Materials
Human SMC growth medium (M231), smooth muscle growth supplement, trypsin/ethylenediaminetetraacetic acid (EDTA) solution, trypsin neutralizer solution, and HASMCs were obtained from Cascade Biologics (Portland, OR). Fetal bovine serum (FBS), antibiotic-antimycotic mixture, mouse TNF-α kits, oligofectamine, and 2’,7’-dichlorofluorescein diacetate were obtained from Life Technology (Grand Island, NY). Small interfering RNA (siRNA) oligonucleotides against RAGE and antibodies against human TNFR1 (sc-8436), MSX2 (sc-17729), BMP2 (sc-6895), RUNX2 (sc-10758), CD68 (sc-9139), β-actin (sc-47778), and anti-hnRNP c1/c2 (sc-32308) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against human, Nox subunit p47 (#610354), and caveoli-1 (#610406) were obtained from BD Biosciences (San Jose, CA). Unless otherwise specified, all other chemicals and reagents obtained from Sigma-Aldrich (St. Louis, MO).
Animal study
Weaned male low-density lipoprotein receptor knockout (LDLR−/−) mice (Jackson Labs #002207; C57BL/6J background) were fed with a normal diet (Picolab Rodent Diet 20 #5053: 5% fat, 21% protein, 3.3% sucrose, and 28% starch) for 2 months then the animals were randomized into 3 groups: 1) normal diet group, 2) high fat diet (HFD) Harlan Teklad, Diet TD88137 (21% milk fat (42% fat calories), 34% sucrose, and 0.15% cholesterol)) group and 3) HFD + sitaglptin group. Animals in group 2 are gavaged with 100 µL of distilled water daily. Animals in group 3 were given 100 mg− 1Kg− 1day− 1 of sitagliptin by gavaging various amount of sitagliptin solution (25 mg/mL) (BioVision Research Products, Milpitas, CA). All mice were kept in microisolator cages under a 12 h day/night cycle. The entire animal was given free access to chow and water. All animal study protocols complied with the Guide for the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital (IACUC no.2020 − 265, Taipei, Taiwan) and the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (8th edition, 2011). The LDLR−/− mice were sacrificed after 24 weeks of treatments by exsanguination under anesthesia (100 mg− 1kg− 1 ketamine–HCl and 20 mg− 1kg− 1 xylazine via IP injection) after 6 hours fasting. The animals were considered as adequately anesthetized when no attempt to withdraw the limb after pressure could be observed. The thoracic cavity was opened for blood and aorta (from heart to diaphragm) sample collections.
Histology and immunohistochemistry
Aorta samples were cut into 4 sections and processed for histological staining as described in our previous study[30]. Paraffin sections (5 µm) from the dissenting aorta were stained using various agents for semi-quantification of atherosclerotic lesion size and severity (hematoxylin and eosin (H&E) staining) and aortic calcium deposition (alizarin red S staining). Immunohistochemical (IHC) staining of RAGE and VSMC actin (SM α-actin) was performed as previously described [31].
Cell cultures and cell viability assay
HASMCs were purchased from Life Technology (Grand Island, NY. catalog number C0075C). The cells were grown and passaged as described previously[30]. Briefly, the HASMCs were grown in M231 medium containing SMC growth supplement and a 1% antibiotic-antimycotic mixture in an atmosphere of 95% air and 5% CO2 at 37°C in plastic flasks. At confluence, the cells were subcultured at a ratio of 1:3, and passages 3 through 8 were used. The cytotoxicity of S100A12 protein and sitagliptin on HASMC cell viability were measured with the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay.
Soluble DPP-4 activity measurement
Serum DPP-4 activity was measured using the Enzyme-linked immunosorbent assay (ELISA)-Kit from Abcam (Abcam, Cambridge, MA, USA, ab22287) according to the manufacturer’s instructions.
Quantification of aorta or cultured HASMC calcium deposit
Cultured cell Ca content was determined using a BioChain Calcium Kit (BioChain, Hayward, CA, USA) as previously described[32]. Briefly, the 6 well plate cultured cells were counted and demineralized with 250 µl 0.6 N HCl for 12 h. A working reagent was prepared by mixing 75 µl reagent A and 75 µl reagent B and was equilibrated to room temperature before use. A volume of 50 µl diluted standards or samples were transferred to each well of a clear-bottom 96-well plate. Then, 200 µl working reagent was added, and the solution was mixed by light tapping. After incubation for 3 min at room temperature, absorbance was measured at 570–650 nm with a 96-well reader. The units of these results are ug/mL calcium. The other 6 well plate cultured cells counted and solubilized in 200 µl lysis solution containing 0.1 N NaOH and 0.1% sodium dodecyl sulfate (SDS) at room temperature for 5 min. Protein concentration was measured with a Bio-Rad DC Protein Assay Kit. The calcium content of the cultured cells normalized to the protein content was reported.
The aortic segments from experimental mice were extracted using 0.6 N HCl for 24 h and the calcium content of the extracts were determined using a BioChain Calcium Kit (BioChain, Hayward, CA, USA) as previously described[30]. The results were expressed as µg/mg of wet aortic tissues.
Extraction of cellular proteins
Nuclear protein extracts were prepared as previously described[33]. In brief, after cells were washed twice using a ice-cold PBS, they were scraped off the plates with a cell scraper and dispersed in 1 mL ice-cold buffer A (10 mM HEPES/NaOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/mL aprotinin, 2 µg/mL pepstatin, and 2 µg/mL leupeptin). Cells were harvested after centrifugation at 500 ⋅ g for 10 min at 4°C then cells were re-suspended in 80 µl buffer B (buffer A containing 0.1% Triton X-100) by gentle pipetting. The cell lysates were allowed to stand on ice for 10 min and then centrifuged at 12,000 ⋅ g for 10 min at 4°C. Nuclear pellets were re-suspended in 70 µl ice-cold buffer C (20 mM HEPES/NaOH, pH 7.9; 1.5 mM MgCl2; 1 mM DTT; 0.2 mM EDTA; 420 mM NaCl; 25% glycerol; 0.5 mM PMSF; 2 µg/mL aprotinin; 2 µg/ mL pepstatin; 2 µg/mL leupeptin), incubated on ice for 30 min with intermittent mixing, and then centrifuged at 15,000 ⋅ g for 30 min at 4°C.
Cell membrane fractions were prepared as described previously[30] with some modifications. Briefly, HASMCs were lysed in a lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 1 mM PMSF, 10 µg/mL aprotinin, and 0.5 µg/mL leupeptin; pH 7.5). The cell lysates were centrifuged at 3000 ⋅ g for 20 min. Pellets were re-suspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, and 1 mM PMSF; pH 7.5) and designated as the membrane fraction.
Total cell lysates were prepared in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, and 1 mM PMSF; pH 7.5). The protein concentrations were determined with Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, CA), and the samples were stored at − 70°C.
Western blot analysis (see previous comment)
Western blot analysis was used to quantify the HASMC nuclear and cytosol of the Nox p47phox, NF-κB p65, nuclear MSX-2, BMP-2 and RUNX2. Proteins of interest were isolated by SDS polyacrylamide gel and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Bedford, MA). The PVDF were blocked with 5% milk solution (Skimmed instant milk powder with PBS-T) then probed with anti-p47phox, anti-TR1, anti-p65, goat anti-MSX-2, BMP-2, or RUNX2 (1:1000) antibodies. Then they were incubated with horseradish peroxidase-conjugated secondary antibodies. The proteins were visualized using an enhanced chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ). Anti-β-actin (1:5000), anti-caveolin-1 (1:1000), and anti-hnRNP c1/c2 (1:1000) antibodies were used as loading controls. Protein expression levels were quantified as optical densities using ImageQuant software v.5.2.
Nox activity assay and hydrogen peroxide determination
Nox activity was determined with superoxide-dependent lucigenin chemiluminescence, as previously described[30]. Confluent HASMCs in 6-well plates were pretreated with various concentrations of antioxidant reagents followed by treated TNF-α + S100A12 with or without sitagliptin for 1 day. Cell membrane extract (40 µg) and 5 µM dark-adapted lucigenin were added to a 96-well luminometer plate and adjusted to a final volume of 250 µl with oxidase assay buffer before 100 µM NADPH was added. Relative light units (RLUs) were measured with a luminometer (Dynatech ML2250, Dynatech Laboratories Inc., VA). Light emission was recorded every 3 minutes for total 30 minutes and expressed as mean RLUs/min.
The ROS production in HASMCs was determined by fluorometric assay using dichloro-dihydro-fluorescein diacetate (DCFH-DA) as the probe. This method is based on the oxidation by H2O2 of nonfluorescent DCFH-DA to fluorescent 2’,7’- dichlorofluorescin. Confluent HASMCs in 24-well plates were pretreated with various concentrations of antioxidant reagents followed by treated TNF-α + S100A12 with or without sitagliptin for 1 day. The cells were washed with PBS, then 250 µL of serum-free M231 containing 10 µM DCFH-DA was added to the well for 30 minutes. The fluorescence intensity (relative fluorescence units) was measured at 485 nm excitation and 530 nm emission using a fluorescence microplate reader after the plates are incubated for 45 min at 37 ℃.
siRNA transfection
siRNA oligonucleotides against RAGE were suspended in RNase-free water at a concentration of 10 µM. Cells were seeded one day before transfection to ensure HASMC were 85–95% confluent on the day of transfection. For transfection, the regular cell culture medium was replaced with a serum-free medium without antibiotics. The cells were transfected with siRNA using oligofectamine at a ratio of 1 siRNA: 2 oligofectamine (µg:µl) at a final concentration of 25–50 nM siRNA. The cells were incubated with the siRNA–oligofectamine complex for 5 h. Then, the serum-free medium was replaced with a normal medium (containing 10% FBS) without antibiotics, and the cells were incubated for 48 h before further analysis.
Statistical analyses
Data were expressed as means ± standard deviation (SD). Statistical evaluation was performed using Student’s t-test or one-way analysis of variance, followed by Dunnett’s test. A P value of < 0.05 was considered significant.