Model preparation
The DM accompanied with arterial media calcification (AMC) model was prepared as described previously [16]. All experiments were approved by the Animal Care and Research Committee of Southeast University, and all animal procedures were performed in accordance with the Guidelines of Animal Experiments from the Committee of Medical Ethics, the National Health Department of China (1998). Briefly, six-week old male Wistar rats (160–180 g, Experimental Animal Center in Shanghai, China) were divided into four groups. The first group was the DM group, and was fed a high fat diet (HFD, 50% carbohydrates, 30% fat (20% lard and 10% soybean oil), 11% protein, 2.5% cholesterol, 6.5% fiber and other ingredients) (Animal Center, Health Science Center, Southeast University) for 8 weeks, followed by a single dose of streptozotocin (STZ, 25 mg·kg-1, intraperitoneally, Sigma). Animals were considered to be diabetic when the blood glucose levels were over 16.7 mmol·L-1. All of the rats were maintained on a standard chow for 1 week after the start of the STZ administration. A portion of the diabetic rats were then switched to treatment with a vehicle buffer (0.1 mol·L-1 sodium citrate buffer, pH 4.5) and a standard chow diet (60% carbohydrates, 22% protein, 10% fat, 8% fiber and other ingredients) (n = 10). For the second group (the DM+VDN group), half of the diabetic rats were treated with a single dose of vitamin D3 (300 000 U/kg, intramuscularly, Sigma) and nicotine (25 mg·kg-1, 5 ml·kg-1, PO, Sigma) to induce AMC (n = 10). Nicotine administration was repeated at 7 p.m. on the same day. For the third group (the VDN group), 15-week old healthy rats were fed with a standard chow diet and were administered with vitamin D3 and nicotine (n = 10). Rats were then allowed to recover for 8 weeks. The CON group included age-matched rats that were only given a vehicle buffer and a standard rat chow diet (n = 10). Please see Figure 1 for a depiction of the treatment protocols.
Metabolic parameters and specimen preparation
Rats were starved for twelve hours before sacrifice. All rats were anesthetized intraperitoneally with sodium pentobarbital (65 mg·kg-1). Blood samples were collected for measurement of plasma variables. Measurement methods for total cholesterol (TC), triglyceride (TG) concentrations, plasma glucose and insulin were described previously [16].
The thoracic aortas were isolated. A portion of the thoracic aortas were frozen at −80°C for subsequent calcium content and ALP protein expression.
Assessment of malondialdehyde content and superoxide dismutase activity in aortas and serum samples
Prior to sacrificing the rats, serum samples were collected in vials for analysis of malondialdehyde (MDA) content and Cu/Zn superoxide dismutase (SOD) activity (Nanjing Jiancheng Biotechnology Institute, China). After sacrifice, a portion of the fresh aortas were collected in a buffer solution and were subsequently homogenized. Next, the homogenized tissue was centrifuged and supernatants were collected for the analysis of MDA content and Cu/Zn SOD activity. MDA content and Cu/Zn SOD activity were measured as described previously [17]. Briefly, MDA content was detected by the thiobarbituric acid method with a maximal absorbance at 532 nm, and Cu/Zn SOD activity was measured by the xanthine oxidase method with an absorbance at 550 nm by spectrometry.
Determination of aorta AGEs
The levels of AGEs were analyzed by a fluorescent method. Supernatants from aorta homogenates were measured on a Hitachi 650–60 fluorescence spectrometer with excitation and emission wavelengths of 380 nm and 452 nm, respectively. Standard curves created with different concentrations of AGE-BSA (0, 1, 5, 10, 50, 100 μg·mL-1) were used to determine the linear range of the assays. For these assays, 1U of AGEs was equivalent to 1 μg·mL-1 of AGE-BSA.
Preparation of AGE-BSA
Briefly, bovine serum albumin (BSA) and D-glucose were dissolved in sodium phosphate buffer (PBS). The final concentrations were 50 mmol·L-1 of glucose and 5 g·L-1 of BSA. The solution was sterilized by ultrafiltration, then was incubated at 37°C for 90 days, and finally was dialyzed against PBS. As a control, BSA was incubated in parallel without D-glucose. No endotoxin was detectable in these preparations.
Induction of calcification of rat VSMCs in vitro
VSMCs were isolated from one-week old Sprague–Dawley rat thoracic aortas (Experimental Animal Centre, Southeast University, Nanjing, China) and cultured as described previously [9]. Passages of 3–5 VSMCs were used for the study. At sub-confluence, calcification of the cells was induced in DMEM with 1% FCS in the presence of 10 mM β-glycerophosphate (β-GP, Sigma, USA). This medium was replaced twice a week. Cells were harvested for analysis at Day 3 of the treatment. In separate experiments, sub-confluent VSMCs cultured in six-well culture plates were pre-incubated with a neutralizing antibody to RAGE (100 μg·mL-1, R&D) or 10 μmol·L-1diphenyleneiodonium (DPI, Sigma), an inhibitor of ROS for 2 h. Next, AGE-BSA was added to the medium containing 10 mmol·L-1 of β-GP for 24 h prior to the measurement of gene and protein expression, or for 72 h prior to the measurement of calcium content. For all data shown, individual experiments were repeated at least three times.
Quantification of calcium deposition
Analysis of calcium content in rat thoracic aortas was performed according to previously described procedures [18]. Briefly, portions of aortas were dried at 55°C and calcium was extracted overnight at 4°C using 10% formic acid. Cells were decalcified with 0.6 M HCl for 24 h. The calcium content in acid extracts was examined colorimetrically by the O-Cresolphthalein Complexone method (Calcium kit; Sigma) [10, 19]. After decalcification, cells were washed three times with PBS and solubilized with 0.1 M NaOH containing 0.1% SDS. A BCA assay kit (Pierce) was used to determined protein concentration. Aorta calcium content was expressed as μg/mg of dry tissue, and cell calcium content was normalized to total protein content.
Examination of ALP activity
Cellular proteins were solubilized with 1% Triton X-100 in 0.9% NaCl. After centrifugation, the supernatants were assayed for ALP activity by using an ALP Assay Kit (Sigma, USA) as described previously [20]. One unit was defined as the activity producing 1 nmol of P-nitrophenol for 30 min. The results were normalized to total cellular protein levels as previously described [21].
Western blot analysis
Total proteins of aortas and cells were extracted in the RIPA lysis buffer (Beyotime, China). After centrifugation, supernatants were collected for the determination of protein concentrations. Equal protein samples were loaded on a 10% SDS–PAGE gel and transferred to PVDF membranes (Bio-Rad). The membranes were blocked with 4% milk-TBST, incubated overnight with the primary antibody, washed with TBST, and then incubated with the secondary horseradish peroxidase-labeled antibody (Santa Cruz, USA, dilution 1:1000). Immunodetection was performed with the Enhanced Chemiluminescence Kit (Amersham Biosciences, Piscataway, NJ). The following antibodies were used: anti-RAGE monoclonal antibody (diluted 1:250, R&D), anti-ALP monoclonal antibody (diluted 1:500, Santa Cruz Biotechnology Inc), anti-Cu/Zn SOD monoclonal antibody (diluted 1:500, Santa Cruz Biotechnology Inc), and anti-Nox1monoclonal antibody (diluted 1:500, Santa Cruz Biotechnology Inc).
Measurement of intracellular ROS generation
VSMCs calcification was induced with different concentrations of AGE-BSA for 2 h to analyze ROS. In separate experiments, VSMCs cultured in 24-well culture plates were pre-incubated with a neutralizing antibody to RAGE, DPI, or 100 μmol·L-1 tempol ( a SOD mimetic, R&D) for 2 h, followed by treatment with 100 mg·L-1AGE-BSA for another 2 h to determine ROS levels. ROS generation was evaluated using the fluorescent technique with dihydroethidine (DHE, Genmed Scientifics Inc. USA) according to the manufacturer’s instructions. VSMCs were incubated with DHE (diluted 1:100 with dilution reagents) for 20 min at 37°C in the dark and were then washed with PBS. Fluorescence was determined using a microscope (Nikon, Japan) at excitation and emission wavelengths of 540 nm and 590 nm, respectively. Fluorescence intensity was semi-quantitatively analyzed by NIS-Elements F 3.0.
Immunohistochemistry
Immunohistochemistry was performed to detect RAGE. Briefly, after dewaxing and hydration of sections, tissue slides were treated with 3% H2O2 for 10 min, then were incubated with the primary antibody against RAGE (R&D, 1:20) overnight at 4°C, followed by incubation with the secondary antibody conjugated to horseradish peroxidase. The antibodies were detected by the diaminobenzidine method which produces a brown color. Adjacent sections treated with non-immune IgG provided controls for antibody specificity. The area of the aorta with brown staining was quantified. The analysis was performed blindly by two examiners for the same slides.
Von kossa staining
The presence of mineral deposits was confirmed by Von kossa staining. Paraffin sections from aortas were dewaxed and hydrated. The slices were immersed in 1% silver nitrate for 30 min under an intense sunbeam, and were then washed 3 times with deionized water. Subsequently, 5% sodium thiosulfate (Sigma) was added for 5 min to remove un-reacted silver. The calcium phosphate salts were visualized as a black staining.
Statistical analysis
Data were presented as the mean ± SD. A one-way ANOVA followed by a post hoctest and chi-square test were performed for statistical comparisons. P< 0.05 was considered to be significant.