AML12 cells culture, treatment, exosomes isolation and characterization
The AML12 cells (iCell Bioscience Inc, Shanghai) were cultured in DMEM/F12 (iCell Bioscience Inc, Shanghai) containing exosome-depleted serum (C38010050, ViVaCell, Shanghai), and treated with TMAO (T1362, Tokyo Chemical Industry Co., Ltd.) at a physiological concentration of 50 μmol/L (TMAO-Exos). The untreated group served as control (Control-Exos). After 24 hours, exosomes were isolated and purified from the culture supernatant using differential centrifugation. Briefly, the medium was collected and centrifuged at 300×g for 10min, 2000×g for 10min at 4℃ and then again at 10000×g for 30 min at 4℃. The supernatant was then passed through a 0.22-mm filter (Millipore) and ultracentrifuged at 110000×g for 70 min at 4℃. The pellets were then washed with phosphate-buffered saline (PBS) followed by a second ultracentrifugation at 110000×g for 70 min at 4℃ and then resuspended in PBS. The protein levels of the exosomes were measured using a BCA protein assay kit (23228, Thermo Scientific). The ultrastructure and size distribution of the exosomes were identified by transmission electron microscopy (JEM1200-EX, Japan) and nanoparticle tracking analysis (Nanosight NS300, Malvern, UK) respectively. Protein marker of CD81 (Servicebio, Wuhan, China) was detected by western blotting. Exosomes were labelled with DiI (Beyotime Biotechnology) for in vitro tracer experiment.
Endothelial cells culture and treatment
Human aortic endothelial cells (HAECs, iCell Bioscience Inc, Shanghai) were cultured in endothelial cell medium (ScienCell) supplemented with 5% fetal bovine serum, 1% growth factors, and 1% penicillin/streptomycin. Cells were treated with Control-Exos and TMAO-Exos at a concentration of 1: 100 (v/v) for 48 hours.
The procedure was performed by standard protocols as previously described . The exosomal protein concentration was determined using a BCA protein assay kit (23228, Thermo Scientific). Then the samples were separated by SDS-PAGE and transferred onto the Millipore polyvinylidene difluoride membranes. Primary antibody of CD81 was purchased from Servicebio, and the final antibody concentration was 1:1000. The expressions of CD81 were detected with enhanced chemiluminescence reagent (Millipore, USA).
Quantitative polymerase chain reaction
Quantitative polymerase chain reaction (qPCR) was performed by standard protocols as previously described . Briefly, total RNA was extracted from treated HAECs using TRIzol reagent (Invitrogen, USA) and concentration was measured using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). Then RNA was reverse transcribed into cDNA using the Color Reverse Transcription Kit (EZBioscience, USA). qPCR was performed on Bio-Rad CFX-96 (Bio-Rad, USA) with Color SYBR Green qPCR Master Mix (EZBioscience, USA). The mRNA expressions of interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and Caspase-3 were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) by using the 2^-△△CT method. The qPCR primers used in the study were listed in Table 1.
Cells apoptosis was evaluated by using the Hoechst 33342 staining solution (Sigma). Briefly, the treated HAECs were washed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes, and then washed twice with PBS again. Then Hoechst 33342 staining solution (10μg/mL) was added to cover cells, after incubating for 8 minutes in the dark, cells were washed twice with PBS. After that, cells were observed and imaged by a fluorescent microscope (Leica DMI4000B, Germany).
Exosomal miRNA expression profiling
Total RNA was isolated using TRIzol reagent (Invitrogen, USA), RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA) and RNA degradation and contamination was monitored on 1% agarose gels. After RNA quantification and qualification, 1 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample and then library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq 6000 platform and 50bp single-end reads were generated. After sequencing, clean data (clean reads) were obtained by removing reads containing ploy-N, with 5’ adapter contaminants, without 3’ adapter or the insert tag, containing ploy A or T or G or C and low-quality reads from raw data. At the same time, Q20, Q30, and GC-content of the raw data were calculated. Then, chose a certain range of length from clean reads to do all the downstream analyses. miRNA expression levels were estimated by TPM (transcript per million) and differential expression analysis between the control-Exos and TMAO-Exos samples (three biological replicates) was performed using the DESeq (v1.22.1).
Target genes prediction and functional enrichment analysis
Potential target genes were predicted from miRDB and miRBase databases. DAVID database was used for investigating the functional annotation of the target genes. GO analysis was performed to elaborate the biological functions and KEGG pathway enrichment was used to explore the relevant signal pathways, and networks were performed on Cytoscape platform (v3.8.2) . STRING database (v11.0)  was used for analyzing the protein-protein interactions, and networks were performed on Cytoscape platform. P-value ≤ 0.05 was considered statistically significant.
Data were presented as mean ± standard error of the mean (SEM). Statistical analysis was conducted using the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). The comparisons between two groups were performed with independent t-test. P < 0.05 was considered statistically significant.