Ethics statements
All animal care and surgical procedures were performed with approval from the Nanjing Medical University Animal Care and Use Committee (IACUC-1709019).
Cell culture and identification
Umbilical cord MSCs from healthy donors were purchased from the Clinical Center of Reproductive Medicine in Nanjing. ucMSCs, H9C2 cells and human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, USA) supplemented with 10% foetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, 100 µg/Ml streptomycin and 110 mg/mL sodium pyruvate. All cells were incubated at 37℃ in a humidified atmosphere containing 5% CO2. ucMSCs used for experiments were between passages 3 and 6.
Umbilical cord MSCs were characterized by the expression of cell surface markers. They were washed with 2% FBS/phosphate-buffered saline (PBS) and incubated at 4℃ for 30 minutes with 5 µl of a monoclonal antibody specific for CD31, CD34, CD45, CD73, CD44 and CD105 (BD Biosciences, San Jose, CA, USA). Unstained ucMSCs were used as controls. FACS Canto II (BD Biosciences, San Jose, CA, USA) was used for cytometry analysis.
Exosome extraction and identification
Exosomes were isolated as previously reported.10 ucMSCs were cultured to 80% confluence in the complete medium, washed three times with PBS, and subsequently cultured with exosomes free DMEM for 48 hours. The conditioned medium was collected and centrifuged at 1500 g for 30 min to remove apoptotic bodies and cell debris followed incubation with RiboTM Exosome Isolation Reagent (RiboBio,China) for 12 hours at 4℃. The supernatant was centrifuged at 2,000 g for 30 min. The supernatant was discarded, and the pellet was suspended in PBS and stored at -80℃. BCA kit (Thermo, USA) was used to analyze concentration. Western blotting was used to identify surface markers of exosomes including TSG101 (14497), CD63 (25682) and CD81 (66866) (Proteintech, USA). Transmission electron microscopy (TEM, HITACHI, H-600IV, Japan) Nanoparticle Tracking Analysis (NTA, Malvern Instruments, UK) were used to determine particle morphology and the particle size distributions of isolated exosomes.
To evaluate whether exosomes could be absorbed by cells, 1 µmol Dil (a red fluorescent cell linker for general cell membrane labeling) was used for labeling exosome, and then Dil labeled exosomes were incubated with target cells for 6 hours and 24 hours. Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI). Confocal images were taken by Zeiss laser-scanning confocal microscope (LSM5 Live, Carl Zeiss, Germany).
RNA extraction and quantitative real-time PCR (qRT-PCR)
RNA was extracted from exosomes and cells by TRIzol (Invitrogen, Carlsbad, CA, USA) according to the protocols of manufacturer. Spectrophotometer (NanoDrop-2000, ThermoFisher Scientific) was to inspect the quantity and quality of RNA. Then miRNAs were reverse transcribed by miRNAs reverse transcription kit (Applied Biosystems) by using thermal circulatory apparatus (Applied Biosystems, Foster City, CA, USA). qRT‐PCR was conducted using a SYBR® Green PCR Master Mix (Applied Biosystems) following the instructions. PCR cycling conditions were 95 °C for 5 minutes, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. Gene expression data were standardized with the values for Cel-mir-39 (exosomal) and U6 (cellular). The sequences of primers used in the study were shown in Supplementary Table 1.
Western blotting
Cells were lysed in a lysis buffer (Cell Signaling Technology, USA) supplemented with protease inhibitors (Calbiochem, USA) at 4 °C for 30 min, while the exosomes were lysed in 20 µL lysis buffer at 4 °C for 10 min. Total protein concentration was quantified using the BCA protein assay kit (Pierce, USA). Western blotting was performed according to the standard protocol as previously described.12 Antibodies used were as follows: phosphorylated-AKT (p-AKT) (4060, Cell Signaling Technology), AKT (4691, Cell Signaling Technology); Cleaved caspase-3 (29034, Signalway Antibody), Bcl-2 (ab196495, Abcam), vascular endothelial growth factor (VEGF, ab52917, Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174, Cell Signaling Technology) and horseradish peroxidase-conjugated secondary antibody (Biosharp, China). The bands were visualized by using enhanced chemiluminescence reagents and analyzed with a gel documentation system (iBrightCL1000, Invitrogen and Image Lab Software version 3.0).
Exosomal miRNA sequencing
The miRNAs sequencing was carried out in MIF-Exo, MSC-Exo and siMIF-Exo. Exosomal miRNA-seq analysis was performed by RiboBio (Guangzhou, China) using the Illumina HiSeq 2500 instrument. Differentially expressed miRNAs were identified through |log2(fold change)| ≥ 1 and P-value < 0.05 with the threshold set for up and down regulated genes. Bioinformatics analyses including differentially expressed miRNA analysis, prediction of target genes of miRNA, gene ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were also performed by RiboBio.
miRNAs transfection
Transfection of miR-133-3p mimics (50 nmol/L), miR-133-3p inhibitors (100 nmol/L) and their negative controls (50–100 nmol/L) were carried out using Lipofectamine 2000 (Invitrogen, USA) according to the instructions of manufacturer. Briefly, cardiac cells were cultured to 70% confluence. miR-133a-3p mimics, miR-133a-3p inhibitor and their negative controls were mixed with a transfection reagent, then added to the cell culture at a final concentration of 50–100 nmol/L. Transfection efficiency was determined by performing qRT-PCR. The sequences of miRNAs transfected were shown in Supplementary Table 2.
Lentiviral constructions and infection
The lentivirus construction in this study was obtained from GENECHEM (Shanghai, China). Two lentiviruses recombinant vectors were constructed. One is pLenti-EF1a-P2A-Puro-NRCMV-MIF-3Flag, used as MIF (overexpression of MIF) virus; and the other, hU6-MCS-Ubiquitin-EGFP-IRES-puromycin, served as siMIF (knocking down of MIF) virus. ucMSCs were seeded in 24 wells plates, when reached 50% confluence, they infected with MIF overexpression lentivirus or MIF knockdown lentivirus. Pools of stable transductions were generated by selection using puromycin (0.75 µg/ml) for three days. Fluorescent signals were viewed under the microscope. The MIF expression was evaluated by western blotting.
Co-culture experiment
HUVECs or H9C2 cardiomyocytes were seeded in 6 wells or 24 wells plates, once reached 70% − 80% confluence, cells were incubated with PBS, MIF-Exo, MSC-Exo, and siMIF-Exo (100 µg/mL) for 24 hours. After incubation, the cells were subjected to hypoxia and serum deprivation (H/SD) for another 12 hours, and they were evaluated by further experiments.
Tube formation of HUVECs assay
Angiogenesis of HUVECs was assessed by using the capillary tube forming assay. Then HUVECs (30,000 cells/well) were seeded in 96 wells plates covered with growth factor reduced Matrigel (356230; BD Biosciences, San Jose, CA, USA). After 6 hours, capillary like tube formation was photographed. Tube length was quantified by Image J software (National Institutes of Health, NIH).
EdU and migration assay
For proliferation evaluation, HUVECs were labeled with EdU for detection as described previously.12 HUVECs (2 × 104 cells/well) were seeded in 24 wells plates and they were incubated with EdU labeling reagent (Invitrogen, in 1:1,000 dilution) for 24 hours. Then cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and treated with Triton X-100 for another 20 min, and then washed three times with PBS. Click‐iT EdU Alexa Fluor 555 Imaging Kit (Invitrogen) was used according to manufacturer's instructions. Finally, cells were stained with 4′, 6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich, St. Louis, MO, USA). Images were analyzed by Image J software.
For migration evaluation, treated HUVECs were cultured in 6 wells plates at the density of 2 × 105 cells/well with 1 mL test medium. After 24 hours, washed with PBS three times, and then scratched using a P200 pipette tip. Twelve hours later, cell migration was observed by microscopy and analyzed by using Image J software.
Flow cytometry analysis of apoptosis
Apoptosis was assayed by flow cytometry as described previously.40 For apoptosis analyzes, cells were harvested with trypsin, fixed with ice cold 70% ethanol. Apoptosis was assayed with Annexin V-fluorescein isothiocyanate and propidium iodide staining (3801660; Sony Biotechnology, San Jose, CA, USA). Data were analyzed with a CELL Quest kit (BD Biosciences).
TUNEL analysis of apoptosis
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used for cell and tissue apoptosis. A TUNEL apoptosis detection kit (Roche, USA) was used to assay apoptosis. All cell nuclei were stained with DAPI. Apoptotic cells were dyed with TUNEL positive nuclei. Samples were examined with a microscope (Zeiss LSM510 META, German). The percentage of apoptotic nuclei was calculated for further analysis.
MI model establishing and exosomes injection
Our animal study protocol conforms to the Guide for the Care and Use of Laboratory Animals [National Institutes of Health, (NIH) Bethesda, MD, USA] and is approved by the Institutional Animal Care and Use Committee of the Nanjing Medical University for Laboratory Animal Medicine. Sprague Dawley rats (male, 6–8 weeks) were provided from Animal Core Facility of Nanjing Medical University (Nanjing, China). MI models were performed as previously described.10 Rats were aneesthesia with intraperitoneal injection of sodium pentobarbital (50 mg/kg), then the left anterior descending coronary artery was ligated at 1.5 mm below the level of the inferior margin of the left auricle. After MI models successful establishing, exosomes (50ug) were equally divided into 4 portions for injection at the border of the infarction area.
Assessment of cardiac function
Cardiac function was evaluated with transthoracic echocardiography (Vevo 2000 high-resolution micro-imaging system) 14 days and 28 days after exosomes therapy using isoflurane inhalation (1.5%-2%). Left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were analyzed using the Vevo 2000 workstation software.
Masson trichrome staining
Slides from paraffin embedded heart tissues were stained by Masson’s trichrome to detect fibrosis. Infarct size was evaluated as the average ratio of fibrosis area to the total ventricular area. Images were captured by scanning electron microscope (SU8010, Japan) and analyzed with Image J software.
Immunofluorescence
Immunofluorescence was performed as previously described.12 In brief, heart tissues were collected, fixed with 4% PFA, embedded in paraffin, and sectioned. For Immunofluorescence analyses, heart sections were stained with primary antibodies against CD31 (ab7388, Abcam, Cambridge, United Kingdom) and anti-Actin (A2066, Sigma-Aldrich). DAPI was used for nuclear counterstaining.
Statistical analysis
Continuous variables and categorical variables were described by means ± SEM and percentages. Independent Sample t-test was used to compare continuous variables between the two groups. Statistical differences among more than two groups were assessed by One-way ANOVA with the Bonferroni test. Chi-squared test was used for comparison between dichotomous variables. A value of P < 0.05 was considered statistically significant.