Animal experiments
Male BALB/c mice were obtained from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). To induce type 2 diabetes mellitus, the mice were housed in laboratory cages and fed with a high-fat (HF) diet (40% fat, Research Diets, Inc., NJ, USA) [14] for 3 weeks. Subsequently, the mice received 75 mg/kg and 150 mg/kg of intravenous STZ, 5 days apart. Feed was not withheld from any of the animals at the time of STZ administration. After induction, blood glucose was measured daily by tail-vein sampling using a ACCUCHEK glucometer (Roche, Basel, Switzerland). Animals with a blood glucose level more than 11.1 mmol/L (200 mg/dL) were included in this study. Six mice were treated with cilostazol for 2 months, where cilostazol was diluted from 0.5% CMC (carboxymethyl cellulose sodium salt), which was used as a vehicle control. Another six mice were treated with 0.5% CMC for 2 months. Finally, the animals were sacrificed and blood and ascending aorta samples were taken. The Animal Ethics Board of National Defense Medical Center (Taipei, Taiwan) approved all animal experimental procedures.
Immunohistochemistry assay
Animal tissue samples in paraffin blocks were cut into 4-μm sections, which were dewaxed and subjected to microwave antigen retrieval. Endogenous peroxidase activity and nonspecific binding were blocked by incubation with 3% hydrogen peroxide and non-immune serum, respectively. Slides were then incubated with anti-RAGE antibody (Millipore, #051050, MA, USA) at 1:250 at 4 °C overnight, and anti-rabbit secondary antibody for 1 h. Diaminobenzidine hydrochloride (Dako, Carpinteria, CA, USA) was then added to localize positive staining sequentially by light microscopy. The sections were counterstained with hematoxylin and cover slipped.
Protein isolation from frozen/OCT-embedded samples
The tissue sections were moved to a 2-ml tube containing ceramic beads (2.8 mm, Bertin Technologies, France). Lysis buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS, protease inhibitor) was added, and the samples were homogenized by vortex and grinding in a homogenizer (Precellys®24, Bertin Technologies). The samples were kept on ice for 30 min to complete the lysis reaction followed by centrifugation (4 °C, 13000 g, 15 min) to collect the supernatant. The samples were then collected and stored at − 80 °C.
Cell culture and reagents
Smooth muscle cell lines from rat thoracic aorta, A7r5 (RRID:CVCL_0137), were purchased from the Bioresource Collection and Research Center (BCRC, Taiwan). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), and incubated at 37 °C in 5% CO2. The cells were seeded at a density of 1.5 × 106 cells on a 10 cm2 dish in DMEM with 10% FBS for 24 h. After serum starvation for 24 h in DMEM with 0.5% FBS, the cells were stimulated by high glucose (HG). A7r5 cells were grown in 30 mmol/L glucose, and controls received 25 mmol/L mannitol and 5 mmol/L glucose. Cilostazol (kindly provided by Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan) was dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the culture medium was less than 0.1%, which had no effect on VSMCs. The cells were incubated for 24 h with 100 and 200 μM cilostazol after high glucose stimulation. All of the chemical compounds, N-acetylcysteine (NAC), U0126 and PDTA were purchased from Cell Signaling (Danvers, CO, USA).
siRNA transfection
Knockdown of RAGE was performed using specific single or pooled siRNAs purchased from Dharmacon RNAi Technologies (Thermo, MA, USA). SiGENOME non-target siRNAs served as negative controls, and transfection was carried out according to the manufacturer’s protocol.
Intracellular ROS
A7r5 cells were plated in a 6-well plate, grown to confluence, and harvested by trypsinization. Cell pellets were washed with PBS and centrifuged for 5 min at 1000×g at room temperature. The cell pellets were resuspended in 10 μmol/L of CM-H2DCFDA by gently pipetting up and down. The cells were then incubated in a cell incubator in the dark for 45 min, followed immediately by flow cytometry analysis (BD Bioscience).
Cell proliferation assay
A7r5 cells were cultured at a density of 1.5 × 105 cells/well in a 24-well plate. The cells were exposed to various stimuli. A methylene blue dye assay was used to evaluate the effect of high glucose and cilostazol on cell growth. The resulting cell growth was measured at 540 nm and calculated graphically in comparison with the growth of the controls.
Cell adhesion assay
A7r5 cells (1 × 106 cells/ml) were cultured in normoglycemic (5 mmol/L glucose) and hyperglycemic (30 mmol/L glucose) conditions in a 6-well culture plate. The cells were Incubated for 24 h in a CO2 incubator. THP-1 was fluorescence-labeled with calcein-AM by incubating the cells (1 × 107 cells/ml) with 5 μ calcein-AM in RPMI 1640 for 30 min at 37 °C in a CO2 incubator. The cells were washed three times with PBS to remove excess dye and resuspended in phenol red-free RPMI 1640 (with 10% FBS) at a density of 1 × 106 cells/ml. After high glucose and cilostazol treatment, the A7r5 cells were co-cultured with calcein-AM-labeled cells (1 × 106 cells/ml in 6- well) in a CO2 incubator at 37 °C for 1 h. The A7r5 cells were then washed four times with PBS to remove the non-adherent calcein-AM-labeled cells and replaced with 1.0 ml of PBS. The fluorescence of each well was measured using a fluorescence microscopy with excitation and emission wavelengths of 480 nm and 530 nm, respectively.
Migration assay
The migration ability of A7r5 cells was examined using 24-well culture insert-based assays (BD Biosciences, Franklin Lakes, NJ, USA). The culture insertion, with a pore size of 8 μm, was pre-coated to a density of 100 μg/insert of gelatin (Sigma, MO, USA). Cells were suspended in medium containing 10% NuSerum (Corning, New York, U.S), and 2.5 × 104 cells were added to the insert. After incubating for 10 h at 37 °C, the cells that migrated through a Fluoro-Blok membrane (Corning, New York, U.S) were stained with propidium iodine, and fluorescence images were taken. The cells were then counted with Image J software.
Western blot analysis
Whole cell lysates for Western blotting were harvested in RIPA buffer (1% SDS and 10 mM Tris buffer, pH 7.4) containing protease and phosphatase inhibitors (Thermo, Wilmington, DE, USA). Protein concentrations in the supernatants were determined using a Pierce BCA Protein Assay Kit (Thermo, Rockford, IL, USA). Thirty micrograms of protein were separated on 5–15% gradient SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) by wet blotting using an electroblotter (Hoefer system). Membranes were blocked for 1 h at 25 °C with 2% bovine serum albumin or 5% skimmed milk in Tris-buffered saline and Tween 20 (TBST). The membranes were incubated with appropriate dilutions of the primary antibodies: RAGE antibody (SC-74473 [1:1000 dilution]; Santa Cruz, Dallas, USA), I-CAM antibody (SC-8439 [1:1000 dilution]; Santa Cruz, Dallas, USA), V-CAM antibody (SC-13160 [1:1000 dilution]; Santa Cruz, Dallas, USA), FAK antibody (3285 [1:1000 dilution]; Cell Signaling, Danvers, MA, USA), p65 antibody (3033 [1:1000 dilution]; Cell Signaling, Danvers, MA, USA), IκBα antibody (9242 [1:1000 dilution]; Cell Signaling, Danvers, MA, USA), GAPDH antibody (5174 [1:2000 dilution]; Cell Signaling, Danvers, MA, USA), Phospho-JNK antibody (9255 [1:1000 dilution]; Cell Signaling, Danvers, MA, USA), JNK antibody (9252 [1:1000 dilution]; Cell Signaling, Danvers, MA, USA), Phospho-ERK antibody (9101 [1:1000 dilution]; Cell Signaling, Danvers, MA, USA), ERK antibody (9102 [1:1000 dilution]; Cell Signaling, Danvers, MA, USA), MMP2 antibody (13,405 [1:1000 dilution]; Millipore, Danvers, MA, USA), β-actin antibody (600–501 [1:3000 dilution]; Novus, CO, USA), overnight at 4 °C. After being washed in TBST three times, the membranes were incubated for 60 min with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies at 25 °C. Signals were visualized using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence assay. Band intensities were determined using a UVP (ChemStudio series imagers) imaging system.
Statistical analysis
Values are expressed as means ± SEM. Immunoblot data are expressed as means ± SEM of band intensity relative to the controls. All experiments were repeated for three times. Groups were analyzed for differences by one-way ANOVA followed by Tukey’s test. Significance was considered at P < 0.05. All statistical analyses were performed using SPSS software (version 20.0; Chicago, II, USA).