Animals and experimental protocol
Animal experiment 1
The animal experiment 1 and 2 were conducted according to the guidelines and ethical standards of the Animal Care and Use Ethics Committees of Sun Yat-Sen University (IACUC-DB-16-072). Eight-week-old male C57BL/6J mice and high-fat (HFD) feed (0.15% cholesterol, 21% lard, 78.85% basic feed, D12492) were purchased from Guangdong Province Medical Animal Center. The mice received a HFD for 2–8 weeks before the thoracic aortic PVAT was removed to examine the expression of CRP by polymerase chain reaction and western blot analyses.
Animal experiment 2
Transgenic mice expressing human CRP only in adipose tissue via the mouse Fabp4 promoter (specific expression in fat tissue)  were purchased from Cyagen Bioscience Inc (Guangzhou, China). Transgenic mice and wild-type (WT) littermates on a C57BL/6N background were used for the experiments performed in this study. To examine the effects of transplanted PVAT on injured arteries, neointimal hyperplasia in injured arteries was examined at 4 weeks after the vascular injury. The intimal and medial areas were measured, and the ratio of neointima/media area was calculated. Mice received a standard chow diet (STD).
Femoral artery injury and adipose tissue transplantation
Femoral artery injury
As described in detail previously , the mice were anesthetized with a mixture of ketamine (8 mg/100 g) and xylazine (1.2 mg/100 g) administered intraperitoneally. The femoral artery, vein, and nerve bundle were exposed, and 6-0 silk sutures were placed in the proximal and distal ends of the femoral vascular bundle. The profunda femoris artery was isolated, and 6-0 silk sutures were placed beneath both ends of the artery before a loop was created at the distal end. The exposed profunda femoris artery was dilated by local application of one drop of 1% lidocaine hydrochloride. Transverse arterioctomy was performed in the profunda femoris artery with vannas scissors. A guide wire was inserted into the artery via the cut, and the femoral artery was stretched to reduce the angle between the profunda femoris artery and femoral artery. The wire was inserted into the proximal ligation along the long axis of the artery and was left in place for 2 min to denude the artery. Then the wire was removed, and the silk suture was looped at the proximal portion.
Adipose tissue transplantation
Mice were sacrificed, and after exposure of the thoracic aorta, the PVAT around the thoracic aorta was carefully removed with microforceps under a surgical microscope. The PVAT was placed in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) with containing 1% antibiotics (R&D Systems, Inc., Minneapolis, MN, USA). The left femoral artery was denuded using a guide wire as described above, and then after removal of endogenous PVAT, the injured femoral artery was wrapped with transplanted PVAT.
Quantification of neointimal hyperplasia
Four weeks after surgical intervention, the mice were killed by intraperitoneal administration of an overdose of nembutal, and then the mice were perfused via the left ventricle with 0.9% NaCl solution followed by 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). The femoral artery and transplanted PVAT were carefully excised, postfixed in 4% paraformaldehyde overnight at 4 °C, and embedded in paraffin. Cross-sections (5 μm) were cut and then stained with hematoxylin and eosin (H&E), and the neointimal and medial areas of the artery were measured utilizing image ImageJ 1.48 software (National Institutes of Health).
Immunohistochemical and immunofluorescene analyses
Paraffin-embedded sections of the femoral artery and transplanted PVAT (5 μm thick) were deparaffinized and blocked with 0.5% horse serum. The sections were then incubated with primary antibody against human C-reactive protein (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA), CD68 (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA), CD31 (R&D Systems, Inc., Minneapolis, MN, USA) or isotype-specific antibody, followed by incubation with goat anti-mouse immunoglobulin (Ig) [F(abʹ)2] conjugated with peroxidase (Amersham Pharmacia Biotech Inc, Piscataway, NJ, USA) as the secondary antibody at room temperature for 60 min. The sections were then counterstained with hematoxylin. Peroxidase activity was visualized by incubation with a 3,3-diaminobenzidine solution and observed under light microscopy. The quantity of CD68+ in adventitia around injured femoral artery was determined according to a previously reported method with minor modification . The quantity of adventitial macrophages was counted manually the number of CD68-related antigen positive cell at 400 times magnification by two independent observers.
For immunofluorescene analysis, Paraffin-embedded sections of perivascular adipose tissue (5 μm thick) were deparaffinized and blocked with 0.5% horse serum. After permeabilisation, sections were incubated with primary a antibodies over night at 4 °C. The antigen was detected with an anti-rabbit Alexa488 (green) and anti-mouse Alexa555 (red) conjugated secondary antibody (1/500). DAPI (blue) was used for nuclei staining. After permeabilisation, cells were incubated with antibodies to rabbit anti-TIP47 (perilipin 3/TIP47,adipocytes marker, Abcam Inc, Abcam, CA, USA) and mouse anti-CD68 (macrophage marker, Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA). Antigen was detected with fluorescently labeled secondary antibodies as described above. Specimens were analyzed by confocal microscopy.
Flow cytometric analysis
After the animals were sacrificed, the perivascular and subcutaneous adipose tissues were carefully removed and completely immersed in D-Hanks buffer containing 100 U/mL penicillin and 0.1 mg/mL streptomycin. 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, Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 60 min at 37 °C with gentle shaking. After neutralization of the collagenase, the floating adipocytes were separated by centrifugation at 1200 rpm for 5 min. The resulting pellet of vascular stromal cell components was resuspended in PBS and filtered through a 70-μm cytoscreener. Finally, the cell suspensions were probed with mouse anti-CD11b-FITC antibody (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) via incubation for 20 min in darkness, and the expression of CD11b was detected by FACS Aria cell sorting system (LSRII FACS; BD Bioscience, Franklin Lakes, NJ, USA) and results were analyzed by Flowjo10 software.
Gel-free quantitative proteomic profiling in adipose tissue of CRPTG mice
After the animals were sacrificed, the perivascular adipose tissues were carefully removed and grinded to powder in liquid nitrogen, and the blood samples were collected. Proteins were extracted in lysis buffer (8.4 M urea, 2.4 M thiourea, 5% CHAPS, 50 mM DTT, and 1% IPG buffer) for 30 min on ice, and then cells were further broken using an ultrasonic cell disruptor, followed by centrifugation at 14,000 rpm for 1.5 h at 19 °C using a TL-100 ultracentrifuge (Beckman, Palo Alto, CA, USA). Finally, the middle layer of aqueous liquid was retained.
Trypsin digestion and labeling of adipose tissue samples with TMT Protein pellets were suspended in 100 mL of 200 mM TEAB and digested overnight at 37 °C with 2.5 μg sequencing grade modified trypsin (Promega, Madison, WI, USA). Six digested samples were individually labeled with TMT6 reagents according to the manufacturer’s instructions. Three littermate control samples and three CRPTG mouse samples were used in this experiment.
Liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis and database searches Mass spectrometric analysis of the TMT-labeled samples was performed on an Ultimate 3000 Dionex LC system (Dionex, Sunnyvale, CA, USA) connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) operated according to a higher energy collisional dissociation model. The detailed steps were described previously . Protein identification and quantification based on LC–MS/MS data were performed with Proteome Discoverer 1.4 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) interfaced with Uniprot (mouse_81798_20161104.fasta).
Isolation and purified of CD14+ human monocytes
The isolation of human monocytes were conducted according to ethical standards of Ethics Boards of Sun Yat-Sen Memorial Hospital of Sun Yat-sen University (EBSYSM-16-022). Heparinized peripheral blood obtained from healthy volunteers was mixed with PBS. After being let stand for 1 h at room temperature, the upper leukocyte-enriched plasma layer was harvested and centrifuged, after which the cell pellet was resuspended in PBS and placed onto a Ficoll (ICN Biomedicals, Irvine, CA, USA) and centrifuged at 700g for 30 min. Red blood cells and polymorphonuclear leukocytes (PMN) are dense and centrifuge through the medium while peripheral blood mononuclear cells (PBMCs; mainly monocytes and lymphocytes) band over Ficoll and can be recovered at the interface. The recovered mononuclear cells were washed three times with PBS buffer. Isolation of CD14+ monocytes was performed using magnetic bead-based separation with CD14 MicroBeads, the MiniMACS separator and LS type columns (Miltenyi Biotec, Germany). Purified CD14+ monocytes were resuspended in RPMI 1640 medium containing 10% FBS (both form Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The purity of CD14+ isolation was > 90%, as determined by flow cytometry.
Cell migration assay
Cell migration experiment was performed using a co-culture system. Primary human peripheral blood mononuclear cells (PBMCs) were seeded on the upper side of 8.0-µm Transwell membrane plates (Corning, Inc., NY, USA) at a density of 5 × 104 cells/well after serum starvation for 12 h. Culture medium alone (control), 3T3-L1 adipocyte conditioned medium (CM) only, CM of 3T3-L1 adipocyte treated with CRP (free of sodium azide; Sino Biological Inc., Beijing, China), or CM of 3T3-L1 adipocyte treated with CRP plus anti-CXCL7 blocking antibody (R&D Systems, Inc., Minneapolis, MN, USA) or isotype-match IgG was introduced in the lower wells of the transwell membrane plates for 12 h. Migrating cells remaining on the transwell membrane were fixed, then stained using 10% crystal-violet (Sigma-Aldrich, St. Louis, MO, USA), and counted under a light microscope.
As described in detail previously , recombinant human CRP (free of sodium azide; Sino Biological Inc. Beijing, China) at different concentrations was mixed with 10% poly-lysine (FN), and 200 μL of the suspension was added to the wells of a 96-well plate for incubation at 4 °C overnight. The wells were then blocked by incubation of 3% bovine serum albumin in PBS at 37 °C for 1 h. The concentrations of primary human PBMCs and THP-1 cells (ATCC, Manassas, USA) were adjusted to 1 × 105 cells/mL with serum-free medium. THP-1 cells were treated with 200 nM phorbol ester (PMA) with or without anti-CD16/32 or anti-CD64 neutralizing antibody (R&D Systems, Inc., Minneapolis, MN, USA) at 37 °C for 1 h before being seeded in a 96-well plate coated with CRP and allowed to incubate at 37 °C for 1 h. Then non-adherent cells were washed away with PBS, and adherent cells remaining on the plate were fixed and stained using 10% crystal-violet. The cells remaining on the plate were counted under light microscopy.
Enzyme-linked immunosorbent assay for CXCL 7
3T3-L1 preadipocytes (obtained from ATCC) were maintained in DMEM containing 10% FCS. Adipocyte differentiation was initiated by adding differentiation medium that contained 10 μg/mL insulin, 0.25 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine. After 48 h, the medium was replaced with medium containing 10 μg/mL insulin, and cells were maintained in this medium until use. Fully differentiated 3T3-L1 cells were starved for 12 h, and then were stimulated with different concentrations of exogenous CRP recombinant protein. The supernatants were collected 24 h later. The concentration of CXCL7 in cell supernatants and plasma sample from mice 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
For preparation of the protein extracts, the cells were rinsed twice with ice-cold PBS, centrifuged, and resuspended in lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min on ice. Protein concentrations were assayed using Coomassie Plus reagent (Pierce) according to the manufacturer’s instructions, and 40 or 100 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% BSA and probed with CXCL7, TNF-ɑ and MCP-1antibodies (all purchased from R&D Systems, Inc., Minneapolis, MN, USA) Protein bands were detected by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence substrates (PerkinElmer, Boston, MA, USA).
Quantitative real-time polymerase chain reaction analysis
Total RNA was extracted using Trizol reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNA was synthesized on DNaseI-treated total RNA templates (0.5 μg) using an iscriptTMcDNA synthesis kit (Takara Bio, Inc., Shiga, Japan). Gene expression was assessed by quantitative real-time polymerase chain reaction (QPCR) using SYBR Green intercalating dye (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and mouse primers. The primer sequences for mouse CRP were: sense: 5′- TTCCCAAGGAGTCAGATACTTCC-3′, and antisense: 5′-TCAGAGCAGTGTAGAAATGGAGA-3′. The comparative threshold cycle method was used to calculate the fold amplification as specified by the manufacturer. The amplified PCR products were separated by gel electrophoresis in a 2% agarose gel and visualized with ethidium bromide. Each sample was replicated at least three times.
The in vitro data are representative of independent experiments performed in triplicate. The statistical analysis was conducted using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). The statistical significance of the differences among groups was tested using one-way analysis of variance or the Mann–Whitney test, multiple comparison between the groups was performed using S-N-K method and P < 0.05 was considered indicative of a significant difference. Error bars are indicative of standard error of mean.