EPCs were isolated, cultured and characterized according to previously described techniques (10–12). The present study was approved by the Ethics Committee of Sir Run Run Shaw Hospital of Zheijang University (Hangzhou, China) and written informed consent was obtained from 30 healthy individuals (20–55 years old; 50:50 male:female). The date range of the recruitment was from September 2014 to December 2014. All the samples were collected in the biomedical research center of Sir Run Run Shaw Hospital. To obtain EPCs, mononuclear cells were isolated from human peripheral blood via density-gradient centrifugation (400 × g; 35 min; room temperature) with Ficoll separating solution (Cedarlane Laboratories, Burlington, ON, Canada). The mononuclear cells were subsequently placed on fibronectin (Merck KGaA, Darmstadt, Germany)-coated plates and incubated with endothelial growth medium-2 (EGM-2MV; Lonza Group, Ltd., Basel, Switzerland). After 7 days of incubation at 37°C and 5% CO₂, 10⁶ of cells grew into EPCs, which exhibited a spindle shape. The EPCs were detached using trypsin and subsequently collected for further experiments. Human umbilical vein endothelial cells (HUVECs) were obtained from ScienCell Research Laboratories, Inc. (San Diego, CA, USA) and cultured in endothelial cell medium supplemented with 10% fetal bovine serum (both ScienCell Research Laboratories, Inc.). For experimental treatments, cells (passages 3–8) were grown to 70–90% confluence.

Total RNA was extracted from EPCs using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNA synthesis was performed with 1 µg total RNA using a PrimeScriptTM RT Master Mix (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. The reaction mixture was incubated under the following condition: RT, 37°C for 15 min; and inactivation of RT with heat treatment, 85°C for 5 sec. RT-qPCR was conducted using the SsoFastTM EvaGreen Supermix with Low ROX (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The reaction mixture was incubated under the following thermocycling condition: Polymerase activation, 95°C for 30 sec; denaturation, 95°C for 5 sec; annealing/extension, 55°C for 30 sec; melt curve analysis, 65–95°C in 0.5°C increments for 2 sec. Data analysis was performed with an ABI 7500 cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the 2^−ΔΔCq method, as previously described (14). GAPDH was used as the endogenous control for mRNA expression. The primers used were as follows: GAPDH forward, 5′-GGGTGTGAACCATGAGAAGT-3′ and reverse, 5′-GACTGTGGTCATGAGTCCT-3′; VEGF forward, 5′-TCTTGGGTGCATTGGAGCCT-3′ and reverse, 5′-AGCTCATCTCTCCTATGTGC-3′; and IL-8 forward, 5′-CCTGATTTCTGCAGCTCTGT-3′ and reverse, 5′-AACTTCTCCACAACCCTCTG-3.

RAC-α serine/threonine-protein kinase (Akt) and endothelial nitric oxide synthase (eNOS) siRNA, in addition to the scramble sequence negative control siRNA, were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). When 60–80% confluence was reached, cells were transfected with 150 pmol Akt siRNA, eNOS siRNA or negative control siRNA with HiPerFect Transfection Reagent (Qiagen GmbH, Hilden, Germany) incubated for 24 h (15–25°C). The effectiveness of Akt and eNOS knockdown was confirmed with western blotting. The Akt siRNA sequence was as follows: Forward, 5′-GCACUUUCGGCAAGGUGAUTT-3′ and reverse, 5′-AUCACCUUGCCGAAAGUGCTT-3′. The eNOS siRNA sequence was as follows: Forward, 5′-CAGUACUACAGCUCCAUUATT-3′ and reverse, 5′-UAAUGGAGCUGUAGUACUGTT-3. The scramble siRNA sequence was as follows: Forward, 5′-GACCCAAAUUUGACAUGAUTT-3′ and reverse, 5′- AUC AUG UCA AAU UUG GGU CTT-3′.

A density of 4×10⁵ transfected EPCs were seeded in 6-well plates and treated with 0, 10, 100 and 1,000 ng/ml Tβ4 (ProSpec-Tany TechnoGene, Ltd., East Brunswick, NJ, USA) for 37°C 24 h. Conditioned media were subsequently collected for the VEGF ELISA (cat. no. DVE00; Abcam, Cambridge, UK).

The tube formation assay was performed using a Matrigel matrix™ basement membrane (BD Biosciences, Franklin Lakes, NJ, USA) to investigate HUVEC angiogenesis under treatment with different types of EPC-CM (8). Matrigel solution was thawed at 4°C overnight and subsequently diluted with EBM-2 (Clonetics^™; Lonza Group, Ltd., Basel, Switzerland) to solidify in a 96-well plate at 37°C for 1 h. A total of 2×10⁴ HUVECs were seeded on the matrix for further incubation with either 2 ml EPC-CM, Tβ4-EPC-CM or Tβ4-EPC-CM with 0.4 µg/ml VEGF antibody (cat. no. MAB293; R&D Systems, Inc., Minneapolis, MN, USA) at 37°C for 6 h. The CCM was the supernatant of different group of EPCs obtained following centrifugation at 150 × g for 5 min. Tube formation was assessed using a light microscope (magnification, ×100) and five random fields were selected for each assay. The average branch point numbers that indicated tube formation were compared using Image Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA; 8th Edition, 2011) and were approved by the Institutional Animal Care and Use Committee of Zhejiang University (Hangzhou, China). The rat myocardial infarction (MI) model was established as previously described (10). A total of 40 male Sprague-Dawley rats (8 weeks; weighing 200–250 g) were purchased from the Experimental Animal Center of Zhejiang University (Hangzhou, China). Experimental rats were anesthetized and intubated for artificial ventilation. A left thoracotomy was subsequently performed. The left anterior descending coronary artery (LAD) was ligated with a 6-0 suture with the fourth intercostal space open. Sham-operated (sham) rats received surgery without LAD ligation. Rats in the EPC group (EPCs) were intramyocardially injected with 1×10⁶ human EPCs re-suspended in 150 µl EGM-2, while rats in the Tβ4-EPC group (Tβ4-EPCs) received the same amount of Tβ4 pretreated human EPCs (EPCs were pretreated with 1,000 ng/ml Tβ4 for 24 h). Rats receiving 150 µl blank EGM-2 without cells were set as the control group. A total of three sites in the border zone of the infarcted heart received three injections. The rats were sacrificed 4 weeks following this procedure.

Immunohistochemical analysis of VEGF (1:100; cat. no. ab46154; Abcam) staining was performed, in which 5 µm fresh frozen sections (−20°C) of rat cardiac tissue were incubated with the primary antibody at 37°C for 2 h. The process was followed by incubation with specific horseradish peroxidase (HRP)-conjugated secondary antibodies at 37°C for 10 min (1:100; cat. no. SPN-9001; OriGene Technologies, Inc., Beijing, China) and DAB at 37°C for 2 h. Sections were rinsed in PBS and subsequently counterstained with 0.5% hematoxylin (cat. no. ZLI-9608; OriGene Technologies, Inc.) at room temperature for 1 min, dehydrated and mounted. A total of five random fields were selected for assessment, and the area of myocardial fibrosis and the expression of VEGF were quantified using Image-Pro Plus 6.0.

Rat cardiac tissue was harvested for western blot analysis. The protein was extracted using radioimmunoprecipitation buffer (Thermo Fisher Scientific, Inc.) and protein concentration was determined with a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). A total of 50 µg protein sample was loaded per lane on a 10% Tris-glycine gradient gel, and subsequently transferred onto polyvinylidene difluoride membranes and blocked with 5% non-fat milk for 1 h at room temperature. Following this, membranes were incubated with the following primary antibodies (1:1,000) overnight at 4°C: VEGF (cat. no. Ab46154; Abcam), eNOS [cat. no. 32027; Cell Signaling Technology (CST), Inc., Danvers, MA, USA], Akt (cat. no. 2920; CST, Inc.) and GAPDH (cat. no. 5174; CST, Inc.). The membrane was subsequently incubated with goat anti-rabbit IgG-HRP secondary antibodies (1:5,000; cat. no. 7074; CST, Inc.) at room temperature for h. Proteins were visualized with enhanced chemiluminescence reagent (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and Image Quant LAS-4000 (GE Healthcare Life Sciences, Shanghai, China). The grayscale values of the bands were examined using Image Multi-Gauge software 2.0 (Fujifilm, Tokyo, Japan).

Data are presented as the mean ± standard error of the mean of least three independent experiments. Statistical analyses between groups were performed using SPSS software version 19.0 (IBM Corp., Armonk, NY, USA) using one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The present study investigated the effect of Tβ4 on the expression of VEGF in EPCs. Treatment with Tβ4 significantly increased the expression of VEGF in EPCs at the transcriptional (Fig. 1A) and protein (Fig. 1B) levels. This pro-secretory effect was dose-dependent, the highest VEGF mRNA/protein concentrations were observed at 1,000 ng/ml Tβ4 for 24 h. At the transcriptional and protein levels, the fold changes in expression were 2.65 and 2.54-fold, respectively, compared with the control group.

The present study investigated the effect of EPC-CM on the angiogenesis of HUVECs in vitro by performing a tube formation assay (Fig. 2A). It was demonstrated that EPC-CM enhanced the angiogenesis of HUVECs compared with the control group, and pretreatment with 1,000 ng/ml Tβ4 further augmented the effect of EPC-CM. Furthermore, the presence of VEGF antibody reversed the pro-angiogenic effect of Tβ4-pretreated EPC-CM (Fig. 2B).

To further investigate whether the Akt/eNOS pathway was involved in Tβ4-induced VEGF secretion in EPCs, Akt and eNOS siRNAs were used to knock down the expression of Akt and eNOS. Western blot analysis was performed to confirm that Akt and eNOS expression was effectively reduced compared with the scramble control (Fig. 3A). In addition, the levels of VEGF in EPC-CM were significantly decreased following knockdown of Akt or eNOS, which indicated that the Akt/eNOS pathway was involved in Tβ4-induced VEGF secretion (Fig. 3B).

Immunohistochemical analysis demonstrated that VEGF expression in cardiac tissue was significantly augmented at 4 weeks post-MI compared with the sham group. The expression of VEGF was significantly higher in the EPC transplantation alone group compared with the untreated control group, and it was significantly higher in the Tβ4-EPC-transplanted MI hearts at 4 weeks post-MI compared with the control and EPC groups (Fig. 4A and B). Western blot analysis indicated that VEGF expression was significantly augmented at 4 weeks post-MI. VEGF expression in the EPCs and Tβ4-EPCs groups increased significantly compared with the control group, and the expression of VEGF in the Tβ4-EPCs group was significantly higher compared with the EPCs group (Fig. 4C).