Animal study
All the experiments were performed strictly following the regulations of the First Affiliated Hospital of Jinzhou Medical University. 5 C57BL/6 mice and 15 ApoE-/- were from Beijing Experimental Animal Institute, Chinese Academy of Medical Sciences and raised for one-week adaptive feeding. Then, mice were fed with high-fat fodder daily which contained 10% lard, 1% cholesterol and 0.5% cholate in the CAS group while normal fodder was used to feed each C57BL/6 mouse in the blank group. After another 28 days, 5 ApoE-/- mice were intravenously injected with SIRT1 activator, SRT 2104 (100mg/kg) and another 5 ApoE-/- were injected with inhibitor Selisistat (10mg/kg) in tail once every two days, feeding as previously, which continued for 21 weeks (Shanghai Yuanye Biotech, China). Thereafter, mice were kept as previously treated till the 30W. Weight changes of each mouse were recorded on a weekly basis for each group and the lipid levels of TC, TG, HDL and LDL in the blood sampled from the postorbital venous plexus were detected using medical automatic biochemical analyzer (Hitachi, Tokyo, Japan) at Week 30 before sacrifice. Coronary artery was taken from each sacrificed animal, followed by HE staining and Immunohistochemistry methods.
HE staining
Coronary artery tissues were fixed using 4% formaldehyde and embedded in paraffin. Serial section was conducted with thickness of 5μm for each tissue slice and distance of 30μm from one slice to another utilizing the lab microtome. After dewaxing using xylene twice, each for 30 min, absolute ethyl alcohol (5min), 95% ethyl alcohol(5min), 80% ethyl alcohol (5min) and double distilled water (5min) washed the sections. Hematoxylin and Eosin Staining Kit was purchased from Beyotime (Shanghai, China) and used strictly according to the producer’s recommendations. The stained sections were dehydrated using pure ethyl alcohol and xylene was used to make the sections transparent. Finally, neutral gum was used to seal the sheet. The stained tissues were observed and pictured under microscope
IHC method
The glass slides containing 5-μm-thick tissue samples were put into the 40°C water bath after the bubbles were driven away. The glass slide was then dragged out and dried in the incubator at 37°C, dewaxed normally. The tissue slices were then washed with water for 10 min and soaked in 3%H2O2 solution for 10 min followed by washed by water twice. Citrate buffer was added and the slide was stewed under medium-fire option in a microwave oven till boiling and then cooled and heated till boiling for the second time. After cooling to room temperature, the citrate buffer was dumped and water washed the slide for twice and then PBS washed the slide twice for 5 min. After cleaning PBS around the tissues, 900µl PBS and100µl FBS (Cat#:20140421, Gibco, USA) was immediately used to seal the locus. Primary antibodies against CD34(µg/ml, ab237972) and VEGFR2(1:70, ab39638) were commercially provided by Abcam, Shanghai, China. After cleaning the serum around the tissue samples, the primary antibody solution was added after respective dilution separately and the slides were put into a fridge at 4°C for a night. The second day, the secondary antibody IgG H&L (HRP) (ab6721, 1:1000, Abcam, Shanghai, China) was added after the slides were washed using PBS for three times and the slides were then kept in an incubator at 37°C for half an hour. DAB kit was used to color the antigens in tissue samples and operated under microscope (ZSGB, Beijing, China). Hematoxylin was then added to redye the nuclear (Beyotime, Shanghai, China). The slides were washed with water and then soaked in alcohol with increasing concentrations (50%, 70%, 85%, 95% and 100%) for 1 min respectively. Neutral balsam was dripped to seal the slides. Inverted microscope was used to observe the stained tissue samples and images were taken and IOD values were evaluated using Image J tool (NIH, USA).
EPC separation and culture
All the mice were executed by cutting the necks and then were put in 75% alcohol for 5 min. Under aseptic condition, we separated the thighbone and wiped out the muscle tissues. 1 ml DMEM/F12 was injected into the one end of the thighbone to wash for three times and the bone marrow tissues were washed out and then grinded using 200-mesh stainless steel screen in order to split the tissues into cells. 2ml DMEM/F12 serum-free culture was used to centrifuge the cells and the supernatant was abandoned and then 2ml DMEM/F12 resuspended cells and then were added into the lymphocyte separating medium and centrifuged at 1500r/min for 30 min. Pipet aspirated the cloud-like cell band in middle. Cells were washed with DMEM/F12 twice and cultured at 37°C with 5% CO2. The medium was changed every three days. After 14 days, the cells grew and merged to 80%-90% as observed under microscope. Cellular morphology was imaged under microscope.
Immunofluorescence method for CD34+ and VEGFR2+ cells identification
Cells at logarithmic phase were seeded onto 24-well plates and cultured for 24h in the incubator at 37°C with 5% CO2. PBS washed cells twice and after aspiration of PBS,300μL 4% triformol was used to fix the cells. After 30min, cold PBS washed the cells for three times (5 min each time). 300 μL 0.05% Triton-X -100 (diluted with PBS) was added into each well and the plate was put still at 4°C for 5 min. Thereafter, 300 μL 10% donkey serum (Gibco, USA) was added into each well for 2h after cells were washed 3 times using cold PBS. The primary antibodies were diluted using 1% donkey serum. 300 μL primary antibodies after dilution was added into each well overnight at 4°C. The primary antibodies were exactly the same ones used above. The second day, after the plate was taken out and put still at room temperature for 30min, cold PBS washed cells for three times (5 min each time). 300μL 1% IgG H&L(FITC) antibody (ab7079, Abcam, Shanghai, China) after diluted with PBS was added into each well and the culture was incubated for 2h at 4°C. Thereafter, the cells were washed using cold PBS for three times and then 300 μL DAPI (1:1000 dilution rate and the final concentration, 1 μg/ml) was added into each well and the plate was incubated for 5 min at 4°C. Cold PBS washed cells for three times and 3μl protective agent was dropped on the slides and the fluorescence was observed and pictured under the fluorescence microscope.
Cell treatment
EPCs at logarithmic phase from the blank, CAS Control, SIRT1 inhibitor and SIRT1 agonist groups were selected for further study and cells from the Control group was further treated with autophagy inhibitor 3Ma (5mmol/L) or wnt pathway inhibitor IWP-2(20umol/L) for 12h. Cells from Control, 3Ma and IWP-2 groups are ready for further analysis.
RT-PCR
Cells were seeded into 6-well plates for RT-PCR assay. Total RNAs were extracted by adding 1ml Trizol (R0016, Beyotime, Shanghai, China) into each well according to producer’s recommendations. cDNA was synthesized using BeyoRT™ III cDNA (Beyotime, Shanghai, China). SYBR Premix Ex Taq Kit was used to measure the relative expression of SIRT1 against internal control of β-actin . The primer sequence was listed (Table1). Each group was detected for three repeated times. The 2 –ΔΔCt method quantified the relative expression levels in each group.
MTT
Cells were planted into 96-well plates. After cultured for 24, 48 and 72h, cell culture in the plates were added with MTT solution (Beyotime, diluted to 5mg/ml MTT solution,10ul per well). The plates were incubated for another 4 hours. Thereafter, 100ul Formazan was added into each well of the plate and the cell culture continued till the Formazan was totally dissolved as confirmed under microscope observation. OD values were measured at the wavelength of 570nm using a micro-plate reader. Each group was repeated for three independent times.
Colony formation
Cell culture medium was abandoned and cells were thereafter washed with PBS. 0.02 EDTA and 0.25% trypsin was added into each well for digestion. DMEM with 10%FBS was added to stop the trypsinization. 200 cells from each group were seeded into the 12-well plates, complemented with 2 ml complete medium. The plates were incubated at 37。C and under 5%CO2 environment for 1-2 weeks. When the cell number in each colony reached 50 at least, the complete medium was abandoned and PBS embathed the cells. 500ul 4% paraformaldehyde was used to fix the cells and the plates were incubated for another 30 min at 4。C. After cells were washed again using PBS, 500ul 0.1% crystal violet was added into each well. After the plates dried, colony images were taken on a Huawei smart phone. All the experiments were done for three times. Colony numbers were counted and further analyzed using Graphpad 8 (Graphpad Prism, CA, USA).
Scratch
Cells were collected from each group and incubated in 96-well plates (10^5). Wounds in parallel were generated using a 20-μl pipette. PBS washed the scratched cells away and then serum-free medium was added and the plates were incubated at 37oC with 5% CO2. Images were taken at 0, 24,48h and Image J was used to analyze the scratch width and relative migration distances at 24h and 48h were calculated based on the width values measured at 0,24,48h. Each group was evaluated for three independent times.
Western Blot
RIPA was added into the cell culture to lyse the cells (Beyotime, Shanghai, China). Then we centrifuged the cells and collected supernatants. Proteins were segregated utilizing SDS-PAGE and thereafter transferred to PVDF membrane(Beyotime, Shanghai, China). The membranes were put into Blotto, shaken and blocked for 2h at room temperature and blotted. The primary antibodies were added into Blotto solution and the membranes were incubated in the mixed culture and incubated overnight at 4。C. After this, the membranes were put in 1X TBST solution and rinsed shaking for 5 min for 4 times. HRP IgG antibody was used as a secondary antibody and mixed in Blotto. The membranes were incubated inside the mixture for 1.5h at room temperature. ECL was used to develop and film the blots of the immunoreactive proteins. Image J Software (NIH, USA) analyzed the relative grey values of the blots against GADPH. The antibodies were used after diluted as below.
Statistical analysis
Statistical presentations were presented in bar or line graphs using mean and standard deviation (sd) values derived from three repetitions of each experiment. Graphpad 8 (Graphpad Prism, CA, USA) was used to perform all the analysis. Single comparison between two groups was analyzed using student-t method while multiple comparisons were made among different groups based on mean and sd values of the results in each group using One-way ANOVA method (for single index) together with Tukey’s multiple comparison test while using Two-way ANOVA method (for multiple index) coupled with Sidak’s multiple comparison test in weight curve, MTT viability analysis. General alpha level is set 0.05.