VSMCs from the thoracic aorta of Wistar Kyoto (WKY) rats were kindly provided by Professor KH Chen at the National Institutes of Health (Bethesda, USA). Cells from passages 3 to 9 were used throughout this study. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand island, NY, USA) at 37 ℃ in 5% CO2. The culture medium was changed every 2 to 3 days.
Fluorescence colocalization detection
VSMCs were plated on cover slips in 6-well plates at 2 × 105 cells/well, serum starved for 24 h and then treated with MRSP-His at various time points (6 h, 12 h, 18 h, 24 h) at 37 ℃ with or without 10% FBS. Cover slips were processed for immunofluorescence with MitoTracker CMXRos (Invitrogen, Carlsbad, CA) and MRSP, and colocalization analysis, as previously described. First, viable cells were stained with MitoTracker CMXRos, followed by fixed and rupture of cell membranes. Then cells were blocked and probed with primary antibody overnight at 4 ℃. The antibody used was anti-His tag antibody (AE003, Abclonal, Boston, USA), and the secondary antibody was Alexa-Fluor 488 (green, Servicebio Biotechnology, Wuhan, China). Then the coverslips were stained with DAPI (Servicebio Biotechnology, Wuhan, China) to stain the nuclei. Images were obtained on a fluorescence microscope (Olympus, Japan), and a colocalization analysis was conducted by Image J.
Cells were seeded in 6-well plates at 2 × 105 cells/well and synchronized by serum deprivation for 24 h. Then, VSMCs were incubated with different concentrations (25 and 50 μM) of MRSP over a time series (12 and 24 h) with or without 10% FBS. The pro-apoptotic effects in different groups were demonstrated by Annexin V-FITC/PI flow cytometric assays. The assay was carried out according to the manufacturer's instructions (Beyotime, Beijing, China). Briefly, the treated VSMCs were harvested with trypsin, washed in PBS, centrifuged at 2000g for 5 min and resuspended in binding buffer at a concentration of 106 cells per 1 ml, followed by incubation with 5 μl of Annexin V-FITC for 15 min and subsequent incubation with 5 μl of PI solution for 5 min at room temperature in the dark. The cell suspension was analyzed within 30 min and at least 10,000 stained cells of each sample were analyzed by flow cytometry. The apoptosis ratio was determined as the sum of early and late apoptotic rates. The experiments were carried out in triplicates.
Measurement of DNA fragmentation by cell death detection ELISA kit
VSMCs were treated as described above. The apoptotic response was measured by the Cell Death Detection ELISA kit according to the manufacturer's instructions (Roche Applied Science, USA). Briefly, VSMCs (1 × 104 cells/well) in 96-well microtiter plates were incubated with different concentrations of MRSP for various periods of time, and the absorbance was examined at 405 nm. The experiments were carried out in triplicates.
VSMCs were treated as described above, fixed in 4% paraformaldehyde solution, washed three times with PBS, incubated for 5 min with 5% Triton-X 100, washed three times with PBS, and then labeled with the DeadEnd Colorimetric TUNEL System (Beyotime, Beijing, China) one hour at 37 ℃, followed by incubating with DAPI for 5 min. Images were captured by a fluorescence microscope. Apoptosis of the neointima was determined following the manufacturer's instructions. In brief, 5 μm sections were deparaffinized, rehydrated and incubated with proteinase K, followed by incubation with fresh 3% hydrogen peroxide and then incubation with TUNEL reaction mixture for 60 min at 37 ℃. After blocking, the anti-fluorescein antibody was applied to the sections for 30 min at 37 ℃ in the dark. The apoptosis ratio was represented by the TUNEL-positive rate, which was equal to the number of TUNEL-positive cells divided by the total cell number * 100%. The experiments were carried out in triplicates.
Cytochrome c release
Firstly, Cells were seeded in 6-well plates at 2 × 105 cells/well and synchronized by serum deprivation for 24 h. Then, VSMCs were incubated with HIV1TAT or nothing for 12 h, which detected the difference between the group of HIV1TAT alone and the control group. Then VSMCs were treated as described above. For determination of the effect of MRSP on cytochrome c release, mitochondrial and cytosolic proteins were separated using the Cell Mitochondria Isolation Kit (Beyotime, Beijing, China). The expression level of cytochrome c was analyzed by western blots. The expression of cytochrome c oxidase 4 (COX4, A11631, ABclonal, Boston, USA), a mitochondrial marker was monitored to determine the purity of the cytosol and mitochondrial components. The experiments were carried out in triplicates.
Western blot assay
VSMCs were treated as described above. Protein levels were measured by western blot analysis as described previously. Cell or tissue proteins were extracted using RIPA buffer (Boster Biological Technology, Wuhan, China) with a protease inhibitor mixture, and protein concentrations were determined using a bicinchoninic acid (BCA) kit (Boster Biological Technology, Wuhan, China). Equal amounts of proteins (20 ug) were loaded, separated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes and blocked with 5% BSA in TBST for 2 h at room temperature. Then, the membranes were washed and incubated overnight with primary antibodies. The primary antibodies included Mfn-2 (1:1000, #9482, CST, Boston, USA), PI3K (1: 1000, #4249, CST, Boston, USA), phospho-PI3K (1: 1000, #4228, CST, Boston, USA), Akt (1: 1000, #4691, CST, Boston, USA), phospho-Akt (1: 1000, #4060, CST, Boston, USA), ERK1/2 (1:1000, #4695, CST, Boston, USA), p-ERK1/2 (1:1000, #4370, CST, Boston, USA), p38 (1:1000, #9212, CST, Boston, USA), p-p38 (1:1000, #9211, CST, Boston, USA), JNK (1:1000, #9252, CST, Boston, USA), p-JNK (1:1000, #9255, CST, Boston, USA), Bcl2 (1: 1000, A0208, ABclonal, Boston, USA), Bax (1: 1000, #2772, CST, Boston, USA), cleaved caspase-3 (1: 1000, #9664, CST, Boston, USA), cleaved caspase-8 (1: 1000, 13423-1-AP, Proteintech, Chicago, IL, USA), cleaved caspase-9 (1: 1000, #9507, CST, Boston, USA), cytochrome c (1:1000, A4912, ABclonal, Boston, USA) and β-actin (1:10,000, AC004, ABclonal, Boston, USA). After that, the membranes were incubated with peroxidase-conjugated secondary antibodies, and ECL (Boster Biological Technology, Wuhan, China) and then visualized by autoradiography.
Rat carotid artery balloon denudation injury
All animal procedures were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals under approval by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University Science and Technology, Wuhan, China.
Male Sprague–Dawley rats (280–320 g, Institutional Animal Research Committee of Tongji Medical College, Wuhan, China) were used. Animals were housed in a room at a controlled temperature of 23 ℃ with a 12:12-h dark–light cycle. The rats had free access to water and rodent chow diet throughout the experiment. Animals were randomly divided into three groups (n = 12 each group): sham, model, and MRSP (10 mg/rat). The animals were intraperitoneally anesthetized with ketamine hydrochloride (70 mg/kg) and xylazine (5 mg/kg) and heparinized with 100 U/kg heparin sodium through the tail vein. The 2F Fogary balloon embolectomy catheter (Baxter Healthcare Corp, IL, USA) was inserted through an incision in the left external carotid artery into the origin of the common carotid artery. The balloon was distended, and the procedure was repeated 3 times with rotation to produce a substantial vascular injury. Immediately following balloon injury, carotid artery surface was painted with 200 μl of 25% Pluronic F-127 gel solution (Sigma-Aldrich, St Louis, MO, USA), a thermosensitive amphiphilic polymer, containing MRSP or nothing. The control rats that underwent sham surgery were treated as described above without balloon-injury. The left external carotid artery was ligated after removal of the catheter, and the wound was closed. All groups were maintained for 1 or 2 weeks on a normal chow diet after surgery.
One week and two weeks after balloon injury, the rats were euthanized and carotid arteries were acquired for further exploration.
Samples were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and sectioned into 4 μm thick slices. Then routine hematoxylin and eosin staining (HE, Beyotime, Beijing, China) was performed. Cross-sectional areas of the lumen, neointima, and media were observed by HE staining, and morphometric analysis was carried out using image analysis software. The intima-to-media ratio (I/M ratio), which represents neointimal proliferation, was then calculated.
After carotid artery slide deparaffinization and rehydration, endogenous peroxidase deactivation was achieved by 3% H2O2 for 10 min, followed by washing with PBS and antigen retrieval. Then antigen blocking serum was applied for 30 min, followed by overnight incubation at 4℃ with a primary anti-Bax (1:200) antibody, anti-Bcl (1:200) antibody, and anti-cleaved-caspase3 (1:200) antibody. After that, these slides were incubated with the biotinylated secondary antibody for 60 min at room temperature. Subsequently, 3′3-diaminobenzidine solution (DAB kit) was used for visualization. For quantitative analysis of the results of immunohistochemical staining, the mean optical density value in each region was calculated as: IOD/total area.
All data are expressed as means ± SD. The error bar is generally the results of three independent experiments under one condition and is represented by SD. A One-way analysis of variance (ANOVA) followed by the Boneferroni procedure was performed to determine the statistical significance. The comparison of two groups was tested by Student's unpaired t test. Differences with p < 0.05 were considered statistically significant.