Collection of blood in DVT patients
EDTA-anticoagulant venous blood was collected from four DVT patients (two males and two females; mean age, 56 ± 15 years) after total hip arthroplasty and four healthy volunteers (two males and two females; mean age, 56.5 ± 12.5 years). Blood samples were centrifuged at 3000 × g for 10 min at 4°C to remove cells. The plasma was collected and immediately frozen in liquid nitrogen for 15 min; 250 µL aliquots were stored at − 80°C prior to analysis. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Shantou University Medical College (China), and all participants provided written informed consent.
Isolation and evaluation of exosomes
The plasma sample was centrifuged at 20,000 × g for 30 min at 4°C. The supernatant was then centrifuged at 100,000 × g for 70 min at 4°C. The resulting supernatant was discarded and the pellets were washed in a large volume of phosphate-buffered saline (PBS) and centrifuged at 100,000 × g for 70 min at 4°C. The resulting pellet was collected and resuspended in PBS. After separation, the ultrastructure, concentration, and size distribution of exosomes were analyzed by electron microscopy and nanoparticle tracking analysis (NTA).
Transmission electron microscopy (TEM)
The purified plasma exosomes were resuspended in PBS, dropped into the electron microscopy grid, which was switched to the absorb mode for 10 min, and negatively stained with 2% phosphotungstic acid (pH 6.8) for 5 min. After air-drying under an incandescent lamp, the plasma exosomes were examined under an electron microscope (Hitachi-H7650, Tokyo, Japan) at 120 kV.
Nanoparticle tracking analysis
The size distribution and number of isolated exosomes were examined with the Nanosight N3000 system and the data were analyzed using NTA 3.2 Dev Build 3.2.16.
Proteomic analysis of exosomes
Total proteins were extracted from plasma exosomes using radioimmunoprecipitation lysis buffer (BioSharp, HeFei, China), and the concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of each protein sample were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane before incubation overnight with primary antibodies against CD63 and CD9 (1:1500; Abcam, Cambridge, MA, USA), SHIP1, CDKN1B, and SOCS1 (1:800; Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin and phosphorylated NF-κB p65 (1:1000; Cell Signaling Technology, Beverly, MA, USA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:4000; Cell Signaling Technology). The protein bands were visualized using IMAGISOLANE LAS4000 Mini (GE Healthcare, Piscataway, NJ, USA). Image-Pro Plus 6.0 software (Media-controlnetics, Silver-Spring, MD, USA) was used to measure the scale values of protein bands, and the relative expression of the indicated proteins was normalized to the internal control β-actin.
Studies of an animal model of venous thromboembolism (VTE)
Adult male Sprague–Dawley rats (8–10 weeks, 300–400 g) were purchased from the Experimental Animal Central of Southern Medical University (Guangzhou, China, 44002100019745). All protocols were approved by the Animal Experimentation Ethics Committee of Shantou University Medical College and carried out in accordance with the Guide for the Care and Use of Laboratory Animals. The experimental animals were anesthetized with 1% sodium pentobarbital (300–400 μL/100 g body weight).
Establishment of the rat model of DVT
An inferior vena cava (IVC) ligation model was established as described previously . The IVC and all visible side branches (usually two or three) were ligated with nonreactive 4 − 0 silk sutures. After 2 days, the IVC and associated thrombus in each group were removed, weighed, and measured for thrombus length. The IVC and its branches in the control group rats were not ligated.
Plasma exosome isolation and characterization
Rat blood was collected into 5-mL EDTA anticoagulant tubes and plasma was obtained by centrifugation at 3000 × g for 10 min at 4°C to remove cells and debris. The supernatant was then centrifuged at 20,000 × g for 30 min at 4°C and the resulting supernatant was centrifuged at 100,000 × g for 70 min at 4°C. The supernatant was discarded and the pellets were washed in a large volume of PBS and centrifuged at 100,000 × g for 70 min at 4°C. Finally, the pellet was collected and resuspended in PBS. After separation, the ultrastructure, concentration, and size distribution of the plasma exosomes were analyzed by electron microscopy and NTA.
Hematoxylin and eosin (HE) staining
Tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned (thickness, 4 mm) for dewaxing and dehydration. HE staining was performed as previously described .
After the cells or tissues were fixed with 4% paraformaldehyde and sectioned, in situ nick end labeling of nuclear DNA fragments was performed using a TUNEL apoptosis assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The samples were then analyzed under a laser scanning confocal microscope (Olympus, Japan).
Western blot analysis
Proteins were extracted from exosomes or tissues using radioimmunoprecipitation lysis buffer (Biosharp, China) and concentrations were determined using the BCA protein assay kit (Absin, China). Equal amounts of protein samples were separated by 10% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and incubated overnight with the following primary antibodies (1:1000): mouse monoclonal antibodies against GAPDH (Proteintech, China) and CD63 (Abcam) and rabbit monoclonal antibodies against Alix (Abcam), MPO (Proteintech), and TSG101 (Abcam). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:4000; Invitrogen, Chicago, IL, USA) at room temperature for 1 h. Protein bands were visualized using an OPTIMAX X-Ray Film Processor (PROTEC, Germany).
For immunofluorescence staining, tissues were fixed with 4% paraformaldehyde, and antigen repair was achieved by incubation with 0.25% citrate (ShiFeng Biology, Shanghai, China) for 16 h at 60°C. Sectioned tissues were blocked with goat serum at room temperature for 0.5 h before incubation overnight at 4°C with the following primary antibodies (1:500): mouse monoclonal antibody against CD31 (Proteintech) and rabbit monoclonal antibody against MPO (Proteintech). Sections were incubated for 1 h at room temperature with fluorescently labeled anti-rabbit or anti-mouse secondary antibody (1:1000, Invitrogen). Nuclei were stained with DAPI for 10 min. The images were captured under a laser scanning confocal microscope (Olympus).
Uptake of exosomes by human umbilical vein endothelial cells (HUVECs)
Exosomes were fluorescently labeled using the PKH67 kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. After washing with PBS (Gibco, Grand Island, NY, USA), exosomes were centrifuged at 100,000 × g for 70 min. Exosomes (1 µg) were then co-cultured with 100,000 HUVECs for 4 h at 37°C and 5% CO2. The uptake of PKH67-labeled exosomes by HUVECs was analyzed under a laser scanning confocal microscope (Olympus).
HUVECs were purchased from American Type Culture Collection and cultured in endothelial cell complete medium containing endothelial cell growth supplement (AllCells, Alameda, CA, USA) at 37°C in a humidified atmosphere containing 5% CO2.
HUVECs were seeded in 96-well plates with 5000 cells in each well. Exosomes (0.05 µg) were added into each well and co-cultured with HUVECs for 72 h. Subsequently, 10 µL CCK-8 solution was added to each well and the cells were incubated for 3 h at 37°C. Absorbance was measured at 450 nm using a microplate reader (BioTek, Carlsbad, CA, USA) and data were analyzed using Gen 5 software.
Experimental data are described as the mean ± standard deviation, and a two-tailed Student’s t-test (GraphPad Prism software Version 5.0) was used to assess statistical significance. P < 0.05 was considered to indicate statistical significance.