The HUVECs and VSMCs were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The HUVECs were maintained in F12 K (Hyclone, Logan, Utah, USA) with 10% fetal bovine serum (Sigma, St. Louis, MA, USA) and 1% penicillin and streptomycin. The VSMCs were maintained in F12 (Hyclone, Logan, Utah, USA) with 10% fetal bovine serum and 1% penicillin and streptomycin. These cells were maintained under standard cell culture conditions of 37 °C, 5% CO2 and 95% humidity.
Isolation of exosomes from HUVECs
HUVECs were seeded in 55 cm2 culture flasks. After incubation in exosomes-free medium with normal glucose (5 mmol/l, NG) or high glucose (30 mmol/l, HG) conditions for 48 h, the cell culture supernatant were collected. The supernatant containing exosomes were centrifuged at 2000g for 20 min, 10,000g for 30 min and 14,000g for 20 min. Then, exosomes were isolated with Exosome Isolation Kit (SBI, Palo Alto, CA, USA) according to the manufacturer’s protocol. All centrifugations were done at 4 °C. Exosomes were stored at − 80 °C or used for the downstream experiments.
Identification of exosomes
The size and morphology feature of exosomes were examined by transmission electron microscopy as described previously in detail . Briefly, exosomes suspension was mixed with an equal volume of 4% paraformaldehyde and deposited on Formvar-carbon-coated EM grids. Images were acquired with a transmission electron microscope (Hitachi, Tokyo, Japan). Exosomal surface marker protein CD63 was identified by Western blot.
VSMCs calcification was assessed by Alizarin Red Staining. Following co-culture with HUVEC-Exos for 10 days, VSMCs were washed twice with PBS and fixed with 4% paraformaldehyde. The cells were exposed to 0.2% Alizarin red (pH 8.3, Solarbio, Beijing, China). Subsequent to washing with PBS, cells were visualized by phase microscopy using an inverted microscope (Olympus Corporation, Tokyo, Japan). The ALP protein expression level was detected using Western blot.
After incubation with HUVEC-Exos for 48 h, VSMCs were fixed in 2% formaldehyde and 0.2% glutaraldehyde for 10 min at room temperature and then washed with PBS. VSMCs senescence was determined with senescence-associated β-galactosidase (SA-β-gal) Staining Kit (Solarbio, Beijing, China) according to the manufacturer’s protocol. The p21 protein expression level was assayed by Western blot.
Mitochondrial membrane potential assay
The mitochondrial membrane potential was assessed by flow cytometry detection of JC-1 fluorescence (Sigma, St. Louis, MO, USA). After culturing with HUVEC-Exos for 48 h, VSMCs (5 × 105) were harvested by centrifugation (5 min at 500g) and then resuspended in 200 μl of RPMI medium without fetal bovine serum. According to the manufacturer’s protocols, cells were left for 20 min at 37 °C in a humidified atmosphere. After this incubation, JC-1 was added to a final concentration of 2.5 μmol/l, and cells were shaken in the dark at 37 °C for 15 min. Afterwards, cells were counted in a BD FACS Calibur (BD Bioscience, San Jose, CA, USA).
SOD, LDH and MDA determination
As indicators of cellular damage, malondialdehyde (MDA) content and lactate dehydrogenase (LDH) activity were determined using commercial kits (Beyotime, Beijing, China) according to the manufacturer’s instructions. The activity of oxidative stress indicator (superoxide dismutase, SOD) was detected using commercial kit (Beyotime, Beijing, China) according to the manufacturer’s instructions.
Exosomes labeling and uptake
Exosomes were labeled with the red fluorescent dye CellTracker DiD (AAT Bioquest, Sunnyvale, CA, USA) as described in previous study . Exosomes labeling with CellTracker DiD were performed following the manufacturer’s procedures. Exosomes from 1.5 × 108 HUVECs were resuspended in 200 μl PBS with 12 μl/ml diluted CellTracker DiD. After 20 min of incubation at room temperature, VSMCs were incubated with the CellTracker DiD-labeled HUVEC-Exos at 37 °C for 2 h. VSMCs were then washed with PBS and fixed with 4% paraformaldehyde for 20 min. After washing with PBS, nuclei were stained with DAPI (Invitrogen, Carlsbad, CA, USA). The signals were analyzed with a fluorescence microscope.
Proteomic analysis of HUVEC-Exos
The HUVEC-Exos samples were processed for iTRAQ-based quantitative proteomic analysis by Jingjie PTM BioLab (Hangzhou, China). We compared the proteomic content of HUVEC-Exos in HG with NG conditions, using high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS). Gene Ontology (GO) analysis was performed to classify all identified proteins into three categories (cell component, molecular function and biological process) using the UniprotKB database (http://www.uniprot.org/), InterProScan (http://www.ebi.ac.uk/interpro/) and GO annotation (http://geneontology.org/). Differentially expressed proteins were identified with a cutoff of absolute fold change ≥ 1.3. For each category, a two-tailed Fisher’s exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins. The GO with a corrected p value < 0.05 was considered significant.
VSMCs and HG-HUVEC-Exos were co-cultured in glass coverslips that had been placed in 6-well culture dishes for 48 h, and then were washed with PBS prior to fixation with 4% formaldehyde. After fixation, cells were washed with PBS and then blocked for 20 min with goat serum. Mitochondria were labeled with fluorescent mitochondrial indicator (ATT, Sunnyvale, CA, USA) according to the manufacturer’s protocol. The cells were washed with PBS and incubated overnight at 4 °C with the VCAN antibody (R&D, Minneapolis, Minnesota, USA) at a concentration of 1:200. Cells were then washed with PBS and incubated with Alexa Fluor goat anti-rabbit IgG at a concentration of 1:100 for 40 min at 37 °C. After that, cell nuclei were stained with DAPI. Images were captured using a Leica TSC-SP5 laser confocal scanning microscope.
Western blot analysis
Cells were lysed in a buffer containing 50 mmol/l TrisHCl, 150 mmol/l NaCl, 10 mmol/l EDTA, 1% Triton X-100, 0.2% NaN3, 10 μg/ml Aprotini and protease inhibitors. The lysates were centrifuged at 10,000g for 5 min and supernatants were collected. Protein concentrations were determined, and equal amounts of protein were submitted to SDS-PAGE and transferred onto 0.2 μm PVDF membranes (Millipore, Temecula, CA, USA). After transfer to PVDF membranes, the membranes were incubated with antibodies that recognize proteins, such as p21 (Cat. No. 60214-1-Ig, 1:1000 dilution), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, alpha subunit (HADHA) (Cat. No. 60250-1-Ig, 1:500) (Proteintech, Rosemont, IL, USA); CD63 (Cat. No. ab59479, 1:500 dilution), ALP (Cat. No. ab67228, 1:500 dilution), VCAN (Cat. No. ab19345, 1:500 dilution), cytochrome oxidase-4 (Cox-4) (Cat. No. ab110261, 1:1000 dilution) and GAPDH (Cat. No. ab125247, 1:4000 dilution) (Abcam, Cambridge, MA, USA) at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Cat. No. A32727, Thermo Fisher, Waltham, MA, USA) for 1 h at room temperature. The reaction was visualized with chemiluminescence.
Knockdown of VCAN with siRNA
The specific small interfering RNA (siRNA) and negative control siRNA were synthesized and purchased from Gene Pharmagps (Shanghai, China). The knockdown of the versican (VCAN) gene was performed using siRNA with the following target sequences: VCAN sense, 5′-GAGGCUGGAACUGUUAUUATT-3′; VCAN antisense, 5′-UAAUAACAGUUCCAGCCUCTT-3′; negative control sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; negative control antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. The HUVECs were seeded in six-well plates at 70% confluence. Either VCAN siRNA or negative control siRNA (2 μmol/l) was added to the cells, which underwent transfection using Lipofectamine 3000 Kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s protocol. Following transfection for 6 h, the cells were exposed to HG condition.
Real-time PCR analysis
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The reverse transcription reaction was performed using 1 μg of RNA and a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, Ontario, CA), according to the manufacturer’s protocol. For real-time PCR amplification, the cDNA were amplified using SYBR GreenPCR Master Mix (ABI, New York, NY, USA) and 0.4 μmol/l of each primer pair. Amplification was undertaken using an ABI 7900 real-time PCR system (ABI, New York, NY, USA). Quantitation of the data was performed via the 2−ΔΔCT method, using GAPDH gene expression as an endogenous reference. The primer sequences used for the real-time PCR analysis were as follows: VCAN forward, 5′-GTAACCCATGCGCTACATAAAGT-3′; VCAN reverse, 5′-GGCAAAGTAGGCATCGTTGAAA-3′; GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′; GAPDH reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′.
Results were presented as mean ± SEM, and analysis was performed with Statistical Product and Service Solutions (version 13.0). Differences between groups were evaluated by one-way analysis of variance (ANOVA), followed by the Bonferroni post hoc test to assess the significance of differences between two groups. The data were based on three independent experiments. A level of p < 0.05 was considered significant.