3.1. Isolation and Culture of MSCs
All animal procedures were undertaken in accordance with the Guidelines for the Care and Use of Laboratory Animals, published by the National Academy Press (NIH Publication No. 85–23, Revised 1996). The experiments were approved by the Animal Care and Use Committee of Zhongshan Hospital, Fudan University.
MSCs were isolated from four-week-old Sprague-Dawley (SD) rats weighing 100 to 120 g by flushing femurs and tibias with Dulbecco’s modified Eagle’s medium (DMEM; GIBCO), 10% Fetal Bovine Serum (FBS; GIBCO) and penicillin/streptomycin. The bone marrow (BM) solution was cultured at 37 °C in an atmosphere of 5% CO2. The culture medium was changed every 3–4 days. Non-adherent hematopoietic cells were removed during this process, and the adherent, spindle-shaped MSCs were cultured and expanded. Passage 4 cells were used for subsequent experiments.
3.2. Adenoviral Transduction and Labeling of MSCs
An adenoviral vector carrying a green fluorescent protein (GFP) (GeneChem) for cell transduction was used to establish and identify a stable population of MSCs. Transduction was facilitated by transfecting Passage 4 MSCs with purified adenovirus cells that had been exposed to 7 μg/mL polybrene. The transfected cells were incubated for 24 h at 37 °C with 5% CO2. The medium was removed, replaced with fresh medium and incubated for a further 24 h. Cells were then sorted by flow cytometry for GFP to ensure that a homogenous population of GFP+ MSCs was obtained (data were not shown).
3.3. In vitro Exposure to H2O2 and HDL
MSCs were incubated for 6 h with DMEM supplemented with 1% FBS prior to experimental interventions. The starved MSCs were then respectively exposed to H2
(0 to 500 μM) or HDL (0 to 200 μg/mL; Merck) for 24 h. The experiments were undertaken in four groups: a control group; a HDL group (MSCs incubated with HDL 100 μg/mL for 24 h); a H2
group (incubation with 400 μM H2
for 24 h); a HDL + H2
group (pre-incubation with HDL 100 μg/mL for 24 h, followed by 24-h exposure to 400 μM H2
). For inhibitor experiments, three additional groups were set as follows: MSCs were pre-incubated with PI3K inhibitor (LY294002, 25 μM) or vehicle (DMSO) for 1 h, and then successively treated with HDL and H2
as above; the third group was pre-cultured with LY294002 alone. The concentrations of HDL, H2
and LY294002 used in these experiments were determined from pilot studies and based on similar methods described in the literature [9
3.4. MSC Viability Assay
MSC viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cells were cultured in 24-well plates and treated according to the protocol. After 24 h, Cell Titer 96® Aqueous One Solution Reagent (Promega) was added to each well according to the manufacturer’s instructions. Cell viability was evaluated after 2-h incubation with this reagent, by testing absorbance at 490 nm using a plate reader.
3.5. TUNEL (Terminal Dexynucleotidyl Transferase-Mediated dUTP Nick end Labeling) Assay
Cell apoptosis was investigated using an In Situ Cell Death Detection kit (Roche). In brief, MSCs were fixed in 3.7% buffered formaldehyde, pretreated with 3.0% H2O2 and exposed to TdT enzyme at 37 °C for 1 h. The cells were then incubated with digoxigenin-conjugated nucleotide substrate at 37 °C for 30 min. Nuclei exhibiting DNA fragmentation were identified after staining with 3,3-diamino benzidine (DAB) for 5 min.
The MSCs were counterstained with hematoxylin and viewed by light microscopy. Five fields at high magnification (×200) were randomly chosen to assess the apoptosis index, which was calculated as a percentage of labeled cells to total nuclei.
3.6. Caspase-3 Activity Assay
Caspase-3 activity was determined in MSC lysates (200 μg) using Caspase-3/CPP32 Fluorometric Assay kit (BioVision). The fluorogenic CPP32/caspase-3 substrate was labeled with the fluorochrome 7-amino-4-methyl coumarin. In this assay, the amount of fluorescence produced upon cleavage was proportional to the amount of caspase-3 activity present in the sample.
3.7. Senescence-Associated β-Galactosidase Staining
MSCs cultured in six-well plates were rinsed with phosphate-buffered saline (PBS), fixed and then incubated with freshly prepared senescence-associated β-Galactosidase (SA-β-Gal) staining solution (Beyotime, China) at 37 °C overnight. Five fields at high magnification (×200) were randomly chosen to count the number of blue cells (SA-β-Gal positive cells). At least 500 cells in each group were examined and the percentages of blue cells presented.
3.8. Western Blot Analysis
MSCs from each group were rinsed with cold PBS and lysed using RIPA buffer containing 1 mM phenylmethanesulfonylfluoride on ice for 30 min. The lysates were transferred to 1.5 mL Eppendorf tubes and centrifuged at 4 °C and 15,000 rpm for 30 min. The protein component was separated by 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked in 5% skimmed milk for 2 h at room temperature, and were incubated overnight at 4 °C with anti-phospho-Akt (Cell Signaling), anti-Akt (Cell Signaling), anti-p16INK4a. Following a 30 min wash, the membranes were incubated for 1 h with a secondary antibody conjugated to horseradish peroxidase-conjugated IgG (Jackson). Protein expression was determined using an enhanced chemiluminescence system and quantified by densitometry (Image System; Bio-Rad).
3.9. Measurement of ROS
The production of intracellular ROS was measured using an oxidation-sensitive fluorescent dihydroethidium (DHE) probe (Vigorous). The assay was based on the principle that DHE crosses cell membranes and is rapidly oxidized in the presence of ROS resulting in the formation of a highly fluorescent form of oxidative ethidium.
In this assay MSCs were washed three-times with PBS and incubated with 10 μM DHE in phenol-red-free MEM medium (Invitrogen) at 37 °C in the dark. After 15 min incubation, the cells were rinsed and the medium was replaced. The fluorescence level, as an indicator of ROS production, was detected using fluorescence microscopy. The excitation and emission filters were 488 nm and 590 nm, respectively.
3.10. Myocardial Infarction and MSCs Transplantation
The MI model was developed in SD rats (220 ± 30 g), as previously described [22
]. MSCs (1 × 106
in 100 μL, derived from male rats) (MSCs group) or MSCs preconditioned with HDL (HDL-MSCs group) were intramyocardially injected into female rats 10 min after ligation-induced infarction. The MSCs were injected into 3 to 5 injection sites into the anterior aspect of the viable myocardium that bordered the area of infarction. For the experiments concerning cardiac remodeling, two additional groups were set as follows: rats received intramyocardial injection of 100 μL of PBS without MSCs (PBS group) or received thoracotomy only but were not ligated (Sham group).
3.11. Tracking of the GFP+ MSCs Injected in MI Hearts
To observe the donor MSCs survival in vivo, rats were sacrificed four days after cell implantation. Hearts were harvested and sliced transversally above the ligation suture. Tissue fixation was minimized to reduce autofluorescence. The sample was embedded at optimum cutting temperature and cut by a cryostat from the base (above the ligation) towards the apex until the ligation suture was reached. Beginning at this point, 10 μm sections were collected throughout the entire lesion. The sections were stained using DAPI (4,6-diamidino-2-phenylindole), and the number of GFP+ MSCs was estimated under fluorescent microscopy at a magnification of ×200.
Three photomicrographs were taken from the infarct border zone of each section. Six sections were selected for counting, giving a total of 18 high power fields per rat. The numbers of GFP+ MSCs were counted and averaged.
3.12. Real-Time Polymerase Chain Reaction
Real-time polymerase chain reaction (PCR) was performed for quantification of the male sry DNA levels on Day 4 after cell transplantation. Rats were sacrificed and hearts were excised, frozen in liquid nitrogen and powdered. DNA purification was performed using Genomic DNA Isolation kit (Qiagen). The concentration of the purified DNA was determined by spectrophotometry. Real-time PCR was performed using Takara SYBR Premix Ex TaqTM in a Bio-Rad iQ5 optical module. Primers for amplification of rat Y chromosome sry and β-actin genes were synthesized (Invitrogen) and were listed as follows: sry, forward, 5′-GAG GCA CAA GTT GGC TCA ACA-3′; reverse, 5′-CTC CTG CAA AAA GGG CCT TT-3′. β-actin, forward, 5′-CCA TTG AAC ACG GCA TTG-3′; reverse, 5′-TAC GAC CAG AGG CAT ACA-3′. The cycling conditions were: 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C, 30 s at 59.5 °C, 30 s at 72 °C. Melting curves were acquired at the end of the reaction by gradually raising the temperature by 1 °C/min from 59.5 °C to 95 °C over a time period of 35 min.
Cardiac remodeling and left ventricular function were assessed by transthoracic echocardiography 4 weeks after MI, using a Vevo 770 high-resolution imaging system (Visual Sonics) with a 17.5-MHz probe. After the induction of light general anesthesia, hearts were imaged in two dimensional and M-modes. The recordings were obtained in the para-sternal long-axis view at the level of the greatest LV diameter. The LV internal end-diastolic diameter (LVIDd) and LV internal end-systolic diameter (LVIDs) were measured from M-mode recordings according to the leading-edge method. All echocardiographic measurements were averaged from at least five separate cardiac cycles.
3.14. Statistical Analysis
Statistical analysis was undertaken using SPSS version 16.0. All data were expressed as means and standard errors (SE). Between-group of differences was analyzed using Student’s t-test. Comparisons between more than two groups were performed using one-way analysis of variance (ANOVA) with Bonferroni’s correction. Values of p < 0.05 were considered statistically significant.