2.1 Animals
Syrian golden hamsters and C57BL/6 mice were originally purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). LDL receptor knockout (LDLR-/-) hamsters were generated by CRISPR/Cas9 genetic editing system in our lab and bred at SPF animal facility of Hebei ex&invivo Biotechnology Co. as described previously[9]. LDLR-/- mice were provided by genetically modified animal model platform of Key Laboratory of Molecular Cardiovascular Sciences of National Science and Technology Ministry. All animals were fed with normal chow diet (20% protein and 4% fat; purchased from Beijing Ke’ao company, Beijing, China) or HCHF diet (0.5% cholesterol, 15% lard (w/w) based on chow diet) and water ad libitum. All animals were kept in a temperature-controlled environment on a light-dark cycle of 12L: 12D. The experimental procedures were handled according to the guidelines of the laboratory animal care (NIH publication no.85Y23, revised 1996) and approved by the Animal Care and Use Committee of the Peking university health science center (LA2015-012).
Female golden hamsters at the age of 12 months and female C57BL/6 mice at the age of 6 months were used in this study in order to induce atherosclerotic lesions more quickly. Thirty LDLR-/- hamster were divided into four groups: chow diet (CD), n=5; chow diet and ezetimibe (CD+EZE), n=5; HCHF diet (HFD), n=10; HCHF diet and ezetimibe (HFD+EZE), n=10. Twenty wild type (WT) hamster as the control were also divided into four groups as above identically (n=5). The WT or LDLR-/- mice were divided into HFD and HFD+EZE groups (n=10) as the controls of lipid lowering investigation for hamsters. Ezetimibe was administrated by gavage dissolved in saline and the saline as the control. The dose of ezetimibe for hamster was 2 mg/kg/day and for mice was 3 mg/kg/day which is hamster equivalent dose based on body surface area. Plasma was collected after 1 week and 2 weeks treatments for determination of plasma lipids levels and lipoprotein profiles. After 40 days, the hamsters were anesthetized by sodium pentobarbital and euthanized for tissues harvesting.
2.2 Plasma lipids analysis
Anticoagulated blood was collected after overnight fast after 2 weeks of HCHF diet and centrifuged (4000 rpm, 4℃, 10 minutes) to separate plasma. The total cholesterol and triglycerides contents were measured using commercially available assay kits from Biosino Biotechnology and Science, Inc. (Beijing, China). Fractions of plasma lipoproteins was separated and collected using a ÄKTA fast protein liquid chromatography (FPLC) system (Amersham Biosciences). 100μL pooled plasma from 5 aliquots of each group were followed by elution with buffer at a constant flow rate of 1 mL/min. 500μL per fraction were collected for triglycerides and cholesterol concentrations measurement. The plasma lipoproteins in LDLR-/- HFD group were too large to be processed with the column. This situation has not been encountered in other animal models during separation of plasma components. Because too many chylus in LDLR-/- hamster plasma after HCHF diet would damage the column (superpose 6 HR10/30, GE), the pooled plasma was centrifuged (15000 rpm, 30min) and the lower phase was used for loading to remove the very large particles, which accounted for approximately 50% of the estimated lipid concentrations in the pre-treatment samples. The triglycerides and cholesterol concentrations of total plasma and the lower phase were calculated to evaluate the quantity of large particles removed.
2.3 Cell culture
Human umbilical vein endothelial cells (HUVEC, 8000, ScienCell) were maintained in endothelial cell medium (1001, ScienCell) at 37 °C under 5% CO2 environment. The cells were subcloned into 6-well plates and equilibrated with 0.2% BSA-ECM for 6 h. Then after added with 5% serum from different hamsters for 24 hours, the mediums were discarded and the cells were washed 3 times with PBS. The protein was extracted by RIPA Lysis Buffer (R0020, Solarbio) to prepare the samples for subsequent western blot analysis.
2.4 Western blot analysis
1 μL plasma or 20 μg protein were prepared with the buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) by heating at 95°C for 10 min. The samples were loaded to 10% or 6% SDS-PAGE gels and electrophoresed at 110 V. Proteins were transferred into nitrocellulose membrane for 90 min at 220 mA. Following blocking with 5% BSA, the membrane was hybridized with the following antibodies respectively: ApoB, ApoE, or ApoAI (ab20737, ab20874, ab20453, rabbit polyclonal IgG, Abcam). Plasma analysis was controlled by same volume of loading plasma and GAPDH (ab8245) as loading control for cell experiments. The target bands were visualized by incubation with horseradish peroxidase conjugated secondary antibodies followed by enhanced chemiluminescence detection (Molecular Imager Gel Doc XR System, Bio-Rad, Hercules, CA, USA).
2.5 Pathological analysis
Tissues were fixed in 4% paraformaldehyde for 24 h and equilibrated in 20% sucrose for 24 h. For oil red O (Sigma-Aldrich, St. Louis, MO, USA) staining, heart and liver were embedded in OCT, frozen at an approximate temperature of −20 °C and sectioned at 7 μm with a freezing microtome (Leica) for preparation. The quantitative analysis of atherosclerosis was represented as the percentage of en face lesions area ratio to whole area of full length of aorta, and the total area of aortic root lesions by image J software. For H&E, Sirius red and immunohistochemical staining, the tissue samples from aortic arch of all of hamsters after en face analysis were embedded in paraffin and sectioned at 3 μm with a Leica microtome, and then following a standard protocol referring to our pathological platform. Immunohistochemical staining was performed with VCAM-1 and α-SMA antibody (BA3840 and A03744, rabbit polyclonal IgG, Boster).
2.6 Tissue lipid analysis
Lipids extraction was referred to modified method of Bligh and Dyer[22]. Briefly, 100 mg tissues were homogenized in 1mL cold PBS. Then the lipids were extracted in 5 mL glass tubes to avoid polymer contamination by vortex with the same volume of chloroform/methanol (v:v=2:1) for 90 seconds and then centrifuged at 2000 rpm for 20 minutes. The chloroform layer at lower phase was transferred using glass syringe and the rest was repeated the above operations. The collected chloroform layer was dried under nitrogen. Lipids dissolved with 3% TritonX-100 for analysis. The cholesterol and triglycerides contents were measured with the kits described above.
2.7 Quantitative real-time PCR assay
The total RNAs were extracted with Trizol reagent (Invitrogen, 12183555) and 50 μg RNA was reverse-transcribed by a commercial RT kit (Invitrogen, 18091200). Real-time PCR were performed using the AriaMx Real-Time PCR System with Top Green PCR Master Mix (TransGen Biotech, AQ131-01). The primers used in real-time PCR were listed in table1 and GAPDH as the internal reference.
2.8 Quantification and Statistical Analysis
All data were expressed as the mean ± SEM, and statistical tests are specified in figure legends. GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA) was used for all statistical analyses. P-values of 0.05 or less were considered statistically significant.