Ethics statement
All Kunming mice (6-week-old, female, weight 180‐220 g) were purchased from Shanghai Sippr‐Bk Laboratory Animals (Shanghai, China) and maintained under a standard environment. This study was approved by the animal Ethics Committee of Shanghai Xuhui District Central Hospital, and all experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication no. 85–23, revised 1996).
Cell isolation, culture and construction of calcification model
The mouse primary vascular smooth muscle cells (VSMCs) were isolated from Kunming mice as previously described(16). The VSMC phenotype was confirmed by western blot using the specific antibody against α-smooth muscle actin (α-SMA). VSMCs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, and 1% penicillin‐streptomycin at 37°C with 5% CO2. To induce calcification, VSMCs at passage 3 were treated with 10 mM β-glycerophosphate (Sigma, St Louis, MO, USA) for total six days as previously reported(6). After washing with PBS for twice, the quantification of calcium deposition was evaluated by using Alizarin Red staining.
Cell transfection
MiR-151-3p mimics/inhibitor and the corresponding negative controls (miR-NC and inhibitor NC) were purchased from GenePharma Co. Ltd. (Shanghai, PR, China). 50 nM of mimics/inhibitor, and negative controls were transfected into mouse primary VSMCs by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After transfection for 48 h, the transfection efficiency was confirmed by qRT-PCR. Then the exosomes were extracted and used for the subsequent experiments. miR-NC: 5′-ACAAAGUUCUGUGAUGCACUGA-3′, inhibitor NC: 5′-ACAAAGUUCUGUGAUGCACUGA-3′, miR mimics: 5′-UCGAGGAGCUCACAGUCUAGUAU-3′, miR inhibitor: 5′-ACTAGACTGTGAGCTCCTCGA-3.
The isolation and characterization of exosomes
The isolation of exosomes from VSMCs, transfected VSMCs or calcific VSMCs was performed by using the Total Exosome Isolation Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The protein concentration of the exosome was determined by using a BCA protein assay kit (Thermo Fisher Scientific). The exosomes were characterized by detection of exosome-specific biomarkers (including CD63, HSP70 and Tsg101) using western blot, and the morphology of the exosomes was evaluated by using transmission electron microscopy (TEM).
Co-culture of exosomes and VSMCs
To explore the effect of exosomes on the calcium deposition in VSMCs, the general VSMCs were treated with 10 μg/ml of exosomes extracted from calcific VSMCs for 24 h. To further determine the role of miR-151-3p, the exosomes were isolated from miR-151-3p mimics/ inhibitor and corresponding negative controls transfected with VSMCs, and 10 μg/ml of these exosomes were used to stimulate calcific VSMCs for 24 h. Finally, the calcium deposition was evaluated.
Quantification of calcium deposition
The quantification of calcium deposition was performed by using Alizarin Red staining as previously described(17). In brief, VSMCs received different treatments were fixed with 4 % paraformaldehyde at 4 °C for 10 min and then stained with 2 % Alizarin Red solution (sodium alizarinsulfonate; Sigma) at room temperature, pH 4.2 for 10 min. After washing with PBS for twice, the stained cells were imaged using a microscope. In addition, the semi-quantitative analysis of calcification was evaluated as the OD570 value by using a spectrophotometer.
QRT-PCR
Total RNA from exosomes was extracted by using a Total Exosome RNA kit (Invitrogen, Carlsbad, CA, USA). And that from VSMCs or artery samples of mice was extracted by using TRIzol reagent (Invitrogen). The total RNA was reversely transcribed into cDNA using SuperScript™ First‐Strand Synthesis kit (ThermoFisher Scientific). Then quantitative reverse transcription-polymerase chain reactions (qRT-PCRs) were performed on a 7500 fast PCR detection system using the QuantiTect SYBR Green PCR Kit (Qiagen). The relative expression levels of target genes were analyzed by using 2−ΔΔCT method, with GAPDH and U6 as the internal controls, respectively. The primers used for qRT-PCR were as follows: miR-151-3p forward: 5′-GGATGCTAGACTGAAGCTCCT-3′, reverse: 5′-CAGTGCGTGTCGTGGAGT-3′; U6 forward: 5′-CTCGCTTCGGCAGCACA-3′, reverse: 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH forward: 5′-TGTTCGTCATGGGTGTGAAC-3′, reverse: 5′-ATGGCATGGACTGTGGTCAT-3′.
Western blot
Total protein of exosomes, VSMCs and artery samples of mice was extracted by using RIPA lysis buffer. Approximately Equal amounts of protein were separated by 10% SDS-PAGE and transferred into PVDF membranes. Then the membranes were incubated with the specific primary antibody against α-SMA (ab5694, 1:1000, Abcam), Runx2 (#8486, 1:1000, Cell Signaling Technology), OPN (ab8448, 1:1000, Abcam), BMP-2 (ab14933, 1:1000, Abcam), CD63 (ab134045, 1:1000, Abcam), HSP70 (#BM4335; 1:1000, Boster Biological Technology, Wuhan, China), Tsg101 (ab125011, 1:1000, Abcam), Atg5 (#9980, 1:1000, Cell Signaling Technology), p62 (#16177, 1:1000, Cell Signaling Technology), LC3B/A (#4108, 1:1000, Cell Signaling Technology), and the internal reference GAPDH (ab9485, Abcam, 1: 1000) at 4 °C overnight. Then the membranes were exposed to horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. The protein bands were visualized by the enhanced chemiluminescence detection kit (ECL, thermo scientific, USA), and the relative grey values of targets were determined by using Image JO software.
Luciferase reporter assay
The putative binding site between miR-151-3p and Atg5 was predicted by using StarBase (http://starbase.sysu.edu.cn/index.php). The wild type (WT) and mutant type (MUT) of Atg5 fragments containing the putative miR-151-3p binding site were synthesized by Sangon Biotech (Shanghai, China) and cloned into pmirGLO luciferase reporter vector (OBIO, Shanghai, China) to generate the recombinant reporter vectors Atg5 WT and Atg5 MUT. VSMCs were co-transfected with Atg5 WT/MUT and miR-151-3p mimics/inhibitor by using Lipofectamine 2000 reagent. After transfection for 48 h, the relative luciferase activity was measured by using dual luciferase reporter system (Promega), and normalized against Renilla luciferase activity.
Animal model
The animal model with soft tissue calcification was constructed by using Vitamin D3 as previously described(18). A total of 30 female Kunming mice were divided into five groups: control group (without any treatment), Vitamin D3 group, Vitamin D3 + Exosome group (the exosomes were extracted from general VSMCs), Vitamin D3 + miR-151-3p mimic Exosome group (the exosomes were extracted from miR-151-3p mimics transfected VSMCs), and Vitamin D3 + miR-151-3p inhibitor Exosome group (the exosomes were extracted from miR-151-3p inhibitor transfected VSMCs). Six mice in each group. The mice in Vitamin D3 group received a dose of 500 000 IU/kg body weight Vitamin D3 (Sigma, St Louis, MO, USA) through subcutaneous injection, and mice in control group received equal amount of saline, followed by three days of maintaining to induce soft tissue calcification as previously reported. On the day 4, 15 μl of exosomes (500 μg/mL) derived from general VSCMs, or miR-151-3p mimics/inhibitor transfected VSMCs were intravenously injected into mice by the jugular vein. Mice in control group and Vitamin D3 group received the equal amount of PBS. After 7 days, the mice were sacrificed by cervical dislocation, and the artery samples were dissected from the mice for the subsequent western blot and qRT-PCR analysis.
Statistical analysis
All data were presented as mean ± standard deviation (SD) from more than three biological replicates. Statistical analysis was performed by using SPSS 18.0 software. The difference between two groups was tested by Student's t-test, and the difference among multiple groups was determined by one-way analysis of variance. P < 0.05 was defined as the significant threshold.