Isolation and cultivation of EPCs
Animals care and experimental procedures were approved by the Animal Research Committee of the Department of Laboratory Animals, Xiangya School of Medicine, Central South University. EPCs were isolated from the bone marrow of Sprague-Dawley (SD) rats (150-250 g) as previously described . Briefly, total bone marrow-derived mononuclear cells (MNCs) were isolated from both femurs and tibias of male SD rats by density gradient centrifugation using Histopaque-1077 (Sigma Aldrich, St. Louis, MO, US). Isolated cells were subsequently maintained in complete EGM-2 medium supplemented with EGM-2-MV BulletKit (Lonza, Walkersville, MD, USA) and 10% fetal bovine serum (FBS) (ScienCell Research Laboratories, Inc. Carlsbad, CA, USA). Cells were maintained at 37 ℃ in a humidified atmosphere (5% CO2). After 4 days, the unattached cells were removed by washing with phosphate buffer saline (PBS) for three times and replaced with fresh media and culture medium was changed every 2-3 days.
Identification of EPCs
After 7 days of culture, EPCs were determined by doubly positive for staining cells with FITC-labeled Ulex europaeus lectin I (UEA-1) and Dil-conjugated acetylated low-density lipoprotein (Dil-ac-LDL). More concretely, cells were incubated with 2.4 μg/ml Dil-labeled acetylated low-density lipoprotein (LDL) (Yiyuan biotechnology, Guangzhou, China) at 37 ℃ for 3 h and then fixed with 4% paraformaldehyde for 10 min. After washing with PBS, the cells were incubated with 15 μg/ml FITC-UEA-I (lectin) (Sigma Aldrich, St. Louis, MO, US) at 37 ℃ for 1 h. After staining, images were obtained by fluorescence microscope (DMi8, Leica). EPCs were also identified by flow cytometry. The surface markers investigated were FITC-conjugated CD34 (1:50, Bioss, Beijing, china), PE-conjugated CD133 (1:50, Novus Biologicals, Littleton, CO, USA), PE-conjugated CD31 (1:50, BD Biosciences, San Jose, CA, USA) and FITC-VEGFR-2 (1:30, Abcam, Cambridge, UK), blank served as negative control.
Carotid artery injury
Carotid artery injury (CAI) was induced as described previously . Briefly, 350-400 g male SD rats were anesthetized by an intraperitoneal injection of 10% chloralic hydras (4 mL/kg), and the surgical procedures were performed under sterile conditions. Using a dissecting microscope, the bifurcation of the left carotid artery was exposed via a midline incision of the ventral side of the neck. Two ligatures were placed proximally and distally around the external carotid artery. The distal ligature was then tied off. After temporary occlusion of the internal and common carotid artery by the vascular clamp, a V-shaped arteriotomy was performed in the ligatures of the external carotid artery, to introduce a 2F arterial embolectomy catheter (Edwards Lifesciences, California, USA) that was slightly pumped 1.5 kPa gas into the balloon and completely fills out the vessel. The catheter was passed along the common carotid artery in a rotating manner for three times. After removal of the catheter, the proximal ligature of the external carotid artery was tied off. Normal blood flow was reassured, and the skin was closed with single sutures using 6-0 silk. After induction of arterial injury and 24 h later, each rat received 2×106 EPCs by intravenous tail vein injection which have transfected with adenovirus vectors expressing human omentin-1 (Ad-omentin-1) or green fluorescent protein (Ad-GFP) for 72 h.
Construction and infection of Adenoviral Vector
Adenovirus vector expressing human omentin-1 (Ad-omentin-1) and adenovirus-carrying green fluorescent protein (Ad-GFP) used as a control were both constructed by Vigene Biosciences (Jinan, China) using pAd-EF1a-GFP adenovirus vector. Adenovirus was added to the cells at 70%–80% confluence with a multiplicity of infection (MOI) of 100, and then incubated in EGM-2 medium without FBS and antibiotics for 12 h. After the transfection, the medium was removed, and the cells were maintained in the complete medium for 48 h before further treatment.
For immunohistochemistry, we used antibodies to TNF-α (1:200, Santa Cruz, Texas, USA) and Ki-67 (1:200, Abcam, Cambridge, UK). Paraffin section was rehydrated and endogenous peroxidase activity was blocked for 30 minutes by endogenous peroxidase blocking buffer (Beyotime Biotechnology, Shanghai, China). Primary antibody was incubated at 4 °C overnight, followed by 30 minutes for biotinylated secondary antibody (1:500, Abcam, Cambridge, UK). All specimens were counterstained with hematoxylin-eosin (HE) staining solution (Beyotime Biotechnology), then neutral gum sealed piece for storage. Images were obtained after scanning the section through Pannoramic MIDI (3D-Histech, Budapest, Hungary) and analyzed with ImageJ software.
Cell proliferation assay
Cell proliferation was assessed with the cell counting kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) and 5-ethynyl-2’-deoxyuridine (EdU) proliferation assay (RiboBio Co., Guangzhou, China). For the CCK-8 test, cells were plated onto 96-well plates (3×103 cells/well) in a triplicate pattern. After treating with recombinant human omentin-1 protein (Novus Biologicals) with 0-500 ng/ml. For combined treatments with omentin-1 and TNF-α, omentin-1 (300 ng/ml) was added to the medium for 6 h before TNF-α (10 ng/ml) treatment and then continuously incubated with 37 °C for 24 h. Cells were added with 100 μl of fresh medium supplemented with 10 μl of CCK-8 solution for another 2 h at 37 °C. The optical density (OD) at 450 nm was measured. For the proliferation of EdU assay, cells were plated with 4×103 cells/well in a triplicate pattern, and then the proliferation was assessed by the EdU assay kit according to the manufacturer's instructions. The EdU positive cells were viewed under fluorescence microscopy (DMi8, Leica) and the number calculated by counting at least three random separate fields. These images were analyzed by ImageJ software.
Cell apoptosis assay
We used low FBS medium to induce EPCs apoptosis. In brief, cells were washed with PBS, the culture medium was replaced with EGM-2 added with omentin-1 (0-500 ng/ml) and then the cells were placed at 37 °C for 24 h. For reversal of apoptosis by omentin-1, omentin-1 (300 ng/ml) was added to the EPCs medium for 6 h before TNF-α (10 ng/ml) treatment and then continuously incubated with for 37 °C 24 h. The apoptosis cells were assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's instructions. After treatment, cells were harvested and washed in ice-cold PBS, then resuspended in 100 μl of binding buffer, 5 µl Annexin V-FITC solution was added to the cells and incubated for 15 min at room temperature in the dark. This was followed by further incubation with 3 μl PI solution for 10 min, and then they were analyzed immediately by bivariate flow cytometry using a Cytek Dxp Athena flow cytometer with Flowjo CE software and analyzed using FlowJo software (Ver X.0.7; FlowJoLLC, Eugene, OR). Approximately 3-4×105 cells were analyzed in each sample.
In vitro tube formation assay
EPCs were incubated in 6-well plates and cells were treated as described above. After treatment, EPCs (4×103 cells/well) were seeded in 96-well plates that had been pre-coated with Matrigel Matrix (70 μl/well) (CORNING Life Sciences, Tewksbury, MA, USA). After incubation for 6 h in serum-containing media, images of tube morphology were captured by an inverted microscope and analyzed using ImageJ software.
Western blot analysis
EPCs were lysed for 30 minutes on ice in RIPA lysis buffer (Beyotime Biotechnology), and the protein concentration was determined by BCA Protein Assay Kit (Beyotime Biotechnology). Total protein (50 to 100 μg) was resolved by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane (Immobilon-P, Millipore, Bredford, MA, USA), and subjected to immunoblot analysis. The primary antibodies for p38 MAPK (1:1000, Cell Signaling Technology, MA, USA), P-p38 MAPK (1:1000, Cell Signaling Technology), CREB (1:1000, Cell Signaling Technology), P-CREB (1:1000, Cell Signaling Technology) and horseradish peroxidase-conjugated secondary antibody (Santa Cruz, Texas, USA) were used. The proteins were detected using ChemiDoc XRS+ Imaging System (Bio-Rad, Hercules, California, USA) with enhanced chemiluminescence reagents (Millipore), and bands were analysed with Image J software normalized by GAPDH (Abcam).
Data were expressed as mean ± standard deviation (SD). Comparisons between two groups were measured using Student's t test, and comparisons of multiple groups were performed with one-way analysis of variance (ANOVA). All statistical analyses were performed using the SPSS 13.0 software package (SPSS, Inc., Chicago, IL, USA), and a two-tailed P value <0.05 was considered to be statistically significant.