Study design
A single-blind randomized study was conducted to reveal the effect of EPCs on endothelial repair and the potential role of the CXCR7/AKT/keap-1/Nrf2 axis in regulating EPC functional activity. The reendothelialization rate at day 7 and 14 and I/M ratio at day 21 after EPCs transplantation were recognized as our primary outcome. Totally 125 rats were randomly divided into two groups: diabetic group and normal group. Then, the diabetic group was divided into six groups: control group, diabetic group, normal groups, CXCR7 group, CXCR7-EPCsNrf2-WT group, and CXCR7-EPCsNrf2-KD group. After groups and subgroups were divided, diabetic rat model was established and proved by rapid glucose meter. Then, EPCs were isolated from the bone marrow of normal or diabetic rats (10 rats each group) and used in subsequent in vitro and in vivo experiments. A carotid artery injury model was established in diabetic rats (totally, 105 rats) for subsequent in vitro study (the details are shown in supplement data 1). Finally, the results of the in vivo and in vitro studies were assessed by an analyst who was blinded to the experimental procedure.
Diabetic rat model
Ten-week-old male Sprague-Dawley (SD) rats (200–250 g) were injected intraperitoneally with 55 mg/kg streptozotocin (STZ; Sigma-Aldrich, St. Louis, USA). On days 7 and 14, rats with fasting blood glucose levels higher than 14 mmol/L were considered diabetic and included in subsequent experiments. If fasting blood glucose levels were lower than 14 mmol/L, another STZ injection was conducted to assure that diabetic rat model was constructed successfully.
EPCs isolation and culture
EPCs were isolated from the bone marrow of normal rats and rats with diabetes. The isolation, culture, and identification of EPCs were conducted as described in a previous study [23]. In brief, bone marrow was isolated from the femur and tibia and subjected to digestion, grinding, filtration, and resuspension in 10 mL of phosphate-buffered saline (PBS). EPCs were isolated from the cell suspension by Ficoll gradient centrifugation (1500×g) for 10 min at room temperature and cultured in endothelial basal medium (Lonza Group Ltd.) containing growth factors, based on the manufacturer’s instructions.
Fluorescent staining, immunocytochemistry, and flow cytometry were conducted to reveal the characteristics of the EPCs used in our study. In brief, fluorescent staining was used to detect the uptake of DiI-conjugated acetylated low-density lipoprotein (ac-LDL) (DiI-ac-LDL; Molecular Probes; Thermo Fisher Scientific, Inc.) and binding of FITC-UEA-l (Sigma-Aldrich; Merck KGaA). For immunocytochemistry, cells were fixed, incubated with primary antibodies overnight, and then incubated with secondary antibodies for 1 h. The cells were then washed three times and visualized using a fluorescence microscope (Leica AF6000). Antibodies specific for CD31 (1:100, Cat No. ab222783), CD34 (1:100, Cat No. ab81289), and vWF (1:100, Cat No. ab216566) were obtained from Abcam (Cambridge, Cambs., UK). For flow cytometry, cells were fixed and were then incubated with the following primary antibodies: BB515-conjugated mouse anti-human CD31 (Cat No. 565408), APC-conjugated mouse anti-human CD34 (Cat No. 560940), PE-conjugated mouse anti-human VEGFR-2 (Cat No. 560872), and FITC-conjugated mouse anti-human CD45 (Cat No. 554883). The antibodies were obtained from BD Biosciences (San Jose, CA, USA). Nonspecific fluorescence was assessed by incubation of similar cell aliquots with isotype-matched mouse monoclonal antibodies. Cells were washed with PBS and analyzed by using a GuavaeasyCyte™ Flow Cytometer (Millipore, Billerica, MA, USA).
Lentiviral transduction and RNA interference
Lentiviral transduction and RNA interference were conducted to upregulate or downregulate, respectively, the expression of CXCR7 to confirm the association between CXCR7 expression and EPC dysfunction.
Recombinant lentivirus encoding CXCR7 was constructed using the pLVX-EGFP-3FLAG-Puro vector (Shanghai Sunbio Medical Biotechnology, Shanghai, China). In brief, EPCs were seeded into 24-well plates at a density of 1 × 105 cells/well and incubated overnight. EPCs were then transduced overnight with purified lentiviral vectors that expressed recombinant CXCR7 at a multiplicity of infection of 25 in the presence of 3 μg/mL polybrene (Sigma, MO, USA). After 24 h of infection, the medium was replaced with 2 mL of fresh medium. The lentiviral transduction efficiency was determined by assessing lentiviral expression of green fluorescent protein (GFP). Apparent GFP expression was observed 48 h after transduction and peaked 72 h after transduction. The levels of CXCR7 expression were confirmed by Western blot analysis.
RNA interference was conducted to downregulate the expression of CXCR7 in EPCs. The sequences of the RNA interference and negative control (NC) constructs were as follows: CXCR7 siRNA sense, 5′-GGAAGAUCAUCUUCUCCUATT-3′ and antisense, 5′-UAGGAGAAGAUGAUCUUCCGG-3′; NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. Transfection was performed as described previously [24]. In brief, siRNA transfection was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocols. EPCs were plated into 6-well plates at a density of 5 × 105 cells/well and incubated overnight. The diluted siRNA constructs and Lipofectamine 2000 reagent were mixed at a ratio of 1:1 (4 pmol siRNA to 4 μL of Lipofectamine 2000) and incubated for 20 min at room temperature. Finally, 400 μL of the mixture was added to each well to achieve a final volume of 2 mL, and the cells were continuously incubated for 48 h before starting subsequent experiments.
Nrf2 expression in CXCR7-primed EPCs was knocked down to reveal the detailed mechanism underlying the regulation of EPC functional activity by the SDF-1/CXCR7 axis. In brief, EPCs were infected with lentiviruses containing shRNA against Nrf2 or nonsense shRNA (Genomeditech, Shanghai, China). The sequences of the Nrf2 and nonsense shRNA constructs are as follows:
Nrf2-shRNA 1, 5′- GGGTAAGTCGAGAAGTGTTTG -3′
Nrf2-shRNA 2, 5′- GGACCTAAAGCACAGCCAACA -3′
Nrf2-shRNA 3, 5′- GCAAGAAGCCAGATACAAAGA -3′
Nonsense shRNA, 5′- TTCTCCGAACGTGTCACGTAG -3′
Transfection was performed following a previously described protocol. After transfection for 48 h, the expression of Nrf2 was determined by Western blot analysis. Then, EPCs were infected with lentiviruses containing the shRNA against Nrf2 with the highest knockdown efficiency (lowest expression of Nrf2) or with nonsense shRNA determined by western blot analysis.
EPC adhesion assay
The adhesion of EPCs to human umbilical vein endothelial cells (HUVECs, Cell Resource Center of Shanghai Institute for Biological Sciences, Chinese Academy of Sciences; Cat No. 3131C0001000200023) was assessed by plating these cells in 24-well plates. First, HUVECs were plated in 24-well plates to form a monolayer. Then, the nonattached cells were washed away with PBS. DAPI (10 μg/mL) was used for staining HUVECs. EPCs were cultured in medium containing DiI dye (4 mg/mL) for 30 min at 37 °C following the manufacturer’s protocol. DiI-labeled EPCs were digested and harvested after three washes with PBS. DiI-labeled EPCs were then added to the plate and were then incubated with the HUVEC monolayer for 2 h, after which nonattached EPCs were washed away with PBS. Adherent EPCs were counted in five random fields under an Olympus microscope at × 400 magnification.
Repair capacity of EPCs in vitro
The repair capacity of EPCs in vitro was assessed using a scratch assay. In brief, EPCs were plated in 96-well plates to form a monolayer after 48 h of culture. Then, the confluent monolayer was scratched using a p200 pipette tip (1 mL) containing serum-free medium. The wound area was imaged under a microscope (magnification, × 100; Olympus Corporation) at baseline (0 h) and after 24 h, and the data were then analyzed using ImageJ software (National Institutes of Health).
Western blot analysis
EPCs were incubated for 24 h before protein extraction with a protein extraction kit (Solarbio, Beijing, China) and quantification with a bicinchoninic acid protein assay kit (Solarbio). Protein extracts were subjected to SDS-PAGE (KeyGEN, Nanjing, China) followed by transfer onto polyvinylidene fluoride membranes (Roche, IN, USA). The following primary antibodies (all obtained from Abcam (Life Technologies, CA, USA)) were used: anti-CXCR7 (1:1500, Cat No. ab138509), anti-GAPDH (1:10000, Cat No. ab8245), anti-p-Akt (1:5000, Cat No. ab38449), anti-Keap-1 (1:1000, Cat No. ab139729), anti-Nrf2 (1:1000, Cat No. ab92946), anti-HO-1 (1:2000, Cat No. ab189491), and anti-NQO-1 (1:1000, Cat No. ab80588). Subsequently, the membranes were incubated with secondary antibodies (1:1000, Cat No. bs-0346R-HRP; Beijing Boaosen Biotechnology Co., Ltd.) for 2 h at room temperature. Protein bands were visualized using an Epson Photo 1650 scanner (Seiko Epson Corp., Japan).
Rat model and treatment regimens
The vascular injury rat model was used to assess the effect of putative EPCs on intimal repair. After establishment of the rat model of type I diabetes described above, the vascular injury model was established as described previously [23]. In brief, the vascular endothelium was injured by a guidewire as used in percutaneous transluminal angioplasty. The operation was performed under anesthesia with pentobarbital sodium. Then, the bifurcation of the left carotid artery was exposed, and the common, internal, and external carotid arteries were separated to temporarily restrict blood flow. The common carotid artery was denuded by three repeated passages of the 0.38-mm flexible angioplasty guidewire through the external carotid artery. The denuded segment encompassed a total length of 5 mm from the carotid bifurcation. The external carotid artery was permanently ligated after the guidewire was removed, and the temporary ligatures were released to allow restoration of blood flow followed by skin suture.
Then, 1.5 mL of saline (control group) or a cell suspension (1 × 106 cells/mL) of normal EPCs (derived from normal rats; normal group), diabetic EPCs (derived from rats with diabetes; diabetic group), CXCR7-EPCs (derived from rats with diabetes, CXCR7-EPC group), CXCR7-EPCsNrf2-WT (derived from normal rats; CXCR7-EPCNrf2-WT group), or CXCR7-EPCsNrf2-KD (derived from normal rats; CXCR7-EPCNrf2-KD group) was injected into the circulation via the tail vein immediately and 12 h after establishment of the vascular injury model.
Assessment of reendothelialization and neointimal hyperplasia
The reendothelialization rate was assessed by Evans blue staining on days 7 and 14 after treatment. In brief, 0.5 mL of 0.5% Evans blue dye was injected intravenously via the tail vein 30 min before sacrifice. Cardiac perfusion was then performed via the bilateral jugular vein with formaldehyde fixation for 5 min followed by washing with PBS until the effluent ran clear. The common carotid artery was harvested 4 mm from the bifurcation after measurement by a Vernier caliper and opened longitudinally using microscissors. Then, an image of the vessel was acquired with a stereomicroscope (DVM6, Leica). Digital images were analyzed using NIH ImageJ 1.63 software. The total endothelial area in all groups was first measured and analyzed. After confirming that the total endothelial area did not differ significantly among the groups, the Evans blue-stained and unstained areas were measured to calculate the reendothelialization rate (unstained area/total area).
Neointimal hyperplasia was assessed using hematoxylin and eosin (HE) staining and Masson trichrome staining on day 21 after treatment. Denuded arteries were harvested from rats and immersed in formalin for 24 h. The neointimal thickness was assessed using the intimal area-to-medial area ratio (I/M) in HE-stained axial sections. A pathologist blinded to the treatment regimen assessed all specimens using NIH ImageJ 1.63 software.
Statistical analysis
Data are expressed as the mean ± standard deviation values. One-way or multi-way analysis of variance (ANOVA) with post hoc LSD comparisons with polynomial contrasts was used to determine significant differences between pairs of subgroups at each time point. p values < 0.05 were considered to be statistically significant. SPSS 20.0 software (IBM Corp., Armonk, NY, USA) was used to perform statistical analysis.