Animals
Sprague‒Dawley (SD) rats have been used on this research. MSCs have been obtained from male SD rats (60–80 g), and feminine SD rats have been used for AMI modeling (200–220 g). The rats have been housed in an SPF animal room at a temperature of twenty-two ~ 25 °C and humidity of 55 ~ 60% with a 12 h/h darkish/gentle cycle. All animals acquired humane care, and the experimental protocol was authorized by the Huazhong College of Science and Know-how Animal Care and Use Committee.
Preparation of the CMBs
The cationic microbubbles (CMB) coated with 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] ldl cholesterol (DC-CHOL) (Avanti Polar Lipids Inc., Alabaster, AL, USA) with an octafluoropropane (C3F8) fuel core (Foshan Huate Fuel Co. Ltd., Foshan, China) have been ready by the thin-film hydration and sonication technique as described in our earlier research [19]. Briefly,
DPPC, DC-CHOL and DSPE-PEG2000 have been weighed and dissolved in a flask with chloroform at a molar ratio of 10:4:1. A lipid movie was shaped by eradicating the natural solvent from the phospholipid answer with a rotary evaporator underneath excessive vacuum strain. The combination was incubated underneath vacuum and dry situations in a single day. Phosphate-buffered saline (PBS) was added, and the combination was then hydrated on a rotary evaporator. The combination was sonicated with octafluoropropane (C3F8) fuel to arrange the CMB suspension. The C3F8 fuel was bubbled by way of the answer for 1 min. The combination was then sonicated (20 kHz, 33–42 W). The lipid shell of the impartial microbubbles (NMBs) on this research consisted of DPPC and DSPE-PEG2000 at a molar ratio of 5:2. After preparation, PBS was used to regulate the focus of the microbubbles to 1 × 109 microbubbles/ml. Subsequently, the MB answer was sterilized by 60Co-γ radiation and saved at 4 °C till additional use.
Characterization of the CMBs
A lightweight microscopy picture of the CMBs was obtained with an optical microscope (mannequin IX70, Olympus Inc., Melville, NY, USA). The floor morphologic traits of the CMBs have been decided by transmission electron microscopy (TEM, Hitachi H-7000FA, Japan). The scale distribution, focus and zeta potential of the CMBs have been measured utilizing a Zetasizer NANO ZS system (Malvern Devices Ltd., Malvern, UK). Bubble stability in answer was analyzed and measured, and the imaging potential of the CMBs at totally different concentrations was assessed in vitro with a do-it-yourself agarose phantom. The imaging properties of the CMBs for rat hearts have been evaluated in vivo. Photographs have been acquired and analyzed utilizing a medical IU22 ultrasound scanner (Philips Medical Techniques, Amsterdam, Netherlands).
Cell tradition and conditioned medium assortment
MSCs from the femoral and tibial bone marrow of SD rats have been remoted and cultured as beforehand described [20]. Briefly, 3-week-old wholesome male rats (60–80 g) have been anesthetized (1% pentobarbital sodium, 40 mg/kg) and sacrificed by cervical dislocation. The bone marrow cavities of the tibia and femur have been flushed with serum-free Dulbecco’s Modified Eagle Medium (DMEM) with F12 (Gibco, Grand Island, NY, USA), and the flushing fluid was collected. After centrifugation, the cells suspended in DMEM/F12 medium containing streptomycin (100 g/mL), penicillin (100 U/mL) and 10% FBS (fetal bovine serum; Gibco) have been cultured at 37 °C in moist air containing 5% CO2. The medium was modified each two days to take away nonadherent cells. MSCs have been noticed utilizing an optical microscope (mannequin IX70, Olympus Inc., Melville, NY, USA). The phenotypic properties of MSCs have been recognized by movement cytometric evaluation for the expression of the everyday markers CD90 and CD29 (BD Biosciences) and the absence of the hematopoietic markers CD45 (eBioscience) and CD34 (Santa Cruz Biotechnology). MSCs at passages 3 and 4 have been used for the next experiments. The cells have been seeded at 5 × 105 cells per 10-cm plate to arrange conditioned medium (CM) from MSCs. When the MSCs reached 80% confluence, they have been positioned in serum-free medium for twenty-four h. The CM was then collected for in vitro experiments. Equally, MSCs have been cultured with H2O2 for six h earlier than MSC-CM use.
Cell migration assay
The impact of PDGF-BB on MSC migration was examined utilizing Transwell cell migration assay and in vitro scratch wound-healing assays. MSCs have been seeded in 6-well plates. When the cells reached 90% confluence, MSCs have been scratched with a 200-µl pipette and washed with PBS. MSCs have been incubated with or with out PDGF-BB at totally different concentrations (R&D Techniques) in serum-free DMEM/F12 for twenty-four h. Photographs of the scratched gaps have been captured at 0 and 24 h with an inverted microscope (IX70, Olympus Inc., Melville, NY, USA), and the cell migration space was measured utilizing ImageJ software program (NIH, USA).
Subsequent, MSCs have been subjected to Transwell assays as described beforehand [21]. Briefly, MSCs (1 × 105 cells per properly) have been handled with PDGF-BB on the optimum focus (R&D Techniques) or car and have been then resuspended in DMEM/F12 with 1% FBS and seeded into the higher chamber of the Transwell system (8.0 μm pore measurement, Merck, USA). DMEM/F12 containing 10% fetal bovine serum (500 µL) with or with out 100 ng/ml SDF-1 (PeproTech, USA) was added to the decrease chamber. After 24 h of tradition within the cell incubator, the noninvading cells on the higher floor have been swabbed with a cotton swab. The inserts have been mounted in 4% paraformaldehyde at room temperature for 30 min and stained with crystal violet for 20 min. The variety of MSCs throughout the membrane was calculated in 5 randomly chosen areas underneath an optical microscope (IX70, Olympus Inc., Melville, NY, USA). In some experiments, a suspension of PDGF-BB-primed MSCs containing 50 µM LY294002 (a PI3K inhibitor, Abcam) or 44 nM AMD3100 (a CXCR4 inhibitor, Abcam) was added to the higher chamber to neutralize PI3K/Akt and CXCR4 bioactivity, respectively.
Cell apoptosis evaluation by movement cytometry
Cell apoptosis was analyzed utilizing an Annexin V-FITC Apoptosis Detection Equipment (eBioscience) following the producer’s directions. Briefly, the cells have been collected, washed with PBS and resuspended in 200 µl binding buffer. Subsequently, the cells have been blended with 5 µl Annexin V-FITC at room temperature for 10 min following incubation with 10 µl propidium iodide (PI). Lastly, early and late apoptosis have been analyzed utilizing flow-assisted cell sorting (FACS) (FACS Calibur, BD).
Rat mannequin of acute Myocardial Infarction
The AMI mannequin was established in feminine SD rats as beforehand described [22, 23]. Briefly, rats (200–220 g) have been 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 (LAD) coronary artery was ligated 2–3 mm from the tip of the left auricle with a 6/0 suture to set off AMI. Profitable ligation of the LAD was confirmed by the looks of a Q wave and S-T section elevation on an electrocardiogram. Sham-operated management rats underwent the identical surgical procedures besides that the suture positioned underneath the left coronary artery was not tied.
Cell labeling
MSCs have been labeled with DiR (Caliper Life Sciences, Hopkinton, MA, USA) or inexperienced GFP utilizing a lentiviral vector (Genechem Ltd, Shanghai, China) following the producer’s directions to allow cell tracing after transplantation in vivo.
MSC supply by UTMD
The distribution and homing of MSCs after UTMD-mediated supply have been then evaluated. SD rats have been randomly subjected to the next 4 remedies: Sham, PBS (1 mL) infusion solely; MI, PBS (1 mL) infusion solely; MI-MSC, 2 × 106 DiR- or GFP-labeled MSCs suspended in 1 mL of PBS infusion; and MI-MSC-UTMD, 2 × 106 DiR- or GFP-labeled MSCs suspended in 1 mL of PBS infusion. SD rats have been subjected to MI after which underwent UTMD adopted by intravenous injection of MSCs. MSCs have been labeled with DiR or GFP utilizing a lentivirus earlier than focused supply mediated by UTMD. Rats within the MI-MSC-UTMD group underwent UTMD adopted by cell transplantation. Rats within the different teams didn’t obtain UTMD.
The results of UTMD mixed with PDGF-BB pretreatment on MSC remedy have been additionally evaluated within the rat MI mannequin. SD rats have been divided into 4 teams: (i) sham, PBS (1 mL) infusion solely; (ii) MI, PBS (1 mL) infusion solely; (iii) MI-MSC-vehicle, 2 × 106 DiR- or GFP-labeled MSCs suspended in 1 mL of PBS infusion; and (iv) MI-PDGF-BB-MSC, 2 × 106 DiR- or GFP-labeled MSCs suspended in 1 mL of PBS infusion. MSCs have been pretreated with PDGF-BB (50 ng/ml) for twenty-four h and labeled with GFP utilizing a lentivirus earlier than supply by way of UTMD.
Rats within the teams handled with MSCs underwent UTMD adopted by cell transplantation. CMBs (120 µl/rat) have been subsequently diluted in saline to a complete quantity of 500 µl/rat. A micropump was used to infuse 0.5 mL of CMBs at a fee of 15 ml/h throughout ultrasound irradiation. UTMD was carried out utilizing a therapeutic ultrasound system (Sonitron 2000 V, Japan) at 1 MHz, 20% responsibility ratio and a couple of.0 W/cm2 output depth for 2 minutes directed to the anterior left ventricular wall. The acoustic window for every rat was confirmed by a diagnostic ultrasound system (IU22, Philips, Bothell, MA, USA). After UTMD, 2 × 106 DiR- or GFP-labeled MSCs with or with out PDGF-BB pretreatment have been injected by way of the caudal vein. Rats within the different teams with out MSC remedy additionally acquired UTMD previous to the 1 mL PBS injection. MSC transplantation mediated by UTMD was carried out at 2-day intervals between Day 1 and Day 5 post-MI. A dose of furosemide (0.4 mg/kg) was injected earlier than MSC transplantation to forestall congestive coronary heart failure attributable to quantity overload.
Ex vivo bioluminescent imaging
The histological distribution of DiR-labeled MSCs was monitored utilizing bioluminescent imaging (BLI) at 3 days after MI. All rats used for ex vivo imaging, have been fed an alfalfa-free weight loss program to cut back fluorescent noise. DiR-labeled MSCs have been intravenously injected into MI rats following UTMD. At 3 days post-MI, three rats from every group have been sacrificed, and the foremost organs have been harvested, together with the hearts, livers, spleens, lungs and kidneys. Then, ex vivo photographs of the organs have been captured and quantified utilizing a small animal imaging system (In-Vivo FX PRO, Bruker, USA) to visualise cell homing and distribution.
Detection and monitoring of GFP-labeled MSCs
A multimodal analysis technique was utilized to evaluate the retention of GFP-labeled MSCs comprising movement cytometry, qPCR and immunofluorescence staining. The movement cytometry assay was carried out following the transplantation of MSCs. The center samples have been finely minced, and a cell suspension was obtained by digestion with an answer of 1 mg/ml collagenase/dispase and 200 µg/ml DNase in alpha-MEM containing 5% FBS at 37°C with fixed agitation. Following digestion, the cells have been washed, and GFP-labeled cells have been quantified utilizing a Novocyte Circulate Cytometer (ACEA Bioscience, USA). The proportion of constructive cells was measured, and the variety of constructive cells was normalized to the overall variety of cells within the pattern. To validate the Novocyte Circulate Cytometer-based findings, quantitative real-time PCR of the Y-chromosome Sry-gene in infarcted myocardium was carried out as beforehand described [24]. Briefly, tissues have been processed, nucleic acids have been extracted, and gDNA concentrations and purities have been measured by UV absorbance. The male rat Sry gene sequence was detected on the background of feminine rat gDNA utilizing a TaqMan PCR equipment (Utilized Biosystems). The goal gene primer sequences have been 5’-CATCGAAGGGTTAAAGTGCCA-3’ and 5’-ATAG TGTGTAGGTTGTTGTCC-3’. The survival fee of transplanted cells in every group was calculated as beforehand described [24]. As well as, immunofluorescence staining was carried out to judge GFP-positive MSCs within the coronary heart. The hearts have been harvested and quickly frozen in liquid nitrogen. Serial 5 μm sections have been ready, stained with a-actin (Sigma, USA) and DAPI (Sigma, USA), and noticed underneath a laser scanning confocal microscope (mannequin IX70, Olympus Inc., Melville, NY, USA). The numbers of GFP-positive cells on every slide have been analyzed utilizing ImageJ software program (NIH, USA).
Echocardiography
Transthoracic echocardiography was carried out to judge left ventricular operate earlier than and 30 days after MI. A commercially obtainable echocardiographic system (GE, VIVID7, USA) outfitting a ten MHz transducer was employed by an investigator blinded to group designation.
Briefly, rats from totally different remedy teams have been anesthetized with 1% pentobarbital sodium (40 mg/kg) and positioned on the experimental platform. Two-dimensional guided M-mode tracings have been recorded from the parasternal long-axis view of the left ventricle. The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of the rats have been measured. Variations within the LVEF and LVFS have been calculated by subtracting the values at endpoints from baseline values. Dimension knowledge are introduced as the common measurements of three cardiac cycles.
Masson staining
Thirty days after MI, rats from totally different remedy teams have been anesthetized and perfused with regular saline. The center of every rat was eliminated rapidly, sectioned and stuck in 4% paraformaldehyde answer. Paraffin-embedded slides have been stained with Masson’s trichrome. Scar measurement (%) was measured as the proportion of fibrotic space within the whole left ventricular space. The determine (Masson trichrome stain, unique magnification ×200) was processed with ImageJ software program to calculate the % fibrosis within the peri-MI space. The thickness (µm) of the infarcted cardiac wall was calculated because the imply of three equidistant measurements on every part. Fluorescence microscopy (mannequin IX70, Olympus Inc., Melville, NY, USA) was used to seize photographs, and ImageJ software program (NIH, USA) was used for the evaluation.
Western blotting
Whole proteins have been extracted from MSCs or myocardial infarct tissue with radioimmunoprecipitation assay lysis (RIPA) buffer containing protease and phosphatase inhibitors for Western blotting as beforehand described [25]. The concentrations of proteins have been decided with a Bradford protein assay equipment (Bio-Rad, Richmond, CA, USA) utilizing BSA as a protein normal. Proteins have been separated by SDS 10% PAGE and transferred to polyvinylidene fluoride membranes (Amersham Biosciences, GE Healthcare, France). The membranes have been blocked in 3% milk for 1 h after which incubated with major antibody at 4 °C in a single day. Following major antibodies have been utilized on this research: 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 inner reference. HRP-linked rabbit IgG (#G1213, Servicebio) was used as a secondary antibody. The density of the respective bands was quantified utilizing a densitometer with Amount One software program (Bio-Rad).
TUNEL staining
A TUNEL apoptosis equipment (Roche Utilized Science, South San Francisco, CA, USA) was used to find out cardiomyocyte apoptosis in line with the producer’s instructions. Cardiomyocytes have been stained with cardiac troponin I (Abcam), and nuclei have been stained with DAPI (Sigma Aldrich, USA). Sections have been imaged utilizing a confocal microscope (IX70, Olympus Inc. Melville, NY, USA). The overall variety of nuclei and TUNEL-positive nuclei have been quantitated utilizing Picture-Professional Plus evaluation software program. The common variety of constructive cells per high-power discipline was calculated to evaluate cardiomyocyte apoptosis.
Immunofluorescence staining
Immunofluorescence staining of tissue sections was carried out as beforehand described [26]. Briefly, coronary heart tissues have been collected, mounted with 4% PFA, embedded in paraffin, and sectioned. For the immunofluorescence analyses, coronary heart sections have been stained with major antibodies in opposition to α-SMA (Abcam) and CD31 (BD Bioscience). DAPI was used for nuclear counterstaining. Photographs have been obtained with a fluorescence microscope (IX70, Olympus Inc. Melville, NY, USA). Quantification of all knowledge was carried out utilizing Picture-Plus picture evaluation software program.
Statistical evaluation
All experiments have been carried out at the least thrice, and consultant outcomes are proven. All outcomes are summarized because the imply ± normal error (M ± SEM). Unbiased two-sample t checks or one-way ANOVA have been used to judge the importance of the variations between totally different teams. The info have been analyzed utilizing SPSS 26.0 software program. P < 0.05 was thought-about to point significance.
