Mesenchymal stem cell isolation and differentiation
MSCs were isolated, characterized, and differentiated from Yucatan microswine femurs as reported previously by our group . All animal procedures were in compliance with applicable federal, state, and local laws and regulations, and institutional policies. The Institutional Animal Care and Use Committee of Creighton University approved the animal research protocol. Cells used for experiments in this study were between passages 3 and 5. The isolated MSCs were CD14–CD45–CD44+CD90+CD105+. The growth media used to harvest and culture MSCs was Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. The differentiation media (DM) used for differentiation was endothelial growth media 2 (EGM-2) containing 2 ng/ml, 25 ng/ml, or 50 ng/ml recombinant human VEGF-A165 (Peprotech, Rocky Hill, NJ, USA), and/or 2 ng/ml, 25 ng/ml, or 50 ng/ml recombinant human Ang II (Sigma; St. Louis, MO, USA). Basic EGM-2 was used as negative control DM. Stimulation began when MSCs were at 50% confluency and continued for 10 days. The cell cultures were maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide. Media containing varying concentrations of VEGF-A, Ang II, and ATR inhibitor were changed every 48 hours.
Fluorescence-activated cell sorting characterization of naïve MSCs and ECs
Briefly, cells (1 × 106/ml) were washed with phosphate-buffered saline (PBS) containing 4% fetal bovine serum and incubated with primary antibodies conjugated to fluorophore (fluorescein isothiocyanate for 30 minutes at 4°C in the dark. The antibody concentrations followed the specifications of the manufacturer. The cells were further washed three times in PBS and resuspended in 750 μl FACS-FIX. Flow cytometry was performed on a BD FACSAria I/II System (BD Biosciences, San Jose, CA, USA). Naïve MSCs highly expressed stem cell markers CD44, CD90 and CD105. The cells from the same gate were negative for the macrophage marker CD14 and the hematopoietic stem cell marker CD45. After differentiation, MSCs were analyzed for the EC markers platelet endothelial cell adhesion molecule-1 (PECAM)–fluorescein isothiocyanate (ebiosciences, San Diego, CA, USA), vascular endothelial cadherin (VE-cadherin)–fluorescein isothiocyanate (ebiosciences), and von Willebrand factor (vWF)–fluorescein isothiocyanate (ebiosciences).
For small interfering RNA (siRNA)-mediated knockdown experiments, MSCs were transfected using the Amaxa Nucleofector II Device with the MSC Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer’s optimized protocol for MSCs. For each nucleofection sample, we harvested 2.5 × 105 cells (cell counter; Beckman Coulter, Brea, CA; USA). Briefly, MSCs were washed in Dulbecco’s modified Eagle’s medium supplemented with 200 U/ml penicillin G sulfate plus 200 mg/ml streptomycin sulfate. Nucleofector medium (100 μl) containing siRNA (5 to 50 nM; Origene, Rockville, MD, USA) was administered in a single square wave pulse in a 4 mm diameter cuvette. After electroporation, MSCs were washed with PBS three times, then transferred into DM or growth media and incubated at 37°C. For transfection control, an equivalent amount of scrambled siRNA was used in either DM. At 72 hours post transfection, cell cultures were harvested and protein lysates were isolated for western blot analysis.
Protein fractions were isolated using the active motif kit (Active Motif, Carlsbad, CA, USA). Total protein lysates were quantified by Bradford assay. Proteins were separated by 10% SDS-PAGE, transferred onto a nitrocellulose membrane, and blocked overnight in blocking solution (1× TBS, pH 7.6, 0.1% Tween-20, and 5% w/v nonfat dry milk). The membrane was then incubated with antibodies specific for VEGF-A (Ab105846, 1:500; Abcam; Cambridge, MA, USA). As a loading control, the membrane was probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000, NB300-221; NOVUS Biological; Littleton, CO, USA). The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody (1:1,000) in blocking solution for 1 hour at room temperature. Horseradish peroxidase activity was detected by incubating the membrane in chemiluminescence solution (Bio-Rad, Hercules, CA, USA). The exposure time was adjusted to keep the integrated optical densities within a linear and nonsaturated range. Densitometric analysis was carried out using a UVP Bioimaging system (UVP, Minneapolis, MN, USA).
Total RNA was isolated from naïve MSCs using Trizol reagent (Sigma) according to the manufacturer’s instructions. The yield of RNA was quantified using a Nanodrop (Thermo-Scientific, Rockford, IL, USA). First-strand cDNA synthesis was performed following the manufacturer’s instructions (Improm II reverse transcription kit; Promega, Madison, WI, USA) using oligo dT primers. Real-time quantitative PCR was performed using SYBR Green Master Mix and a Real-time PCR system (CFX96; BioRad Laboratories, Hercules, CA, USA). The following primers are reportedly specific to swine : AT1R-F, 5′-GGCCAGTGTTTTTCTTTTGAATTTAGCAC-3′ and AT1R-R, 5′-TGAACAATAGCCAGGTATCGATCAATGC-3′; AT2R-F, 5′-GTTCCCCTTGTTTGGTGTAT-3′ and AT2R-R, 5′-CATCTTCAGGACTTGGTCAC-3′; and GAPDH-F, 5′-CCCATCACCATCTTCCAGGAG-3′ and GAPDH-R, 5′-GTTGTCATGGATGACCTTGGCC-3′.
Primers were obtained from Integrated DNA Technologies (Coralville, IA, USA). The specificity of the primers was confirmed by running a melting curve (data not shown). The thermocycler conditions were as follows: 5 minutes at 95°C for initial denaturation, and 40 cycles of 30 seconds at 95°C, 30 seconds at 55 to 60°C (depending upon the primer annealing temperatures), and 30 seconds at 72°C. Fold expression of mRNA transcripts relative to controls was determined after normalizing to GAPDH.
Angiotensin receptor blockade
To analyze the effects of AT1R and AT2R on differentiation, MSCs were pre-incubated with 5 μM Telmisartan (an AT1R blocker) or 5 μM PD123319 (an AT2R blocker) for 1 hour. Pretreated MSCs were then cultured in DM containing 1 μM of either ATR blocker. In preliminary experiments, titration was performed for AT1R-specific and AT2R-specific antagonists (1 to 20 μM) for 24 hours to determine the optimal concentration that had no effect on cell viability or proliferation. Cells were then detached by with 0.25% trypsin/ethylenediamine tetraacetic acid, counted, and evaluated by annexin V/PI assay. Florescence-activated cell sorting analysis revealed that >95% of cell were viable at concentrations of ATR blockers between 1 μM and 5 μM. MSCs were then induced to undergo differentiation with differentiating media containing the indicated concentrations of VEGF-A alone or in combination with Ang II and/or ATR antagonists.
Cells were incubated in blocking solution containing PBS, 0.25% Triton X-100, 10 mg/ml bovine serum albumin, and 5% normal goat serum (Jackson Laboratories, West Grove, PA, USA) for 1 hour at room temperature. The cells were incubated with primary antibodies selective for anti-AT1R (Ab9391, 1:1,000; abcam) or anti-AT2R (Ab19134, 1:1,000; abcam) for 1 hour at room temperature. After washing with PBS containing 0.1% bovine serum albumin three times for 5 minutes each, a secondary antibody (affinity purified goat anti-rabbit Cy2 and Cy3 antibody, 1:500, Jackson laboratories; West Grove, USA) was applied to the sections for 1 hour in the dark to visualize AT1R-labeled and AT2R-labeled cells (Jackson Immunolabs, West Grove, PA, USA). Negative controls were run in parallel either using rabbit pre-immune serum PAC-767 (Pacific Immunology, Ramona, CA, USA) instead of primary antibody or by complete omission of primary antibody. Negative control was absent of staining. Sections were washed with PBS with 0.1% bovine serum albumin three times for 5 minutes and dipped into distilled water for 2 seconds. Fluorescence was preserved by sealing specimens with a solution of equal parts of PBS and glycerol containing 10 mg/ml n-propyl gallate, and 1.5 mg/ml 4′,6-diamidino-2-phenylindole. To prevent the escape of the mounting medium from the cover slips, a single layer of nail polish was placed around the edges. Pictures were taken within 1 hour of mounting using an Olympus DP71 camera (Olympus, St Louis, MO, USA).
After stimulation for 10 days, MSCs were harvested and an angiogenesis assay was performed according to the manufacturer’s protocol (Chemicon, Temecula, CA, USA). Polymerized EC matrices were prepared by incubating 100 μl ECMatrix gel solution into each well of a 24-well plate at 37°C for 1 hour. The stimulated cells were seeded at a concentration of 1 × 104 cells on EC matrices. EGM-2 medium (300 μl) was added to each well and maintained at 37°C and 5% carbon dioxide incubation for 6 hours. The formation of capillary tubes was analyzed using an inverted phase contrast microscope (CKK41; Olympus).
Data are presented as the mean ± standard deviation from three to six independent experiments. For each experiment, MSC cultures were isolated from the femoral bone of separate pigs. Data were analyzed using GraphPad Prism, GraphPad Software; La Jolla, California, USA. Multiple group comparisons were performed by Bonferroni’s multiple comparison tests using one-way analysis of variance. P <0.05 was accepted as statistically significant.