which belongs to the same genus as CBM, exhibited inhibitory activity on inflammation and proliferation in various cells (Cheon et al., 2009; Li et al., 2009; Yuan et al., 2009). In the present study, we found that CBMFF contained components such as thymol and eugenol that can exert anticancer, antioxidant, antimicrobial, anti-inflammation, and vasorelaxant effect (Table 1). These findings imply that CBMFF may exert a biological effect on VSMC function. However, there are no reports demonstrating that CBMFF, especially obtained by a steam distillation, affects VSMC proliferation and migration. Although other pharmacological activities have been reported for CBMFF, our study for the first time demonstrated the effect of the CBMFF on VSMC migration and proliferation. As described above, PDGF-BB plays a crucial role in VSMC proliferation and migration and is an important stimulator of atherosclerotic lesion development (Arita et al., 2002; Zhou et al., 1999). PDGF-BB is also a mitogen and chemo-attractant for VSMCs and it is known to bind to and activate PDGFR-b on VSMCs in the vascular wall (Holycross et al., 1992; Owens et al., 2004; Zhou et al., 1999). PDGF-BB binding to PDGFR-b causes PDGFR dimerization and transphosphorylation that in turn initiates a series of downstream signals including phosphorylation of p38 MAPK and ERK1/2, ultimately leading to proliferation and migration (Holycross et al., 1992; Kim & Yun, 2007; Pukac et al., 1988). In the present study, PDGF-BB-induced migration and proliferation were decreased by the PDGFR inhibitor in VSMCs. These results imply that
Figure 5. Effect of Chrysanthemum boreale Makino flower floral water on PDGF-BB-stimulated sprout growth of aortic rings. (A) Aortic rings (1 mm) were derived from rats, embedded in Matrigel, and cultured. The rings were treated with or without PDGF-BB (10 ng/mL) in the absence or presence of steam-distilled floral water of C. boreale Makino flower (CBMFF: 0.1–100 lg/mL). Responses were observed on day 5. (B) Statistical results were obtained from panel A. The level in vehicle (0.1% DMSO)-treated rings is expressed as 100% (n ¼ 6). Each value is expressed as the mean ± SD. Two-way ANOVA, *p50.05 versus the PDGF-BB (10 ng/mL)-stimulated state.
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PDGF-stimulated activation of PDGFR-b participates in MAPK-mediated migration and proliferation in VSMCs. In the present study, we tested the effect of the CBMFF on PDGF-stimulated PDGFR-b activation and found that PDGFBB increased PDGFR-b phosphorylation, and this response was significantly inhibited by treatment with CBMFF in a dose-dependent manner. Therefore, it is possible that CBMFF may inhibit PDGFR-b phosphorylation to abolish its downstream signals, probably linked to MAPK pathway, which results in the inhibition of migration and proliferation in VSMCs in response to PDFG-BB, although the mechanism by which the CBMFF inhibits PDGFR-b phosphorylation remains to be elucidated. Many studies have shown that MAPK pathways participate in various cellular functions including proliferation and migration (Stork & Schmitt, 2002). Activation of MAPK in VSMCs is a critical response in stimulating proliferation and migration (Choi et al., 2009) and was involved in PDGFstimulated migration and proliferation in VSMCs (Bornfeldt et al., 1994; Holycross et al., 1992; Pukac et al., 1988). PDGF increased the phosphorylations of p38 MAPK and ERK1/2, and elevated the migration and proliferation in RASMCs (Lee et al., 2007a). p38 MAPK acts as a mediator in cellular responses, including migration and proliferation in VSMCs (Kavurma & Khachigian, 2003). In our previous and current studies, we demonstrated that PDGF-BB stimulated p38 MAPK phosphorylation and also induced VSMC migration and proliferation that were inhibited by the treatment with p38 MAPK inhibitor (Lee et al., 2007a, 2008a,b), indicating that PDGF-BB-induced migration and proliferation in VSMCs is mediated by p38 MAPK pathway. Similarly, the present study demonstrated that PDGF-BB stimulated VSMC migration and proliferation, as well as p38 MAPK phosphorylation, and these responses were attenuated by CBMFF. It is reported that the flower extract of Chrysanthemum indicum inhibits the activation of MAPKs including p38 MAPK in macrophages (Cheon et al., 2009), although this is a different type of cell. Based on these results, it is possible that CBMFF may exhibit inhibitory activity on RASMC migration and proliferation in response to PDGFBB, probably via the suppression of p38 MAPK phosphorylation. Similar to p38 MAPK, ERK1/2 is also reported as an important signaling molecule that is involved in PDGF-BBinduced migration and proliferation and was phosphorylated by PDGF-BB stimulation in VSMCs (Kavurma & Khachigian, 2003). These PDGF-BB-stimulated responses in VSMCs were attenuated by ERK1/2 inhibition as demonstrated in our previous study (Won et al., 2008), as well as the present study. Therefore, these reports suggest that ERK1/2 could be a mediator in PDGF-BB-induced migration and proliferation in VSMCs. Moreover, the flower extract of Chrysanthemum indicum is known to suppress ERK1/2 phosphorylation, as well as proliferations, in cancer cells in response to isoproterenol (Yuan et al., 2009). The present study demonstrated that the phosphorylation of ERK1/2 was induced in response to PDGF-BB in VSMCs and this response was decreased by treatment with CBMFF. CBMFF also inhibited VSMC migration and proliferation in response to PDGF-BB. These findings imply that CBMFF evokes the inhibition of
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ERK1/2 signal and this event may contribute to the downregulation of PDGF-BB-stimulated migration and proliferation in VSMCs. Therefore, it can be assumed that the inhibitory activity of CBMFF on PDGF-BB-induced migration and proliferation may occur through a signal pathway mediated by MAPKs, especially p38 MAPK and ERK1/2, although the present study did not directly demonstrate CBMFF-stimulated interactions between activities of two MAPKs and two cellular responses, migration and proliferation, in VSMCs in response to PDGF-BB.
Conclusions This study has demonstrated that CBMFF dose dependently inhibited PDGF-BB-increased migration and proliferation in RASMCs. PDGF-BB-induced phosphorylations of PDGF-b receptor, p38 MAPK, and ERK1/2 in RASMCs were also decreased by the treatment with the CBMFF. Moreover, the CBMFF also attenuated PDGF-BB-stimulated sprout outgrowth of aortic rings. Based on these findings, we concluded that CBMFF may inhibit vascular responses, VSMC proliferation, and migration, probably through inhibition of the PDGFR-b-mediated MAPK pathway. Therefore, the steam-distilled floral water of CBMF may be useful as a promising agent candidate with anti-atherosclerotic property. However, further research is required to isolate and identify a key bioactive component with anti-atherosclerotic activity from the extract.
Declaration of interest The authors declare that they have no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant nos. A103017 and HN12C0054) and this research was supported by Bioindustry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs.
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DOI: 10.3109/13880209.2014.941882
Chrysanthemum boreale Makino in vascular smooth muscle cells
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ORIGINAL ARTICLE
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Chrysanthemum boreale flower floral water inhibits platelet-derived growth factor-stimulated migration and proliferation in vascular smooth muscle cells Do-Yoon Kim1*, Kyung-Jong Won2*, Mi-So Yoon1, Ho-Jin Yu1, Joo-Hoon Park1, Bokyung Kim2, and Hwan Myung Lee1 1
Department of Cosmetic Science, College of Natural Science, Hoseo University, Asan, Chungnam Prefecture, Republic of Korea and Department of Physiology, School of Medicine, Konkuk University, Chungju, Chungbuk Prefecture, Republic of Korea
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Abstract
Keywords
Context: Chrysanthemum boreale Makino (Compositae) (CBM) is a traditional medicine that has been used for the prevention or treatment of various disorders; it has various properties including antioxidation, anti-inflammation, and antitumor. Objective: The present study was designed to explore the in vitro effect of CBM flower floral water (CBMFF) on atherosclerosis-related responses in rat aortic smooth muscle cells (RASMCs). Materials and methods: CBMFF was extracted from CBM flower by steam distillation and analyzed using gas chromatography–mass spectrometry. The anti-atherosclerosis activity of CBMFF was tested by estimating platelet-derived growth factor (PDGF)-BB (10 ng/mL)-induced proliferation and migration levels and intracellular kinase pathways in RASMCs at CBMFF concentrations of 0.01–100 lM and analyzing ex vivo aortic ring assay. Results: Gas chromatography–mass spectrometry showed that the CBMFF contained a total of seven components. The CBMFF inhibits PDGF-BB-stimulated RASMC migration and proliferation (IC50: 0.010 lg/mL). Treatment of RASMCs with PDGF-BB induced PDGFR-b phosphorylation and increased the phosphorylations of MAPK p38 and ERK1/2. CBMFF addition prevented PDGF-BBinduced phosphorylation of these kinases (IC50: 008 and 0.018 lg/mL, for p38 MAPK and ERK1/ 2, respectively), as well as PDGFR-b (IC50: 0.046 lg/mL). Treatment with inhibitors of PDGFR, P38 MAPK, and ERK1/2 decreased PDGF-BB-increased migration and proliferation in RASMCs. Moreover, the CBMFF suppressed PDGF-BB-increased sprout outgrowth of aortic rings (IC50: 0.047 lg/mL). Discussion and conclusion: These results demonstrate that CBMFF may inhibit PDGF-BB-induced vascular migration and proliferation, most likely through inhibition of the PDGFR-b-mediated MAPK pathway; therefore, the CBMFF may be promising candidate for the development of herbal remedies for vascular disorders.
Anti-atherosclerosis, chemotherapy, natural products, vascular disease
Introduction Vascular smooth muscle cell (VSMC) migration and proliferation are important in the development progression of vascular neointima in atherosclerosis (Lee et al., 2012; Wang et al., 2012). These events were stimulated by various factors, including proinflammatory cytokines and peptide growth factors (Ross, 1999). Platelet-derived growth factors (PDGF)
*These authors contributed equally to this work. Correspondence: Hwan Myung Lee, Assistant Professor, Department of Cosmetic Science, College of Natural Science, Hoseo University, Asan-city, Chungnam Prefecture 336-795, Republic of Korea. Tel: +82 41 540 9551. Fax: +82 41 540 9538. E-mail: [email protected] Bokyung Kim, Professor, Department of Physiology, School of Medicine, Konkuk University, Chungju-city, Chungbuk Prefecture 380-701, Republic of Korea. Tel: +82 43 840 3726. Fax: +82 43 851 9329. E-mail: [email protected]
History Received 9 April 2014 Accepted 2 July 2014 Published online 21 October 2014
are a family of dimeric growth factors that are important to the regulation of VSMC proliferation and migration (Dammanahalli et al., 2012; Jiang et al., 2010; Won et al., 2008). The individual PDGF monomers may be one of four proteins: PDGF-A, B, C, or D (Fredriksson et al., 2004). In their dimeric form, PDGFs bind to and activate a PDGF receptor (PDGFR) that consists of two receptor tyrosine kinase subtypes, PDGFR-a and PDGFR-b, localized to the plasma membrane of cells. Once bound to a PDGF, PDGFRs activate diverse signaling molecules and regulatory proteins that contain Src homology 2-domains and thereby elicit various cellular responses like actin reorganization, proliferation, differentiation, or VSMC migration (Jiang et al., 2010; Won et al., 2011). Mitogen-activated protein kinases (MAPKs) are also signaling molecules important for regulating various cellular processes in cells, including proliferation, differentiation, and migration (Kim et al., 2009; Lee et al., 2008a,b).
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MAPK activation is induced by diverse stimuli, including growth factors, hormones, and cytokines (Lee et al., 2009). PDGFs can activate some MAPKs, including extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stressactivated protein kinase/c-Jun N-terminal kinase (Fukai et al., 1998). PDGF-induced activation of MAPK pathways is known to trigger VSMC migration and proliferation (Eguchi et al., 2001). Specifically, PDGFs stimulate the phosphorylation of p38 MAPK and ERK1/2 (Lee et al., 2007a), which are known to initiate migration or proliferation and growth, respectively, in VSMCs (Dubey et al., 2000; Gan et al., 2013). Chrysanthemum boreale Makino (Compositae) (CBM) is a perennial plant that is widely distributed in East Asia countries such as Korea, Japan, and China (Kim et al., 2010). CBM and other related species such as C. indicum Linne´ (Composite) and C. lavandulaefolium Makino (Fisch.) Mak. are traditional oriental medicines that have been used to treat a variety of disorders such as pneumonia, colitis, stomatitis, and carbuncle (Kim et al., 2003). CBM contains various pharmacologically active components, including flavonoids, sesquiterpene lactones, chrysanthemin, and essential oils (Kim et al., 2003). The components isolated from CBM exhibit diverse pharmacological activities including antibacterial, antitumor, anti-angiogenesis, anti-hypertension, and anti-inflammatory activities. CBM flowers (CBMF) are known to have antipyretic and anti-vertigo effects (Lee et al., 2003; Perry, 1980). Although some herbal medicines have been used for the prevention or treatment of cardiovascular diseases like atherosclerosis (Mashour et al., 1998), there are no reports of CBMF being used to treat vascular disorders. Since there are many reports about other herbal extracts inhibiting VSMC migration and proliferation that are key events in neointima formation (Choi et al., 2009; Won et al., 2009), we here explored whether CBMF floral water (CBMFF) could affect PDGF-BB-induced proliferation and migration of rat aortic smooth muscle cells (RASMCs).
Materials and methods Materials PDGF-BB was purchased from R&D Systems (Minneapolis, MN) and Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, phosphate buffered saline (PBS), trypsin-ethylenediamine tetraacetic acid (EDTA), and Hank’s balanced salt solution (HBSS) were from Hyclone (Logan, UT) or Invitrogen (Carlsbad, CA). Matrigel and type I collagen were purchased from BD Bioscience (Franklin Lakes, NJ), and collagenase was from Wako (Richmond, VA). Bovine serum albumin (BSA) and elastase were purchased from Sigma (St. Louis, MO) and the Diff-Quik stain kit was from Sysmex Corp (Kobe, Japan). AG1296, SB203580, and PD98059 were purchased from Tocris Bioscience (Bristol, UK). The antibodies used included anti-p38 MAPK, anti-phospho p38, anti-ERK1/2, anti-phospho ERK1/2, anti-PDGFR-b (Cell signaling, Beverly, MA), anti-phospho-tyrosine(4G10) (Upstate, Lake Placid, NY) and anti-b-actin (Sigma, St. Louis, MO) antibodies.
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Preparation of CBMFF Fresh CBMFs (20 kg) were collected from the farm of Hoseo University, Republic of Korea, in October 2010 and CBMFF was isolated by steam distillation for 2 h. In brief, the procedure comprises a passage of vapor from a boiler into a chamber holding plants, followed by condensation of the solute-containing steam by contact with cold water. The different densities of the oil and floral water (10 L) enabled the phases to effectively separate. The collected CBMFF were dried by freeze-drying, and the resulting extracts were dissolved in dimethyl sulfoxide (DMSO) and stored at 20 C. Analysis of CBMFF and compound identification CBMFF obtained by steam distillation of CBMF was dissolved in methanol and analyzed on a gas chromatograph (Agilent 6890N)-mass spectrometer (Aglient 5975i MS) (Agilent technologies, Palo Alto, CA) equipped with a DB5-MS column (30 m 250 lm, 0.25 lm film thickness; J&W Scientific, Folsom, CA). The samples were introduced by split mode at a split ratio of 1:10. The injector temperature was set at 280 C. The oven temperature was programmed as follows: isothermal at 50 C for 2 min, rising at 10 C/min to 300 C and held at this temperature for 10 min. Helium was used as the carrier gas at a rate of 1 mL/min. The interface temperature was 300 C. Effluent of the GC column was introduced directly into the source of the MS via a transfer line (230 C). Mass range was 50–800 m/z. Compounds were tentatively identified by comparison of mass spectra of each peak with those of an authentic sample in the Wiley7Nist05.L library. Cell isolation and culture All animal investigations were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996) and approved by the Animal Subjects Committee and by the Institutional Guidelines of Konkuk University, Korea. RASMCs were isolated from the aortas of male Sprague–Dawley (SD) rats (6 weeks old, 160–180 g, n ¼ 8) by collagenase and elastase treatment and were subsequently cultured in DMEM containing 10% FBS, 100 U/mL penicillin, 100 lg/mL streptomycin, and 200 mM L-glutamine. For all experiments, the RASMCs (used at passages 3–8) were grown to 70–80% confluence in DMEM and starved of FBS for 24 h. Immunoblotting After treatment with CBMFF and PDGF-BB, RASMCs were lysed with cold extraction buffer (20 mM HEPES [pH 7.5], 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, 2.5 mM 4-nitrophenylphosphate, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], and 1 complete proteinase inhibitor cocktail tablet [Roche, Indianapolis, IN]). Cell lysates were centrifuged at 17 000 g for 15 min at 4 C and the supernatants were collected as protein samples. Protein concentrations were determined using the protein assay reagents (Bio-Rad, Hercules, CA).
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DOI: 10.3109/13880209.2014.941882
Chrysanthemum boreale Makino in vascular smooth muscle cells
Protein homogenates were diluted 1:1 (v/v) with SDS loading buffer (40 mM Tris-HCl pH 6.8, 8 mM ethylene glycol tetraacetic acid, 4% 2-mercaptoethanol, 40% glycerol, 0.01% bromophenol blue, and 4% SDS) and boiled for 5 min. Samples (30–50 lg/lane) were separated on 12–14% polyacrylamide SDS gels and transferred electrophoretically to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membrane was blocked for 2 h at room temperature (RT) with PBS containing 0.05% Tween-20 and 5% fat-free dried milk and incubated overnight at 4 C with selective antibodies diluted 1000- to 2000-fold. Immune complexes were detected with horse radish peroxidase-conjugated antibodies (Amersham Pharmacia Biotech, Piscataway, NJ), followed by visualization on photographic film. Band intensities were quantified with Quantitation software package (Bio-Rad, Berkeley, CA). Immunoprecipitation To measure the activation of PDGFR, RASMCs were lysed in extraction buffer and lysates containing 200–300 lg of proteins were incubated with 4 lg/mL of anti-PDGFR-b antibody for 5 h at RT. The immunocomplex was precipitated by incubation with protein A-agarose beads (Roche, Branford, CT) at overnight at 4 C. Beads were subsequently washed with PBS containing 0.1% Tween-20, resuspended in SDS sample buffer, and boiled for 5 min. Protein samples collected were immunoblotted with anti-phospho-tyrosine (4G10) antibody as per the protocol described above. Migration assay Cell migration assays were performed in 48-well Boyden microchemotaxis chambers (Neuro Probe, Cabin John, MD). Polycarbonate membranes with 8 lm pores (Neuro Probe, Cabin John, MD) were coated with a 0.1 mg/mL solution of type I collagen from rat tail tendon (BD Bioscience, San Jose, CA) in distilled water and then dried for 60 min. RASMCs were harvested using trypsin–EDTA and resuspended in DMEM that contained 0.1% BSA. PDGF-BB, CBMFF, and test inhibitors were loaded in the bottom chamber and the membrane was laid over an aliquot of approximately 5 104 cells. The chamber was then incubated for 90 min at 37 C, fixed, and stained using a Diff-Quik staining kit (BD Bioscience, San Jose, CA). All samples used in the experiments contained 0.1% DMSO, which did not affect RASMC migration. The number of cells migrating through the membrane was determined by counting the cells in four randomly chosen regions of each well under a microscope at 400-fold magnification. Proliferation assay RASMC proliferation activity was measured via a 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay using a WelCountÔ cell viability assay kit (WelGENE, Korea). In brief, the cells were seeded at 2 103 cells per well in DMEM onto a 96-well plate coated with 0.1 mg/mL type I collagen. After incubating for 12 h, the DMEM was replaced with serum-free DMEM followed by incubation for an additional 6 h. After removing the medium, the remaining cells were treated with CBMFF, test inhibitors
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and PDGF-BB and incubated for 48 h. To measure proliferation, 20 lL of XTT solution was added to each well, followed by incubation for 4 h at 37 C in an atmosphere of 5% CO2. The plates were then thoroughly shaken, and the absorbance at 450 nm and 690 nm was measured using a microplate reader (Bio Tek, Seoul, Korea). Aortic ring assay Ex vivo migration and proliferation of the RASMCs were measured by an aortic ring assay using Matrigel according to a previously reported method with slight modifications (Nicocia & Ottinetti, 1990). The endothelium and adventitium of the aorta from four 5-week-old SD rats were removed enzymatically, and the vessels were cut into 1 mm rings. The rings were embedded onto 48-well plates coated with Matrigel, and then PDGF-BB and CBMFF suspended in FBS-free DMEM were simultaneously added. Following growth for 5 d, the rings were stained with Diff-Quik, photographed, and the length of the sprouts analyzed using Scion Image software (Institutes of Health, Houghton, MI). Statistical analysis Data are expressed as mean ± standard deviation (SD). Statistical evaluation of data was performed with Graphpad Prism version 5.0 (GraphPad Software, San Diego, CA) using a two-way analysis of variance (ANOVA). Values of p50.05 were considered statistically significant.
Results Identification of compounds in CBMFF To analyze the compounds of CBMFF in the absence of hydrophobic essential oil, we performed a GC-MS analysis and found that the floral water contained seven compounds (Table 1). Among them, 1,2-cis-1,5-trans-2,5-dihydroxy-4methyl-1-(10-hydroxy-1-isopropyl)cyclohex-3-ene (1.29%) was the highest content and all other compounds exhibited the following descending order: cis-4-hydrocy-2-methyl-5-(1hydrocy-1-isopropyl)-2-cyclohexen-1-one (1.29%), thymol (1.09%), methyl ester of ricinoleic acid (0.91%), eugenol (0.48%), cis-carveol (0.35%), and 2,4-pentadienenitrile (0.33%). CBMFF possessed not only bioactive compounds such as thymol and eugenol that have anticancer, antioxidant, antimicrobial, anti-inflammation, and vasorelaxant activities but also compounds with unknown activity. Effects of CBMFF on migration and proliferation in response to PDGF-BB The effect of CBMFF on RASMC migration and proliferation in response to PDGF-BB was assessed using a modified Boyden chamber and an XTT assay, respectively. At a concentration of 10 ng/mL, PDGF-BB induced RASMC migration (473.47 ± 21.97% of control, n ¼ 6; Figure 1). PDGF-BB-induced migration was suppressed by treatment with CBMFF (0.01–100 lg/mL) in a dose-dependent manner. On one hand, the maximum suppression was observed at a CBMFF concentration of 100 lg/mL (302.56 ± 20.62% of control, n ¼ 6). PDGF-BB treatment at 10 ng/mL also
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Table 1. Phytochemical components of the Chrysanthemum boreale Makino flower floral water. Compounds name (CAS no.) 2,4-Pentadienenitrile (1615-70-9) cis-Carveol (1197-06-4) Thymol (889-83-8)
RT
% of area
3.52 11.09 11.90
0.33 0.35 1.09
Activity Unknown Unknown Anti-cancer Anti-septic Anti-oxidant
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Anti-microbial
Eugenol (97-53-0)
12.77
0.48
Vasorelaxant Anti-inflammation Anti-cancer Anti-oxidation Anti-microbial
1,2-cis-1,5-trans-2,5-Dihydroxy-4-methyl-1-(10hydroxy-1-isopropyl)cyclohex-3-ene (87096-70-6) cis-4-Hydrocy-2-methyl-5-(1-hydroxy-1-isopropyl)-2cyclohexen-1-one (97762-02-2) Methyl ester of ricinoleic acid (141-24-2)
15.57
2.14
Vasorelaxant Unknown
15.80
1.29
Unknown
22.47
0.91
Unknown
References
Deb et al. (2011) and Hsu et al. (2011) Beena et al. (2013) Archana et al. (2009), Meeran and Prince (2012), and Satooka and Kubo (2011) Elissondo et al. (2008), Rivas et al. (2010), Wattanasatcha et al. (2012) Peixoto-Neves et al. (2010) Lee et al. (2007b) and Yeh et al. (2011) Manikandan et al. (2011), Pal et al. (2010), and Pisano et al. (2007) Devi et al. (2010), Ito et al. (2005), and Nagababu et al. (2010) Ali et al. (2005), Pei et al. (2009), and Shah et al. (2013) Damiani et al. (2003)
RT, retention time.
Figure 1. Effect of Chrysanthemum boreale Makino flower floral water on PDGF-BB-induced migration in RASMCs. RASMCs were treated with PDGF-BB (10 ng/mL) in the presence or absence of steam-distilled extract floral water of Chrysanthemum boreale Makino flower (CBMFF: 0.01–100 lg/mL) for 90 min. Cell migration was analyzed using a Boyden chamber assay. Cell migration in the quiescent state was considered as 100% (n ¼ 6). Results are represented as the mean ± SD. *p50.05 compared to the PDGF-BB-stimulated state by a two-way ANOVA.
elevated RASMC proliferation (232.03 ± 13.86% of control, n ¼ 6; Figure 2), and this response was suppressed by treatment with CBMFF in a dose-dependent fashion (0.01– 100 lg/mL). The maximum inhibition was observed at a CBMFF concentration of 100 lg/mL (118.58 ± 3.78% of control, n ¼ 6). On the other hand, CBMFF (0.01–100 lg/ mL) did not affect RASMC migration and proliferation at PDGF-BB-untreated states (Figures 1 and 2).
Figure 2. Effect of Chrysanthemum boreale Makino flower floral water on PDGF-BB stimulated proliferation in RASMCs. RASMCs were treated with or without steam-distilled floral water of Chrysanthemum boreale Makino flower (CBMFF: 0.01–100 lg/mL) and then stimulated by PDGF-BB (10 ng/mL) for 48 h. Cell proliferation was analyzed by performing the XTT assay. Cell proliferation in the quiescent state was expressed as 100% (n ¼ 6). Each value is expressed as the mean ± SD. *p50.05 compared to the PDGF-BB-stimulated state by a two-way ANOVA.
Effects of CBMFF on phosphorylation of PDGFR-b in RASMCs PDGFs normally bind to a cell surface protein tyrosine kinase receptor-like PDGFR-b, and this binding event leads to activation of the receptor and associated signaling proteins (Chen et al., 2006). Accordingly, to better understand the mechanism by which the CBMFF affects PDGF-BB activity, we examined the effect of the CBMFF on the activation of
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DOI: 10.3109/13880209.2014.941882
Chrysanthemum boreale Makino in vascular smooth muscle cells
PDGF-linked pathway proteins. As shown in Figure 3, treatment of RASMCs with PDGF-BB at 10 ng/mL for 10 min increased the level of phosphorylated PDGFR-b (369.83 ± 39.38% of control, n ¼ 4). Treatment with the CBMFF (0.01–100 lg/mL) dose dependently inhibited PDGF-BB-induced phosphorylation of PDGFR-b (n ¼ 4). This inhibitory response was significant starting at treatment with 0.01 lg/mL of CBMFF (265.69 ± 18.93% of control) and reached a maximum at 100 lg/mL (106.42 ± 11.70% of control). Moreover, we also tested the effects of PDGFR-b inhibition on PDGF-BB-induced migration or proliferation in RASMCs. Treatment with 10 lM of AG1296 suppressed PDGF-BB (10 ng/mL)-stimulated RASMC migration (97.46 ± 19.46% of control, n ¼ 8; Figure 3C) and RASMC proliferation (101.53 ± 10.27% of control, n ¼ 10; Figure 3D). Effects of the CBMFF on phosphorylation of PDGFR-b pathway kinases The activation of MAPKs is also known to be important for PDGF-stimulated VSMC migration and proliferation (Lee et al., 2007a). To investigate the mechanism of inhibitory effects of CBMFF on VSMC responses, we
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treated RASMCs with PDGF-BB and CBMFF. PDGF-BB (10 ng/mL) increased phosphorylation of p38 MAPK (351.32 ± 16.34% of control, n ¼ 4; Figure 4). The PDGF-BB-induced response was inhibited by CBMFF in a dose-dependent manner (0.01–100 lg/mL) (n ¼ 4) and reached a maximum by treatment with 100 lg/mL of CBMFF (101.42 ± 4.63% of control). Moreover, the elevated ERK1/2 phosphorylation in response to PDGF-BB (10 ng/ mL) was suppressed by the treatment with CBMFF in a dosedependent manner (0.01–100 lg/mL) (n ¼ 4; Figure 4), which was reached a maximum at a CBMFF concentration of 100 lg/mL (101.71 ± 5.77% of control; n ¼ 4). Notably, total protein levels of p38 MAPK and ERK1/2 were not affected by either the PDGF-BB or the CBMFF (Figure 4A). Moreover, to determine correlation between MPAKs, migration and proliferation in response to PDGF-BB in RASMCs, RASMCs were stimulated with PDGF-BB after treatment with inhibitors of MAPKs. As shown in Figure 4(D), PDGF-BB (10 ng/mL)-stimulated RASMC migration was inhibited by treatment with 30 lM of p38 MAPK inhibitor SB203580 (93.64 ± 10.11% of control, n ¼ 8) or 30 lM of ERK1/2 inhibitor PD98059 (99.58 ± 18.63% of control, n ¼ 8). PDGF-BB (10 ng/mL)-induced proliferation was also
Figure 3. Effect of Chrysanthemum boreale Makino flower floral water on PDGF-BB-induced phosphorylation of PDGF receptor-b. (A) RASMCs were incubated in the absence or presence of steam-distilled floral water of Chrysanthemum boreale Makino flower (CBMFF: 0.01–100 lg/mL) for 30 min, and then treated with PDGF-BB (10 ng/mL) for 10 min. The cell lysates were immunoprecipitated with anti-PDGFR-b antibody, and then immunoblotted with anti-phospho-tyrosine (4G10) antibody. The total expression of PDGFR-b was determined by immunoblotting with an antiPDGFR-b antibody. (B) A statistical graph was obtained from panel A. The ratio of phosphorylated/non-phosphorylated PDGFR-b in the basal state is expressed as 100% (n ¼ 4). (C) Effect of PDGFR inhibitor on PDGF-BB-induced RASMC migration. Cells were preincubated with PDGFR inhibitor AG1296 (10 lM) or steam-distilled extract floral water of Chrysanthemum boreale Makino flower (CBMFF; 100 lg/mL) and then they were stimulated with 10 ng/mL PDGF-BB from 90 min. (D) Effect of PDGFR inhibitor on PDGF-BB-induced RASMC proliferation. Cells were treated for 48 h with or without PDGF-BB (10 ng/mL), AG1296 (10 lM), or steam-distilled extract floral water of Chrysanthemum boreale Makino flower (CBMFF; 100 lg/mL). Cell migration (C) and proliferation (D) were examined using Boyden chamber assay and a XTT assay, respectively, as described in the Methods section. Migration and proliferation in the quiescent state are expressed as 100% (n ¼ 8), respectively. Two-way ANOVA, *p50.05 versus the PDGF-BB-stimulated state. IB, immunoblotting; IP, immunoprecipitation; p-PDGFR-b, phosphorylated PDGFR-b; Ab con, antibody control.
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Figure 4. Effects of Chrysanthemum boreale Makino flower floral water on phosphorylation of MAPKs in RASMCs. (A) After treatment without or with the steam-distilled floral water of Chrysanthemum boreale Makino flower (CBMFF: 0.01–100 lg/mL) for 30 min, RASMCs were stimulated with PDGF-BB (10 ng/mL) for 10 min. The cell lysates were immunoblotted with each MAPK antibody. The phosphorylation of p38 MAPK and ERK1/2 was detected using phospho-specific antibodies. The total expression of protein kinases and b-actin was measured using non-phospho-specific antibodies. (B) and (C) Statistical graphs of the phosphorylation levels of MAPKs were obtained from panel A. The basal levels of each phosphorylation are considered as 100% (n ¼ 4). (D) Effects of inhibitors of ERK1/2 and p38 MAPK on PDGF-BB-induced RASMC migration. Cells were incubated for 90 min with p38 MAPK inhibitor SB 203580 (30 lM), ERK1/2 inhibitor PD 98059 (30 lM), or steam-distilled extract floral water of C. boreale Makino flower (CBMFF; 100 lg/mL), and were then stimulated with 10 ng/mL PDGF-BB. Cell migration was examined using the Boyden chamber assay as described in the Methods section. (E) Effects of kinase inhibitors on the PDGF-BB-stimulated RASMC proliferation. Cells were treated with or without PDGF-BB (10 ng/mL), SB 203580 (30 lM), ERK1/2 inhibitor PD 98059 (30 lM), or steam-distilled extract floral water of C. boreale Makino flower (CBMFF; 100 lg/mL), for 48 h. Cell proliferation was tested using a XTT assay. Migration (D) and proliferation (E) in the quiescent state are expressed as 100% (n ¼ 8) Results are represented as the mean ± SD. Two-way ANOVA, *p50.05 compared with the PDGF-BBstimulated state. p-p38 MAPK, phosphorylated p38 MAPK; p-ERK1/2 MAPK, phosphorylated ERK1/2.
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Chrysanthemum boreale Makino in vascular smooth muscle cells
suppressed by treatment with p38 MAPK inhibitor SB203580 at a concentration of 30 lM (103.52 ± 9.25% of control, n ¼ 8) or ERK1/2 inhibitor PD98059 at a concentration of 30 lM in RASMCs (91.97 ± 12.31% of control, n ¼ 8) (Figure 4E).
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Influence of CBMFF on PDGF-BB-stimulated sprout outgrowth of aortic rings To explore whether the CBMFF affects vascularization ex vivo, we treated aortic rings with CBMFF and analyzed sprout outgrowth using an aortic ring assay. Treatment of aortic rings with 10 ng/mL of PDGF-BB increased sprout outgrowth (358.03 ± 23.01% of control, n ¼ 6; Figure 5). This PDGF-BB-elevated aortic sprout outgrowth was inhibited by CBMFF in a dose-dependent manner (0.1–100 lg/mL) and this effect revealed maximum at a 100 lg/mL concentration of CBMFF (110.26 ± 15.40% of control, n ¼ 6). However, the vehicle in which CBMFF was suspended did not affect aortic sprout outgrowth.
Discussion In the present study, we have evaluated whether CBMFF, especially isolated by a steam-distilled extraction method, can affect RASMC migration and proliferation. We found that the CBMFF inhibited the migration and proliferation in response to PDGF-BB. Furthermore, to verify these in vitro findings, we performed ex vivo aortic ring assay that tests aortic sprout formation. The aortic ring assay revealed that CBMFF attenuated PDGF-BB-induced aortic sprout growth. Migration and proliferation of VSMC participated in the development of atherosclerosis (Lee et al., 2012; Wang et al., 2012). These findings collectively indicate that CBMFF inhibits VSMC migration and proliferation and
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may regulate vascular disorders such as atherosclerosis. CBMFF is known to have various biological properties including anticancer, inhibition of angiogenesis, and antiinflammation (Kim et al., 2010; Lee et al., 2003; Perry, 1980). Flower extract of Chrysanthemum indicum Linne´, which belongs to the same genus as CBM, exhibited inhibitory activity on inflammation and proliferation in various cells (Cheon et al., 2009; Li et al., 2009; Yuan et al., 2009). In the present study, we found that CBMFF contained components such as thymol and eugenol that can exert anticancer, antioxidant, antimicrobial, anti-inflammation, and vasorelaxant effect (Table 1). These findings imply that CBMFF may exert a biological effect on VSMC function. However, there are no reports demonstrating that CBMFF, especially obtained by a steam distillation, affects VSMC proliferation and migration. Although other pharmacological activities have been reported for CBMFF, our study for the first time demonstrated the effect of the CBMFF on VSMC migration and proliferation. As described above, PDGF-BB plays a crucial role in VSMC proliferation and migration and is an important stimulator of atherosclerotic lesion development (Arita et al., 2002; Zhou et al., 1999). PDGF-BB is also a mitogen and chemo-attractant for VSMCs and it is known to bind to and activate PDGFR-b on VSMCs in the vascular wall (Holycross et al., 1992; Owens et al., 2004; Zhou et al., 1999). PDGF-BB binding to PDGFR-b causes PDGFR dimerization and transphosphorylation that in turn initiates a series of downstream signals including phosphorylation of p38 MAPK and ERK1/2, ultimately leading to proliferation and migration (Holycross et al., 1992; Kim & Yun, 2007; Pukac et al., 1988). In the present study, PDGF-BB-induced migration and proliferation were decreased by the PDGFR inhibitor in VSMCs. These results imply that
Figure 5. Effect of Chrysanthemum boreale Makino flower floral water on PDGF-BB-stimulated sprout growth of aortic rings. (A) Aortic rings (1 mm) were derived from rats, embedded in Matrigel, and cultured. The rings were treated with or without PDGF-BB (10 ng/mL) in the absence or presence of steam-distilled floral water of C. boreale Makino flower (CBMFF: 0.1–100 lg/mL). Responses were observed on day 5. (B) Statistical results were obtained from panel A. The level in vehicle (0.1% DMSO)-treated rings is expressed as 100% (n ¼ 6). Each value is expressed as the mean ± SD. Two-way ANOVA, *p50.05 versus the PDGF-BB (10 ng/mL)-stimulated state.
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PDGF-stimulated activation of PDGFR-b participates in MAPK-mediated migration and proliferation in VSMCs. In the present study, we tested the effect of the CBMFF on PDGF-stimulated PDGFR-b activation and found that PDGFBB increased PDGFR-b phosphorylation, and this response was significantly inhibited by treatment with CBMFF in a dose-dependent manner. Therefore, it is possible that CBMFF may inhibit PDGFR-b phosphorylation to abolish its downstream signals, probably linked to MAPK pathway, which results in the inhibition of migration and proliferation in VSMCs in response to PDFG-BB, although the mechanism by which the CBMFF inhibits PDGFR-b phosphorylation remains to be elucidated. Many studies have shown that MAPK pathways participate in various cellular functions including proliferation and migration (Stork & Schmitt, 2002). Activation of MAPK in VSMCs is a critical response in stimulating proliferation and migration (Choi et al., 2009) and was involved in PDGFstimulated migration and proliferation in VSMCs (Bornfeldt et al., 1994; Holycross et al., 1992; Pukac et al., 1988). PDGF increased the phosphorylations of p38 MAPK and ERK1/2, and elevated the migration and proliferation in RASMCs (Lee et al., 2007a). p38 MAPK acts as a mediator in cellular responses, including migration and proliferation in VSMCs (Kavurma & Khachigian, 2003). In our previous and current studies, we demonstrated that PDGF-BB stimulated p38 MAPK phosphorylation and also induced VSMC migration and proliferation that were inhibited by the treatment with p38 MAPK inhibitor (Lee et al., 2007a, 2008a,b), indicating that PDGF-BB-induced migration and proliferation in VSMCs is mediated by p38 MAPK pathway. Similarly, the present study demonstrated that PDGF-BB stimulated VSMC migration and proliferation, as well as p38 MAPK phosphorylation, and these responses were attenuated by CBMFF. It is reported that the flower extract of Chrysanthemum indicum inhibits the activation of MAPKs including p38 MAPK in macrophages (Cheon et al., 2009), although this is a different type of cell. Based on these results, it is possible that CBMFF may exhibit inhibitory activity on RASMC migration and proliferation in response to PDGFBB, probably via the suppression of p38 MAPK phosphorylation. Similar to p38 MAPK, ERK1/2 is also reported as an important signaling molecule that is involved in PDGF-BBinduced migration and proliferation and was phosphorylated by PDGF-BB stimulation in VSMCs (Kavurma & Khachigian, 2003). These PDGF-BB-stimulated responses in VSMCs were attenuated by ERK1/2 inhibition as demonstrated in our previous study (Won et al., 2008), as well as the present study. Therefore, these reports suggest that ERK1/2 could be a mediator in PDGF-BB-induced migration and proliferation in VSMCs. Moreover, the flower extract of Chrysanthemum indicum is known to suppress ERK1/2 phosphorylation, as well as proliferations, in cancer cells in response to isoproterenol (Yuan et al., 2009). The present study demonstrated that the phosphorylation of ERK1/2 was induced in response to PDGF-BB in VSMCs and this response was decreased by treatment with CBMFF. CBMFF also inhibited VSMC migration and proliferation in response to PDGF-BB. These findings imply that CBMFF evokes the inhibition of
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ERK1/2 signal and this event may contribute to the downregulation of PDGF-BB-stimulated migration and proliferation in VSMCs. Therefore, it can be assumed that the inhibitory activity of CBMFF on PDGF-BB-induced migration and proliferation may occur through a signal pathway mediated by MAPKs, especially p38 MAPK and ERK1/2, although the present study did not directly demonstrate CBMFF-stimulated interactions between activities of two MAPKs and two cellular responses, migration and proliferation, in VSMCs in response to PDGF-BB.
Conclusions This study has demonstrated that CBMFF dose dependently inhibited PDGF-BB-increased migration and proliferation in RASMCs. PDGF-BB-induced phosphorylations of PDGF-b receptor, p38 MAPK, and ERK1/2 in RASMCs were also decreased by the treatment with the CBMFF. Moreover, the CBMFF also attenuated PDGF-BB-stimulated sprout outgrowth of aortic rings. Based on these findings, we concluded that CBMFF may inhibit vascular responses, VSMC proliferation, and migration, probably through inhibition of the PDGFR-b-mediated MAPK pathway. Therefore, the steam-distilled floral water of CBMF may be useful as a promising agent candidate with anti-atherosclerotic property. However, further research is required to isolate and identify a key bioactive component with anti-atherosclerotic activity from the extract.
Declaration of interest The authors declare that they have no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant nos. A103017 and HN12C0054) and this research was supported by Bioindustry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs.
References Ali SM, Khan AA, Ahmed I, et al. (2005). Antimicrobial activities of eugenol and cinnamaldehyde against the human gastric pathogen Helicobacter pylori. Ann Clin Microbiol Antimicrob 4:1–7. Archana PR, Nageshwar Rao B, Ballal M, Satish Rao BS. (2009). Thymol, a naturally occurring monocyclic dietary phenolic compound protects Chinese hamster lung fibroblasts from radiation-induced cytotoxicity. Mutat Res 680:70–7. Arita YK, Kihara S, Ouchi N, et al. (2002). Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factorBB-binding protein and regulates growth factor-induced common postreceptor signal in vascular smooth muscle cell. Circulation 105: 2893–8. Beena, Kumar D, Rawat DS. (2013). Synthesis and antioxidant activity of thymol and carvacrol based Schiff bases. Bioorg Med Chem Lett 23:641–5. Bornfeldt KE, Raines EW, Nakano T, et al. (1994). Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J Clin Invest 93:1266–74. Chen X, Aravindakshan J, Yang Y, et al. (2006). Aberrant expression of PDGF ligands and receptors in the tumor prone ovary of follitropin receptor knockout (FORKO) mouse. Carcinogenesis 27:903–15. Cheon MS, Yoon T, Lee do Y, et al. (2009). Chrysanthemum indicum Linne´ extract inhibits the inflammatory response by suppressing
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DOI: 10.3109/13880209.2014.941882
Chrysanthemum boreale Makino in vascular smooth muscle cells
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