Animals

The animal handing and experimental procedures were in accordance with the NIH regulations for the care and use of animals in research, and were approved by the Animal Ethics and the Use Committee of the Second Affiliated Hospital of Guangzhou Medical University. The 8 to 12-week-old wild-type (WT) C57BL/6J male mice were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). ASMase knockout (ASMase−/−) mice on a C57BL/6 background were obtained from Cyagen Biosciences Inc (supplementary Fig. 1). As previously described, all the mice were housed in a 12 h dark/light cycle with unlimited access to food and water. After an initial adaptation period, the mice were randomly assigned to five groups: 1. WT mice (control group), 2. WT mice with TNF-α treatment (WT-TNF-α group), 3. WT mice with TNF-α and amitriptyline treatment (WT-AMI-TNF-αgroup), 4. ASMase−/− mice (ASMase−/− group), and 5. ASMase−/− mice with TNF-α treatment (ASMase−/−-TNF-α group) (n = 5 per group). The mice in group 3 received 5.0 mg/kg of amitriptyline by intragastric administration for 30 days. During the last four days, the mice in groups 2, 3, and 5 were intraperitoneally injected with recombinant mouse TNF-α protein (50349 Sino Biological, Beijing, China) at a dose of 30 μg/kg body weight for 4 consecutive days. Saline was used as a negative control during the same period. After the experiment, all mice were fasted for 12 h and anaesthetized by inhalation of 2.0% isoflurane followed by cervical dislocation.

Vascular Tone Measurement

The thoracic aortic rings (2–3 mm in length) were removed immediately after the mice were killed and the surrounding tissues were gently removed and immersed in oxygenated modified physiological saline solution (PSS) containing (in g/L) NaCl, 7.598; MgSO4, 0.29; KCl, 0.35; KH2PO4, 0.26; NaHCO3, 1.25; glucose, 1.0; CaCl2, 0.178; and Na2-EDTA, 0.01 (pH 7.4). The rings were then cut and mounted onto a 4-channel wire myograph system (610M; DMT, Aarhus. Denmark) in a 5-mL organ chamber filled with PSS and gassed with 95% O2 and 5% CO2 at 37°C. The artery ring was stretched to a resting tension of 9 mN and acclimated for 30 mins; the PSS was replaced every 15 min. The solution was replaced twice with 60 mmol/L of high potassium physiological saline solution (KPSS) containing (in g/L) NaCl, 4.37; MgSO4, 0.29; CaCl2, 0.178; KCl, 4.47; KH2PO4, 0.26; NaHCO3, 1.25; glucose, 1.0; and Na2-EDTA, 0.01 (pH 7.4) to test for maximal contraction. After adequate washing with PSS and equilibration, the aortic tissues were examined for endothelium-dependent relaxation under cumulative concentrations of acetylcholine (10−10 to 10−6 mol/L) and endothelium-independent relaxation under cumulative concentrations of sodium nitroprusside (SNP, 10−11 to 10−7 mol/L) after submaximal contraction with phenylephrine (0.1 μmol/L). For analysis of the potential roles of NOS in changes in relaxation, the NOS blocker L-NAME (10 μmol/L) was added to the bath for 30 min before the application of Ach, and then Ach-induced vasodilatation was re-examined.

Cell Culture and Treatment

HUVECs were obtained from Cell Applications Inc. (Supplementary Fig. 2). Human THP-1 monocytes were grown in suspension at a cell density between 105 and 106 cells/mL in RPMI 1640 with 10% FBS and 1% penicillin/streptomycin. HUVECs were cultured in EBM-2 as described previously [31]. After HUVECs reached 70–80% confluence, they were starved in FBS-free EBM-2 for 12 h and then preincubated with AMI (0.625–5 μM) for 1 h before being incubated with TNF-α (20 ng/ml) for 24 h. AMI was also present in the treatment medium alongside TNF-α.

Acid Sphingomyelinase Activity Assay

An ASMase assay kit (ab190554, Abcam) was used to assay ASMase activity in HUVECs. Briefly, the cells were lysed with mammalian cell lysis buffer (ab179835, Abcam) and then the samples were reacted with ASMase assay reagents according to the protocol recommended by the manufacturer. After the incubation, fluorescence from each sample was detected at Ex/Em=540/590 nm using a microplate reader.

Cell Viability Assay

The viability of HUVECs was determined using the Cell Counting Kit-8 (CCK-8, Dojindo, CK04-11). HUVECs were seeded in 96-well plates and allowed to reach 70% confluence. After treatment with AMI for 24 h, the cells were incubated in 100 μL of fresh serum-free medium containing 10 μL CCK-8 reactive solution for 3 h. The absorbance of the supernatant was detected at 450 nm according to the manufacturer’s instructions.

Western Blotting

The protein levels of ICAM-1, VCAM-1, MCP-1, phospho-eNOS, phospho-transcription factor nuclear factor-κB (NF-κB), phospho-p44/42 mitogen-activated protein kinase (MAPK), phospho-SAPK/JNK MAPK, and phospho-P38 MAPK were analyzed using western blotting. As described previously [32], cells were washed with PBS twice and then lysed in 65 μL of ice-cold RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher, USA). Total protein (10–20μg) was separated by SDS-PAGE and transferred to PVDF membranes (0.45μm, Millipore, USA). Then, the membranes were blocked with 5% nonfat milk in Tris-buffered saline Tween (TBST) at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies against VCAM-1 (1:1000, Cell Signaling Technology, USA, 13662), ICAM-1 (1:1000, Cell Signaling Technology, USA, 67836), MCP-1 (1:1000, Cell Signaling Technology, USA, 39091), phospho-eNOS (Ser1177, 1:1000, Cell Signaling Technology, USA, 9570), phospho-p44/42 (Thr202/Tyr204,1:1000, Cell Signaling Technology, USA, 9101), phospho-SAPK/JNK (Thr183/Tyr185,1:1000, Cell Signaling Technology, USA, 4668), phospho-P38 (Thr180/Tyr182,1:1000, Cell Signaling Technology, USA, 4511), phospho-NF-κB (Ser536,1:1000, Cell Signaling Technology, USA, 3033), ASMase (1:1000, ABclonal, China, A6743), β-actin (1:1000, Cell Signaling Technology, USA, 4970), and GAPDH (1:1000, Cell Signaling Technology, USA, 3261). After incubation with the secondary anti-rabbit antibody (1:5000, Jackson, USA, 144208), immune complexes were detected with an ECL western blotting substrate (Affinity, USA, KF005). Densitometry was performed using ImageJ (1.52v, USA) software.

Immunofluorescence

The expression of ICAM-1 was analyzed by immunofluorescence, which was performed as previously described [33]. In brief, vascular tissues from mice were fixed with 4% paraformaldehyde, fixed in paraffin, and sectioned into slices. Then, the tissues were blocked with BSA. The slices were incubated with the primary antibody overnight at 4 °C, and then incubated with the secondary antibody for 60 min at room temperature. Then, the slices were incubated with DAPI for 10 min at room temperature. Fluorescence images were captured by inverted fluorescence microscopy. The primary antibodies against ICAM-1 (cat.gb11306) and CD31 (cat.gb12063) were purchased from Wuhan Servicebio Technology Co., Ltd (Wuhan, China).

Nitric Oxide (NO) Detection

According to a previously described method [34], the NO level in the supernatant was measured by the nitrate reductase method according to the instructions of the NO determination kit (No. A012, Nanjing Jiancheng, China). Absorbance was detected using a spectrophotometer at a wavelength of 550 nm.

Proliferation Assay

The effects of AMI on cell proliferation were determined by EdU staining (Riobio, C10310-3). Cells (5×103/well, five replicates per group) were seeded in 96-well plates and incubated with 10 nM EdU for 4 h. Cells were washed, fixed, and stained according to the manufacturer’s instructions. Cells were observed and counted using a fluorescence microscope.

Adhesion of Monocytes Assay

The measurement of adhesive monocytes was performed according to a previously described method [35]. HUVECs were cultured in 24-well glass chamber slides. THP-1 cells were labeled with 5 μg/ml calcein-AM in EBM-2 medium containing 5% FBS. Calcein-AM-labeled THP-1 cells (4.0×105 cells/mL) were seeded on AMI-treated and TNF-α-treated ECs and coincubated for 45 min. Images were obtained using a fluorescence microscope.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISAs were performed on the cell supernatant to determine the levels of ceramide (11268, Ruxinbio Quanzhou, China), sICAM-1 (1113542, Dakota, China), and sVCAM-1 (1114062, Dakota, China), according to the manufacturer’s instructions.

Transwell Assay

The EC migration assay was performed using Costar Transwell cell culture chambers (354234, Corning, USA) according to the manufacturer’s instructions. HUVECs were preconditioned and seeded (4×104 cells per well) in the top chambers with 8.0-μm pore polycarbonate membrane inserts. Cells were then incubated in serum-free medium in the upper chamber, and medium with 3% FBS was added to the bottom chambers. Cells were incubated for 16 h, and cells in the lower layer were washed, fixed, stained with crystal violet solution (Sigma-Aldrich), and observed under a microscope.

Tube Formation

Matrigel (356234, Corning) was used to coat the 96-well plates and allowed to solidify (37°C, 45 min), prior to seeding with HUVECs. Cells (3×105/ml) were incubated at 37 °C for 6 h in serum free EBM-2 medium. Images of the tube network were obtained using a microscope, and the total length of the tubes were analyzed using Image J software (Bethesda, MD, USA, USA).

Detection of ROS Generation by DHE Fluorescence Staining

The ECs were incubated with dihydroethidium (DHE, 20 μM) in EBM-2 for 45 min, followed by three washes with PBS. Quantification of fluorescent images was performed with ImageJ software (Bethesda, MD, USA, USA).

Statistical Analysis

Data are presented as the mean ± standard error of mean (SEM). Statistical analysis was performed using SPSS 21.0 software (IBM Corporation, Armonk, NY, USA). Student’s t test was employed to compare data between two groups or multigroup comparisons were performed through one-way analysis of variance, followed by a post hoc Tukey test. A P < 0.05 denoted a statistically significant difference.