Animals
Sprague-Dawley (SD) rats were used in the study. MSCs were obtained from male SD rats (60-80g), and AMI models were created in female SD rats (200-220g). All animals received humane care and the experimental protocol was approved by the Huazhong University of Science and Technology Animal Care and Use Committee.
Preparation of the CMBs
The cationic microbubbles (CMB) consisting of a coating of 1,2-distearoyl-sn-glycerin-3-phosphate choline (DSPC), 1,2-distearoyl-sn-glycerin-3- phosphate ethanolamine-n-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000) and 3-[N-(N', N'-dimethyl-lamino-ethane)-carbamoyl] cholesterol (DC-CHOL) (Avanti Polar Lipids Inc., Alabaster, AL, USA) with a octafluoropropane (C3F8) gas core (Foshan Huate Gas Co. Ltd., Foshan, China) were prepared by the thin-film hydration and sonication method as described in our previous study [19]. The lipid shell of the neutral microbubbles (NMB) in this study consisted of DPPC and DSPE-PEG2000. After preparation, PBS was used to adjust the concentration of the microbubbles to 1×109 microbubbles/ml. Subsequently, the microbubbles solution was sterilized by 60Co-γ radiation and stored at 4°C until further use.
Characterization of the CMBs
A light microscopic image of the CMBs was taken with an optical microscope (model IX70, Olympus Inc., Melville, NY, USA). The surface morphologic characteristics of the CMBs were detected by transmission electron microscopy (TEM, Hitachi H-7000FA, Japan). The size distribution, concentration and zeta potential of the CMBs were measured using a Zetasizer NANO ZS system (Malvern Instruments Ltd., Malvern, UK). Bubble stability in solution was analyzed, measured, and the imaging ability of the CMBs at different concentrations was assessed with a homemade agarose phantom in vitro. Also, the imaging properties of the CMBs for rat hearts were evaluated in vivo. The images were acquired and analyzed using a clinical IU22 ultrasound scanner (Philips Medical Systems, Amsterdam, Netherlands).
Cell culture and conditioned medium collection
MSCs from femoral and tibial bone marrow of SD rats were isolated and cultured as previously described [20]. Briefly, 3-week-old healthy male rats (60-80g) were anesthetized (1% pentobarbital sodium, 40mg/kg) and killed with cervical dislocation. The bone marrow cavities of tibia and femur were flushed with serum-free DMEM/F12 medium (Gibco, Grand Island, NY, USA), and the flushing fluid was collected. After centrifugation, the cells suspended in DMEM/F12 medium containing streptomycin (100g/mL), penicillin (100 U/mL) and 10% FBS (fetal bovine serum; Gibco) were cultured at 37°C in wet air containing 5% CO2. The medium was changed every two days to remove nonadherent cells. MSCs were observed using an optical microscope (model IX70, Olympus Inc., Melville, NY, USA). The phenotypic properties of MSCs were identified by flow cytometric analysis for the expression of the typical markers, CD90 and CD29 (BD Biosciences), and the absen ce of the haematopoietic markers CD45 (eBioscience) and CD34 (Santa Cruz Biotechnology). MSCs at passage 3, 4 were used for experiments. To prepare CM from MSCs, the cells were seeded at 5×105 cells per 10-cm plate. MSCs reached 80% confluence and were then placed in serum-free medium for 24h. The conditioned medium (CM) was then collected for in vitro experiments. Similarly, MSCs and H2O2 were co-cultured for 6h before the obtained MSC-CM could be used.
Cell migration assay
The effect of PDGF-BB on MSCs migration was examined using both the transwell cell migration assay and the in vitro scratch wound-healing assay. MSCs were seeded in 6-well. When the cells grew to 90% confluence, MSCs were scratched with a 200-µl pipette and washed with PBS. MSCs were incubated with or without PDGF-BB (50 ng/ml, R&D Systems) in serum-free DMEM/F12 for 24 h. Images of scratched gaps were captured at 0 and 24 h with an inverted microscope (IX70, Olympus Inc., Melville, NY, USA), and the cell migration area was measured using ImageJ software (NIH, USA).
Next, MSCs were subjected to transwell assays as described previously [21]. Briefly, MSCs (1×105 cells per well) were treated with PDGF-BB (50 ng/ml, R&D Systems) or vehicle, and were then resuspended in DMEM/F12 with 1% FBS, and seeded into the upper chamber of the transwell system (8.0 µm pore size, Merck, USA). DMEM/F12 containing 10% fetal bovine serum (500 µL) with or without 100ng/ml SDF-1 (PeproTech, USA) was filled in the lower chamber. After 24 h of culture in the cell incubator, the non-invading cells on the upper surface were swabbed with a cotton swab. The inserts were fixed in 4% paraformaldehyde at room temperature for 30 min and stained with crystal violet for 20 min. The number of MSCs across the membrane was calculated in five randomly selected regions under an optical microscope (IX70, Olympus Inc., Melville, NY, USA). In some experiments, the suspension of PDGF-BB-Primed MSC containing 50 µM LY294002 (a PI3K inhibitor, Abcam) or 44 nM AMD3100 (an CXCR4 inhibitor, Abcam) was added into the upper chamber to neutralize PI3K/Akt and CXCR4 bioactivity respectively.
Cell apoptosis analysis by Flow cytometry
Cell apoptosis was analyzed using Annexin V-FITC Apoptosis Detection Kit (eBioscience), following the manufacturer’s instructions. Briefly, the cells were collected and washed with PBS and then resuspended in 200 µl binding buffer. Consequently, cells were mixed with 5 µl Annexin V-FITC at room temperature for 10 min following the incubation with 10 µl propidiumIodide (PI). Finally, early and late apoptosis were analyzed using FACS cytometry (FACS Calibur, BD).
Rat model of acute myocardial infarction
The AMI model was established in female SD rats as previously described [22–23]. Briefly, rats (200–220 g) were anesthetized with 1% Pentobarbital Sodium (40 mg/kg, administered intraperitoneally), intubated and ventilated. then, the left thoracic cavity was opened, and the left anterior descending coronary artery was ligated 2–3 mm from the tip of the left auricle with a 6/0 suture to trigger AMI. Successful ligation of the LAD was confirmed by the appearance of a Q wave and S-T segment elevation on an electrocardiogram. Sham-operated control rat underwent the same surgical procedures except that the suture placed under the left coronary artery was not tied.
Cell labelling
To trace the cells after transplantation in vivo, MSCs were labeled with DiR (Caliper Life Sciences, Hopkinton, MA, USA) or green GFP using a lentiviral vector (Genechem Ltd, Shanghai, China) following to the manufacturer’s protocol.
MSCs delivery by UTMD
Rats in those groups treated with MSCs, underwent UTMD followed by cell transplantation. CMBs (120 µl/rat) were subsequently diluted in saline to a total volume of 500 µl/rat. A micropump was used to infuse 0.5 mL of CMBs at a rate of 15 ml/h during ultrasound irradiation. UTMD was performed using a therapeutic ultrasound system (Sonitron 2000V, Japan) at 1 MHz, 20% duty ratio and 2.0 W/cm2 output intensity for two minutes directed to the anterior left ventricular wall. The acoustic window for each rat was confirmed by a diagnostic ultrasound system (IU22, Philips, Bothell, MA, USA). After UTMD, 2×106 DiR or GFP labelled MSCs were injected through the caudal vein. Rats in other groups without MSCs treatment, also received UTMD prior to receiving 1 mL PBS injection. MSC transplantation mediated by UTMD was performed at 2-day intervals. To prevent congestive heart failure because of the volume overload, a dose of furosemide (0.4 mg/kg) was injected before MSC transplantation.
Ex Vivo Bioluminescent Imaging
The histological distribution of DiR-labelled MSCs were monitored using BLI at 3 days after MI. To reduce fluorescent noise, all rats used for ex-vivo imaging were fed an alfalfa-free diet. DiR-labeled MSCs were intravenously injected into MI rats following UTMD. At 3 days post-MI, three rats from each group were sacrificed, and major organs including hearts, livers, spleens, lungs and kidneys were harvested. Then ex vivo images of the organs were captured and quantified using a small animal imaging system (In-Vivo FX PRO, Bruker, USA) to locate cell homing and distribution visually.
Detection and tracking of GFP labelled MSCs
To assess the retention of GFP labelled MSCs, a multimodal evaluation strategy was applied comprising Flow Cytometry, qPCR and Immunofluorescence staining. The flow cytometry assay was carried following transplantation of MSCs. Here, heart samples were finely minced and a cell suspension was obtained by digestion with a solution of 1 mg/ml Collagenase/Dispase and 200 µg/ml DNase in alpha-MEM containing 5% FBS, at 37°C, with constant agitation. Following digestion, cells were washed, GFP labeled cells were quantified by a Novocyte Flow Cytometer (ACEA Bioscience, USA). Percentage of positive cells was measured and the number of positive cells was normalized to total number of cells in the sample. To validate Novocyte Flow Cytometer-based findings, quantitative real-time PCR of the Y-chromosome sry-gene in infarcted myocardium was performed as previous described [24]. Briefly, tissues were processed, nucleic acids were extracted and gDNA concentrations and purities measured by UV absorbance. The male rat SRY gene sequence was detected in a background of female rat gDNA using a Taqman PCR kit (Applied Biosystems). The target gene primer sequences were 5'-CATCGAAGGGTTAAAGTGCCA-3' and 5'-ATAG TGTGTAGGTTGTTGTCC-3'. The survival rate of transplanted cells in each group was calculated as described in a previous study [24]. In addition, to evaluate GFP positive MSCs in heart, the immunofluorescence staining was also performed. The hearts were harvested and rapidly frozen in liquid nitrogen. Serial sections were prepared at 5µm thickness, stained with a-actin (Sigma, USA) and DAPI (Sigma, USA), and observed under a laser scanning confocal microscope (model IX70, Olympus Inc., Melville, NY, USA). The numbers of GFP-positive cells in each slide were analyzed using ImageJ software (NIH, USA).
Echocardiography
Transthoracic echocardiography was performed to evaluate left ventricular function before and 30 days after MI. A commercially available echocardiographic system (GE, VIVID7, USA) outfitting a 10 MHz transducer was employed by an investigator blinded to group designation.
Briefly, rats from different treatment groups were anesthetized with 1% pentobarbital sodium (40 mg/kg) and placed on the experimental platform. Two-dimensional guided M-mode tracings were recorded from parasternal long-axis view of the left ventricle. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of rats were measured. Dimension data are presented as the average of measurements of three cardiac cycles.
Masson staining
Thirty days after MI, rats from different treatment groups were anesthetized and perfused with normal saline. The heart of each rat was taken out quickly, sectioned and fixed in 4% paraformaldehyde solution. Paraffin-embedded slides were stained by Masson's trichrome. Infarcted area was measured as the percentage of fibrotic area in the total left ventricular area. The thickness of infarcted cardiac wall was calculated as the mean of three equidistant measurements on each section. Fluorescence microscopy (model IX70, Olympus Inc., Melville, NY, USA) was used to catch images and ImageJ software (NIH, USA) to analyze them.
Western Blot Analysis
Total proteins were extracted from MSCs or myocardial infarct tissue with RIPA buffer containing protease and phosphatase inhibitors for western blotting as previously described [25]. The concentrations of proteins were determined with a Bradford protein assay kit (Bio-Rad, Richmond, CA, USA) using BSA as a protein standard. Proteins were separated by SDS 10% PAGE and transferred to polyvinylidene fluoride membrane (Amersham Biosciences, GE Healthcare, France). The membrane was sealed in 3% milk for 1 h and then incubated with primary antibody at 4°C overnight. Following primary antibodies were utilized in this study: PI3K (#AF6241, Affinity), p-PI3K (#AF3242, Affinity), AKT (#10176-2-AP, proteintech), p-AKT (#AF0016, Affinity), ERK (#11257-1-AP, proteintech), p-ERK (#28733-1-AP, proteintech), CXCR4 (#AF5279, Affinity), Caspase 3 (#9662, Cell Signaling), cleaved-Caspase 3 (#9664, Cell Signaling), Bax (#BA0315-2, BOSTER), BCL-2 ((#MA00040, BOSTER), VEGF (#BA0407, BOSTER), bFGF (# CY3239, Abways), and IGF-1 (ab182408, Abcam) with GAPDH (#GB11002, Servicebio) as internal reference. An HRP Linked Rabbit IgG (#G1213, Servicebio) was used as a secondary antibody. The density of the respective bands was quantitated using a densitometer with software Quantity One (Bio-Rad).
TUNEL staining
TUNEL apoptosis kit (Roche Applied Science, South San Francisco, CA, USA) was applied to determine cardiomyocyte apoptosis according to manufacturer’s protocol. Cardiomyocytes were stained with Cardiac Troponin I (Abcam) and nucleus were stained with DAPI (Sigma Aldrich, USA). Sections were imaged using confocal microscope (IX70, Olympus Inc. Melville, NY, USA). The total number of nuclei and the number of TUNEL-positive nuclei were quantitated using Image-Pro Plus analysis software. The average number of positive cells per square millimeter (mm2) was calculated to assess cardiomyocyte apoptosis.
Immunofluorescence Staining
Immunofluorescence staining of tissue sections was performed as described previously [26]. 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 α-SMA (Abcam)and CD31 (BD Bioscience). DAPI was used for nuclear counterstaining. Images were taken with the fluorescence microscope (IX70, Olympus Inc. Melville, NY, USA). Quantification of all data was performed with Image Plus image analysis software.
Statistical analysis
All experiments were performed at least three times and representative results are shown. All results are summarized as the mean ± standard deviation (M ± SD). Independent two sample t-test or one-way ANOVA were used to evaluate statistical significance of differences between different groups. The data were analyzed using SPSS 26.0 software. P < 0.05 was considered statistically significant.