10 to 12 weeks old male BKS.Cg-m+/+Leprdb/J (db/db; Jackson Lab, Bar Harbor, ME) mice (diabetes mellitus type 2) or male C57BLKS/J mice (healthy litter mate control; Jackson Lab, Bar Harbor, ME) were used as BMMC donors. In the db/db mice elevations of plasma insulin begin at 10 to 14 days and elevations of blood sugar at four weeks. These homozygous mutant mice are polyphagic, polydipsic, and polyuric. Delayed wound healing, increased metabolic efficiency, peripheral neuropathy and myocardial disease are seen in C57BLKS-Leprdb homozygotes. Blood glucose levels were measured with FreeStyle glucose meters and FreeStyle test strips (Abbott Laboratories, Abbott Park, IL) before surgery or harvesting BMMCs. In db/db mice, diabetes was confirmed when the blood glucose level after 4 hours of fasting was > 240 mg/dl. Female BKS.Cg-m+/+Leprdb/J mice were used as recipients for all in vivo experiments. Diabetes was confirmed as described above. Animal care was provided in accordance with the Stanford University School of Medicine guidelines and policies for the use of laboratory animals.
Preparation of BMMCs
Bone marrow cells were harvested from the long bones of 10- to 12-week male BKS.Cg-m+/+Leprdb/J mice (db/db BMMCs) or age matched C57BLKS/J mice (control BMMCs) by flushing with PBS using a 25-gauge needle. After passing through a 70 μm strainer, the isolate was centrifuged at 1200 rpm for 5 minutes, washed, and resuspended into 5 ml of saline. To acquire the mononuclear fraction, the bone marrow isolate was centrifuged for 35 minutes at 1900 rpm using a 14 ml tube with 5mL Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ) gradient and 5 cell/saline suspension. BMMCs were washed again and counted using a Hemacytometer (Fischer Scientific, Pittsburgh, PA). BMMCs were prepared freshly for application.
Flow cytometry analysis
The BMMCs’ identity was investigated by fluorescent-activated cell sorting (FACS) with antibodies recognizing Sca-1 (progenitor cells), Mac-1 (macrophages), GR-1 (granulocytes), CD45 (hematopoietic cells), CD34 (EPCs), and CD133 (EPCs; Becton, Dickinson and Company. San Jose, CA). BMMCs (1 × 106) were incubated in 2% fetal bovine serum/PBS at 4°C for 30 minutes with 1 μl of monoclonal fluorescein isothiocyanate (FITC)-conjugated antibodies and processed through a FACSCalibur system with CellQuest software (Becton, Dickinson and Company, San Jose, CA) according to the manufacturer’s protocol. For each antibody, the experiment was repeated independently three times.
Quantification of apoptotic cells was performed using an Annexin-V-FITC Apoptosis Detection Kit (CD106 (Becton, Dickinson and Company, San Jose, CA) according to the manufacturer’s instructions. 1×106 BMMCs cells from both db/db and control mice were plated in 2 ml of medium (IDIM with 10% FBS and 1% P/S-L-G, all from Invitrogen Corporation (Gibco), Carlsbad, CA) into a 6-well plate. They were then incubated at 37°C under normoxia conditions (78% CO2/21% O2) and hypoxia conditions (95% CO2/5% O2), using Nillups-Rothenberg Modular Incubator Chamber (Del Mar, CA). Afterwards, cells were collected, washed, and resuspended in 500 μl of binding buffer, and 5 μl of Annexin-V-FITC with 10 μl of propidium iodide (PI) was added and incubated at room temperature for 10 minutes. Analyses were performed with FACSCalibur and CellQuest software (Becton, Dickinson and Company, San Jose, Ca) according to manufacturer’s protocol. The cells in the FITC-positive and PI-negative fraction were regarded as apoptotic cells. This experiment was repeated three times.
Cell proliferation assay
Proliferation was determined by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 1×105 BMMCs cells from both db/db (n=2) and control (n=2) mice were plated in 100 μl medium (IDIM with 10% FBS en 1% P/S-L-G (all from Invitrogen, Carlsbad, CA) into a 96-well plate and were incubated under normoxia and hypoxia conditions as described above for 32 hours. 20 μl of MTT was added to each well, and the plate was further incubated for another 4 hours in same conditions. Absorbance was determined with a multi-well absorbance reader (Genios, Tecan Systems Inc., San Jose, CA) at 490 nm with included manufacture software (Magellon v6.2). Experiments were carried out as quadruplicates. Absorbance values were expressed as percentages.
Surgical model for myocardial infarction
10- to 12-week old female diabetic db/db mice were randomized into three groups receiving either 2.5×106
db/db BMMCs (n=8, db/db group), 2.5×106
control BMMCs (n=8, control group), or phosphate buffered saline (n=6, PBS group). Animals were intubated with a 20-gauge angiocath (Ethicon Endo-Surgery, Inc., Cincinnati, OH) and placed under general anesthesia with 2–3% mixture of isoflurane and 100% O2
using a rodent ventilator (Harvard Apparatus, Holliston, MA). Myocardial infarction (MI) was created by ligation of the left anterior descending (LAD) artery with 8-0 ethilon suture through a left anterolateral thoracotomy as described before (25
). Two doses were injected into the infarcted area via the lateral and medial infarct zones using a Hamilton syringe with a 29-gauge needle. A total volume of 50 μl was injected containing 2.5×106
cells or PBS alone, depending on group randomization. Surgery was performed by a single experienced micro-surgeon (G.H.).
Echocardiographic determination of left ventricular contractility
Echocardiography was performed at postoperative days 7, 14, and 35. Animals received continuous inhaled anesthetic (1.5–2% isoflurane) for the duration of the imaging session. Two-dimensional transversal-targeted M-mode traces were obtained at the level of the papillary muscles using a 14.7-MHz transducer on a Sequoia C512 Echocardiography system (Siemens, Malvern PA). Analysis of the M-mode images was performed using DicomWorks 1.3.5 (http://dicom.online.fr
) analysis software. Left ventricular end-diastolic diameter (EDD) and end-systolic diameter (ESD) were measured by a blinded investigator (R.J.S.) and used to calculate fractional shortening (FS) by the following formula: FS = (EDD−ESD)/EDD as described (26
Measurements of hemodynamics with pressure-volume loops
Invasive, steady-state hemodynamics measurements were conducted by closed-chest pressure-volume loop analysis prior to sacrifice at week 5. The animal was placed under general anesthesia as described above. After midline neck incision, a 1.4-F conductance catheter (Millar Instruments, Houston TX) was advanced retrogradely through the right carotid artery into the left ventricle. Surgery was performed by a single blinded micro-surgeon (G.H.) with over 6 years of experience with this model. The measurements of segmental conductance were recorded, allowing extrapolation of the left ventricular volume, which was coupled with pressure. These data were analyzed by a blinded investigator (K.E.A.v.d.B.) using PVAN 3.4 Software (Millar Instruments, Houston, TX) and Chart/Scope Software (AD Instruments, Colorado Springs, CO).
TaqMan PCR for male specific Sry gene
At week 5, animals were sacrificed and hearts were explanted, minced, homogenized in 2 ml DNAzol (Invitrogen, Carlsbad, CA) and 500 ng DNA was processed for Taqman PCR using primers (TaqMan Gene Expression Assays, Alied Biosystems, Foster City, CA) specific for the Sry locus. RT-PCR reactions were conducted in iCylcer IQ Real-Time Detection System (Bio-Rad, Hercules, CA). Detection levels were compared to a standard curve to assess the number of viable cell per sample. Each sample was carried out in sextuplicate and the average was used for the analysis.
Unless otherwise stated, data are presented as mean ± SEM. Comparisons between groups were done by independent sample t-tests or analysis of variance (ANOVA) least significant difference post hoc tests, where appropriate. Differences were considered significant for P-values <0.05. Statistical analysis was performed using SPSS statistical software for Windows.