Sequence analysis of EPC-Exos miRNAs
Total RNA from EPCs and EPC-Exos was isolated by means of a mirVana miRNA isolation kit (Ambion, Austin, TX, USA). One biological repeat with three mixed samples for EPCs and EPC-Exos was used for the sequence analysis. The concentration and quality of RNA samples were detected with an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA), and 50 ng RNA samples were fragmented and used for miRNA sequencing on Genome Analyzer IIx (Illumina, San Diego, CA, USA). After generating the sequences, a BLASTN search was utilized to identify conserved miRNAs, and Rfam was employed (European Molecular Biology Laboratory, Heidelberg, Germany) for deletion of non-miRNA sequences. MiRDeep2 was used to predict novel miRNAs. The length distribution of the cells and exosomal miRNAs was analyzed, and miRNAs with differential expression between cells and exosomes were detected as described previously [30]. Gene Ontology (GO) categories [31] and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses [32] were utilized to investigate specific functional roles of differentially expressed miRNAs. To determine whether the expression changes in miR-218-5p and miR-363-3p were influenced by the number of exosomes secreted from EPCs, GW4869 (an inhibitor of neutral sphingomyelinase 2, 10 µM for 12 h, MCE China) was used to treat EPCs, and then, EPC-Exos were isolated using a Total Exosome Isolation Reagent Kit (4478359, Thermo Fisher, USA).
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA samples were extracted from EPCs, EPCs-Exos and tissues around an infarct using Trizol reagent (Takara, Dalian, Chia). The synthesis of cDNA used for gene and miRNAs was performed using a Bestar™ qPCR RT kit (DBI; #DBI-0) and Bestar™ miRNA qPCR RT kit (DBI; #2220), respectively. The relative mRNA levels of α-SMA, vimentin, CD31, VEGFR-2p53, JMY and miRNAs were determined via real-time quantitative PCR using a 20 µL reaction system. PCR was performed on an ABI PRISM 7300 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) using the following conditions: 95℃ for 2 min, followed by 40 cycles of 94℃ for 20 sec, 58℃ for 20 sec and 72℃ for 20 sec. GAPDH or U6 was used as the internal reference for genes and miRNAs, respectively. Fold changes were determined via 2−ΔΔCt (where ΔCt = (Ct of miRNA of interest) − (Ct of U6), and ΔΔCt = (ΔCt of miRNA of interest) − (ΔCt of U6)) for at least three biological repeats with three technological replicates. All the primer sequences used are listed in Table 1.
Table 1
List of primers for qRT-PCR of the 16 differentially expressed miRNAs.
ID | Sequence (5’-3’) | Tm (℃) |
U6 F | CTCGCTTCGGCAGCACA | 60.42 |
U6 R | AACGCTTCACGAATTTGCGT | 59.69 |
All R | CTCAACTGGTGTCGTGGA | 51.5 |
hsa-miR-218-5p | TTGTGCTTGATCTAACCATGT | |
hsa-miR-218-5p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACATGGTT | 86.3 |
hsa-miR-218-5p F | ACACTCCAGCTGGGTTGTGCTTGATCTAACCA | 75.9 |
hsa-miR-1-3p | TGGAATGTAAAGAAGTATGTAT | |
hsa-miR-1-3p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATACATAC | 82.7 |
hsa-miR-1-3p F | ACACTCCAGCTGGGTGGAATGTAAAGAAGTAT | 70.8 |
hsa-miR-1246 | AATGGATTTTTGGAGCAGG | |
hsa-miR-1246 RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCTGCT | 87.3 |
hsa-miR-1246 F | ACACTCCAGCTGGGAATGGATTTTTGGAGCA | 77.2 |
hsa-miR-122-5p | TGGAGTGTGACAATGGTGTTTG | |
hsa-miR-122-5p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAAACACC | 87.5 |
hsa-miR-122-5p F | ACACTCCAGCTGGGTGGAGTGTGACAATGGTG | 78.6 |
hsa-miR-451a | AAACCGTTACCATTACTGAGTT | |
hsa-miR-451a RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACTCAGT | 85.0 |
hsa-miR-451a F | ACACTCCAGCTGGGAAACCGTTACCATTACTG | 74.6 |
hsa-miR-6087 | TGAGGCGGGGGGGCGAGC | |
hsa-miR-6087 RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCTCGC | 88.6 |
hsa-miR-6087 F | ACACTCCAGCTGGGTGAGGCGGGGGGGCGA | 87.7 |
hsa-miR-363-3p | AATTGCACGGTATCCATCTGTA | |
hsa-miR-363-3p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTACAGATG | 84.6 |
hsa-miR-363-3p F | ACACTCCAGCTGGGAATTGCACGGTATCCATC | 78.1 |
hsa-miR-486-5p | TCCTGTACTGAGCTGCCCCGAG | |
hsa-miR-486-5p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTCGGGGC | 90.8 |
hsa-miR-486-5p F | ACACTCCAGCTGGGTCCTGTACTGAGCTGCCC | 79.5 |
hsa-miR-500a-3p | ATGCACCTGGGCAAGGATTCTG | |
hsa-miR-500a-3p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGAATCC | 87.1 |
hsa-miR-500a-3p F | ACACTCCAGCTGGGATGCACCTGGGCAAGGAT | 82.2 |
hsa-miR-29b-3p | TAGCACCATTTGAAATCAGTGTT | |
hsa-miR-29b-3p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACACTGA | 86.1 |
hsa-miR-29b-3p F | ACACTCCAGCTGGGTAGCACCATTTGAAATCA | 75.7 |
hsa-miR-362-5p | AATCCTTGGAACCTAGGTGTGAGT | |
hsa-miR-362-5p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACTCACACC | 87.3 |
hsa-miR-362-5p F | ACACTCCAGCTGGGAATCCTTGGAACCTAGGTG | 77.6 |
hsa-miR-221-5p | ACCTGGCATACAATGTAGATTT | |
hsa-miR-221-5p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAATCTAC | 83.3 |
hsa-miR-221-5p F | ACACTCCAGCTGGGACCTGGCATACAATGTAG | 75.4 |
hsa-miR-21-3p | CAACACCAGTCGATGGGCTGT | |
hsa-miR-21-3p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAGCCCA | 89.0 |
hsa-miR-21-3p F | ACACTCCAGCTGGGCAACACCAGTCGATGGGC | 83.4 |
hsa-miR-374a-3p | CTTATCAGATTGTATTGTAATT | |
hsa-miR-374a-3p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAATTACAA | 83.9 |
hsa-miR-374a-3p F | ACACTCCAGCTGGGCTTATCAGATTGTATTGT | 71.5 |
hsa-miR-365a-5p | AGGGACTTTTGGGGGCAGATGTG | |
hsa-miR-365a-5p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCACATCTG | 87.3 |
hsa-miR-365a-5p F | ACACTCCAGCTGGGAGGGACTTTTGGGGGCAGA | 83.3 |
hsa-miR-181a-3p | ACCATCGACCGTTGATTGTACC | |
hsa-miR-181a-3p RT | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGTACAAT | 85.5 |
hsa-miR-181a-3p F | ACACTCCAGCTGGGACCATCGACCGTTGATTG | 80.4 |
GAPDH F | TGTTCGTCATGGGTGTGAAC | 56.7 |
GAPDH R | ATGGCATGGACTGTGGTCAT | 57.8 |
ACTA2 F | CCCTTGAGAAGAGTTACGAGTT | 55.1 |
ACTA2 R | ATGATGCTGTTGTAGGTGGTT | 54.5 |
Vimentin F | CCTTGAACGCAAAGTGGAAT | 57.8 |
Vimentin R | AGGTCAGGCTTGGAAACATC | 56.4 |
CD31 F | CCTGCGGTATTCAAAGACAAC | 57.5 |
CD31 R | GGGACCAGATCCTTCATTCAC | 58.1 |
KDR F | AGAGTGGCAGTGAGCAAAGG | 57.6 |
KDR R | CATAGACATAAATGACCGAGGC | 56.9 |
TP53 F | CAGCACATGACGGAGGTTGT | 58.8 |
TP53 R | TCATCCAAATACTCCACACGC | 58.3 |
JMY F | AGCAGGAAATCGACACTCTGT | 56.3 |
JMY R | TCCTCGGGATCATCGGTCTC | 61.8 |
Luciferase reporter assay
Through TargetScan (http://www.targetscan.org) and the UCSC (https://genome.ucsc.edu/) website, we successfully predicted the putative targets of miR-218-5p and miR-363-3p as the p53 promoter region and the 3’ UTR of junction-mediating and regulatory protein (JMY), respectively. The interaction between the p53 promoter region and miR-218-5p, and the 3’ UTR of JMY and miR-363-5p was evaluated using a Dual-Luciferase Reporter Assay System (Promega, Madison, USA) as instructed by the manufacturer. For the p53 promoter region, wild-type and mutant sites were constructed and subcloned into pGL3-Basic vector (Promega, E1751, Madison, USA). For the 3’ UTR of JMY, wild-type and mutant sites were constructed and subcloned into psi-Check2 vector (Promega, C8021, Madison, USA). Using Lipofectamine™ 2000 transfection reagent (cat. #52887; Invitrogen, Carlsbad, USA), CFs were co-transfected with pGL3-Basic or pGL3-Basic-p53 promoter together with miR-218-5p and pRL-SV40, or CFs were co-transfected with psi-Check2 or psi-Check2-3’UTR-JMY together with miR-363-3p. Caenorhabditis elegans Cel-miR-39 mimic served as the negative control. Forty-eight hours after transfection, the cells were washed twice with phosphate-buffered saline and lysed using passive lysis buffer. Luciferase activity was evaluated with the Dual-Luciferase Reporter Assay System (Promega). The primers used for vector construction are listed in Table 2.
Table 2
List of primers for the wild-type and mutant promoter of p53 and 3’UTR of JMY construction in the luciferase assay.
ID | Sequence (5’-3’) |
JMY-P1 | JMY-F1:CCGCTCGAGGCAGGTTATCACTTTCCAGTCGTTC |
| JMY-R1:ATTTGCGGCCGCTAAAAGCACGGAAACTTCAAAACCT |
| JMY-MUT-F1:ACATGAATTTTACATCGTGCCAGTTCCTGACTGCAAT |
| JMY-MUT-R1:ATTGCAGTCAGGAACTGGCACGATGTAAAATTCATGT |
JMY-P2 | JMY-F2:CCGCTCGAGTAAATGATGCTACCCATACAGTGAC |
| JMY-R2:ATTTGCGGCCGCAAAAGGCAACAGTCAAAACGA |
| JMY-MUT-F2:CCATTTACTAGCTTTCGTACCGATTATAAAAGGTAG |
| JMY-MUT-R2:CTACCTTTTATAATCGGTACGAAAGCTAGTAAATGG |
TP53 | TP53-F: GGGGTACCACCGAGTCCCGCGGTAATTC |
| TP53-R: CCCTCGAGATCTCCTTCACAACCCTTATCACTC |
| TP53-MUT-F: CCGCGGTAATTCTTATCAGCTCTGCACCGCCCC |
| TP53-MUT-R: GGGGCGGTGCAGAGCTGATAAGAATTACCGCGG |
Isolation of EPC-Exos, cell culture and transfection
The preparation and culture of EPCs and cardiac fibroblasts were described previously [23]. Briefly, for preparation and culture of EPCs, human peripheral blood (1:1 with phosphate-buffered saline (PBS)) was overlaid onto separation medium (GE Healthcare, Piscataway, NJ, USA) with endothelial growth factors and cytokines. The medium was centrifuged at 2,000 r/min for 30 min, and the mononuclear cells were collected. After washing with PBS, the mononuclear cells were placed into a 25 cm2 culture bottle with DMEM (Dulbecco's modified Eagle's medium, GIBCO BRL, Grand Island, NY, USA) containing 10% FBS (fetal bovine serum, GIBCO). The FBS was pre centrifuged to remove any exosomes (100,000 g for 3 h). After 72 h, the nonadherent cells were removed, and the medium was refreshed every three days. The cells were incubated in a 37 °C constant temperature incubator with 5% CO2. EPCs at passage 3–6 were utilized for the following experiments. For isolation of EPC-Exos, EPCs were harvested at 90% confluency, washed with PBS three times and cultured in EGM-2 (endothelial cell growth medium-2, Lonza, CC3162) containing 10% exosome-depleted FBS for 24 h. The culture medium from the EPCs was collected and centrifuged at 3,000 g for 30 min, followed by 100,000 g for 90 min at 4 °C. The supernatant was centrifuged at 100,000 g for 1 h at 4 °C to obtain the exosomes. The morphology of the exosomes was examined via transmission electron microscopy (Hitachi H-7650, Japan). For functional assays, 10,000 EPCs (in 100 µL) per well were seeded in 6-well plates for 24 h and transfected with 100 pM miRNA mimic or inhibitor of miR-6087, miR-218-5p or miR-363-3p (synthesized by GenePharma, Suzhou, China) using Lipofectamine 2000 (Invitrogen, CA) (Table 3), and 24 h later, the EPC-Exos were isolated, and qRT-PCR detection was performed to assess transfection efficiency. A human cardiac fibroblast cell line was purchased from PriCells (HUM-CELL-0016, Wuhan, China) and cultured in DMEM with 10% FBS to remove weakly attached and unattached cells. The CFs (1×105 cells per well) were seeded onto 35-mm plates, cultured for three days, and then co-cultured with 0.5 µg EPC-Exos transfected with miRNA mimic or inhibitor for 48 h before further functional assays. For P53 suppression and JMY overexpression in CFs, 1×105 cells per well were seeded onto 35-mm plates, cultured for three days and then transfected with 2 µg si-P53 or OE-JMY and cultured for 48 h before further functional assays. si-NC and OE-NC acted as controls. In addition, the cultured CFs were transfected with 100 pM miRNA mimic or inhibitor of miR-218-5p and miR-363-3p to directly detect the role of miR-218-5p and miR-363-3p in angiogenesis and cell proliferation.
Table 3
List of miRNA mimic or inhibitor.
miRNA | Sequence |
miR-218-5p mimic | 5’-UUGUGCUUGAUCUAACCAUGU-3’ |
| 3’-AUGGUUAGAUCAAGCACAAUU-5’ |
miR-218-5p inhibitor | 5’-ACAUGGUUAGAUCAAGCACAA-3’ |
miR-363-3p mimic | 5’-AAUUGCACGGUAUCCAUCUGUA-3’ |
| 3’-CAGAUGGAUACCGUGCAAUUUU-5’ |
miR-363-3p inhibitor | 5’-UACAGAUGGAUACCGUGCAAUU-3’ |
cel-miR-39 mimic | 5'-UCACCGGGUGUAAAUCAGCUUG-3' |
| 3'-AGCUGAUUUACACCCGGUGAUU-5' |
miR-6087 mimic | 5’-UGAGGCGGGGGGGCGAGC-3’ |
| 3’-UCGCCCCCCCGCCUCAUU-5’ |
miR-6087 inhibitor | 5’-GCUCGCCCCCCCGCCUCA-3’ |
Cell proliferation assay
A volume of 100 µL containing 8,000 CFs harboring si-NC, si-p53, OE-NC or OE-JMY plasmid were co-cultured with EPC-Exos in 96-well plates for 24 h, and the cells were transfected with siRNA or plasmid for another 24 h. CCK8 (Cell Counting Kit-8) assays (Solarbio, Beijing, China) were utilized to evaluate cell viability after the addition of 0.5 µg exosomes for another 24, 48 or 72 h. Cell viabilities were calculated via measurement of optical density at 450 nm using a spectrophotometric plate reader (BioTek, USA). Cultured cells (2×104/mL) in a volume of 200 µL were treated with 50 µM BrdU (5-bromo-2´-deoxyuridine) labelling solution (B23151, Invitrogen, CA). The cells were incubated at 37 °C for 2 h, fixed with 4% formaldehyde in PBS for 15 min and permeabilized with Triton X-100 for another 20 min. Cells were incubated in 1 M HCl for 10 min on ice and neutralized with 0.1 M phosphate/citric acid buffer (pH 7.4). The cells were blocked with 0.3% Triton X-100 and 10% normal goat serum for 1 h and incubated with a monoclonal rat anti-BrdU antibody (1:100, Accurate) overnight at 4 °C, followed by blocking with 5% BSA. Fluorescence-labelled secondary antibodies were added (1:1,000, Alexa Fluor 488 goat anti-rat IgG; Molecular Probes) and incubated with the cells for 1 h at room temperature. The nuclei were stained with Hoechst 33258. Immunostaining was visualized with a fluorescence microscope (Olympus inverted microscope IX71) and quantitated using flow cytometry. Cells within the field of view under the same magnification and with the same cell seeding density before the experiment were analyzed in the study.
Cell phagocytosis assay
CFs (1×104/mL in a volume of 100 µL) co-cultured with EPC-Exos were seeded in a 24-well plate coated with a gelatin-based coating solution (6950, Cell Biologics) for 10 min to achieve 90% confluence. The cells were washed with 1% bovine serum albumin (BSA) and incubated in culture medium containing 0.05% FBS at 37 °C for 2 h. After removal of the cell culture medium, the cells were incubated with 10 µg/mL Dil-Ac-LDL (acetylated low-density lipoprotein, labelled with 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine, L-35353, Alexa 594, Life Technologies) at 37 °C for 4 h. After that, the cells were incubated with anti-Dil-Ac-LDL and anti-UAE-1 (Ulex europaeus Agglutinin I), and the staining was detected as described previously [23].
Immunofluorescence staining
The CFs co-cultured with EPC-Exos were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% BSA, the cells were incubated with the following primary antibodies: anti-α-smooth muscle actin (SMA, Abcam, ab5831), anti-vimentin (Abcam, ab92547), anti-CD31 (Dako, M0823), anti-vascular endothelial growth factor receptor 2 (VEGFR-2, Abcam, ab10972), anti-p53 (Abcam, ab131442) or anti-JMY (Abcam, ab217953) overnight at 4 °C. The cells were incubated for 30 min with the appropriate secondary antibody conjugated to fluorescein isothiocyanate (FITC). The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Briefly, the cells were washed twice with PBS and incubated for 5 min in the dark with 1:750 solution of stock DAPI (2 mg/mL). After rinsing with PBS, the slides were allowed to air dry at room temperature, and the cells were observed under an Afv10i confocal microscope (Olympus, Japan).
Western blotting and tube-like structure formation assay
Cells and tissues were collected and snap-frozen in liquid nitrogen using the ultrasonic cell-break method for five sec on ice. After centrifugation at 12,000 g for 30 min, the supernatant was collected. Protein lysates (30 µg) were loaded onto SDS-PAGE gels and transferred to PVDF membranes. The PVDF membranes were blocked with 5% BSA and incubated overnight with monoclonal antibodies against α-SMA (1:1,000, Santa Cruz, CA, USA), vimentin (1:1,000), CD31 (1:2,000), VEGFR-2 (1:2,500), p53 (1:1,500), JMY (1:3,000), collagen-1/3 (1:2000) or Timp1-4 (1:3000). The membranes were probed with secondary antibody (1:4,000; Abcam). Immunoreactivity was determined via enhanced chemiluminescence (Millipore, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10,000) was utilized as a control.
For detection of tube-like structure formation, Matrigel (300 µL) was plated into the bottom of 6-well plates at 37 °C for 30 min, and 10,000 CFs were seeded on the Matrigel and treated with exosomes containing miRNAs for 24 h. An inverted microscope (Olympus) was used to assess tube formation.
Animal experimental model
Three-month-old healthy male SD (Sprague-Dawley) rats (230–250 g) were used for experimentation in accordance with German federal law regarding the protection of animals and the 'Guide for the Care and Use of Laboratory Animals' (National Institutes of Health publication 8th Edition, 2011). The protocol was approved by the Committee for the Ethics of Animal Experiments of The First Affiliated Hospital of Sun Yat-sen University. The rats were fixed on a board and anaesthetized via ether inhalation. Surgery was performed under sterile conditions. After non-invasive oral endotracheal intubation, we disinfected the chest of the rats and lightly pressed on the right side of the thorax, extruding the heart and inserting a needle 2 mm below the root of the left atrial appendage to a needle depth of 0.5 mm. We threaded the surface of the myocardium near the pulmonary arterial cone with 6/0 suture, rapidly ligated the left anterior descending artery, closed the thorax and finally sutured the incisions layer by layer. The rats were allowed to move freely after surgery and were injected with 400,000 U penicillin to prevent infection. Post-operative medication was also used to alleviate any discomfort.
After the operation, when the animals recovered spontaneous breathing, rats whose electrocardiogram (ECG) showed significantly increased J points and/or tall and biphasic or inverted T waves and/or Q waves were considered to be MI rats. The animals that met the above ECG requirements were randomly divided into three groups: MI group (model), MI with exosomes containing miR-218-5p mimic group and MI with exosomes containing miR-363-3p mimic group, with ten rats in each group. Pooled exosomes containing miR-218-5p mimic or miR-363-3p mimic (300 µg) were first resuspended in 150 µL of PBS and injected intramyocardially with an insulin syringe through a 21-gauge needle into the border of the visually recognizable ischemic area at 24 h after surgery. The sham operated group without any surgery and the control model rats were injected with 150 µL of PBS. After eight weeks, echocardiography was used to detect cardiac function in the rats. Capillary density was observed and calculated under an optical microscope.
Histological Evaluation
The heart tissues of rats in the different groups were immediately dissected, fixed in 10% buffered formalin and processed in a paraffin tissue processing machine. Seven-micrometer sections were stained with hematoxylin and eosin (H&E, G1005, Servicebio, China), Masson's trichrome (G1006, Servicebio, China) and Van Gieson (G1046, Servicebio, China), and the tissues were assessed. Representative photomicrographs were observed under a microscope. For measurement of infarct volumes, the excised left ventricles of the hearts were frozen and sectioned from the apex to the base into 3 mm slices. The slices were immersed in 2% TTC (2,3,5-triphenyltetrazolium chloride, T8877, Sigma-Aldrich) solution in PBS for 30 min at 37 °C in the dark. The slices were fixed in 10% formaldehyde, and the area of the ischemic damage was measured using a morphometric program (Digi Cell 4.0).
Immunohistochemistry
Heart tissues slices were blocked by incubation with 3% H2O2 after deparaffinization and rehydration and washed with 0.05 M ethylenediaminetetraacetic acid (EDTA) followed by 4% paraformaldehyde. The tissues were incubated in 5% dry milk and 0.5% goat serum for 20 min. Then, 5 µm sections were incubated with anti-von Willebrand factor (anti-vWF) antibody in the presence of 10% rabbit serum overnight at room temperature. The sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody for another two hours. Diaminobenzidine (DAB) staining (K5007, DAKO) was used for examination. The slides were counterstained with Harris hematoxylin to stain cell nuclei. The heart tissue slices were also incubated with anti-α-SMA antibody and the respective secondary antibody. The slides were counterstained with DAPI to stain cell nuclei.
Statistical Analysis
All the data are presented as the means ± standard deviation (SD). Each in vitro experiment was performed in triplicate. All the data produced by qRT-PCR were based on at least 3 biological repeats with at least 3 technical repeats. Statistical significance between/among different treatment groups was calculated using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test or an unpaired Student t-test, as appropriate, using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to be statistically significant.