Isolation and characterization of peripheral blood EPCs (PB-EPCs)
For the isolation of EPCs, peripheral blood (10 ml/kg) was obtained from New Zealand white rabbits by puncturing the ear vein. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation with Ficoll-Plaque Plus (GE Healthcare, America). Mononuclear cells were then washed and plated on six-well plates that were coated with human fibronectin (Sigma, America) at a concentration of 3 µg/cm2 or rat tail collagen I (BD, America) at a concentration of 10 mg/cm2. Cells were supplemented with EGM-2 MV BulletKit medium (Lonza Corp, Switzerland). Mononuclear cells were incubated at 37℃, 5% CO2, and fed daily with EGM-2. At 3 to 7 days post-plating, adherent EPC were detached with 0.025% trypsinase containing 0.02% EDTA. The same procedure was performed for the subsequent 3 passages (approximately 1 week of culture). All experiments were performed with cells that were cyropreserved at passage 3.
EPCs isolated from rabbits were characterized based on their ability to take up DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) and bind to Ulex europaeus agglutinin-1 (UEA-1), as previously described. Briefly, P3 cultures of EPCs were seeded on 6-well plates and incubated with 20 μg/ml Alexa Fluor 488-labeled DiI-Ac-LDL (Molecular Probes) for 4 h or with 15 µg/ml rhodamine-labeled UEA-1 (Vector) for 1 hour. After incubation, cells were washed with serum-free medium and detected under fluorescence microscopy. Immunostaining for vascular endothelial growth factor receptor 2 (VEGFR2) and CD31 was performed as previously described. Briefly, EPC cultures were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 minutes, then blocked with 5% bovine serum albumin (BSA) at 37°C for 1 hour. Cells were then incubated with mouse anti-mCherry (1:1000; Biolegend) at 4°C overnight. Sections were washed 4 times in PBS and stained for 2 h in the dark at room temperature with Alexa Fluor 488 goat anti-mouse antibody(1:1000; Molecular Probe). After 4 washes with PBS, the slides were mounted in medium containing DAPI (Vectorshield).
Isolation and culturing of adipose stromal cells
Adipose tissue was obtained from the thighs and groins of New Zealand white rabbits and cut into 2-mm pieces. Tissues were then washed twice with PBS containing 2% penicillin/streptomycin and once with DMEM/F12 medium. The cleaned fat blocks were transferred into a 15-ml centrifuge tube and agitated in 1 mg/ml Liberase TL solution prepared in DMEM/F12 medium (Invitrogen) supplemented with 5% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin, for 1 h at 37℃ and 180 rpm. Single cells were obtained by filtering the solution through a 70-μm cell strainer. Samples were then centrifuged at 1000 rpm for 5 min to separate the stromal cell fraction from the adipocytes. The pellet was treated with red blood cell (RBC) lysis buffer to eliminate erythrocytes, then resuspended in EGM-2MV media. The ASC monolayers were split upon achieving 80% confluence and used at passage 3.
Molecular phenotyping of ASCs and EPCs cultured in vitro
Quantitative real-time PCR (qRT-PCR) was performed to measure gene expression in EPCs and ASCs cultured in vitro. ASCs and EPCs were seeded into 6-well plates. Total RNA was extracted from cultured cells and fresh isolated PBMC with Trizol reagent (Invitrogen), then converted into cDNA with a First Strand cDNA Synthesis kit (Thermo Scientific). QRT-PCR was conducted using SYBR Green I Master mix (Applied Biosystems) with an ABI 7500 instrument. The primers for CD31, CD34, CD45, VEGF, VEGFR2, and ANGPT1(Angiopoietin-1) are summarized in Table 1. Relative gene expression was normalized to PBMC levels of GAPDH expression. All experiments were performed in triplicate. The results are expressed as mean±SEM.
Two-dimensional assessment of Matrigel angiogenesis
To test the ability of endothelial progenitor cells-mesenchymal stem cell (EPC-MSC) cocultures to form tubes in vitro, EPCs and ASCs were resuspended in EGM-2 basal medium (10% heat-inactivated FBS, without growth factors), then seeded at cell number ratios of 100:0, 80:20, 67:33, 50:50, 33:67, 20:80, and 0:100 onto 24-well plates precoated with thick Matrix gel (300 µl/well, Matrigel matrix growth factors reduced, BD). The final density of cells was 105 cells/ml/well. The plates were incubated for 6 h at 37℃ and 5% CO2. Tube formation was observed with inverted phase-contrast microscopy. For each sample, a total of 6 pictures were randomly captured from three wells in parallel. Tube length and number of branches were measured with Image-Pro Plus software. The results are expressed as mean±SEM.
To analyze the pro-angiogenic activity of ASCs, ASCs supernatant was collected after 48 h of cultivation and subjected to a two-dimensional angiogenesis assay. EPC supernatant was used as the internal control. EGM-2 basal medium and EGM-2 MV complete medium were used as culture controls. As described previously, 105 EPCs were resuspended in 1 ml of ASC supernatant, EPC supernatant, EGM-2 basal medium, or EGM-2MV complete medium, then seeded into 24-well plates precoated with thick Matrix gel. Tube formation was analyzed with the methods described above.
Coculture of ASCs and EPCs
The expression of angiogenesis markers in EPCs cocultured with ASCs was examined with qRT-PCR. EPCs were resuspended in EGM-2 basal medium, then seeded in in a total volume of 2.7 ml containing 5.4×105 cells in 6-well plates. A total of 3×105 ASCs that had been resuspended in 1.5 ml EGM-2 basal medium were added to each transwell insert (pore size 2 µm, Corning). The same volume of EGM-2 basal medium or EGM-2 MV complete medium was added to a different transwell insert as a control. After coculture for 24 h, the EPCs in the lower chamber were harvested for total RNA extraction with Trizol reagent (Invitrogen). RNA (2 μg) was reverse-transcribed using the First Strand cDNA Synthesis kit (Thermo scientific). QRT-PCR was conducted using SYBR Green I MasterMix (Applied Biosystems) with an ABI 7500 instrument. The primers for VEGF, angiopoietin-1/2, Flt-1, KDR, Tie-1/2, VE-cadherin, CD31, and GAPDH are shown in Table 1. Experiments were performed in triplicate for each gene. The results are expressed as mean±SEM.
EPC transmigration and invasion
The transwell migration assay was used to investigate the chemotactic ability of ASCs cocultured with EPCs. Briefly, 500 μl EPCs (5×104 cells) diluted with EGM-2 basal medium were seeded into the cell culture inserts (pore size 8 µm, Corning) in the 24-well plate. Then, 750 μl of suspended ASCs (7.5×104 cells) or the same volume of control medium (EGM-2 basal medium or EGM-2 MV complete medium) was added to the outer compartment of each well of the 24-well plate. After 8 h of coculture, the culture inserts were carefully removed and fixed in 4% paraformaldehyde. Non-migrated cells on the upper side of the membrane were removed with a cotton swab. The cells that had migrated were stained with crystal violet.
Invasion assays were used to analyze the proteolytic activity and capacity for migration of EPCs cocultured with ASCs. As described above, the EPCs were seeded into BioCoat matrigel-pretreated chambers (pore size 8 μm, Corning). ASCs were cocultured in the outer compartment. Twenty-four hours later, cells that had migrated to the bottom side of the membrane were fixed and stained with crystal violet. All the experiments were performed in triplicate. Cell counts for transmigration and invasion assays were obtained with Image-Pro Plus software and expressed as mean±SEM.
Seeding of an adipose tissue implant with ASCs and EPCs
To evaluate the capacity of ASCs to promote vascularization among EPCs in the setting of fat implantation, implanted adipose tissue was seeded with ASCs and EPCs.
Donor fat was obtained from New Zealand white rabbits that had been anesthetized with pentobarbital (0.01 ml/g body weight at 5 mg/ml in 5% ethanol/PBS, intraperitoneally). The fat tissue obtained was cut into small cubes (approximately 5 mm×5 mm×3 mm) and kept in EGM-2 basal medium in a 24-well plate. To study fat transplant angiogenesis in vivo, fat tissue was embedded in polymerized Matrigel, then mixed with EPCs or EPCs+ASCs and implanted subcutaneously in C57BL/6 nude mice (20 g; Beijing Vital River Laboratory Animal Technology). A total of 4×105 EPCs or EPCs+ASCs (ratio 1:1) were resuspended in 200 µl matrigel, then added to the fat tissue. The tissue and cells that had been embedded in gel were incubated for 30 minutes at 37℃ to allow for solidification. Incisions of 4 mm in length were made on the backs of anesthetized nude mice, and the gel-entrapped tissue was implanted subcutaneously. Thirty days after implantation, mice were sacrificed via gas inhalation (CO2). Fat tissue was harvested and subjected to immunohistochemistry to evaluate vascular network formation. All experiments were performed at Kunming Medical University in compliance with the university's guidelines for the ethical treatment of experimental animals.
All data are presented as means±SEM. SPSS11 software was used for statistical analysis. The independent-sample t-test was used for comparisons between groups. Results with p≤0.05 were considered to be statistically significant.