Isoproterenol was purchased from Sigma-Aldrich (St Louis, MO, USA). MDA kits were purchased from Sigma-Aldrich. TRIzol® for total RNA extraction was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). PrimeScriptTMRT Reagent Ki and SYBR® Premix Ex TaqTM were purchased from TaKaRa (Shiga, Japan). The antibody against VEGFR1 was purchased from Abcam (Cambridge, MA, USA).
This project was approved by the Ethical Committee of Islamic Azad University, Karaj, Iran (ethical code: IR.IAU.K.REC.1399.052). We also carefully follow the national and internationally accepted guidelines for the care and use of animals.
Animals and experimental design
Ten male NMRI mice with a weight between 25 and 30 g were kept in cages under controlled ambient temperature (22 ± 2 °C) and controlled 12:12 h light-dark cycle. The mice were randomly divided into two groups (1) Control (healthy mice) and (2) MI model group (Isoproterenol injection). DL-Isoproterenol (ISO; Sigma), a nonselective beta-adrenergic agonist, was administered subcutaneously at a single dose of 85 mg/kg body weight diluted in 2 ml of saline. Animals were sacrificed 2 days after the post- ISO injection. Furthermore, fifteen healthy NMRI mice were used for aorta isolation.
Preparation of acellular aortic matrices (AAMs)
Fifteen healthy male NMRI mice were sacrificed by Ketamine/Xylazine-induced anesthesia (50 mg/kg ketamine, 5 mg/kg xylazine). The thoracic aorta was isolated and excised from the animal body according to a comprehensive protocol provided by Robbins et al. . The fresh isolated thoracic aorta was flushed twice with the phosphate-buffered solution (PBS, Merck, Germany) containing amphotericin B and antibiotics. Acellular matrices (AMs) were prepared according to Meezan et al. method . Briefly, the aorta was processed with distilled water for 18 h at 4 °C and 4% sodium deoxycholate (Sigma, UK) for 4 h, and 2000 kU deoxyribonucleases I (DNase-I, Sigma, USA) in 1 M NaCl (Merck, Germany) for 3 h. The treatment was repeated twice until the cells were completely removed. Acellular matrices were stored in PBS at 4 °C until used.
To confirm the complete removal of cells from the isolated thoracic aorta tissues, DAPI nuclear staining was performed. To do this, the isolated acellular aortic tissues were fixed by 4% paraformaldehyde for 10 min. After washing with PBS, the tissues were stained with DAPI solution (0.5 µg/ml) (Thermofisher, USA) for 2 min in the dark. The samples were finally visualized by fluorescent microscope (Nikon, Japan).
Preparation of the cells
To isolate cardiomyocytes, both healthy and MI model mice were sacrificed under anesthesia (50 mg/kg ketamine, 5 mg/kg xylazine) and then the hearts were dissected and perfused with (PBS) comprising heparin sodium (Heparodic, Iran) to rinse out the blood. To do this, a 30 gauge needle was inserted into the aorta in the left ventricle, and immediately the right ventricle was cut to allow the blood to flow out. Hearts were finely minced then placed into 10 mL digestion media (Dulbecco’s Modified Eagle’s Medium [DMEM], 100 U/mL collagenase I) and incubated at 37 °C for 40 min. The minced tissue was centrifuged at 1000 × g for 20 min at 4 °C. The cell pellet was solved in 2 mL of fresh digestion media and incubated at 37 °C for 20 min. The cell was diluted with 5 mL of culture media (DMEM, 10% Fetal Bovine Serum, 1% penicillin/streptomycin). The cell suspension was centrifuged at 1000 × g for 20 min at 4 °C. The cell pellet was suspended in 5 mL culture media and plated into T25 culture flasks (5 mL medium per flask). The cells were allowed to attach under standard culture conditions (37 °C, 5% CO2, 100% humidity) for 2 h, then non-adherent cells were removed by washing with PBS, and the culture medium was replaced. The culture media were changed every 2–3 days. Cardiomyocyte cultures were typically reached to proper confluence in 4–7 days. Then, cell cultures were passaged once a week.
Cultures of myocardial cells on the acellular aortic matrices (AAMs)
For experimental purposes, the acellular aortic matrix (AAM) specimens were divided into 1 × 1 cm2 pieces, put into 24-well cell culture dishes, washed with PBS twice, and later incubated in DMEM media at 37 °C and 5% carbon dioxide for 10 min, before the cell seeding process. Isolated cardiomyocytes (at a density of 5 × 104 cells/well) were seeded onto the luminal surface of AAMs and maintained for 14 days in the DMEM medium at 37 °C in 5% CO2. Also, cells were seeded in 24-well plates containing completed medium without AAMs (referred to as 2D culture system). Therefore, four-cell groups were designed to study, including (1) Healthy heart-derived cells cultured as 2D culture system (control cells), (2) MI-derived cells cultured as 2D culture system (MI cells), (3) MI-derived cells cultured on the acellular aortic matrix as 3D culture system (acellular aorta + MI cells), and (4) healthy heart-derived cells cultured on the acellular aortic matrix as 3D culture system (acellular aorta + normal cells).
Cell viability assay
For the cell viability assay, myocytes isolated from healthy and MI model heart of mice were seeded at a density of 1 × 104 cells/well in 96-well plates with (3D culture) and without (2D culture) AAMs and then were incubated for 3 and 14 days. The cell viability was evaluated using by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. MTT solution at 5 mg/ml was added and incubated for 4 h at 37 °C. Next, the contents of all wells were discarded and 200 μl DMSO was added to each well. The absorbance of each well was recorded at a wavelength of 570 nm using an ELISA-reader. The viability percent relative to the control values were measured and calculated for each group. Each experiment was conducted in triplicate to confirm the obtained values.
Molecular studies using real-time PCR
The gene sequences from Hif-1α, VEGF, and β-actin were obtained from the NCBI database, and the sequence of their exons and introns was determined to design the primers used in Real-Time PCR. Primer design was performed using the Gene runner software. Then, designed primers were blasted to verify their accuracy and reproduce only the genes’ mRNA sequences. The sequences of the Real-Time PCR primers were forward TCAGAGCAAGAGAGGCATCC and reverse GGTCATCTTCTCACGGTTGG for β-actin, forward CCTGCACTGAATCAAGAGGTTGC and reverse CCATCAGAAGGACTTGCTGGCT for Hif-1α and forward TCTCAAGTGCAGAGGGGAGG and reverse TCGAAGTAGATGTAGGGAGGT for VEGF. Total RNA was extracted from the two-dimensional (2D) and three-dimensional (3D) (on day 14) cultured cells by using RNX-Plus™ (Cinnagen, Iran) according to the manufacturer’s recommendations. The RNA concentration and quality were then determined using a NanoDrop 2000c (Eppendorf, Germany). cDNA was synthesized from a 1000 ng DNase-treated RNA sample with a Revert Aid™ first-strand cDNA synthesis kit (Fermentase, Lithuania) using Oligo (dT) primers. PCR was performed using Master Mix and SYBR® Green (Applied Biosystems, Life Technologies, Paisley, United Kingdom) in StepOne™ Applied Biosystems according to the manufacturer’s instructions. The primers’ efficiency and specificity, the fidelity of qPCR, and melting curve analysis were determined as before. Thermocycler conditions included an initial step at 95 °C for 15 min, followed by 40 cycles at 94 °C: 20 s, 58–60 °C: 40 s, and 72 °C: 30 s. The β-actin gene was chosen as internal control against which mRNA expression of the target gene was normalized. A reference gene was used to assure the validity of the housekeeping gene and results. The resultant gene expression level was presented as 2−ΔΔCt, in which ΔCt was the difference between Ct values of the target gene and reference gene .
Western blotting analysis
On days 3 and 14, the cells cultured in 2D or 3D conditions were lysed in RIPA (Sigma, UK) for 30 min, and total proteins were obtained. The total protein concentration was examined by Bradford assay utilizing bovine serum albumin as the standard before progressing with the western blot. At first, the protein extracts (30 µg/lane) were electrophoretically isolated on a 10% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was then blocked using blocking buffer (phosphate-buffered saline, PBS, containing 5% non-fat dry milk) overnight at 4 °C. The proteins were probed with mouse monoclonal antibodies against VEGF and VEGFR1 (1:1000 dilution) (Abcam, UK). The membranes were incubated with goat anti-mouse HRP-conjugated secondary antibodies VEGFR1 primary antibodies (1:1000 dilution) (Abcam, UK) against VEGF and at room temperature for 2 h. The bound proteins were recognized using chemiluminescence by enhanced electrochemiluminescence reagents. Ultimately, the analysis of protein bands quantification and densitometry was performed employing ImageJ software (National Institutes of Health, imagej.nih.gov/ij).
Measurement of malondialdehyde (MDA) activity
MDA is secondary lipid peroxidation product and can be examined with thiobarbituric acid (TBA) that can create a pink-colored adduct with the highest absorbance at 532 nm. The MDA was detected using the method described by Schmedes et al. with a minor modification .
Concisely, 0.2 ml cell lysate, 1.5 ml 20% acetic acid (adjusted to pH 3.5), 1.5 ml 0.9% TBA, 0.2 ml 8.1% sodium dodecyl sulfate, and 0.6 ml distilled water were vortex mixed, and the mixture was incubated in a water bath at 95 °C for 50 min. Following cooling down to 25 °C, 5.0 ml butanol: pyridine mixture and 1.0 ml of distilled water (1:15; v/v) were added. After centrifugation at 3000 rpm for 10 min, the absorbance was measured at 532 nm. The MDA concentration was calculated through a molar extinction coefficient of 1.56 × 105 M−1 cm−1, and values were shown as μmol of MDA. The standard sample was a breakdown product of 1, 1, 3, 3-tetra ethoxy propane.
Data were examined utilizing SPSS 23.0 statistical software. One-way analysis of variance followed by the post hoc (Tukey) test was used to compare differences among all groups. Data were represented as mean ± SD. Values were considered statistically significant if the p values were less than 0.05.