Online Supplements for Materials and Methods and Supplementary figures and table
1) Materials and Methods
Animal study
Rat Carotid Artery Balloon Denudation Injury
A previously well-established rat carotid artery balloon injury model was used in this study. Rats were anesthetized with a combination anesthetic (ketamine, 70 mg/kg; xylazine, 7 mg/kg IP; Yuhan Corp, Seoul, Korea). After the left external carotid artery was exposed, heparin (35 IU) is administered systemically via the external jugular vein. A 2F Fogarty embolectomy catheter (Baxter Healthcare Corp, IL) was introduced into an external carotid arteriotomy incision, advanced to the common carotid artery, and inflated with 0.2 mL of saline and withdrawn 10 times with rotation.
Morphometric Analysis
Two weeks after balloon injury, rats were euthanized with a lethal dose of pentobarbital, and carotid arteries were fixed by perfusion at 120 mmHg with 4% formaldehyde via an 18G intravenous cannula placed retrograde in the abdominal aorta. Tissues were then embedded in paraffin, and sections were stained with H&E. The extent of neointimal formation in histologically stained sections was quantified by computed planimetry. The cross-sectional areas of the blood vessel layers, ie, the lumen, intimal, and medial areas, are quantified in 3 different sections (proximal, middle, and distal) using an Image-Pro Plus Analyzer Version 4.5 (Media Cybernetics, MD). The intima-media ratio (IMR) was calculated from the mean of these determinations.
Immunohistochemical Staining for Proliferation
To detect proliferating cells, immunohistochemical staining against proliferating cell nuclear antigen (PCNA) were performed on balloon injured arteries. Briefly, paraffin-embedded samples were sectioned and treated with protease K for 4 minutes, and then endogenous peroxidase was quenched with methanol/peroxidase solution. Specimens were treated with 50 mmol/L Tris HCl (pH 7.6) containing 0.15 mol/L NaCl and 0.1% Tween 20 for 5 minutes, and then incubated in 1:50 diluted anti-PC-10 antibody (Dako, Cincinnati, OH) for PCNA staining. Specimens were then processed by incubation with 1:50 diluted 3,39-diaminobenzidine tetrahydrochloride substrate solution (Dako) and counterstained with Mayer hematoxylin (Dako). Proliferation index was defined as the percentage of PCNA positive cells versus total nucleated cells in 4 different sectors per tissue section.
Immunohistochemical Staining for Endothelial cells
To evaluate the degree of re-endothelialization after balloon injury, we performed immunostaining. Endothelial cells were stained with TRITC-labeled Bandeiraea simplicifolia(BS)-1 lectin (Sigma) diluted 20 μg/ml in PBS, VSMCs were stained with α-SMA (Sigma), and nucleus was stained with DAPI.
Glucose metabolism, adiponectin and inflammatory status
Possible relevant factors affecting the degree of neointimal formation, such as glucose homeostasis, adipocytokines and inflammatory status were evaluated. Plasma glucose concentration was measured using a glucose oxidase method (YSI 2300-STAT; Yellow Springs Instrument, OH) immediately after the blood was drawn. Serum insulin and c-peptide concentration were measured using specific radioimmunoassay kits (Linco, St Louis, MO). The homeostasis model assessment insulin resistance index (HOMA-IR) was calculated to evaluate insulin resistance. Total cholesterol and triglyceride concentrations were determined by enzymatic procedures (Hitachi 747 chemistry analyzer; Hitachi, Tokyo, Japan). Rat adiponectin and high-sensitivity C-reactive protein (hsCRP) were measured by using ELISA kits developed by Adiopogen (Seoul, Korea) and BD Biosciences Pharmingen (Heidelberg, Germany), respectively. Monocyte chemoattractant protein-1 (MCP-1), TNF-α, andinterleukin-6 (IL-6) were also measured by a Multiplex kit (RADPK-81K, Linco). 8-hydroxy-2’-deoxyguanosine (8-OHdG) which is a marker of DNA oxidative damage was measured with an established high-performance liquid chromatography method in urine samples.The value of 8-OH-dG (μg) was normalized to urine creatinine concentration (g).
Cell Study
Rat aortic smooth muscle cells (RAoSMCs) were obtained from Bio-Bud (Seoul, Korea) and cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 4.5g/L glucose and 100 U/mL penicillin-streptomycin. Primary cultures of human umbilical vein endothelial cells (HUVECs; Cambrex, Walkersville, MD) were maintained in endothelial cell growth medium (EGM-2; Cambrex) containing 2% FBS, 0.4% hydrocoritisone, 4% hFGF-B, 0.1% VEGF, 0.1% R3-IGF, 0.1% ascorbic acid, 0.1% hEGF, 0.1% GA-1000, and 0.1% heparin, according to the manufacturer’s instruction. Cells were grown in an atmosphere of 95% air and 5% CO2 at 37°C. Subcultured RAoSMCs from passages 4 to 11 and HUVECs from passages 2 to 9 were used in this experiment.
Cell Proliferation Assessed by MTT Assay
Cell viability was determined by a modified 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT (Sigma) (5 mg/mL) was dissolved in PBS. RAoSMCs were grown in 48-well plates at a density of 2×103 per well, and starved for 48 hours, and then placed in DMEM with 0.5% FBS. The cells were exposed to 10ng/ml of TNF-α and different doses of TMZ for 24, 48 and 72 hours. HUVECs were grown in 48-well plates at a density of 2×103 per well. After 24 hours of starvation, endothelial cell basal medium (EBM-2, Cambrex Co., East Rutherford, NJ) was supplemented with 0.2% FBS. Lysophosphatidylcholine (LysoPC), which is a component of oxidized low-density lipoproteins, is known to inhibit migration and proliferation12, to induce apoptosis13 in HUVECs and to generate ROS in human aortic endothelial cells.14The cells were exposed to the LysoPC (Sigma) and the same dose of TMZ as RAoSMC for 24 and 48 hours. After 0.5 mL of MTT solution (5 mg/mL) in starvation medium was added to each well, the plates were incubated for 4 hours at 37°C in 5% CO2/air. The medium was removed carefully, and the purple dye was dissolved in 0.1 mL dimethyl sulfoxide. After 10-minute incubation, 200 μL from each well was transferred to a 96-well plate, and the absorbance was measured at 570 nm with a spectrophotometer.
Measurement of caspase-3 activity
1104 RAoSMCs were seeded into microplate and incubated in DMEM with 10% FBS, then starved in DMEM with 0% FBS for 24 hr.For TNF-α stimulation, the cells were incubated with 10 ng/mL of TNF-α (R&D systems, Camarillo, CA). The cells were treated with various concentrations of TMZ for 24 h. Caspase-3 activity was measured by using Caspase-Glo 3/7 assay kit (Promega, WI).
Cell Migration Assessed by Wound-Healing Assay
RAoSMCs were grown to confluence in 6-well plates and then starved in DMEM with 0.5% FBS for 48 hours. Each well was divided into a 2×3 grid. A linear wound was made using a 100-1000 μL pipette tip in each hemisphere of the well. After wounding, the medium was changed to starvation medium with reagents immediately. All reagents were mixed in starvation medium. Images were taken of the intersections of the linear wound and each grid line, which resulted in 3 fields per well. Cells were allowed to migrate for 24 hours at 37°C. Each field was measured at 24 hours.
Measurement of intracellular ROS
The endogenous in vitro O2•- production of the cell was determined by staining with dihydroethidium. (Molecular Probes, Eugene, OR) Cells were stained in the dark for 30 min with a 1 μM solution of dihydroethidium at 37°C in a humidified 5% CO2 incubator. Each aliquot was analyzed using flow cytometry. Red fluorescence (dihydroethidium) was evaluated between 590 and 700nm (excitation, 488nm; emission, 535 nm). Images were captured and acquired using AxioCam MR c5 and acquisition software AxioVision v.4.4 (Carl Zeiss, Gottingen, Germany). Intracellular hydrogen peroxides were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), a cell-permeable, oxidation-sensitive probe.15 The dye is cleaved and trapped intracellularly, where it can be oxidatively modified by intracellular ROS and peroxidases to produce fluorescent 2′,7′-dichlorofluorescein (DCF). The cells were incubated with 10μM DCFH-DA.The fluorescence was analyzed by flow cytometry and evaluated at 488 nm excitation and 535 nm emission.
Mitochondria apoptosis analysis
HUVECs were grown and treated on chamber slide. For analysis of mitochondria apoptosis, cells were washed and subsequently stained with 1 mM MitolightTM (Promega) diluted in PBS for 10 min at 24 h after drug treated. Images were captured and acquired using AxioCam MR c5 and acquisition software AxioVision v.4.4 (Carl Zeiss) and analyzed by flow cytometry at 488 nm excitation, 535 nm emission.
Immunoblot analysis
At the termination of culture, harvested cells were solubilized in cell lysis buffer (Cell signaling, Beverly, MA) containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1% Triton, 2.5 mM sodium pyrophate, 1 mM β-glcerophosphate, 1 mM Na3VO4, 1 mg/ml leupetin, and 1 mM PMSF for 30 min at 4℃ and protein lysate concentrations were measured by Bradford protein assay kit (BioRad, Hercules, CA). The same amounts of proteins from whole cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transfer onto methanol-treated PVDF membranes (Millipore Co, Bedford, MA). After blocking the membrane with Tris-buffered saline-Tween 20 (TBS-T, 0.1% tween 20) containing 5% blocking buffer for 1 hr at room temperature, they were washed with TBS-T and incubated primary antibodies, Caspase-3 (Santa Cruz Biotechnology, CA)for 1 hr at room temperature or for overnight at 4°C. The membranes were washed three times with TBS-T for 10 min, and then incubated for 1 hr at room temperature with horseradish peroxidase-conjugated secondary antibodies. After extensive washing, the bands were detected by enhanced chemiluminescence reagent (Santa Cruz Biotechnology).
2) Supplementary figures
Figure legends
Supplementaryfig. 1.In vivo inhibition of neointimal formation after 4weeks of treatment with trimetazidine (TMZ) in OLETF rats. A, H&E stained sections of the three groups (control and TMZ groups: 10 and 20 mg/kg). B, Intima-media ratios (IMRs) in the three groups (n = 10 in each group). The IMR was calculated from the mean areas of intima and media. Treatment with TMZ produced a lower IMR than in controls in an inverselydose-dependent manner (P <0.05 between control and 10 mg/kg or 20mg/kg TMZ-treated groups).
Supplementary fig. 2. Effect of trimetazidine on cell survival. A. Calcein-acetoxymethyl ester (calcein-AM) cell viability assay kit was used (Biotium, Hayward, CA, USA). Cells were was with PBS and incubated with 2μM calcein AM for 30min. The fluorescence was measured using 485 nm excitation wavelength and 530 nm emission wavelength with a Victor 3 instrument (Perkin-Elmer, Boston, MA, USA). B. Cell viability was also measured with Cell Counting Kit-8 (CCK-8, Dojindo, Japan). Absorbance was measured at 450 nm (VersaMax; Molecular Devices, Sunnyvale, CA, USA). Cell Counting Kit-8 (CCK-8) allows convenientassays by utilizing Dojindo’s highly water-solubletetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt] produces a water-soluble formazandye upon reduction in the presence of an electron carrier.WST-8 isreduced by dehydrogenases in cells to give a yellow-coloredproduct (formazan), which is soluble in the tissueculture medium. The amount of the formazan dyegenerated by the activity of dehydrogenases in cells isdirectly proportional to the number of living cells.
Supplementary fig. 3. Effect of TMZ on caspase activity without TNFα treatment. Same protocol was applied without TNFα treatment as Figure 3B.
Supplementary fig. 4. Effects of TMZ on the LysoPC-induced proliferation of rat aortic smooth muscle cells. Cells were incubated with lysoPC (20 μM) and different doses of TMZ for 24, 48 and 72 hours. In MTT assays, cell proliferation was significantly decreased by TMZ treatment. Data are represented as percentages of controls.
Supplementaryfig. 5.Effect of TMZ on matrix metalloproteinase-2 expression and activity.A.Representative zymogram shows the pro-MMP-2 and activated MMP-2 bands. There was an increase in activated MMP-2 in TNF-α treated cells. As the amount of TMZ treated increased from 100 µM to 500 µM, enzyme activity in activated MMP-2 decreased.B.Western Blot of rat aortic smooth muscle cell probed with anti-MMP-2. A significant decrease in activated MMP-2 levels was foundin trimetazidine stimulated cells from 100 µM to 500 µM.
Supplementaryfig. 6. Effects of TMZ on proliferation and migration of vascular smooth muscle cells after platelet-derived growth factor treatment.
Similar to the results after TNF-α stimulation, TMZ treatment reduced PDGF-BB-induced proliferation (A) and decreased PDGF-BB-induced migration of vascular smooth muscle cells (B & C).
Supplementary fig.7. Effects of TMZ on TNF-α-induced proliferation of human umbilical endothelial cells. Endothelial proliferation, which was decreased by TNFα treatment, was recovered with TMZ treatment.
Supplementaryfig.8. Correlations between intima-media ratio (IMR) and adiponectin, TNF-α and 8 OH-dG levels. IMR was positively correlated with proliferation index of vascular smooth muscle cells. There were positive correlations between IMR and TNF-α/8OH-dG levels whereas negative correlation between IMR and adiponectin level.
Figures
Supplementary fig. 1
Supplementary fig. 2
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Supplementary fig. 8
3)Supplementary table
Supplementary table 1. Adiponectin, inflammatory markers, and oxidative stress marker after two weeks treatment of trimetazidine (10 mg/kg and 20 mg/kg) or normal saline (Control) in OLETF rats.Variable / Control / Trimetazidine
10 mg/kg / Trimetazidine
20 mg/kg / P*
Adiponectin (μg/mL) / 12.2 / 5.3 / 14.1 / 4.2 / 15.3 / 4.3 / NS
TNF-α (pg/mL) / 37.2 / 17.4 / 35.4 / 16.8 / 31.2 / 15.8 / NS
IL6 (pg/mL) / 3.7 / 2.1 / 3.6 / 1.8 / 3.6 / 1.7 / NS
MCP-1 (pg/mL) / 0.57 / 0.13 / 0.55 / 0.12 / 0.49 / 0.18 / NS
8OH-dG (μg/L) / 5.8 / 3.8 / 4.8 / 2.1 / 4.7 / 1.9 / NS
Data are shown as the mean±SD. MCP-1, monocyte chemoattractant protein-1; 8OH-dG, 8 hydroxy-deoxyguanosine. *Statistical significance was tested by one-way analysis of variance between groups.
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