L-homocysteine (69453), telmisartan (T8949), was purchased from Sigma-Aldrich. Disposable consumables such as cell culture bottle and 6-well plates were purchased from Guangzhou Jet Bio-Filtration Co., Ltd.
Animals and Treatments
A total of 21 ApoE-/- mice (male, 8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Beijing, China (Ltd. Number: SCXK2016-0006). The mice were randomly divided into three groups: a control group with a standard mouse diet (n = 7), an HHcy group fed with standard mouse diet plus 1.5% methionine (n = 7),17, 18 and a telmisartan gavage group (HHcy+Telmi) treated with telmisartan (10mg/kg/day). Ten Sprague Dawley (SD) rats (male, weighing 150–200 grams) were purchased from Inner Mongolia Medical University Laboratory (Hohhot, China). All animal experiments were approved by the Institutional Animal Care and Use Committee of Inner Mongolia Medical University, Hohhot, China and was conducted in accordance with the Guide for the Care and Use of laboratory animals from the National Institutes of Health(Bethesda, MD, USA).
Tail-cuff measurement of systolic blood pressure
Systolic blood pressure (SBP) was recorded by a computerized non-invasive tail-cuff system (Cat. NO.BP-300A, Chengdu Techman Software CO.,LTD). Measurements were performed in quiet environment to avoid causing mice anxiety. The mice underwent 7 consecutive days of training sessions to become accustomed to the tail-cuff procedure. Three measurements were weekly performed on each mouse, so that the average of total of 3 measurements was represented as the SBP of each mouse.
Measurement of Serum Homocysteine Level
The levels of Hcy, TG, TC, HDL, and LDL were assessed with kits from Beijing Sino-UK Institute of Biological Technology. Hcy concentration was measured using enzyme-linked immunosorbent assay (HY-N0080).
Histology and Immunostaining
The heart and approximately 2 mm of proximal aortas were fresh frozen in OCT compound.20 Tissue samples were cut into 7μm thin slices. Frozen tissue sections were stained with Oil Red O, H&E, Masson, and immunostaining.21 A Masson's trichrome staining kit was used to detect collagen deposition in the atherosclerotic plaque. Analyzed with Image-Pro Plus 6.0 or ImageJ software.20
Cryosections were fixed in 4% paraformaldehyde and subsequently treated with H2O2 and after the primary antibody was blocked in 5% goat serum (1:100) at 4°C overnight. The tissue sections were washed in PBS then incubated with secondary antibody (1:100). They were incubated in diaminobenzidine until a change in color was observed under the microscope and then the nucleus was stained with hematoxylin.22 Immunofluorescence staining procedure was similar to immunostaining.
Primary Rat AFs
Cells were prepared according to common methods19 and with some modifications. SD rat were euthanized with sodium pentobarbital at 120 mg/kg. Thoracic aortas were removed and cleaned under sterile conditions. The adventitia was stripped from the aorta and cut into pieces about 2–3 mm3 in size. Then, the tissue pieces were attached to the 6-well plates and immersed in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 30% fetal bovine serum (FBS, Gibco) and maintained at 37°C, 5% CO2. Cells were collected, and when the cells grew to about 70%–80% fusion, the tissue pieces could be cultured again. AFs were verified by positive immunofluorescent staining of vimentin and ER-TR7. The AFs after passage were cultured in DMEM supplemented with 10% FBS at 37°C, 5% CO2. All AFs used in this study were from early passage with a maximum of four passages.
The human embryonic kidney cell line HEK293A was a generous gift from our laboratory and cultured in DMEM (high glucose) supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2. The cells were confirmed of no mycoplasma contamination before application.
Cell Proliferation Assay
The proliferation of cells was examined with Cell Counting Kit-8 (Cat. NO. K1018, ApexBio Technology LLC). Briefly, cells were seeded in 96-well plates with 5 × 103 cells/well, cultured for 24 hours, and subjected to the indicated treatment.23 The absorbance at 450 nm was measured using an automatic microplate reader (n = 3).
AFs (5x105 cells/well) were inoculated into 12-well plates. After the cells completely covered the bottom wall of the 12-well plate, three vertical lines were drawn in the 12-well plate with a sterile 200-μl pipette tip, and the floating cells were removed by PBS washing.24 DMEM 1000 μl containing 1% FBS and Hcy 200 μmol/L was added into the 6-well plate. The cells were cultured in an incubator at 37°C and 5% CO2 for 48 hours. At the same location, the images of 0 and 48 hours were taken to observe the cell migration. Cell migration analysis was processed by Image-Pro Plus 6.0 software24 (n = 3).
Transwell Matrigel Invasion Assays
Transwell Matrigel invasion assays were performed using a transwell membrane (8-μm pore size, 6.5-mm diameter; Cat. NO. 35488, Corning Incorporated) in a 24-well plate. The Hcy-treated cells were digested with trypsin (0.25%) and then centrifuged and the supernatant was discarded. The cell suspension was prepared with DMEM without serum. The cells (1x105 cells/well) were loaded to the upper side of the chamber (500 μl/well), and the lower chambers contained 750 μl/ of DMEM with 10% FBS as chemoattractant. The cells were cultured in the cell incubator for 6 hours. The filter inserts were then removed from the wells. Cells on the upper surface of the filter were removed using cotton swabs and the membranes were fixed and stained with crystal violet reagent.21,25 Invasion was quantified by counting 5 random fields under a light microscope (Leica, Germany) at x40 magnification.
Detection of Intracellular ROS
Intracellular ROS generation was monitored by using Reactive Oxygen Species Assay Kit (WLA070a, Wanleibio). Cells (1x106 cells/well) were seeded in 6-well plates. Cells were treated with Hcy for 48 hours and stained with 20 µmol/L DCF-DA in culture medium for 30 minutes in the dark. The cells were then harvested and washed in PBS three times and oxidation-induced DCF fluorescence was assayed by fluorescence microscopy.26
Assay of Hydrogen Peroxide Level
Hydrogen peroxide level was assayed using a Hydrogen Peroxide Assay Kit (Cat. NO. A064-1-1, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions.
Western Blot Analysis
Protein extracts (40 μg) were resolved by 10% SDS-PAGE for western blot analysis. The blots were subsequently incubated with primary antibodies (AT1R, 1:300, Abcam, ab124734; p-PKC, 1:1000, Cell Signaling Technology, 9371s; t-PKC, 1:1000, Wanleibio, WL02234; p-ERK1/2, 1:1000, Cell Signaling Technology, 9101s; t-ERK1/2, 1:1000, Cell Signaling Technology, 9102s). The corresponding secondary antibody was added for incubation at room temperature. The protein expressions in different samples were detected using the enhanced chemiluminescence method.
All data were expressed as mean ± standard error of the mean (SEM). All experiments were repeated at least three times. The data were analyzed by GraphPad Prism 5.0 (GraphPad Software, San Diego, California, USA) and SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). The data were analyzed by one-way ANOVA or repeated measurement ANOVA, without assuming sphericity. When multiple measurements were made in the same cell, Greenhouse–Geisser correction was performed. The data that failed to pass the normality test were analyzed by nonparametric test. The difference was significant (P < 0.05).