Animals
Wild-type C57BL/6 N mice (male, 6–8 weeks old) were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed in the specific-pathogen-free (SPF) room with constant temperature (23–24 °C), humidity (55 ± 5%), and light (12 hours light-dark cycle). All the mice had free access to standard mouse chow and tap water. This study and all animal procedures conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85 − 23, 1996, revised 2011) and approved by the Institutional Animal Care and Use Committee of Tongji University (Number: TJBB00122101).
Establishment of mice MI models
Acute myocardial infarction was induced by permanent left anterior descending artery (LAD) ligation as described previously [46]. Briefly, the mice were anesthetized with 2% isoflurane and placed in a supine position, with respiration controlled by a rodent ventilator (Nemi Scientific, Inc., Framingham, MA). Subsequently, a left thoracotomy is performed in the fourth intercostal space of the mice, and an 8 − 0 monofilament nylon suture is passed approximately 2–3 mm below the left atrium for permanent ligation of the LAD. After the operation, EVs derived from mice hearts or kidneys were re-suspended in sterile PBS (2.0 × 109 particles/uL in 50 μL PBS) and intramyocardial injected into the left ventricular wall (border zone) at two different locations (MI + cEVs group or MI + nEVs group), respectively. Mice with 50 μL sterile PBS myocardial injection were set as a control (MI + PBS group). Sham-operated mice underwent the same procedure without coronary artery ligation (Sham group). At the indicated time points, mice were sacrificed by an overdose of anesthesia, and tissues were subsequently harvested for histological analysis. Besides, the ratios of heart weight (HW) to body weight (BW) were also calculated for each mouse on day 28 post-operation.
Echocardiography analysis
Cardiac function was assessed with a Vevo2100 ultrasound system (Visual Sonics, Canada) at different time points (day 3, day 14, and day 28) after MI surgery. Briefly, mice were mildly anesthetized with 1.25% isoflurane and placed on a platform. The left ventricle internal diameters at end-systole (LVESD) and end-diastole (LVEDD) were measured in two-dimensional long-axis views. The left ventricular ejection fraction (EF) and fraction shortening (FS) were calculated for cardiac function assessment.
Histological examination
The isolated fresh heart tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into 5 μm-thick slices for hematoxylin and eosin (H&E) staining and Masson trichrome staining to determine the morphological effects and infarct size. The tissue staining blue were considered to have fibrosis and were calculated as the total infarct circumference divided by the total LV circumference × 100. These data were measured and analyzed by Image J software (National Institutes of Health, USA). For immunofluorescence staining, mouse heart sections were incubated with anti-CD45, anti-F4/80, and anti-CD31 antibody (1:100, Cell Signaling Technology, USA) at 4 °C overnight. After washing with PBS for three times, fluorescein-isothiocyanate-conjugated secondary antibodies (1:1000, Cell Signaling Technology, USA) were incubated at room temperature in the dark for 1 hour. Images were acquired using a fluorescence microscope (Leica, Wetzlar, Germany) and quantified using Image J.
Myocardial apoptosis detection
The effects of EVs on cardiomyocyte apoptosis were detected by Tunel staining. Briefly, mouse hearts were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm-thick sections. Afterwards, an In Situ Apoptosis Detection kit (Yeasen, Shanghai, China) was used to detect cardiomyocyte apoptosis according to the manufacturer’s instructions. Cell nuclei were stained with DAPI (Sigma-Aldrich, USA).
EVs isolation
EVs were isolated by ultracentrifugation, as previously described [47]. In brief, the heart and kidney were perfused with pre-chilled PBS until free of blood. Then, the heart and kidney were removed and rinsed with PBS, minced with sterile scissors, and digested in 0.1% type II collagenase (Sigma-Aldrich, USA) at 37 °C for 30 min with a shaker speed of 300 rpm. Subsequently, the digested tissue was centrifuged at 300 g for 5 min, 2000 g for 10 min, and 10,000 g for 10 min. Thereafter, the supernatant was placed in an ultracentrifuge (Optima L-100XP Ultracentrifuge, Beckman Coulter) and centrifuged at 110,000 g for 70 min. Finally, the pellet was re-suspended in sterile PBS and stored at -80 °C for further use.
Nanoparticle tracking analysis (NTA)
Nanoparticle tracking analysis (NTA) was performed to detect the particle size and range distribution of EVs by Nano Sight LM10 instruments (Malvern Instruments, Inc., UK), following the manufacturer’s protocols. Briefly, EVs were diluted with PBS at 106 − 109 particles/mL, and the light scattered by the EVs with laser illumination was captured by a camera. Meanwhile, the movement of EVs under brownian motion was also captured and saved as a video file. The NTA 3.1 software was used for analyzing particles individually from 10 to 2000 nm, and the Strokes-Einstein equation was used to calculate their particle size distribution.
Transmission electron microscopy (TEM)
For TEM, the fresh-isolated EVs were fixed with 2.5% glutaraldehyde stationary liquid overnight. Then 5–7 μL of EVs suspension solution was loaded on the grid and incubated for 1 min. After removing the excess UA solution from the grid by contacting the grid edge with filter paper, dry it for 10 min at room temperature. EVs samples were placed in an EM grid box for observation by a transmission electron microscope (TEM; Hitachi, HT7700).
Tube formation assay
To detect the effect of cEVs and nEVs on the tube formation ability of endothelial cells. HUVECs (purchased from ATCC) were cocultured with cEVs or nEVs (both 1.0 × 109 particles/mL) for 12 hours, followed by hypoxia treatment with 1% O2 and 5% CO2 for another 12 hours at 37 °C. Then, a tube formation assay was used to detect the tube formation ability of HUVECs following different treatments. In brief, 50 μL of the Matrigel (Yeasen, China) was added to each well of 96-well plates and incubated at 37 °C for 30 min. Then, a total of 3 × 104 HUVECs per well in each group were seeded on the Matrigel-coated wells. 6 hours later, an inverted microscope was used to observe the tube formation of HUVECs. Image J was used to analyze the tube formation ability of HUVECs in each group.
Apoptosis detection assay
Mouse Cardiac Myocytes (MCM) cells (purchased from ATCC) were pretreated with cEVs or nEVs (both 1.0 × 109 particles/mL) for 12 hours, followed by hypoxia treatment with 1% O2 and 5% CO2 for another 12 hours at 37 °C. Then, MCM cells in different groups were washed in ice-cold PBS and collected for the analysis of apoptosis rate with an Annexin V/PI kit (Yeasen, China) according to the manufacturer’s instructions. Briefly, cells were resuspended in 100 μL binding buffer and incubated with 5 μL Annexin V-FITC and 10 μL PI solution for 30 min at room temperature in the dark. Thereafter, another 400 μL of binding buffer was added to resuspend MCM cells. The apoptosis rate of MCM cells in each group was immediately analyzed by flow cytometry (Beckman, Germany).
Detection of reactive oxygen species (ROS)
Mice hearts were perfused with ice-cold PBS, quickly embedded in an optimal cutting temperature compound (OCT), and frozen in liquid nitrogen. 5 μm-thick sections of the mouse heart were then stained with dihydroethidium (DHE, Invitrogen) for 30 min in the dark, followed by DAPI (Sigma-Aldrich, USA) staining for 3 min. Images were acquired using a fluorescence microscope (Leica, Wetzlar, Germany).
Detection of ATP in mice heart
The ATP content in the heart tissue of mice from different groups was determined by an ATP detection assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, heart tissue under the LAD ligation site and above the apex of the heart is separated and mixed with lysis buffer. After centrifugation at 12,000 g for 5 min at 4 °C, the supernatant is collected for subsequent determination. A 20 μL sample or standard is then added to 100 μL of ATP assay solution. The concentration of ATP in the tissue was measured via luminescence spectrometry. Bicinchoninic acid assay (BCA) protein estimation kit (Thermofisher Scientific, USA) was used to detect sample protein concentration to normalize the ATP content.
RNA extraction, library construction and construction of gene expression profiles
Total RNA from heart tissues was extracted using TRIzol reagent (Invitrogen, USA). A high-throughput sequencing service was provided by CloudSeq Biotech (Shanghai, China). In brief, total RNA (1 μg) was used for removing the rRNAs using Ribo-Zero rRNA Removal Kits (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. RNA libraries were constructed by using rRNA-depleted RNAs with the TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). For RNA sequencing, 10 pM libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters, and finally sequenced for 150 cycles on the Illumina NovaSeq 6000 Sequencer according to the manufacturer’s instructions. Then, guided by the Ensembl gtf gene annotation file, Cuffdiff software (part of Cufflinks) was used to get the gene level FPKM as the expression profiles of mRNA, and fold change and p-value were calculated based on FPKM. Differentially expressed mRNA were identified. GO and Pathway enrichment analysis were performed based on the differentially expressed mRNAs. For detection of mRNA expression, the resulting cDNA was amplified by semi-quantitative RT-PCR using SYBR Green MasterMix (Applied Biosystems; Thermo Fisher Scientific, Inc., USA) with three replicates. β-actin was used as the endogenous control. The 2–∆∆CT method was used to calculate the relative expression of different genes. All the primers used in the study were listed in Additional File 1: Table 1.
PPI network construction
To explore the association of mRNAs in cEVs and nEVs, we used the PPI biological network through the STRING database (https://string-db.org/). Cytoscape software was used for the visualization of interactions [48]. In addition, CytoHubba was used to rank the gene and find the top 10 hub genes in functional networks [49].
Western blotting analysis
Heart tissue samples and EVs were homogenized in RIPA buffer containing protease and phosphate inhibitors. Protein quantification was carried out using the BCA protein estimation kit (Thermofisher Scientific, USA) in accordance with the manufacturer’s instructions. The antibodies used in this study were listed as follows: Alix (Abcam, UK), Tsg 101 (Abcam, UK), Calnexin (Abcam, UK), Collagen I (Abclone, China), Collagen III (Abclone, China), Bax (Cell Signaling Technology, Inc. USA), Bcl-2 (Cell Signaling Technology, Inc. USA), Cleaved-Caspase-3 (Cell Signaling Technology, Inc. USA), Caspase-3 (Cell Signaling Technology, Inc. USA), and β-actin (Santa Cruz Biotechnology, USA). Total proteins extracted from tissue samples or EVs were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 3% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour at room temperature, the PVDF membrane was incubated with the primary antibody at 4 °C overnight and the secondary antibody at 37 °C for 1 hour, respectively. Then, specific bands were detected by ECL reagent (Share-Bio, Shanghai) and quantified using Image J software.
Statistical analysis
Results were presented as mean ± SEM. Data normality was determined by the Shapiro-Wilk test. Data obtained from multiple groups were compared using ANOVA, followed by Bonferroni post hoc analysis. A P-value less than 0.05 was considered significant. GraphPad Prism 8.0 (Graph Pad Prism Software Inc., San Diego, CA, USA) was used for statistical analysis.
Add Comment