Mouse ADSC isolation and cell culture
Primary mouse ADSCs from mouse adipose tissue were isolated and cultured as described previously with minor modifications . The fatty tissue around the inguinal region of male C57/BL6 mice, 3–4 weeks old, was separated. After the removal of visible blood vessels, lymph nodes, and fascia, the tissue was finely minced with scissors and digested with collagenase type I (1.25 % w/v) for 60 min at 37 °C with gentle shaking. After collagenase neutralization, the floating adipocytes were separated by centrifugation at 1200 rpm for 5 min. The resulting pellet was resuspended and the cells were plated in tissue culture flasks in Dulbecco’s modified Eagle’s medium with low glucose (DMEM; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 0.1 mg/ml streptomycin (both from Thermo Fisher Scientific, Inc., Waltham, MA, USA), VEGF 10 ng/ml, basic fibroblast growth factor (FGF) 10 ng/ml, and alpha-FGF 10 ng/ml (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a 5 % CO2 humidified atmosphere.
Flow cytometry analysis
Cell apoptosis was detected by an Annexin V-FITC apoptosis detection kit according to the manufacturer’s instructions. The cells were incubated with or without the addition of various concentrations of recombinant human CRP (free of sodium azide; Sino Biological Inc., Beijing, China) for different times and then harvested and rinsed in cold phosphate-buffered saline (PBS). The fraction of apoptotic cells was determined by cell staining in Annexin-V binding buffer with FITC-conjugated Annexin-V and propidium iodide (PI; Sigma-Aldrich). After 15 min of incubation in the dark at room temperature, the samples were analyzed by flow cytometry (LSRII FACS; BD Bioscience, Franklin Lakes, NJ, USA). Apoptotic cells were identified as Annexin-V-positive cells.
For the cell cycle analysis, the cells were trypsinized and fixed in 75 % ethanol for 60 min on ice and stained with PI and Hoechst 33342 (5 μg/ml; Thermo Fisher Scientific, Inc.) in PBS for 30 min. Equal numbers of cells were assessed for ADSCs by flow cytometry analysis.
Cell proliferation assay
ADSCs were seeded in 96-well plates at a density of 4 × 103 cells/well. After 24, 48, and 72 hours, the medium was removed and cells were counted using a Cell Counting Kit-8 (CCK-8; Dojindo, Rockville, MD, USA). Cells were treated with 10 % CCK-8 solution for 4 hours at 37 °C in a humidified 5 % CO2 incubator and the absorbance was measured at 450 nm by a microplate reader.
Cell migration assay
ADSCs were seeded on the upper site of 8.0 μm transwell membrane plates (Corning, Inc., NY, USA) at a density of 5 × 104 cells per well after serum starvation for 12 hours. CRP (25 μg/ml) or plus inhibitors (PD098059, 10 μM; LY294002, 5 μM) in DMEM were introduced on the lower site of transwell membrane plates for 12–24 hours. Migrated cells remaining in the transwell membrane were fixed and then stained using 10 % crystal-violet (Sigma-Aldrich), and cells in the membrane were counted by light microscopy.
Inhibitor and block antibody treatment
After reaching 80 % confluence, ADSCs were seeded in six-well plates (5 × 105 cells/well). The cells were treated with the following reagents for 24 hours: (a) ADSC basal medium as a control; (b) stimulant CRP alone; (c) stimulant plus inhibitors or block antibodies, including ERK inhibitor (PD098059, 10 μM), PI3K inhibitor (LY294002, 5 μM), and nuclear factor-kappa beta (NF-kB) inhibitor (BAY-11-7082, 5 μM) (all from Cell Signaling Technology, Danvers, MA, USA), or anti-CD16 (2 μg/ml; R&D Systems, Inc., Madison, WI, USA), anti-CD16/32 (1 μg/ml; Abcam Inc., Cambridge, UK), and anti-CD64 (1:100, 3 μg/ml; R&D Systems Inc.); or (d) inhibitors and block antibodies alone. Doses of the inhibitors or block antibodies were determined according to previous laboratory characterization and published data. Supernatants and cell extractions were collected 24 hours after treatment.
In-vitro tube formation assay
Tube formation on Matrigel was performed as described previously . A total of 50 μl of chilled Matrigel (BD Bioscience) was added to a 96-well plate and incubated at 37 °C for 30 min. Human umbilical vein endothelial cells (HUVECs; 1 × 104 cells) were suspended in 100 μl of EBM-2 or endothelial growth medium (EGM; LONZA Inc., Basel, Switzerland), and conditioning medium (CM) of ADSCs, CRP-treated CM of ADSCs or plus VEGF-neutralizing antibody (0.15 μg/ml; R&D Systems Inc.), or EBM-2 plus CRP was added to the solidified Matrigel. The CM was harvested after incubation of the ADSCs in EBM-2 for 24 hours. After incubation on Matrigel at 37 °C in a 5 % CO2 chamber, morphological changes were observed under a microscope (Leica, Germany). The five representative fields were photographed. Images were analyzed using Image J software (NIH, Bethesda, MD, USA) to determine the tube lengths.
In-vivo Matrigel plug assay
The animal experiments were conducted according to the guidelines and ethical standards of the Animal Care and Use Ethics Committees of Sun Yat-Sen University (IACUC-DB-16-070). ADSC (100 μl, 1 × 106 cells) or CRP-treated ADSC (24-hour pretreatment with 25 μg/ml CRP without FBS, 1 × 106 cells) suspensions were mixed with 400 μl of ice-cold growth factor reduced phenol red-free Matrigel (BD Bioscience), and Matrigel containing PBS was used as a negative control. The Matrigel mixture was injected subcutaneously into the dorsal area of male nu/nu mice, 4–5 weeks old. Each experimental condition was performed with three mice. At day 7, the Matrigel implants were removed and then fixed with formalin, and the fixed Matrigel plug was embedded in paraffin to prepare sections for hematoxylin and eosin (H & E).
Enzyme-linked immunosorbent assay
The assay was performed for the CM using a mouse angiogenesis array kit (R&D Systems, Inc.) according to the manufacturer’s instructions. VEGF-A production was examined by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Raybiotech, Atlanta, GA, USA) according to the manufacturer’s instructions.
Western blot analysis
To prepare the protein extracts, the cells were rinsed twice with ice-cold PBS and harvested. After centrifugation, the cells were resuspended and extracted in lysis buffer (Thermo Fisher Scientific, Inc.) for 30 min on ice. Protein concentrations were assayed using Pierce Coomassie Plus reagent according to the manufacturer’s instructions, and 40 μg of protein was loaded for separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to polyvinylidene difluoride membranes (Immobilon-P; EMD Millipore Corporation, Billerica, MA, USA). The membranes were blocked in Tris-buffered saline containing 5 % bovine serum antigen (BSA) and probed with HIF-1a, tissue inhibitor of metalloproteinase-2 (TIMP-2), VEGF-A, hepatocyte growth factor (HGF), matrix metalloproteinase (MMP)-2, and MMP-9 (all from R&D Systems Inc.) corresponding antibodies. Reacted bands were detected by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence substrates (PerkinElmer, Boston, MA, USA).
Quantitative real-time PCR
Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, Inc.). The synthesis of cDNA was performed on DNaseI-treated total RNA templates (0.5 μg) using an iscript™ cDNA synthesis kit. Gene expression was assessed by quantitative real-time PCR (qRT-PCR) using SYBR Green intercalating dye (Thermo Fisher Scientific, Inc.) and mouse primers. The primer sequences are presented in Additional file 1: Table S1. The comparative threshold cycle method was used to calculate the amplification fold as specified by the manufacturer. The amplified PCR products were separated by gel electrophoresis in a 2 % agarose gel visualized with ethidium bromide. Each sample was replicated at least three times.
The ADSCs were fixed with 4 % paraformaldehyde (PFA) for 10 min, followed by blocking with 5 % BSA in PBS for 60 min at room temperature. The cells were incubated with the following antibodies at room temperature for 1 hour: rabbit anti-CD16/32 (1:200; Abcam Inc.) and rabbit anti-CD64 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Following a wash in PBS, the cells were incubated in goat anti-rabbit secondary antibodies conjugated with FITC (1:200; Thermo Fisher Scientific, Inc.) in PBS for 1 hour at room temperature. DAPI was used for the nuclear stain. The samples were then washed three times, and mounted in mounting medium (Vector Laboratories, Burlington, CA, USA). Images were obtained using an inverted fluorescence microscope.
The ADSCs were incubated with CRP (25 μg/ml) for 6 hours and then washed and lysed in 1 ml of RIPA buffer. Cell lysates were precipitated with goat antibodies against CRP (2 μg per 100 g of total protein; Santa Cruz Biotechnology, Inc.) that had been preabsorbed by protein G-Sepharose (Biotool Inc., Houston, TX, USA). Immunoprecipitated proteins were washed in RIPA buffer, subjected to SDS-PAGE, and immunoblotted with specific antibodies against CRP (Santa Cruz Biotechnology, Inc.) or FcgRs (1:200; R&D Systems, Inc.).
The in-vitro data are representative of independent experiments performed in triplicate. The statistical analysis was conducted using SPSS software (SPSS, Inc., Chicago, IL, USA). The statistical significance of the differences among groups was tested using one-way analysis of variance or Student’s t test. Error bars are indicative of standard deviation. p < 0.05 or p < 0.01 was considered significant.