2.1 Tissues
In this study, 10 patients with atherosclerotic plaque and normal arterial tissues were all from atherosclerotic patients who underwent carotid endarterectomy at Southern Hospital of Southern Medical University. Arteries without macroscopic evidence of atherosclerosis were collected from individuals who died from a traffic accident or cerebral edema. After washing the specimens with saline, the outer fat tissues and surrounding normal tissues of the plaque were separated. The plaque tissue was separated into appropriate sizes, and immediately placed in liquid nitrogen. The collected tissue was pathologically tested and diagnosed as primary atherosclerosis. The exclusion criteria were patients with diabetes, cancer, congestive heart failure, valvular heart disease, hematological system diseases, autoimmune disease, and/or infections. The basic information of the patients included name, age, and sex. The study was approved by the Committee for Ethical Review of Research Involving Human Subjects, Nanfang Hospital, Southern Medical University, Guangzhou, China (Ethics approval ID: NFEC-2018-142). Informed consent was obtained from the participants or relatives of deceased individuals.
2.2 Cells
Human vascular endothelial cells (HUVECs, ATCC CRL-1730) were obtained from ATCC. HUVECs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). The cells were incubated at 37°C in an atmosphere of 5% CO2. Cells were seeded in 6- or 12-well plates or 60 mm dishes and grown to 60–80% confluence before use.
2.3 Animals
ldlr–/– mice and ugp2+/– mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). All experimental protocols in accordance with the NIH Guide for the Care and Use of Laboratory Animals were approved by the Ethic Committee of Nanfang Hospital, Southern Medical University. All mice had the C57BL/6J background. To generate ldlr and ugp2 double-deficient mice, ugp2+/– mice were crossbred with ldlr–/– mice. Due to embryonic lethality of ugp2-knockout homozygosity (ugp2–/–), ugp2+/– ldlr–/– and ugp2+/+ ldlr–/– mice were used in this study. Six-week-old mice from both groups (5 males and 5 females in each group) were fed a Western high-fat diet for 12 weeks. Thereafter, mice were fasted for 4 hours, then placed under general anesthesia, after which blood was collected from the retro-orbital venous plexus using a capillary tube. The mice were then sacrificed by cervical dislocation, and tissues were collected for analysis.
2.4 Quantitative RT-PCR analysis
Total RNA from cultured cells were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed. Real-time PCR was performed on the lightCycler 480 II (Roche, Pleasanton, CA, USA) with SYBR Green Dye detection (TaKaRa Bio, Mountain View, CA, USA). All samples were assayed in triplicate. The data were analyzed using the ΔΔCt method, with GAPDH as a reference in the mRNA analysis. The primer sequences are listed in Table.1.
2.5 Immunoblot analysis
The total protein was measured by the bicinchoninic acid protein assay kit (P0010-1; Beyotime, China). The cleavage products of each sample were separated by 12% SDSPAGE. The western blots were incubated for 12h at 4℃ with antibodies against UGP2 (Abcam, catalog ab157473), TP53 (Abcam, catalog ab26), Cleaved caspase-3 (CST, catalog #9664), or β-ACTIN (Abcam, catalog ab8227), and then incubated 2h at room temperature with horseradish-peroxidase-conjugated secondary antibodies. Chemiluminescence (ECL Plus Western Blot Detection System; Amersham Biosciences, Foster City, CA, USA) was used in the visualization of proteins.
2.6 Detection of intracellular ROS
Intracellular generation of ROS was detected by fluorescence probe 2, 7-dichlorofluorescin diacetate kit (DCFH-DA, Sigma) as described by the production instruction. HUVECs were treated with 0.5mM H2O2(Sigma)for 24h after transfection. The treated cells were washed three times with PBS and then incubated in 10 mM DCFH-DA for 30 min at 37 °C without light. After washed 3 times with PBS, the samples were assessed on a flow cytometer and analyzed using CellQuest (BD Biosciences, New Jersey, New York, USA) software.
2.7 Detection of apoptosis cells
Cells apoptosis was assessed using an apoptosis detection kit (Kaiji Biotechnology, Nanjing, China). Briefly, cells were collected after treatment, washed twice in ice-cold PBS, and resuspended in binding buffer, then added into 5μl of Annexin V-APC and 5μl of 7-AAD mixed for 15 min at room temperature in the dark. The samples were assessed on a flow cytometer and analyzed using CellQuest (BD Biosciences, New Jersey, New York, USA) software. Apoptotic cells were quantitated by Annexin V binding and 7-AAD uptake. The Annexin V-APC+7-AAD– cell populations were considered to represent early apoptotic cells, and The Annexin V-APC+7-AAD+ cell populations were considered to represent lately apoptotic cells.
2.8 siRNA assay
Control siRNA (negative control), siRNA-UGP2, and siRNA-TP53 were purchased from Ribo Targets (Guangzhou, China). ECs were transfected with 50 nM siRNA. Control samples for all experimental procedures were processed with a non-targeting control mimic sequence of equal concentration. qRT-PCR and Western blotting analysis was performed to observe the efficiency of siRNA protein knockdown. The sequence of siRNA-UGP2, siRNA-TP53, and control siRNA are shown in Table.2.
2.9 Recombinant plasmid construction
A plasmid containing the full-length UGP2 cDNA was purchased from OriGene (Rockville, MD, USA). UGP2 cDNA was amplified by PCR and subcloned into the pcDNA3.1 (+) vector. The correct sequence of the UGP2 cDNA in the recombinant plasmid was verified by sequencing and named pcDNA-UGP2. The plasmid was used to transfect ECs using Lipofectamine 3000transfection reagent.
2.10 ChIP
Human vascular endothelial cells (HUVECs) were crosslinked by incubation in formaldehyde and then incubated with glycine to quench formaldehyde. ChIP was carried out using an anti-TP53 antibody (Abcam, catalog ab26) and the Pierce Agarose ChIP kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. DNA in the chromatin immunoprecipitated by the anti-TP53 antibody or an isotope IgG (Abcam, catalog ab172730) to serve as a control was subjected to quantitative PCR analysis of the UGP2 gene and the housekeeping gene β-ACTIN promotor sequence, respectively. The primer sequences are listed in Table.3.
2.11 Histological and immunohistochemical analyses
Sections (4μm thick) of formalin-fixed, paraffin-embedded human atherosclerotic plaques and normal arterial wall specimens, respectively, were subjected to immunohistochemical staining using an anti-UGP2 antibody (Abcam, catalog ab157473).
To examine the extent of aortic atherosclerotic lesions in the studied mice, the aorta was opened longitudinally along the ventral midline from the iliac arteries to the aortic root, pinned out flat on a blue surface, and stained en face with oil red O (Sigma-Aldrich). Images of stained aortas were captured using a Leica DMI6000 microscope. The oil red O–stained areas were quantified using Image-Pro Plus 6.0 (Media Cybernetics).
Formalin-fixed, paraffin-embedded sections (3μm thick) of mouse hearts with aortic root were stained with H&E, Masson for collagen, von Kossa for calcification, Tunel for apoptosis, and immunohistochemically stained using antibodies for Cleaved caspase-3 (CST, catalog #9664). Frozen sections (3μm thick) of mouse hearts with aortic root were stained with oil red O, and DHE for ROS. Images were acquired using an Olympus BX50 microscope with a digital color camera and analyzed using Optimus software (version 6.2).
2.12 Statistical analysis
Data were analyzed using SPSS version 13.0 (SPSS, Chicago, IL, USA) software. Data are presented as the mean ± SD or median (interquartile range) unless otherwise indicated. The results were analyzed by one-way analysis of variance or unpaired Student’s t-tests when continuous variables were normally distributed. A two-tailed p value < 0.05 was considered statistically significant.