Animal study protocol.
We purchased 30 8–12 weeks old male C57BL/6 wild-type mice (body weight: 25–30 gm) from the Laboratory Animal Center of the Fourth Military Medical University. The mice were anesthetized on a C57BL/6 background with 2% isoflurane. After 25 min, ADSC-Exos (100 µg protein, 50 µL) was administered evenly intramuscularly into five locations along the anterior wall of the left ventricle’s border zone. The slipknot was released after 40 min to reperfuse the myocardium. All animal experiments were approved by the Animal Care and Use Committee of the Fourth Military Medical University and followed the National Institutes of Health guidelines for the use of laboratory animals (National Institutes of Health Pub. No. 85 − 23, Revised 2011). The hearts were collected after 4 weeks and fixed with paraformaldehyde or further analysis.
Adsc Preparation
ADSCs were extracted according to the method described previously2. Under anesthesia, mice inguinal subcutaneous fat was harvested. The adipose tissue was washed several times with sterile phosphate-buffered saline (PBS, Sigma) and then the blood vessels in the adipose tissue were removed with the aid of a dissecting microscope. The remaining adipose tissue was digested with 0.1% type I collagenase (catalog number 17018029, ThermoFisher Scientific, USA) at 37°C for 60 min and then centrifuged at 1000 g for 10 min, non-adherent cells were removed 48 h after the cells were plated. Then, ADSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing 20% Fetal Bovine Serum (FBS) and penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Cells from passage 2 were used for all experiments.
Cell Culture
Microvascular endothelial cells were purchased from the American Type Culture Collection (ATCC). Cell culture medium contains DMEM medium (Life Technologies, Grand Island, NY, USA) and 10% heat-inactivated fetal bovine serum (Hyclone, UT, USA). All the cells were incubated in a 37°C humidified incubator containing 5% CO2.
Echocardiography
Cardiac function of the mice subjected to I/R after 6 h and 4 weeks were evaluated by echocardiography, as previously described2. Mice were anesthetized by inhalation of 1–2% isoflurane, and transthoracic two-dimensional exercise mode echocardiography (VisualSonics) was performed. The Vevo770 software program (VisualSonics) was used to collect and analyze LV end-systolic dimensions (LVESD), LV end-diastolic dimensions (LVEDD) and LV ejection fraction (LVEF) parameters.
He And Masson Staining
Mice were executed and the hearts were isolated. For histological analysis of angiogenesis and fibrosis, HE and Masson staining were performed, a total of 10 sections (7–10 um thick) per heart were prepared2. HE staining of the sections were performed according to the manufacturer's instruction. Masson’s trichrome was used to evaluate fibrosis in post-I/R mice hearts. The percentage of fibrotic area to total heart represents myocardium fibrosis in post-MI mice hearts. Myocardial fibrosis was quantified by means of Image-Pro plus 6.0 software (Media Cybernetics).
Immunofluorescence Staining
All the sections were blocked with 1% goat serum albumin for 1 h and then incubated with mouse monoclonal anti-CD31 primary antibody (Ab955, 1:200; Abcam) at 4°C overnight. The sections were then stained with rabbit anti-mouse secondary antibody (1:1000; Abcam) for 1 h at room temperature. The tissue slices were washed and mounted with medium containing DAPI. All slices were observed by the Olympus FV1000 laser confocal microscope.
Western Blot Analysis
Western Blot Analysis
Protein was extracted from heart or cultured microvascular endothelial cells and ADSCs according to standard Invitrogen protocols (Invitrogen, Carlsbad, CA, USA) as previously described2. Protein quantitation was modified by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) and then separated by SDSPAGE with primary antibodies against The blots were incubated with primary antibodies as follows: HIF-1α(ab179483; Abcam), VEGF (ab32152; Abcam), CD63 (ab217345; Abcam), CD9 (ab223052; Abcam), TSG101 (ab235011; Abcam), β-actin (ab8226;Abcam), Caspase3 (ab184787; Abcam) overnight at 4◦C. The blots were visualized using a chemiluminescence system (Amersham Bioscience, Buchinghamshire, UK). Anti-β actin antibody (Proteintech, IL, United States) was used as a loading control. The signals were quantified by Image J software.
Extraction And Characterization Of Adsc-exos
Cultured ADSCs were plated at 5 × 105 cells in a 10-cm dish. The culture medium was collected and centrifuged at 13,000 g for 30 min to remove cells and cell debris. According to the manufacturer's instructions (System Biosciences, CA, United States), 2 mL of ExoQuick-TCTM exosome precipitation solution was used to isolate ADSC-Exo from 10 mL of culture medium. After overnight incubation at 4°C, the mixture was centrifuged at 10,000 g for 30 minutes at 4°C. After being washed, the exosomes were centrifuged at 10,000 g for 15 minutes at 4°C. Suspended the purified ADSC-Exos with 100 µL PBS and stored at -80°C for further study. The protein extracted from the exosomes was quantified using the Bradford method. And then biomarker CD63 and CD9 were used to characterize the purified ADSC-Exos. The morphology of ADSC-Exo was detected through the transmission electron microscope (Hitachi, Tokyo, Japan). The size of ADSC-Exo was evaluated by Nanoparticle tracking analysis (NTA) analysis.
Wound Healing Assay
Wound healing was used to measure proliferation, microvascular endothelial cells were plated at 3,000 cells/well in 96-well plates and treated with ADSC-Exos or vehicle for 3 days. Then the treated microvascular endothelial cells were seeded onto Culture Insert 2 Well in -Dish 35 mm (no. 81176, Ibidi). After administration, cells were cultured in serum-free medium overnight. Then removed the culture insert to create an ~ 500um cell-free gap, and covered the dish with culture medium. Cellular migration was visualized at the indicated time points. The width of the open area at each time point versus the width at time 0 was used to determine the extent of wound healing ratio.
Assessment Of Apoptosis
TdT-Mediated dUTP Nick End-Labeling (TUNEL) Assay kit (In Situ Cell Death Detection Kit, Roche, CA, USA) was used to detect the apoptosis of cardiomyocytes in MI-treated mice. The percentage of TUNEL-positive nuclei relative to the total nucleus represents the apoptosis level of cardiomyocytes. In addition, the apoptotic microvascular endothelial cells were evaluated using flow cytometry.
Admsc Oxidative Damage Measurement
Oxidative stress is characterized by the production of reactive oxygen species (ROS). They represent the injury of ADSCs in the setting of oxidation stress. The intracellular ROS production of ADSCs was assessed using 10 µM dihydroethidium (DHE; Invitrogen, San Diego, CA, USA). The ADSCs were treated with DHE, incubated in a dark environment, 37℃ and viewed using confocal laser microscopy (Olympus).
Cell Viability Assessment
Cells were harvested 24 h after reoxygenation and incubated in a culture medium containing 10 µL of Cell Counting Kit-8 (CCK-8; Sigma Aldrich, Darmstadt, Germany) solution for another 2 h. Subsequently, the optical density was measured at a wavelength of 450 nm, and the cell proliferation rate was calculated.
Statistical analysis
All data are presented as mean ± the standard error of the mean. Graphpad Prism 8.0 software was used for statistical analyses. For the analysis of two groups, unpaired two-tailed Student t-tests were conducted. When more than two groups were compared, one-way analysis of variance with post hoc analysis was performed. P values of 0.05 were considered statistically significant.