ORIGINAL ARTICLE
Phloretin Inhibits Platelet-derived Growth Factor-BB–induced Rat Aortic Smooth Muscle Cell Proliferation, Migration, and Neointimal Formation After Carotid Injury Dong Wang, MD, Qingjie Wang, MD, Gaoliang Yan, MD, Yong Qiao, MD, and Chengchun Tang, MD
Abstract: Abnormal vascular smooth muscle cell proliferation and migration are key factors in many cardiovascular diseases. Here, we investigated the effects of phloretin on platelet-derived growth factor homodimer (PDGF-BB)–induced rat aortic smooth muscle cell (RASMC) proliferation, migration, and neointimal formation after carotid injury. Phloretin significantly inhibited the PDGF-BB– stimulated RASMC proliferation in a concentration-dependent manner (10–100 mM). Also, PDGF-BB–stimulated RASMC migration was inhibited by phloretin at 50 mM. Pretreating RASMC with phloretin dose-dependently inhibited PDGF-BB– induced Akt and p38 mitogen-activated protein kinases activation. Furthermore, phloretin increased p27kip1 and decreased cyclindependent kinase 2, CDK4 expression, and p-Rb activation in PDGF-BB–stimulated RASMC in a concentration-dependent manner (10–50 mM). PDGF-BB–induced cell adhesion molecules and matrix metalloproteinase-9 expression were blocked by phloretin at 50 mM. Preincubation with phloretin dose-dependently reduced the intracellular reactive oxygen species production. In vivo study showed that phloretin (20 mg/kg) significantly reduced neointimal formation 14 days after carotid injury in rats. Thus, phloretin may have potential as a treatment against atherosclerosis and restenosis after vascular injury. Key Words: phloretin, vascular smooth muscle cell, PDGF-BB, proliferation, migration (J Cardiovasc Pharmacol 2015;65:444–455)
INTRODUCTION Excessive vascular smooth muscle cell (VSMC) proliferation and migration are key factors in many cardiovascular diseases, such as atherosclerosis and restenosis, after percutaneous coronary intervention.1,2 Although a number of
cytokines and growth factors drive these pathological processes, a critical serum growth factor for VSMC proliferation and migration is platelet-derived growth factor homodimer (PDGF-BB).3 PDGF-BB and its receptor PDGFRb are increased in atherosclerotic lesions and balloon catheterinjured arterial tissue compared with that in the normal vessel wall.4,5 PDGF antibodies and PDGF receptor kinase inhibitors suppress VSMC proliferation, migration, and neointimal formation in a rat model of balloon catheter-injured arterial restenosis.6,7 Flavonoids, a subclass of polyphenols, are derived from a variety of vegetables, fruits, and plants. Studies on flavonoids show their inhibitory effects on proliferation and migration of VSMCs, and neointimal formation in an arterial restenosis model.8,9 Phloretin (Fig. 1), a dihydrochalcone bicyclic flavonoid, has been shown to possess modest estrogenic activity.10,11 Furthermore, phloretin is also an inhibitor of glucose cotransporter and glucose transporter, which has been approved to lower blood glucose and inhibit tumor growth.12,13 Recent studies demonstrated that phloretin had various other biological activities, such as anti-inflammatory and antioxidant.14–16 However, the effects of phloretin on vascular bioactivity have not been clarified. Therefore, our study was to elucidate the effects of phloretin on PDGF-BB–induced rat aortic smooth muscle cell (RASMC) proliferation, migration, and neointimal formation after carotid injury, as well as the underlying mechanism.
METHODS Materials and Reagents
Received for publication September 11, 2014; accepted December 17, 2014. From the Department of Cardiology, Zhongda Hospital of Southeast University Medical School, Nanjing, China. Supported by grants to Chengchun Tang from National Natural Science Foundation of China (research Grant 81170105) and National Natural Science Foundation of China (research Grant 81370225). The authors report no conflicts of interest. Reprints: Chengchun Tang, MD, Department of Cardiology, Zhongda Hospital of Southeast University Medical School, No.87 Dingjiaqiao, 210009 Nanjing, Jiangsu, China (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Phloretin, 5-bromo-2-deoxyuridine (BrdU), and 2,7dichlorofluorescin diacetate (H2DCFDA) were purchased from Sigma Chemical Co. (St Louis, MO). Phloretin was dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO was less than 0.05%. DMSO was used in the groups in which phloretin was not treated. Recombinant human PDGF-BB was purchased from PromoKine (Heidelberg, Germany). Cell culture materials and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD). Sources of antibodies are listed below: (1) Anti-Akt, anti-Erk1/2, anti-phospho-Akt (Ser473), antiphospho-Erk1/2 (Thr202/Thr204), anti-PDGF Receptor b, anti-phospho-PDGF Receptor b, anti-phospholipase Cg1
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J Cardiovasc Pharmacol Volume 65, Number 5, May 2015
Phloretin Inhibits Neointimal Formation
PBS and incubated with Alexa Fluor 488-conjugated donkey anti-goat second antibody for 1 hour. Finally, the cells were washed with PBS, stained with DAPI, and observed under an Olympus IX71 microscope (Tokyo, Japan).
Cell Proliferation Assay
FIGURE 1. The chemical structure and molecular weight of phloretin.
(PLCg1), anti-phospho-PLCg1 (Ser1248) (Cell Signaling Technology, Beverly, MA), (2) anti-p-Rb, anti–proliferating cell nuclear antigen (PCNA), anti-MMP2, anti-MMP9, antiICAM-1, anti-VCAM-1—Santa Cruz Biotechnology (Santa Cruz, CA), (3) anti-CDK2, anti-CDK4, anti-p27Kip1, and anti-BrdU—Abcam Inc. (Cambridge, MA, United Kingdom), (4) anti-smooth muscle a-Actin—Sigma Chemical Co. (St Louis, MO), (5) Internal control anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and anti-b-actin—Santa Cruz Biotechnology, and (6) secondary antibodies conjugated with horseradish peroxidase—Pierce (Rockford, IL), secondary antibodies conjugated with Alexa Fluor 488—Cell Signaling Technology.
Animal and Ethics Statement All animals were purchased from the Animal Center of Southeast University. Animal experiments conformed to the Guide for the Care, and Use of Laboratory Animals was published by the US National Institutes of Health (DHWE publication No. 96-01, revised in 2002) and was approved by the Ethics Review Board for Animal Studies of Institute of Southeast University, Nanjing, China. All animals were fed a standard diet of rat chow and were settled in the laboratory animal room with 218C–268C temperature, 40%–70% humidity, and 15–20 Lux lighting.
Cell Culture Primary RASMC were isolated from the thoracic aorta of male Sprague–Dawley rats weighing between 150 and 180 g as described.17 Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium containing 10% FBS, 100 mg/mL penicillin–streptomycin in a humidified atmosphere of 95% air, and 5% CO2 at 378C. We used morphology assay and immunofluorescence staining with smooth muscle a-actin, a marker of smooth muscle cells, to identify the cultured RASMCs. Cells between the third and 8 passages were used for all experiments.
Immunofluorescence Staining Cells were washed 3 times in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 20 minutes. Cells were washed in PBS and then treated with 0.3% Triton-100 for 5 minutes on ice. After washing, cells were blocked with 5% FBS for 30 minutes at room temperature and then incubated with anti-smooth muscle a-actin (1:100) at 48C overnight. Cells were washed 3 times in Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
The proliferation of RASMCs was measured by 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay as described.18 RASMCs (5 · 103) were planted onto a 96-well culture plate and then starved with 0.1% FBS in DMEM/F12 for 24 hours. Cells were treated with PDGFBB (25 ng/mL) and phloretin at concentrations of 10, 30, 50 and 100 mM, respectively. After 24 hours, 20 mL MTT (5 mg/ mL) was added into each well, and cells were incubated for 4 hours. The accumulative formazan crystals were dissolved with 150 mL DMSO. The optical density of the solution was measured at a wavelength of 570 nm with a microplate reader (Thermo Scientific). We also used BrdU immunofluorescent staining assay to evaluate the effect of phloretin on newly synthesized DNA of RASMC. Cells were starved with 0.1% FBS in DMEM/F12 for 24 hours and then treated with PDGF-BB (25 ng/mL) and phloretin (50 mM) for 24 hours. BrdU (10 mM) was added to incubate for 2 hours before the end of culture. Cells were fixed with 4% paraformaldehyde and then treated with 0.1% Triton X-100 in PBS for 5 minutes. After washing, cells were blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Next, cells were treated with 2 mol/L hydrochloric acid for 30 minutes and 0.1 mol/L antipyonin for 5 minutes. Then, cells were incubated with anti-BrdU antibody (1:1000) overnight at 48C and then incubated with Anti-rat (Alexa Fluor 488 Conjugate) secondary antibody for 1 hour in the dark at room temperature. Photos were taken by an Olympus IX71 microscopy. BrdU-positive cells were calculated as the total number of cells per 5 different random fields.
Migration Assay The migration assay was performed using the Transwell system (a 6.5-mm polycarbonate membrane with 8.0-mm pores; Corning, NY) as described.19 Of note, 5 · 104 of cells suspensions, containing fresh serum-free media, were seeded on the upper chamber. PDGF-BB (25 ng/mL) with or without phloretin (50 mM) was added into the bottom chamber as the chemoattractant. The cells were allowed to migrate through the membrane to the lower surface for 6 hours. Cells on the upper surface of the membrane that had not migrated were scraped off with cotton swabs, and cells that had migrated to the lower surface were fixed by 4% paraformaldehyde and stained with 0.1% crystal violet and counted. Migrated cell numbers were calculated as the number of migrated cells per 5 different random fields. At last, the cells were dissolved with 30% acetic acid and then measured under 490 nm using a microplate reader (Thermo Scientific). We also used a wound scratch assay to detect the effect of phloretin on RASMCs migration. GFP-positive transgenic Sprague–Dawley rat was used to obtain GFP+ RASMCs. Cells (1 · 106) were seeded onto the 6-well plates overnight. After serum starvation (0.1% FBS in DMEM/F12) for 24 hours, cell monolayer was wounded using a pipette tip and www.jcvp.org |
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then washed twice with PBS gently. Then, cells were treated with PDGF-BB (25 ng/mL) and phloretin (50 mM) for 8 hours. The cell migration ability was evaluated by the percentage of scratch area per 5 different random fields.
performed with the transcribed cDNA and SYBR Premix Ex Taq II (Takara) in triplicates using the 7300 real-time PCR system (ABI, Foster City, CA) to detect messenger RNA (mRNA) expression. The primer sequences purchased from Invitrogen were showed in Table 1, and the program was as follows: 958C for 30 seconds, 40 cycles at 958C for 5 seconds, and 608C for 31 seconds. The relative mRNA amounts of target genes were normalized to the values of b-actin. The results were expressed as fold changes of threshold cycle (Ct) value relative to the controls using the 22DDCt method.
Western Blotting After serum starvation (0.1% FBS in DMEM/F12) for 24 hours, cells were stimulated with PDGF-BB (25 ng/mL) with or without phloretin (50 mM). Total proteins were prepared at the indicated time points using protein extraction buffer containing protease inhibitor Complete (Roche, IN). Western blotting was carried as described previously.19 In brief, cells lysate with equal amount of protein (50 mg) were separated by 10% SDS–polyacrylamide gel electrophoresis and then transferred electronically to the polyvinylidene difluoride membranes. Membranes were blocked in 5% nonfat milk powder in trisbuffered saline and tween 20 (TBST) (0.1% Tween 20 in 1 · TBS) for 1 hour at room temperature and then incubated with (1) 1:1000 dilutions of anti-PDGF-Rb, anti-Erk1/2, anti-Akt, anti-phospho-PDGF-Rb, anti-phospho-Erk1/2, anti-phosphoAkt, anti-PLCg1, anti-phospho-PLCg1, anti-CDK2, and antiCDK4, (2) 1:200 dilutions anti-p38 MAPK, anti-phospho-p38 MAPK, anti-JNK, anti-phospho-JNK, anti-p-Rb, anti-MMP2, anti-MMP9, anti-ICAM-1, and anti-VCAM-1, and (3) 1:5000 dilutions anti-b-actin and anti-GAPDH at 48C overnight. Finally, the membranes were incubated with secondary antibodies (1:5000) conjugated with horseradish peroxidase for 1 hour at room temperature. Immunoreactive materials were visualized by using Chemiluminescent Substrate kit (Pierce, Rockford, IL). The membranes were scanned, and the sum optical density was quantitatively analyzed by Quantity One software (Bio-Rad, Richmond, CA).
Real-time Polymerase Chain Reaction Total RNA was isolated using an RNAiso Blood (Takara, Otsu, Japan) according to the manufacturer’s instructions. First, 1-mg RNA was reverse transcribed to complementary deoxyribonucleic acid (cDNA) with random primers using PrimeScript RT Master Mix (Takara). Then, quantitative real-time polymerase chain reaction (PCR) was
Intracellular Reactive Oxygen Species Measurement We used the reactive oxygen species (ROS)–sensitive probe H2DCFDA to detect intracellular ROS production. Briefly, cells were serum-starved in DMEM with 0.1% FBS for 24 hours and then subjected to the treatment with phloretin or N-acetyl-cysteine (NAC) for 2 hours. Cells were then challenged with PDGF-BB (25 ng/mL) for 30 minutes. After treatment, cells were then labeled with H2DCFDA (10 mM) and incubated for 30 minutes at 378C. Then, cells were washed gently for 3 times with PBS and immediately observed under an Olympus IX71 microscope with excitation and emission wavelengths of 488 nm and 525 nm, respectively. The fluorescence intensity was measured from 3 random fields for each well by using Image J software (NIH, Bethesda).
Balloon Injury Model in Rat Carotid Artery The surgical intervention was performed as described previously.20 Briefly, rats weighing between 300–350 g were anesthetized with 10% chloral hydrate at a dose of 300 mg/kg (intraperitoneally). Then, expose the right common, external, and internal carotid arteries completely and inject heparin sodium intravenously at a dose of 100 U/kg. Insert a balloon catheter (1.5 mm in diameter, 15 mm in length; Medtronic) into the common carotid artery by a small cut in the external carotid artery after temporary blockage of the internal and common carotid arteries. Next, aerate the balloon and pull the catheter back and forth 5 times using a rotatory movement. Rats were divided into 3 groups: sham (n = 5), saline
TABLE 1. Primer Sequences for Real-time PCR Genes
Primer Sequences
b-Actin ICAM-1 VCAM-1 MMP2 MMP9 TIMP-1 TIMP-2
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Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
50 -CCCATCTATGAGGGTTACGC-30 50 -TTTAATGTCACGCACGATTTC-30 50 -TGCTCAGGTATCCATCCA-30 50 -TTCATCCAGTTAGTCTCCAA-30 50 -CTGGAGATTGAACTACTGAAG-30 50 -TTGACTGCTGTGTAACTTAG-30 50 -ACAACCAACTACGATGATGA-30 50 -TGGATAGTCGGAAGTTCTTG-30 50 -TGGACAGCGAGACACTAA-30 50 -TATGATGGTGCCACTTGAG-30 50 -TCCTCTTGTTGCTATCATTG-30 50 -GCTGGTATAAGGTGGTCTC-30 50 -ACCCTCTGTGACTTTATTGT-30 50 -ATTGATGCTCTTCTCTGTGA-30
Amplicon (bp) 150 123 168 172 102 147 180
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Phloretin Inhibits Neointimal Formation
(n = 6), and phloretin (n = 8). Rats in the sham group were exposed to surgery without vessel injury. Rats in the phloretin group were given an injection of 20 mg/kg phloretin (intraperitoneally) once a day. Rats in the sham and saline group were injected an equal volume of normal saline. The carotid arteries were collected after 2 weeks.
the PDGF-BB alone group (Fig. 3A). However, 50 mM phloretin alone did not decrease absorbance. The phloretin concentration producing 50% inhibition (IC50) was 42.6 mM (Fig. 3A). In addition, the BrdU fluorescence assay revealed that the percentage of BrdU-positive cells increased significantly after PDGF-BB treatment. However, phloretin at 50 mM diminished PDGF-BB–induced proliferation in RASMCs (Fig. 3B, C).
Morphometric Analysis and Immunohistochemistry
Carotid arteries were fixed with 4% paraformaldehyde and were embedded in paraffin. Then, the carotid arteries sections (4 mm) were stained with hematoxylin–eosin, and the photos were taken by a light microscopy (Olympus). The thickness of neointima was measured using Image-Pro Plus 6.0 per 4 different random fields and were normalized to media thickness. Immunohistochemical detection of PCNA was performed as described previously.20 PCNA-positive cells were calculated as the total number of cells within the neointima per 4 different random fields.
Statistical Analysis
Data were presented as means 6 SE. One-way analysis of variance was used for multiple comparisons by SPSS 19.0 (SPSS Inc, Chicago, Il). If there was a significant variation between treated groups, the Tukey test was applied. P , 0.05 was considered statistically significant.
RESULTS Purity Identification of Cultured RASMCs The RASMCs exhibited characteristic spindle shapes and a typical appearance of “valley and hill” using a light microscope (Fig. 2A). As determined through immunofluorescence staining with smooth muscle a-actin, more than 95% of the cells emitted green fluorescence (Fig. 2B).
Effects of Phloretin on PDGF-BB–induced Proliferation of RASMCs The results of the MTT assay showed that absorbance increased significantly after PDGF-BB treatment compared with that in the unstimulated group. Phloretin treatment inhibited PDGF-BB–induced RASMC proliferation in a concentration-dependent manner. Phloretin at 10, 30, 50, and 100 mM inhibited proliferation by about 8%, 35%, 48%, and 70% in PDGF-BB–induced RASMC, respectively, compared with
Effects of Phloretin on PDGF-BB–induced Migration of RASMCs As shown in Figure 4A, B, 25 ng/mL PDGF-BB significantly increased RASMC migration. The 50 mM phloretin treatment decreased absorbance and increased the percentage of scratched area compared with those in the PDGF-BB–stimulated group. However, phloretin alone did not inhibit migration of RASMCs.
Effects of Phloretin on the PDGF-BB–induced PDGFR Signaling Pathway in RASMCs The PDGFR signaling pathway is related to cell proliferation, migration, differentiation, and angiogenesis. Distribution of the PDGFR differs in different tissues and cells. PDGF-Ra is highly expressed in mesenchymal cells; however, the mesenchyme contains more PDGF-Rb, particularly VSMCs. As shown in Figure 5, phloretin had no effect on PDGF-BB–induced PDGF-Rb phosphorylation at 10–50 mM and had no effects on downstream extracellular regulated kinase (ERK1)/2, PLCg1, or JNK phosphorylation. However, phloretin inhibited PDGF-BB–stimulated Akt and p38 MAPK phosphorylation at 10–50 mM in a concentrationdependent manner (Fig. 5B).
Effects of Phloretin on cell Cycle Regulatory Protein Expression Induced by PDGF-BB in RASMCs Cyclins, cyclin-dependent kinases (CDKs), and cyclindependent kinase inhibitors (CKIs) play major roles in regulating the cell cycle. To verify whether the antiproliferative effect of phloretin was related to cell cycle regulatory proteins, we examined CDK2 and CDK4 expression and Rb phosphorylation using Western blot analysis. An important CKI, p27kip1, was also detected. As shown in Figure 6, phloretin reduced upregulation of PDGF-BB–induced CDK2, CDK4, and proliferating cell nuclear antigen, as well as Rb
FIGURE 2. The purity identification of RASMCs. A, Cell morphology images were taken under a microscopy (scale bar, 100 mm). B, Isolated RASMCs were fixed and subjected to immunofluorescence staining with primary antibodies to smooth muscle a-Actin (green). Nucleus was labeled with DAPI (blue) (scale bar, 100 mm). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 3. Effects of phloretin on PDGF-BB–induced RASMC proliferation. A, RASMC viability was detected by the MTT assay. Data are mean 6 SE (n = 4, *P , 0.05 vs. control and &P , 0.05 vs. PDGF-BB alone). B and C, DAPI-labeled cells are blue, and BrdU-positive cells are green. The number of BrdU-positive cells in the different experimental groups was normalized to the total number of cells. Data are mean 6 SE (n = 3, *P , 0.05 vs. control; #P , 0.05 vs. PDGF-BB alone) (scale bar, 50 mm).
phosphorylation at 10–50 mM in a concentration-dependent manner. In contrast, PDGF-BB diminished p27kip1 protein expression, whereas the expression of this protein increased after phloretin treatment.
VSMCs express various adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which bind to their receptors, resulting in monocytes and lymphocytes adhering and migrating to the vessel wall.21 We used Western blotting and real-time PCR analyses to detect ICAM-1 and VCAM-1 protein and mRNA, respectively. As shown in Figure 7A, B, 50 mM phloretin not only inhibited the upregulation of PDGF-BB–induced ICAM-1 and VCAM-1
mRNA but also inhibited upregulation of their proteins. However, phloretin alone exerted no effect compared with that in the control group. Extracellular matrix degradation also plays a major role in cell migration. Matrix metalloproteinases (MMPs) break down the extracellular matrix to promote migration, but their activities are modulated by specific endogenous tissue inhibitors of metalloproteinase (TIMPs).22 MMP2, MMP9, and their inhibitors TIMP-1 and TIMP-2 are now well studied; thus, we examined changes in their levels using Western blotting and real-time PCR. As shown in Figure 7C, 50-mM phloretin inhibited PDGF-BB–induced MMP9 mRNA upregulation but had no effect on MMP2, TIMP1, or TIMP2. In addition, Western blotting showed that the enhanced MMP9 expression was suppressed by phloretin at 50 mM. However, no change in MMP2 was detected (Fig. 7D).
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Effect of Phloretin on the Expression of Adhesion Molecules and Extracellular Matrix Proteins Induced by PDGF-BB in RASMCs
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Phloretin Inhibits Neointimal Formation
FIGURE 4. Effects of phloretin on PDGF-BB–induced RASMC migration. The transwell assay and scratch test were used to evaluate RASMC migration. A, Photographs of migration were taken under a microscope (magnification, ·100). The results are mean 6 SE (n = 3, *P , 0.05 vs. control group and #P , 0.05 vs. PDGF-BB alone group). B, Photographs of the scratch test were taken under a microscope (magnification, ·40). The percentage of scratch area is shown as mean 6 SE (n = 5, *P , 0.05 vs. control group and #P , 0.05 vs. PDGF-BB-alone group).
Effects of Phloretin on the Intracellular ROS Production Induced by PDGF-BB in RASMCs Excessive ROS plays an important role in oxidative stress. Exposed to PDGF-BB for 30 minutes, the level of intracellular ROS was markedly increased in RASMCs compared with that in the control group (Fig. 8). PDGF-BB– induced intracellular ROS activation was inhibited by the pretreatment of phloretin at 1–50 mM in a concentration-dependent manner and also by the antioxidant NAC at 10 mM.
saline group (Fig. 9A). The intima:media ratio in the group treated with 20 mg/kg phloretin decreased significantly compared with that in the saline group (Fig. 9A). The proportion of PCNA-positive cells in the saline group increased observably compared with the sham group. However, in the phloretin-treated group, this proportion decreased approximately 58% compared with that in the saline group (Fig. 9B). These results are the same as those from the in vitro study. Thus, these results indicate that phloretin may inhibit neointimal formation in a carotid artery injury model.
Effects of Phloretin on Neointimal Formation in a Rat Carotid Artery Injury Model Intima and media thicknesses were estimated to test the effects of phloretin on neointimal formation. Hematoxylin and eosin staining of carotid artery sections revealed a barely visible intima in the sham group and a thick neointima in the Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
DISCUSSION This study showed that phloretin could prevent neointimal formation after vascular injury. This conclusion was based on the following evidence. First, phloretin inhibited www.jcvp.org |
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FIGURE 5. Effect of phloretin on PDGF-BB–induced PDGF-receptor b, extracellular regulated kinase (ERK)1/2, Akt, phospholipase C (PLC)g1, p38 MAPK, and JNK phosphorylation in RASMC. Confluent cells were precultured with phloretin in serum-free medium for 24 hours. Then, the cells were stimulated with 25 ng/mL PDGF-BB at 378C for 1 minute to analyze PDGF-Rb, for 5 minutes to analyze ERK1/2, and for 15 minutes to analyze Akt, PLCg1, p38 MAPK, and JNK. Levels of protein phosphorylation were determined using Western blot analyses and quantified by densitometry. The respective total protein levels were used for normalization. Data are mean 6 SE. A, Levels of PDGF-Rb phosphorylation (n = 3). B, Levels of Akt, ERK1/2, PLCg1, p38 MAPK, and JNK phosphorylation (n = 3, *P , 0.05 vs. PDGF-BB-alone group; &P , 0.01 vs. PDGF-BB alone).
PDGF-BB–induced proliferation and migration of RASMCs. Second, phloretin inhibited PDGF-BB–induced Akt and p38 MAPK activation of RAMSCs. Third, phloretin upregulated p27kip1 and downregulated CDK2, CDK4, adhesion molecules, MMP9, and p-Rb phosphorylation induced by PDGF-BB. In addition, phloretin suppressed intracellular ROS production in PDGF-BB–stimulated RASMCs. It is well known that inhibiting excess proliferation and migration of VSMCs is an effective intervention to prevent
atherosclerosis and restenosis.1,23 Therefore, we first detected the effects of phloretin on RASMC proliferation and migration. A 25-ng/mL PDGF-BB treatment was used as a mitogen to promote cell proliferation and migration as previously described.8,20 Phloretin inhibits proliferation of many cell types, such as liver cancer cells, breast tumor cells, and T lymphocytes.24–26 We clearly demonstrated that phloretin inhibited PDGF-BB–induced RASMC proliferation at 10–100 mM in a concentration-dependent manner. These results are in
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Phloretin Inhibits Neointimal Formation
FIGURE 6. Effect of phloretin on the activation of cell cycle regulatory proteins induced by PDGF-BB in RASMC. Levels of CDK2, CDK4, PCNA, p27kip1 proteins, and Rb phosphorylation were determined using Western blot analyses and quantified by densitometry. Total b-actin was used for normalization. Results are mean 6 SE (n = 3, *P , 0.05 vs. PDGF-BB alone and &P , 0.01 vs. PDGF-BB alone).
accordance with those by Kim et al,24 as phloretin displayed an antiproliferative effect at 10–80 mM in human breast epithelial cells. Furthermore, DNA synthesis stimulated by PDGF-BB was also suppressed by phloretin, and this further confirmed the antiproliferative effect of phloretin. This antiproliferative effect was not because of cytotoxicity but because phloretin was not cytotoxic at 50 mM, similar to
the results by Chung et al.27 Only a thin endothelial cell layer is present in a normal vessel wall. However, VSMCs proliferate and migrate from the media to the intima in atherosclerotic lesions and balloon catheter-injured arterial tissue, resulting in neointimal hyperplasia. We found that phloretin inhibited PDGF-BB–induced RASMC migration in the transwell and wound scratch assays. However, no
FIGURE 7. Effects of phloretin on the expression of adhesion molecules and key extracellular matrix proteins induced by PDGF-BB in RASMC. Messenger RNA and protein levels were determined using real-time PCR and Western blot analyses, respectively. GAPDH was used for normalization. Results are mean 6 SE (n = 3–4, *P , 0.05 vs. Control group and #P , 0.05 vs. PDGF-BB alone). A and B, Levels of ICAM-1 and VCAM-1 mRNA and protein. C and D, Levels of matrix MMP2, MMP9, TIMP1, and TIMP2 mRNA and protein. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 8. Effects of phloretin on the intracellular ROS production induced by PDGF-BB in RASMC. The intracellular ROS level was detected by ROS-sensitive probe H2DCFDA. A, Immunofluorescene images were taken under a fluorescence microscopy. ROS was labeled with green. B, Levels of the DCF fluorescence intensity. Data are mean 6 SE (n = 3, *P , 0.05 vs. Control group; #P , 0.05 vs. PDGF-BB alone).
effects on migration were found in the phloretin-alone group. PDGF binds to its receptor-PDGFR, and then the receptor dimerizes and autophosphorylates.28 PDGF receptor kinase inhibitors are effective for treating PDGF-driven diseases, such as cancers, atherosclerosis, and restenosis.29,30 PI3K/Akt, mitogen-activated protein kinases (MAPKs), and PLCg are the major downstream signaling intermediates related to activating PDGFR.21 Suppression of Akt, MAPKs, and PLCg phosphorylation results in antiproliferative effects in VSMCs. Previous studies have shown that phloretin inhibits tyrosine kinase activity,31 as well as Akt and Erk phosphorylation at micromolar levels in cancer cells.25,27 However, we found that phloretin did not decrease PDGFBB–induced PDGF-Rb phosphorylation in RASMCs. Furthermore, no inhibitory effects on downstream signaling intermediates or Erk1/2, JNK, or PLCg1 phosphorylation were observed by phloretin. However, phloretin reduced PDGF-BB–stimulated phosphorylation of Akt (Ser473), a marker of PI3K activation, and p38 MAPK in a concentration-dependent manner at micromolar levels. This reduction in Akt and p38 MAPK phosphorylation may be involved in inhibiting mitogen-stimulated proliferation and migration of RASMCs.
Subsequently, we investigated whether the antiproliferative effect of phloretin was involved in cell cycle regulatory proteins, such as CDKs, cyclins, CKIs, and p-Rb. CDK2 and CDK4 are key mediators during the G0/G1 to S phase progression of the cell cycle and form complexes with cyclin E and cyclin D1, respectively.32 Cyclin-dependent kinase (CDK)–cyclin complexes phosphorylate Rb resulting in dissociation of the p-Rb/E2F complex, and the E2F transcription factor reactivates for cells to enter the S phase. Therefore, inhibition of CDK and cyclin expression and p-Rb phosphorylation may lead to cell cycle arrest. In this study, we found that phloretin not only suppressed PDGF-BB–induced CDK2 and CDK4 expression but also inhibited p-Rb phosphorylation in a concentration-dependent manner. PCNA, a cell proliferative marker induced by PDGF-BB, also decreased after phloretin treatment. These observations may be the underlying mechanism related to the antiproliferative effects of phloretin. A succession of CKIs, including p16ink4, p21cip1, and p27kip1, is the primary negative cell cycle regulators, as they block the activities of several CDK-cyclin complexes. Marra et al33 found that overexpressing p27kip1 forces VSMCs into G1 growth arrest. Drugs upregulating p27kip1 exhibit an antiproliferative effect on VSMCs.33,34 We found that reducing p27kip1 with PDGF-BB was prevented by phloretin, which inhibited
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Phloretin Inhibits Neointimal Formation
FIGURE 9. Effects of phloretin on neointimal formation in a rat carotid artery injury model. Red and yellow bars represent neointima and media, respectively. A, Representative hematoxylin and eosin–stained carotid artery sections from each experimental group. Results are mean 6 SE (n = 5–8; *P , 0.05 vs Sham group; #P , 0.01 vs Saline group) (scale bar, 100 mm). B, Representative immunohistochemical staining for PCNA expression after rat carotid artery injury. Results are mean 6 SE (*P , 0.01 vs. Saline group). Scale bar, 200 mm.
the cell cycle and prevented cell growth. Therefore, our findings demonstrate that phloretin inhibited the proliferation of RASMCs, probably by upregulating p27kip1 and downregulating CDK2, CDK4, and PCNA, as well as by activating p-Rb. Cell adhesion molecules and MMPs are the key regulators of cell migration and neointimal formation.22 Stangl et al35 found that phloretin suppresses cytokinestimulated ICAM-1, VCAM-1, and E-selectin expression in human umbilical vein endothelial cells. However, no evidence has been reported about the effects of phloretin on cell adhesion molecules and MMPs in VSMCs. We found that phloretin inhibited PDGF-BB–induced ICAM-1, VCAM-1, and MMP9 upregulation in RASMCs, but no effect on MMP2 was observed. Suppression of MMPs and cell adhesion molecules inhibits VSMC migration and neointimal formation in a carotid artery injury rat model.18,33,36 Thus, we consider that reducing the increase in ICAM-1, VCAM-1, and MMP9 expression may be involved in suppressing RASMC migration and neointimal formation because of phloretin. In VSMC, PDGF-BB can trigger oxidative stress and induce the production of intracellular ROS. Excessive ROS Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
promotes VSMC proliferation and migration by upregulating cyclins, CDKs, adhesion molecules, and downregulating CKIs. Antioxidants or some natural compound could inhibit PDGF-induced VSMC proliferation and migration by reducing ROS production. Phloretin is an inhibitor of oxidative phosphorylation,37 and previous studies established its antioxidative activity.15 In our study, we found that phloretin reduced PDGF-BB–induced ROS production in a concentration-dependent manner in RASMCs. Although the mechanism by which phloretin inhibits PDGF-BB–induced proliferation and migration in RASMCs is not thoroughly clear, one potential explanation is that phloretin reduces excessive ROS production in RASMCs and thereby prevents the downstream effects of oxidative stress, such as downregulation of p27kip1 expression, upregulation of CDK2, CDK4, ICAM-1, and VCAM-1 expression, and that result in the inhibition of proliferation and migration. To further study the effects of phloretin on neointimal formation, we used a balloon catheter-injured carotid artery restenosis rat model. Previous studies indicated that 10–40 mg/kg (body weight) of phloretin was an effective dosage www.jcvp.org |
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to treat some diseases in vivo.25,38 In this study, we used 20 mg/kg phloretin (intraperitoneally) to treat the rat carotid artery injury model for 2 weeks. We found that neointima began to form when arterial walls were injured for 2 weeks, and neointimal hyperplasia was suppressed after the 20 mg/kg phloretin treatment. PCNA expression decreased in the phloretin-treated group, and that was in accordance with what we found in cultured RASMCs. Thus, our data indicated that phloretin could inhibit neointimal formation through preventing VSMC proliferation and migration. Oxidative stress exists in a balloon injury rat model. Dietary flavonoid consumption acts as a cardiovascular protective agent.39 One of the mechanisms is that flavonoids can reduce intracellular ROS level, prevent superoxide-induced NO inactivation, and increase endothelial nitric oxide synthase activity, and hence helping VSMC to maintain quiescent.40,41 Ullen et al16 found that 2-chlorohexadecanal, an inhibitor of eNOS, induced endothelial cell dysfunction, and cell death was ameliorated by phloretin at 50 mM. However, we only focused on the effects of phloretin on VSMC itself and the effects of phloretin on the recovery of the endothelial cell layer and the NO production within an injured carotid artery model were not revealed. A future work will be performed to fully understand the mechanism. Phloretin, enriched in various fruits, is a component of the human diet. Although little in vivo research about phloretin has been carried out in humans, 2 studies showed that phloretin, as a topical agent, could ameliorate ultravioletinduced photodamage in human skin, and that was attributed to its antioxidant and anti-inflammatory activities.42,43 Moreover, phloretin has been proven effective to treat diseases such as tumor,25 diabetes,44 and colonitis45 in animal models. Therefore, we speculate that phloretin may have a broad spectrum of bioactivities such as lowering blood glucose, antitumor, antioxidant, and anti-inflammatory activities in humans. In summary, our data suggest that phloretin is a reasonable strategy to prevent neointimal formation after artery injury through blockade of oxidative stress, VSMCs proliferation, and migration, which give a new evidence for future studies to test the effect of phloretin on patients with restenosis.
8. Yu JY, Lee JJ, Lim Y, et al. Genistein inhibits rat aortic smooth muscle cell proliferation through the induction of p27kip1. J Pharmacol Sci. 2008;107:90–98. 9. Jiang D, Li D, Wu W. Inhibitory effects and mechanisms of luteolin on proliferation and migration of vascular smooth muscle cells. Nutrients. 2013;5:1648–1659. 10. Miksicek RJ. Commonly occurring plant flavonoids have estrogenic activity. Mol Pharmacol. 1993;44:37–43. 11. Garrett SD, Lee HA, Morgan MR. A nonisotopic estrogen receptor-based assay to detect estrogenic compounds. Nat Biotechnol. 1999;17:1219–1222. 12. Schulze C, Bangert A, Kottra G, et al. Inhibition of the intestinal sodiumcoupled glucose transporter 1 (SGLT1) by extracts and polyphenols from apple reduces postprandial blood glucose levels in mice and humans. Mol Nutr Food Res. 2014;58:1795–1808. 13. Kobori M, Shinmoto H, Tsushida T, et al. Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport. Cancer Lett. 1997;119:207–212. 14. Chang WT, Huang WC, Liou CJ. Evaluation of the anti-inflammatory effects of phloretin and phlorizin in lipopolysaccharide-stimulated mouse macrophages. Food Chem. 2012;134:972–979. 15. Rezk BM, Haenen GR, van der Vijgh WJ, et al. The antioxidant activity of phloretin: the disclosure of a new antioxidant pharmacophore in flavonoids. Biochem Biophys Res Commun. 2002;295:9–13. 16. Ullen A, Fauler G, Bernhart E, et al. Phloretin ameliorates 2-chlorohexadecanal-mediated brain microvascular endothelial cell dysfunction in vitro. Free Radic Biol Med. 2012;53:1770–1781. 17. Funakoshi Y, Ichiki T, Ito K, et al. Induction of interleukin-6 expression by angiotensin II in rat vascular smooth muscle cells. Hypertension. 1999;34:118–125. 18. Kwon JS, Joung H, Kim YS, et al. Sulforaphane inhibits restenosis by suppressing inflammation and the proliferation of vascular smooth muscle cells. Atherosclerosis. 2012;225:41–49. 19. Wang L, Zheng J, Du Y, et al. Cartilage oligomeric matrix protein maintains the contractile phenotype of vascular smooth muscle cells by interacting with alpha(7)beta(1) integrin. Circ Res. 2010;106:514–525. 20. Yoo SH, Lim Y, Kim SJ, et al. Sulforaphane inhibits PDGF-induced proliferation of rat aortic vascular smooth muscle cell by up-regulation of p53 leading to G1/S cell cycle arrest. Vascul Pharmacol. 2013;59:44–51. 21. Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol. 2008;28:812–819. 22. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol. 2001;17:463–516. 23. Otsuka F, Finn AV, Yazdani SK, et al. The importance of the endothelium in atherothrombosis and coronary stenting. Nat Rev Cardiol. 2012;9:439–453. 24. Kim MS, Kwon JY, Kang NJ, et al. Phloretin induces apoptosis in H-Ras MCF10A human breast tumor cells through the activation of p53 via JNK and p38 mitogen-activated protein kinase signaling. Ann N Y Acad Sci. 2009;1171:479–483. 25. Wu CH, Ho YS, Tsai CY, et al. In vitro and in vivo study of phloretininduced apoptosis in human liver cancer cells involving inhibition of type II glucose transporter. Int J Cancer. 2009;124:2210–2219. 26. Lu XY, Zeng YY, Ye YX, et al. Anti-inflammatory and immunosuppressive effect of phloretin. Yao Xue Xue Bao. 2009;44:480–485. 27. Chung MJ, Sohng JK, Choi DJ, et al. Inhibitory effect of phloretin and biochanin A on IgE-mediated allergic responses in rat basophilic leukemia RBL-2H3 cells. Life Sci. 2013;93:401–408. 28. Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev. 1999;79:1283–1316. 29. Levitzki A. PDGF receptor kinase inhibitors for the treatment of restenosis. Cardiovasc Res. 2005;65:581–586. 30. Levitzki A. PDGF receptor kinase inhibitors for the treatment of PDGF driven diseases. Cytokine Growth Factor Rev. 2004;15:229–235. 31. Yang EB, Guo YJ, Zhang K, et al. Inhibition of epidermal growth factor receptor tyrosine kinase by chalcone derivatives. Biochim Biophys Acta. 2001;1550:144–152. 32. Gorenne I, Kavurma M, Scott S, et al. Vascular smooth muscle cell senescence in atherosclerosis. Cardiovasc Res. 2006;72:9–17. 33. Tanner FC, Boehm M, Akyurek LM, et al. Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation. Circulation. 2000;101: 2022–2025.
REFERENCES 1. Lacolley P, Regnault V, Nicoletti A, et al. The vascular smooth muscle cell in arterial pathology: a cell that can take on multiple roles. Cardiovasc Res. 2012;95:194–204. 2. Gomez D, Owens GK. Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc Res. 2012;95:156–164. 3. Raines EW. PDGF and cardiovascular disease. Cytokine Growth Factor Rev. 2004;15:237–254. 4. Ross R, Masuda J, Raines EW, et al. Localization of PDGF-B protein in macrophages in all phases of atherogenesis. Science. 1990;248: 1009–1012. 5. Barrett TB, Benditt EP. sis (platelet-derived growth factor B chain) gene transcript levels are elevated in human atherosclerotic lesions compared to normal artery. Proc Natl Acad Sci U S A. 1987;84:1099–1103. 6. Ishii S, Okamoto Y, Katsumata H, et al. Sunitinib, a small-molecule receptor tyrosine kinase inhibitor, suppresses neointimal hyperplasia in balloon-injured rat carotid artery. J Cardiovasc Pharmacol Ther. 2013; 18:359–366. 7. Jandt E, Mutschke O, Mahboobi S, et al. Stent-based release of a selective PDGF-receptor blocker from the bis-indolylmethanon class inhibits restenosis in the rabbit animal model. Vascul Pharmacol. 2010;52:55–62.
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34. Marra DE, Simoncini T, Liao JK. Inhibition of vascular smooth muscle cell proliferation by sodium salicylate mediated by upregulation of p21 (Waf1) and p27(Kip1). Circulation. 2000;102:2124–2130. 35. Stangl V, Lorenz M, Ludwig A, et al. The flavonoid phloretin suppresses stimulated expression of endothelial adhesion molecules and reduces activation of human platelets. J Nutr. 2005;135:172–178. 36. Cho A, Reidy MA. Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury. Circ Res. 2002;91:845–851. 37. De Jonge PC, Wieringa T, Van Putten JP, et al. Phloretin—an uncoupler and an inhibitor of mitochondrial oxidative phosphorylation. Biochim Biophys Acta. 1983;722:219–225. 38. Yang KC, Tsai CY, Wang YJ, et al. Apple polyphenol phloretin potentiates the anticancer actions of paclitaxel through induction of apoptosis in human hep G2 cells. Mol Carcinog. 2009;48:420–431. 39. Grassi D, Desideri G, Di GP, et al. Tea, flavonoids, and cardiovascular health: endothelial protection. Am J Clin Nutr. 2013;98(6 suppl):1660S–1666S.
40. Hodgson JM, Croft KD. Tea flavonoids and cardiovascular health. Am J Clin Nutr. 2010;31:495–502. 41. Duarte J, Francisco V, Perez-Vizcaino F. Modulation of nitric oxide by flavonoids. Food Funct. 2014;5:1653–1668. 42. Oresajo C, Stephens T, Hino PD, et al. Protective effects of a topical antioxidant mixture containing vitamin C, ferulic acid, and phloretin against ultraviolet-induced photodamage in human skin. J Cosmet Dermatol. 2008;7:290–297. 43. Shin S, Kum H, Ryu D, et al. Protective effects of a new phloretin Derivative against UVB-induced Damage in skin cell model and human Volunteers. Int J Mol Sci. 2014;15:18919–18940. 44. Najafian M, Jahromi MZ, Nowroznejhad MJ, et al. Phloridzin reduces blood glucose levels and improves lipids metabolism in streptozotocininduced diabetic rats. Mol Biol Rep. 2012;39:5299–5306. 45. Lee JH, Regmi SC, Kim JA, et al. Apple flavonoid phloretin inhibits Escherichia coli O157:H7 biofilm formation and ameliorates colon inflammation in rats. Infect Immun. 2011;79:4819–4827.
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Phloretin Inhibits Platelet-derived Growth Factor-BB–induced Rat Aortic Smooth Muscle Cell Proliferation, Migration, and Neointimal Formation After Carotid Injury Dong Wang, MD, Qingjie Wang, MD, Gaoliang Yan, MD, Yong Qiao, MD, and Chengchun Tang, MD
Abstract: Abnormal vascular smooth muscle cell proliferation and migration are key factors in many cardiovascular diseases. Here, we investigated the effects of phloretin on platelet-derived growth factor homodimer (PDGF-BB)–induced rat aortic smooth muscle cell (RASMC) proliferation, migration, and neointimal formation after carotid injury. Phloretin significantly inhibited the PDGF-BB– stimulated RASMC proliferation in a concentration-dependent manner (10–100 mM). Also, PDGF-BB–stimulated RASMC migration was inhibited by phloretin at 50 mM. Pretreating RASMC with phloretin dose-dependently inhibited PDGF-BB– induced Akt and p38 mitogen-activated protein kinases activation. Furthermore, phloretin increased p27kip1 and decreased cyclindependent kinase 2, CDK4 expression, and p-Rb activation in PDGF-BB–stimulated RASMC in a concentration-dependent manner (10–50 mM). PDGF-BB–induced cell adhesion molecules and matrix metalloproteinase-9 expression were blocked by phloretin at 50 mM. Preincubation with phloretin dose-dependently reduced the intracellular reactive oxygen species production. In vivo study showed that phloretin (20 mg/kg) significantly reduced neointimal formation 14 days after carotid injury in rats. Thus, phloretin may have potential as a treatment against atherosclerosis and restenosis after vascular injury. Key Words: phloretin, vascular smooth muscle cell, PDGF-BB, proliferation, migration (J Cardiovasc Pharmacol 2015;65:444–455)
INTRODUCTION Excessive vascular smooth muscle cell (VSMC) proliferation and migration are key factors in many cardiovascular diseases, such as atherosclerosis and restenosis, after percutaneous coronary intervention.1,2 Although a number of
cytokines and growth factors drive these pathological processes, a critical serum growth factor for VSMC proliferation and migration is platelet-derived growth factor homodimer (PDGF-BB).3 PDGF-BB and its receptor PDGFRb are increased in atherosclerotic lesions and balloon catheterinjured arterial tissue compared with that in the normal vessel wall.4,5 PDGF antibodies and PDGF receptor kinase inhibitors suppress VSMC proliferation, migration, and neointimal formation in a rat model of balloon catheter-injured arterial restenosis.6,7 Flavonoids, a subclass of polyphenols, are derived from a variety of vegetables, fruits, and plants. Studies on flavonoids show their inhibitory effects on proliferation and migration of VSMCs, and neointimal formation in an arterial restenosis model.8,9 Phloretin (Fig. 1), a dihydrochalcone bicyclic flavonoid, has been shown to possess modest estrogenic activity.10,11 Furthermore, phloretin is also an inhibitor of glucose cotransporter and glucose transporter, which has been approved to lower blood glucose and inhibit tumor growth.12,13 Recent studies demonstrated that phloretin had various other biological activities, such as anti-inflammatory and antioxidant.14–16 However, the effects of phloretin on vascular bioactivity have not been clarified. Therefore, our study was to elucidate the effects of phloretin on PDGF-BB–induced rat aortic smooth muscle cell (RASMC) proliferation, migration, and neointimal formation after carotid injury, as well as the underlying mechanism.
METHODS Materials and Reagents
Received for publication September 11, 2014; accepted December 17, 2014. From the Department of Cardiology, Zhongda Hospital of Southeast University Medical School, Nanjing, China. Supported by grants to Chengchun Tang from National Natural Science Foundation of China (research Grant 81170105) and National Natural Science Foundation of China (research Grant 81370225). The authors report no conflicts of interest. Reprints: Chengchun Tang, MD, Department of Cardiology, Zhongda Hospital of Southeast University Medical School, No.87 Dingjiaqiao, 210009 Nanjing, Jiangsu, China (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Phloretin, 5-bromo-2-deoxyuridine (BrdU), and 2,7dichlorofluorescin diacetate (H2DCFDA) were purchased from Sigma Chemical Co. (St Louis, MO). Phloretin was dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO was less than 0.05%. DMSO was used in the groups in which phloretin was not treated. Recombinant human PDGF-BB was purchased from PromoKine (Heidelberg, Germany). Cell culture materials and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD). Sources of antibodies are listed below: (1) Anti-Akt, anti-Erk1/2, anti-phospho-Akt (Ser473), antiphospho-Erk1/2 (Thr202/Thr204), anti-PDGF Receptor b, anti-phospho-PDGF Receptor b, anti-phospholipase Cg1
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Phloretin Inhibits Neointimal Formation
PBS and incubated with Alexa Fluor 488-conjugated donkey anti-goat second antibody for 1 hour. Finally, the cells were washed with PBS, stained with DAPI, and observed under an Olympus IX71 microscope (Tokyo, Japan).
Cell Proliferation Assay
FIGURE 1. The chemical structure and molecular weight of phloretin.
(PLCg1), anti-phospho-PLCg1 (Ser1248) (Cell Signaling Technology, Beverly, MA), (2) anti-p-Rb, anti–proliferating cell nuclear antigen (PCNA), anti-MMP2, anti-MMP9, antiICAM-1, anti-VCAM-1—Santa Cruz Biotechnology (Santa Cruz, CA), (3) anti-CDK2, anti-CDK4, anti-p27Kip1, and anti-BrdU—Abcam Inc. (Cambridge, MA, United Kingdom), (4) anti-smooth muscle a-Actin—Sigma Chemical Co. (St Louis, MO), (5) Internal control anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and anti-b-actin—Santa Cruz Biotechnology, and (6) secondary antibodies conjugated with horseradish peroxidase—Pierce (Rockford, IL), secondary antibodies conjugated with Alexa Fluor 488—Cell Signaling Technology.
Animal and Ethics Statement All animals were purchased from the Animal Center of Southeast University. Animal experiments conformed to the Guide for the Care, and Use of Laboratory Animals was published by the US National Institutes of Health (DHWE publication No. 96-01, revised in 2002) and was approved by the Ethics Review Board for Animal Studies of Institute of Southeast University, Nanjing, China. All animals were fed a standard diet of rat chow and were settled in the laboratory animal room with 218C–268C temperature, 40%–70% humidity, and 15–20 Lux lighting.
Cell Culture Primary RASMC were isolated from the thoracic aorta of male Sprague–Dawley rats weighing between 150 and 180 g as described.17 Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium containing 10% FBS, 100 mg/mL penicillin–streptomycin in a humidified atmosphere of 95% air, and 5% CO2 at 378C. We used morphology assay and immunofluorescence staining with smooth muscle a-actin, a marker of smooth muscle cells, to identify the cultured RASMCs. Cells between the third and 8 passages were used for all experiments.
Immunofluorescence Staining Cells were washed 3 times in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 20 minutes. Cells were washed in PBS and then treated with 0.3% Triton-100 for 5 minutes on ice. After washing, cells were blocked with 5% FBS for 30 minutes at room temperature and then incubated with anti-smooth muscle a-actin (1:100) at 48C overnight. Cells were washed 3 times in Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
The proliferation of RASMCs was measured by 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay as described.18 RASMCs (5 · 103) were planted onto a 96-well culture plate and then starved with 0.1% FBS in DMEM/F12 for 24 hours. Cells were treated with PDGFBB (25 ng/mL) and phloretin at concentrations of 10, 30, 50 and 100 mM, respectively. After 24 hours, 20 mL MTT (5 mg/ mL) was added into each well, and cells were incubated for 4 hours. The accumulative formazan crystals were dissolved with 150 mL DMSO. The optical density of the solution was measured at a wavelength of 570 nm with a microplate reader (Thermo Scientific). We also used BrdU immunofluorescent staining assay to evaluate the effect of phloretin on newly synthesized DNA of RASMC. Cells were starved with 0.1% FBS in DMEM/F12 for 24 hours and then treated with PDGF-BB (25 ng/mL) and phloretin (50 mM) for 24 hours. BrdU (10 mM) was added to incubate for 2 hours before the end of culture. Cells were fixed with 4% paraformaldehyde and then treated with 0.1% Triton X-100 in PBS for 5 minutes. After washing, cells were blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Next, cells were treated with 2 mol/L hydrochloric acid for 30 minutes and 0.1 mol/L antipyonin for 5 minutes. Then, cells were incubated with anti-BrdU antibody (1:1000) overnight at 48C and then incubated with Anti-rat (Alexa Fluor 488 Conjugate) secondary antibody for 1 hour in the dark at room temperature. Photos were taken by an Olympus IX71 microscopy. BrdU-positive cells were calculated as the total number of cells per 5 different random fields.
Migration Assay The migration assay was performed using the Transwell system (a 6.5-mm polycarbonate membrane with 8.0-mm pores; Corning, NY) as described.19 Of note, 5 · 104 of cells suspensions, containing fresh serum-free media, were seeded on the upper chamber. PDGF-BB (25 ng/mL) with or without phloretin (50 mM) was added into the bottom chamber as the chemoattractant. The cells were allowed to migrate through the membrane to the lower surface for 6 hours. Cells on the upper surface of the membrane that had not migrated were scraped off with cotton swabs, and cells that had migrated to the lower surface were fixed by 4% paraformaldehyde and stained with 0.1% crystal violet and counted. Migrated cell numbers were calculated as the number of migrated cells per 5 different random fields. At last, the cells were dissolved with 30% acetic acid and then measured under 490 nm using a microplate reader (Thermo Scientific). We also used a wound scratch assay to detect the effect of phloretin on RASMCs migration. GFP-positive transgenic Sprague–Dawley rat was used to obtain GFP+ RASMCs. Cells (1 · 106) were seeded onto the 6-well plates overnight. After serum starvation (0.1% FBS in DMEM/F12) for 24 hours, cell monolayer was wounded using a pipette tip and www.jcvp.org |
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then washed twice with PBS gently. Then, cells were treated with PDGF-BB (25 ng/mL) and phloretin (50 mM) for 8 hours. The cell migration ability was evaluated by the percentage of scratch area per 5 different random fields.
performed with the transcribed cDNA and SYBR Premix Ex Taq II (Takara) in triplicates using the 7300 real-time PCR system (ABI, Foster City, CA) to detect messenger RNA (mRNA) expression. The primer sequences purchased from Invitrogen were showed in Table 1, and the program was as follows: 958C for 30 seconds, 40 cycles at 958C for 5 seconds, and 608C for 31 seconds. The relative mRNA amounts of target genes were normalized to the values of b-actin. The results were expressed as fold changes of threshold cycle (Ct) value relative to the controls using the 22DDCt method.
Western Blotting After serum starvation (0.1% FBS in DMEM/F12) for 24 hours, cells were stimulated with PDGF-BB (25 ng/mL) with or without phloretin (50 mM). Total proteins were prepared at the indicated time points using protein extraction buffer containing protease inhibitor Complete (Roche, IN). Western blotting was carried as described previously.19 In brief, cells lysate with equal amount of protein (50 mg) were separated by 10% SDS–polyacrylamide gel electrophoresis and then transferred electronically to the polyvinylidene difluoride membranes. Membranes were blocked in 5% nonfat milk powder in trisbuffered saline and tween 20 (TBST) (0.1% Tween 20 in 1 · TBS) for 1 hour at room temperature and then incubated with (1) 1:1000 dilutions of anti-PDGF-Rb, anti-Erk1/2, anti-Akt, anti-phospho-PDGF-Rb, anti-phospho-Erk1/2, anti-phosphoAkt, anti-PLCg1, anti-phospho-PLCg1, anti-CDK2, and antiCDK4, (2) 1:200 dilutions anti-p38 MAPK, anti-phospho-p38 MAPK, anti-JNK, anti-phospho-JNK, anti-p-Rb, anti-MMP2, anti-MMP9, anti-ICAM-1, and anti-VCAM-1, and (3) 1:5000 dilutions anti-b-actin and anti-GAPDH at 48C overnight. Finally, the membranes were incubated with secondary antibodies (1:5000) conjugated with horseradish peroxidase for 1 hour at room temperature. Immunoreactive materials were visualized by using Chemiluminescent Substrate kit (Pierce, Rockford, IL). The membranes were scanned, and the sum optical density was quantitatively analyzed by Quantity One software (Bio-Rad, Richmond, CA).
Real-time Polymerase Chain Reaction Total RNA was isolated using an RNAiso Blood (Takara, Otsu, Japan) according to the manufacturer’s instructions. First, 1-mg RNA was reverse transcribed to complementary deoxyribonucleic acid (cDNA) with random primers using PrimeScript RT Master Mix (Takara). Then, quantitative real-time polymerase chain reaction (PCR) was
Intracellular Reactive Oxygen Species Measurement We used the reactive oxygen species (ROS)–sensitive probe H2DCFDA to detect intracellular ROS production. Briefly, cells were serum-starved in DMEM with 0.1% FBS for 24 hours and then subjected to the treatment with phloretin or N-acetyl-cysteine (NAC) for 2 hours. Cells were then challenged with PDGF-BB (25 ng/mL) for 30 minutes. After treatment, cells were then labeled with H2DCFDA (10 mM) and incubated for 30 minutes at 378C. Then, cells were washed gently for 3 times with PBS and immediately observed under an Olympus IX71 microscope with excitation and emission wavelengths of 488 nm and 525 nm, respectively. The fluorescence intensity was measured from 3 random fields for each well by using Image J software (NIH, Bethesda).
Balloon Injury Model in Rat Carotid Artery The surgical intervention was performed as described previously.20 Briefly, rats weighing between 300–350 g were anesthetized with 10% chloral hydrate at a dose of 300 mg/kg (intraperitoneally). Then, expose the right common, external, and internal carotid arteries completely and inject heparin sodium intravenously at a dose of 100 U/kg. Insert a balloon catheter (1.5 mm in diameter, 15 mm in length; Medtronic) into the common carotid artery by a small cut in the external carotid artery after temporary blockage of the internal and common carotid arteries. Next, aerate the balloon and pull the catheter back and forth 5 times using a rotatory movement. Rats were divided into 3 groups: sham (n = 5), saline
TABLE 1. Primer Sequences for Real-time PCR Genes
Primer Sequences
b-Actin ICAM-1 VCAM-1 MMP2 MMP9 TIMP-1 TIMP-2
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Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
50 -CCCATCTATGAGGGTTACGC-30 50 -TTTAATGTCACGCACGATTTC-30 50 -TGCTCAGGTATCCATCCA-30 50 -TTCATCCAGTTAGTCTCCAA-30 50 -CTGGAGATTGAACTACTGAAG-30 50 -TTGACTGCTGTGTAACTTAG-30 50 -ACAACCAACTACGATGATGA-30 50 -TGGATAGTCGGAAGTTCTTG-30 50 -TGGACAGCGAGACACTAA-30 50 -TATGATGGTGCCACTTGAG-30 50 -TCCTCTTGTTGCTATCATTG-30 50 -GCTGGTATAAGGTGGTCTC-30 50 -ACCCTCTGTGACTTTATTGT-30 50 -ATTGATGCTCTTCTCTGTGA-30
Amplicon (bp) 150 123 168 172 102 147 180
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Phloretin Inhibits Neointimal Formation
(n = 6), and phloretin (n = 8). Rats in the sham group were exposed to surgery without vessel injury. Rats in the phloretin group were given an injection of 20 mg/kg phloretin (intraperitoneally) once a day. Rats in the sham and saline group were injected an equal volume of normal saline. The carotid arteries were collected after 2 weeks.
the PDGF-BB alone group (Fig. 3A). However, 50 mM phloretin alone did not decrease absorbance. The phloretin concentration producing 50% inhibition (IC50) was 42.6 mM (Fig. 3A). In addition, the BrdU fluorescence assay revealed that the percentage of BrdU-positive cells increased significantly after PDGF-BB treatment. However, phloretin at 50 mM diminished PDGF-BB–induced proliferation in RASMCs (Fig. 3B, C).
Morphometric Analysis and Immunohistochemistry
Carotid arteries were fixed with 4% paraformaldehyde and were embedded in paraffin. Then, the carotid arteries sections (4 mm) were stained with hematoxylin–eosin, and the photos were taken by a light microscopy (Olympus). The thickness of neointima was measured using Image-Pro Plus 6.0 per 4 different random fields and were normalized to media thickness. Immunohistochemical detection of PCNA was performed as described previously.20 PCNA-positive cells were calculated as the total number of cells within the neointima per 4 different random fields.
Statistical Analysis
Data were presented as means 6 SE. One-way analysis of variance was used for multiple comparisons by SPSS 19.0 (SPSS Inc, Chicago, Il). If there was a significant variation between treated groups, the Tukey test was applied. P , 0.05 was considered statistically significant.
RESULTS Purity Identification of Cultured RASMCs The RASMCs exhibited characteristic spindle shapes and a typical appearance of “valley and hill” using a light microscope (Fig. 2A). As determined through immunofluorescence staining with smooth muscle a-actin, more than 95% of the cells emitted green fluorescence (Fig. 2B).
Effects of Phloretin on PDGF-BB–induced Proliferation of RASMCs The results of the MTT assay showed that absorbance increased significantly after PDGF-BB treatment compared with that in the unstimulated group. Phloretin treatment inhibited PDGF-BB–induced RASMC proliferation in a concentration-dependent manner. Phloretin at 10, 30, 50, and 100 mM inhibited proliferation by about 8%, 35%, 48%, and 70% in PDGF-BB–induced RASMC, respectively, compared with
Effects of Phloretin on PDGF-BB–induced Migration of RASMCs As shown in Figure 4A, B, 25 ng/mL PDGF-BB significantly increased RASMC migration. The 50 mM phloretin treatment decreased absorbance and increased the percentage of scratched area compared with those in the PDGF-BB–stimulated group. However, phloretin alone did not inhibit migration of RASMCs.
Effects of Phloretin on the PDGF-BB–induced PDGFR Signaling Pathway in RASMCs The PDGFR signaling pathway is related to cell proliferation, migration, differentiation, and angiogenesis. Distribution of the PDGFR differs in different tissues and cells. PDGF-Ra is highly expressed in mesenchymal cells; however, the mesenchyme contains more PDGF-Rb, particularly VSMCs. As shown in Figure 5, phloretin had no effect on PDGF-BB–induced PDGF-Rb phosphorylation at 10–50 mM and had no effects on downstream extracellular regulated kinase (ERK1)/2, PLCg1, or JNK phosphorylation. However, phloretin inhibited PDGF-BB–stimulated Akt and p38 MAPK phosphorylation at 10–50 mM in a concentrationdependent manner (Fig. 5B).
Effects of Phloretin on cell Cycle Regulatory Protein Expression Induced by PDGF-BB in RASMCs Cyclins, cyclin-dependent kinases (CDKs), and cyclindependent kinase inhibitors (CKIs) play major roles in regulating the cell cycle. To verify whether the antiproliferative effect of phloretin was related to cell cycle regulatory proteins, we examined CDK2 and CDK4 expression and Rb phosphorylation using Western blot analysis. An important CKI, p27kip1, was also detected. As shown in Figure 6, phloretin reduced upregulation of PDGF-BB–induced CDK2, CDK4, and proliferating cell nuclear antigen, as well as Rb
FIGURE 2. The purity identification of RASMCs. A, Cell morphology images were taken under a microscopy (scale bar, 100 mm). B, Isolated RASMCs were fixed and subjected to immunofluorescence staining with primary antibodies to smooth muscle a-Actin (green). Nucleus was labeled with DAPI (blue) (scale bar, 100 mm). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 3. Effects of phloretin on PDGF-BB–induced RASMC proliferation. A, RASMC viability was detected by the MTT assay. Data are mean 6 SE (n = 4, *P , 0.05 vs. control and &P , 0.05 vs. PDGF-BB alone). B and C, DAPI-labeled cells are blue, and BrdU-positive cells are green. The number of BrdU-positive cells in the different experimental groups was normalized to the total number of cells. Data are mean 6 SE (n = 3, *P , 0.05 vs. control; #P , 0.05 vs. PDGF-BB alone) (scale bar, 50 mm).
phosphorylation at 10–50 mM in a concentration-dependent manner. In contrast, PDGF-BB diminished p27kip1 protein expression, whereas the expression of this protein increased after phloretin treatment.
VSMCs express various adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which bind to their receptors, resulting in monocytes and lymphocytes adhering and migrating to the vessel wall.21 We used Western blotting and real-time PCR analyses to detect ICAM-1 and VCAM-1 protein and mRNA, respectively. As shown in Figure 7A, B, 50 mM phloretin not only inhibited the upregulation of PDGF-BB–induced ICAM-1 and VCAM-1
mRNA but also inhibited upregulation of their proteins. However, phloretin alone exerted no effect compared with that in the control group. Extracellular matrix degradation also plays a major role in cell migration. Matrix metalloproteinases (MMPs) break down the extracellular matrix to promote migration, but their activities are modulated by specific endogenous tissue inhibitors of metalloproteinase (TIMPs).22 MMP2, MMP9, and their inhibitors TIMP-1 and TIMP-2 are now well studied; thus, we examined changes in their levels using Western blotting and real-time PCR. As shown in Figure 7C, 50-mM phloretin inhibited PDGF-BB–induced MMP9 mRNA upregulation but had no effect on MMP2, TIMP1, or TIMP2. In addition, Western blotting showed that the enhanced MMP9 expression was suppressed by phloretin at 50 mM. However, no change in MMP2 was detected (Fig. 7D).
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Effect of Phloretin on the Expression of Adhesion Molecules and Extracellular Matrix Proteins Induced by PDGF-BB in RASMCs
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Phloretin Inhibits Neointimal Formation
FIGURE 4. Effects of phloretin on PDGF-BB–induced RASMC migration. The transwell assay and scratch test were used to evaluate RASMC migration. A, Photographs of migration were taken under a microscope (magnification, ·100). The results are mean 6 SE (n = 3, *P , 0.05 vs. control group and #P , 0.05 vs. PDGF-BB alone group). B, Photographs of the scratch test were taken under a microscope (magnification, ·40). The percentage of scratch area is shown as mean 6 SE (n = 5, *P , 0.05 vs. control group and #P , 0.05 vs. PDGF-BB-alone group).
Effects of Phloretin on the Intracellular ROS Production Induced by PDGF-BB in RASMCs Excessive ROS plays an important role in oxidative stress. Exposed to PDGF-BB for 30 minutes, the level of intracellular ROS was markedly increased in RASMCs compared with that in the control group (Fig. 8). PDGF-BB– induced intracellular ROS activation was inhibited by the pretreatment of phloretin at 1–50 mM in a concentration-dependent manner and also by the antioxidant NAC at 10 mM.
saline group (Fig. 9A). The intima:media ratio in the group treated with 20 mg/kg phloretin decreased significantly compared with that in the saline group (Fig. 9A). The proportion of PCNA-positive cells in the saline group increased observably compared with the sham group. However, in the phloretin-treated group, this proportion decreased approximately 58% compared with that in the saline group (Fig. 9B). These results are the same as those from the in vitro study. Thus, these results indicate that phloretin may inhibit neointimal formation in a carotid artery injury model.
Effects of Phloretin on Neointimal Formation in a Rat Carotid Artery Injury Model Intima and media thicknesses were estimated to test the effects of phloretin on neointimal formation. Hematoxylin and eosin staining of carotid artery sections revealed a barely visible intima in the sham group and a thick neointima in the Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
DISCUSSION This study showed that phloretin could prevent neointimal formation after vascular injury. This conclusion was based on the following evidence. First, phloretin inhibited www.jcvp.org |
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FIGURE 5. Effect of phloretin on PDGF-BB–induced PDGF-receptor b, extracellular regulated kinase (ERK)1/2, Akt, phospholipase C (PLC)g1, p38 MAPK, and JNK phosphorylation in RASMC. Confluent cells were precultured with phloretin in serum-free medium for 24 hours. Then, the cells were stimulated with 25 ng/mL PDGF-BB at 378C for 1 minute to analyze PDGF-Rb, for 5 minutes to analyze ERK1/2, and for 15 minutes to analyze Akt, PLCg1, p38 MAPK, and JNK. Levels of protein phosphorylation were determined using Western blot analyses and quantified by densitometry. The respective total protein levels were used for normalization. Data are mean 6 SE. A, Levels of PDGF-Rb phosphorylation (n = 3). B, Levels of Akt, ERK1/2, PLCg1, p38 MAPK, and JNK phosphorylation (n = 3, *P , 0.05 vs. PDGF-BB-alone group; &P , 0.01 vs. PDGF-BB alone).
PDGF-BB–induced proliferation and migration of RASMCs. Second, phloretin inhibited PDGF-BB–induced Akt and p38 MAPK activation of RAMSCs. Third, phloretin upregulated p27kip1 and downregulated CDK2, CDK4, adhesion molecules, MMP9, and p-Rb phosphorylation induced by PDGF-BB. In addition, phloretin suppressed intracellular ROS production in PDGF-BB–stimulated RASMCs. It is well known that inhibiting excess proliferation and migration of VSMCs is an effective intervention to prevent
atherosclerosis and restenosis.1,23 Therefore, we first detected the effects of phloretin on RASMC proliferation and migration. A 25-ng/mL PDGF-BB treatment was used as a mitogen to promote cell proliferation and migration as previously described.8,20 Phloretin inhibits proliferation of many cell types, such as liver cancer cells, breast tumor cells, and T lymphocytes.24–26 We clearly demonstrated that phloretin inhibited PDGF-BB–induced RASMC proliferation at 10–100 mM in a concentration-dependent manner. These results are in
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Phloretin Inhibits Neointimal Formation
FIGURE 6. Effect of phloretin on the activation of cell cycle regulatory proteins induced by PDGF-BB in RASMC. Levels of CDK2, CDK4, PCNA, p27kip1 proteins, and Rb phosphorylation were determined using Western blot analyses and quantified by densitometry. Total b-actin was used for normalization. Results are mean 6 SE (n = 3, *P , 0.05 vs. PDGF-BB alone and &P , 0.01 vs. PDGF-BB alone).
accordance with those by Kim et al,24 as phloretin displayed an antiproliferative effect at 10–80 mM in human breast epithelial cells. Furthermore, DNA synthesis stimulated by PDGF-BB was also suppressed by phloretin, and this further confirmed the antiproliferative effect of phloretin. This antiproliferative effect was not because of cytotoxicity but because phloretin was not cytotoxic at 50 mM, similar to
the results by Chung et al.27 Only a thin endothelial cell layer is present in a normal vessel wall. However, VSMCs proliferate and migrate from the media to the intima in atherosclerotic lesions and balloon catheter-injured arterial tissue, resulting in neointimal hyperplasia. We found that phloretin inhibited PDGF-BB–induced RASMC migration in the transwell and wound scratch assays. However, no
FIGURE 7. Effects of phloretin on the expression of adhesion molecules and key extracellular matrix proteins induced by PDGF-BB in RASMC. Messenger RNA and protein levels were determined using real-time PCR and Western blot analyses, respectively. GAPDH was used for normalization. Results are mean 6 SE (n = 3–4, *P , 0.05 vs. Control group and #P , 0.05 vs. PDGF-BB alone). A and B, Levels of ICAM-1 and VCAM-1 mRNA and protein. C and D, Levels of matrix MMP2, MMP9, TIMP1, and TIMP2 mRNA and protein. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 8. Effects of phloretin on the intracellular ROS production induced by PDGF-BB in RASMC. The intracellular ROS level was detected by ROS-sensitive probe H2DCFDA. A, Immunofluorescene images were taken under a fluorescence microscopy. ROS was labeled with green. B, Levels of the DCF fluorescence intensity. Data are mean 6 SE (n = 3, *P , 0.05 vs. Control group; #P , 0.05 vs. PDGF-BB alone).
effects on migration were found in the phloretin-alone group. PDGF binds to its receptor-PDGFR, and then the receptor dimerizes and autophosphorylates.28 PDGF receptor kinase inhibitors are effective for treating PDGF-driven diseases, such as cancers, atherosclerosis, and restenosis.29,30 PI3K/Akt, mitogen-activated protein kinases (MAPKs), and PLCg are the major downstream signaling intermediates related to activating PDGFR.21 Suppression of Akt, MAPKs, and PLCg phosphorylation results in antiproliferative effects in VSMCs. Previous studies have shown that phloretin inhibits tyrosine kinase activity,31 as well as Akt and Erk phosphorylation at micromolar levels in cancer cells.25,27 However, we found that phloretin did not decrease PDGFBB–induced PDGF-Rb phosphorylation in RASMCs. Furthermore, no inhibitory effects on downstream signaling intermediates or Erk1/2, JNK, or PLCg1 phosphorylation were observed by phloretin. However, phloretin reduced PDGF-BB–stimulated phosphorylation of Akt (Ser473), a marker of PI3K activation, and p38 MAPK in a concentration-dependent manner at micromolar levels. This reduction in Akt and p38 MAPK phosphorylation may be involved in inhibiting mitogen-stimulated proliferation and migration of RASMCs.
Subsequently, we investigated whether the antiproliferative effect of phloretin was involved in cell cycle regulatory proteins, such as CDKs, cyclins, CKIs, and p-Rb. CDK2 and CDK4 are key mediators during the G0/G1 to S phase progression of the cell cycle and form complexes with cyclin E and cyclin D1, respectively.32 Cyclin-dependent kinase (CDK)–cyclin complexes phosphorylate Rb resulting in dissociation of the p-Rb/E2F complex, and the E2F transcription factor reactivates for cells to enter the S phase. Therefore, inhibition of CDK and cyclin expression and p-Rb phosphorylation may lead to cell cycle arrest. In this study, we found that phloretin not only suppressed PDGF-BB–induced CDK2 and CDK4 expression but also inhibited p-Rb phosphorylation in a concentration-dependent manner. PCNA, a cell proliferative marker induced by PDGF-BB, also decreased after phloretin treatment. These observations may be the underlying mechanism related to the antiproliferative effects of phloretin. A succession of CKIs, including p16ink4, p21cip1, and p27kip1, is the primary negative cell cycle regulators, as they block the activities of several CDK-cyclin complexes. Marra et al33 found that overexpressing p27kip1 forces VSMCs into G1 growth arrest. Drugs upregulating p27kip1 exhibit an antiproliferative effect on VSMCs.33,34 We found that reducing p27kip1 with PDGF-BB was prevented by phloretin, which inhibited
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Phloretin Inhibits Neointimal Formation
FIGURE 9. Effects of phloretin on neointimal formation in a rat carotid artery injury model. Red and yellow bars represent neointima and media, respectively. A, Representative hematoxylin and eosin–stained carotid artery sections from each experimental group. Results are mean 6 SE (n = 5–8; *P , 0.05 vs Sham group; #P , 0.01 vs Saline group) (scale bar, 100 mm). B, Representative immunohistochemical staining for PCNA expression after rat carotid artery injury. Results are mean 6 SE (*P , 0.01 vs. Saline group). Scale bar, 200 mm.
the cell cycle and prevented cell growth. Therefore, our findings demonstrate that phloretin inhibited the proliferation of RASMCs, probably by upregulating p27kip1 and downregulating CDK2, CDK4, and PCNA, as well as by activating p-Rb. Cell adhesion molecules and MMPs are the key regulators of cell migration and neointimal formation.22 Stangl et al35 found that phloretin suppresses cytokinestimulated ICAM-1, VCAM-1, and E-selectin expression in human umbilical vein endothelial cells. However, no evidence has been reported about the effects of phloretin on cell adhesion molecules and MMPs in VSMCs. We found that phloretin inhibited PDGF-BB–induced ICAM-1, VCAM-1, and MMP9 upregulation in RASMCs, but no effect on MMP2 was observed. Suppression of MMPs and cell adhesion molecules inhibits VSMC migration and neointimal formation in a carotid artery injury rat model.18,33,36 Thus, we consider that reducing the increase in ICAM-1, VCAM-1, and MMP9 expression may be involved in suppressing RASMC migration and neointimal formation because of phloretin. In VSMC, PDGF-BB can trigger oxidative stress and induce the production of intracellular ROS. Excessive ROS Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
promotes VSMC proliferation and migration by upregulating cyclins, CDKs, adhesion molecules, and downregulating CKIs. Antioxidants or some natural compound could inhibit PDGF-induced VSMC proliferation and migration by reducing ROS production. Phloretin is an inhibitor of oxidative phosphorylation,37 and previous studies established its antioxidative activity.15 In our study, we found that phloretin reduced PDGF-BB–induced ROS production in a concentration-dependent manner in RASMCs. Although the mechanism by which phloretin inhibits PDGF-BB–induced proliferation and migration in RASMCs is not thoroughly clear, one potential explanation is that phloretin reduces excessive ROS production in RASMCs and thereby prevents the downstream effects of oxidative stress, such as downregulation of p27kip1 expression, upregulation of CDK2, CDK4, ICAM-1, and VCAM-1 expression, and that result in the inhibition of proliferation and migration. To further study the effects of phloretin on neointimal formation, we used a balloon catheter-injured carotid artery restenosis rat model. Previous studies indicated that 10–40 mg/kg (body weight) of phloretin was an effective dosage www.jcvp.org |
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to treat some diseases in vivo.25,38 In this study, we used 20 mg/kg phloretin (intraperitoneally) to treat the rat carotid artery injury model for 2 weeks. We found that neointima began to form when arterial walls were injured for 2 weeks, and neointimal hyperplasia was suppressed after the 20 mg/kg phloretin treatment. PCNA expression decreased in the phloretin-treated group, and that was in accordance with what we found in cultured RASMCs. Thus, our data indicated that phloretin could inhibit neointimal formation through preventing VSMC proliferation and migration. Oxidative stress exists in a balloon injury rat model. Dietary flavonoid consumption acts as a cardiovascular protective agent.39 One of the mechanisms is that flavonoids can reduce intracellular ROS level, prevent superoxide-induced NO inactivation, and increase endothelial nitric oxide synthase activity, and hence helping VSMC to maintain quiescent.40,41 Ullen et al16 found that 2-chlorohexadecanal, an inhibitor of eNOS, induced endothelial cell dysfunction, and cell death was ameliorated by phloretin at 50 mM. However, we only focused on the effects of phloretin on VSMC itself and the effects of phloretin on the recovery of the endothelial cell layer and the NO production within an injured carotid artery model were not revealed. A future work will be performed to fully understand the mechanism. Phloretin, enriched in various fruits, is a component of the human diet. Although little in vivo research about phloretin has been carried out in humans, 2 studies showed that phloretin, as a topical agent, could ameliorate ultravioletinduced photodamage in human skin, and that was attributed to its antioxidant and anti-inflammatory activities.42,43 Moreover, phloretin has been proven effective to treat diseases such as tumor,25 diabetes,44 and colonitis45 in animal models. Therefore, we speculate that phloretin may have a broad spectrum of bioactivities such as lowering blood glucose, antitumor, antioxidant, and anti-inflammatory activities in humans. In summary, our data suggest that phloretin is a reasonable strategy to prevent neointimal formation after artery injury through blockade of oxidative stress, VSMCs proliferation, and migration, which give a new evidence for future studies to test the effect of phloretin on patients with restenosis.
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J Cardiovasc Pharmacol Volume 65, Number 5, May 2015
Phloretin Inhibits Neointimal Formation
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