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Introduction

Human endothelial progenitor cells (EPCs) serve an important role in angiogenesis and endothelial repair (1,2). However, it is difficult to obtain an adequate number of EPCs for transplantation from one single donation. The function of EPCs is impaired in the presence of various cardiovascular disease risk factors, including age, hypertension and diabetes (3,4). Therefore, increasing the number of EPCs obtained from donations and improving their function is critical for EPC transplantation optimization.

In addition to direct differentiation into mature endothelial cells to repair endothelial injury, EPCs are capable of secreting protective paracrine factors to accelerate repair, which is another critical mechanism of EPC transplantation (5). There is increasing evidence to indicate that EPCs secrete various paracrine factors, including stromal cell-derived factor (SDF)-1, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), to improve endothelial cell function and repair various types of wounds in different animal models (6). EPC-conditioned medium (EPC-CM) promotes angiogenesis in rat artery loops, inhibits apoptosis in endothelial cells and increases the capillary density of an ischemic limb (7). Kim et al (8) reported that an injection of EPC-CM alone improves the recovery of diabetic wounds, and even requires a smaller number of cells compared with EPC transplantation.

Thymosin β4 (Tβ4) is a small protein widely distributed in numerous cells and tissues, which mediates multiple biological reactions, including vessel formation and wound healing (9). Previous studies indicated that Tβ4 promotes EPC proliferation, migration and adhesion, and inhibits apoptosis and senescence in vitro (10–12). Since these actions may all contribute to angiogenesis, there has been an increased interest in evaluating the role of Tβ4 in EPC angiogenesis.

VEGF is an important paracrine factor secreted by progenitor cells to promote angiogenesis. Barcelos et al (13) reported that prominin 1 (CD133)+ progenitor cells and the cell-conditioned medium (CCM) of CD133+ cells contain high levels of VEGF-A and interleukin (IL)-8, and application of these cells or CD133+ CCM accelerates the healing process of diabetic ulcers in the lower limbs of rats. In addition, neutralizing antibodies against VEGF-A or IL-8 inhibit the healing effect of CD133+ CCM (13). Wnt family member 7A expression is increased in CD133+ cell-treated wounds compared with either CD133− cell- or collagen-treated wounds. Secreted frizzled-related proteins are Wnt antagonists, and their application abolishes the facilitation of wound closure and reparative angiogenesis by CD133+ CCM (13). These findings indicate that the promotion of angiogenesis is primarily mediated by paracrine factors secreted by progenitor cells. However, whether Tβ4 stimulates EPCs to secrete an increased amount of VEGF to improve endothelial function requires further investigation. In the present study, it was examined whether pre-treatment of Tβ4 was able to augment the volume of VEGF mRNA and secretion of serum VEGF in EPC-CM. Additionally, the present study aimed to determine whether the increased amount of VEGF was able to further promote the angiogenesis of endothelial cells, which may improve the efficacy of therapeutic EPC transplantation.

Materials and methods

Cell culture

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.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

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.

Small interfering (si)RNA transfection

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′.

ELISA

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).

EPC tube formation assay

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).

Experimental animal model

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 staining

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.

Western blot analysis

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).

Statistical analysis

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.

Results

Tβ4 increases the secretion of VEGF from human EPCs

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.

Figure 1. Tβ4 increases VEGF secretion in human EPCs. (A) Treatment with Tβ4 increased the mRNA and (B) protein expression of VEGF in EPCs. n=4. *P<0.05, **P<0.01 vs. control group. Tβ4, thymosin β4; VEGF, vascular endothelial growth factor; EPCs, endothelial progenitor cells.

VEGF is a critical mediator of Tβ4-enhanced angiogenesis

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).

Figure 2. VEGF is a critical mediator of Tβ4-enhanced angiogenesis. (A) EPCs-CM, Tβ4-EPCs-CM and Tβ4-EPCs-CM with VEGF Ab was added to test the angiogenic ability of HUVECs in vitro at the concentration of 1,000 ng/ml for 24 h. Compared with the control group, EPCs-CM enhanced the angiogenic effect. This effect was further promoted in the 1,000 ng/ml Tβ4-EPC-CM-treated group. VEGF Ab reversed the pro-angiogenic effect of Tβ4-EPC-CM. (B) Angiogenesis was quantified by determining the number of branch points in five random fields of view. Magnification, ×100. n=6. *P<0.05, **P<0.01 vs. control group. #P<0.05 vs. EPCs-CM treated group. &P<0.05 vs. Tβ4-EPC-CM group. EPCs, endothelial progenitor cells; Tβ4, thymosin β4; VEGF, vascular endothelial growth factor; CM, conditioned medium; Ab, antibody.

Akt/eNOS pathway signaling is involved in Tβ4-induced VEGF secretion

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).

Figure 3. Akt/eNOS pathway signaling is involved in Tβ4-induced VEGF secretion. (A) Successful knockdown of Akt and eNOS expression by siRNA transfection was confirmed by western blot analysis. **P<0.01 vs. scramble siRNA group. (B) VEGF expression in EPC-CM was decreased following knockdown of Akt or eNOS. n=4. **P<0.01 vs. control group. #P<0.05 vs. Tβ4 + scramble group. Akt, RAC-α serine/threonine-protein kinase; eNOS, endothelial nitric oxide synthase; Tβ4, thymosin β4; VEGF, vascular endothelial growth factor; CM, conditioned medium; siRNA, small interfering RNA; EPCs, endothelial progenitor cells.

Tβ4-pretreated EPC transplantation significantly augments VEGF expression in the border zone of infarcted hearts

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).

Figure 4. Transplantation of Tβ4-pretreated EPCs significantly augments VEGF expression in the border zone of infarcted hearts. (A) VEGF expression was significantly higher in the EPC group compared with the untreated control group, and further increased in the Tβ4-EPCs group in MI hearts at 4 weeks post-MI, light microscopy was used (magnification, ×100). (B) Quantification of the positively stained VEGF area. (C) VEGF expression in the EPC and Tβ4-EPC transplanted group 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. n=6. *P<0.05 vs. sham surgery group. #P<0.05 vs. control group with MI surgery. &P<0.05 vs. EPC transplanted group with MI surgery. Tβ4, thymosin β4; EPCs, endothelial progenitor cells; VEGF, vascular endothelial growth factor; MI, myocardial infarction.

Discussion

Angiogenesis is essential for the survival and functional maintenance of the ischemic myocardium. Numerous known methods of vessel formation have been investigated in normal tissue and tumors, including the recruitment of EPCs that differentiate to endothelial cells, intussusception and sprouting angiogenesis (15). In the present study, it was reported that Tβ4 promoted EPC angiogenesis by increasing the secretion of VEGF, indicating that paracrine functions served an important role in the regulation of angiogenesis. The Akt/eNOS pathway was demonstrated to be involved in the regulation of VEGF expression induced by Tβ4. Furthermore, in vivo, VEGF was highly expressed near the border zone of infarcted hearts following transplantation with Tβ4 pretreated EPCs, suggesting that the ischemic myocardium may have benefited from angiogenesis mediated by VEGF expression. Taken together, the experiments of the present study revealed that Tβ4-pretreated EPCs promoted angiogenesis through activation of the Akt/eNOS pathway, and may provide a novel strategy for the effective transplantation of EPCs in MI.

Research has previously been conducted to determine the way in which EPCs home to ischemic areas and become involved in vasculogenesis, thereby increasing the blood flow to these tissues and preserving cardiac function (16,17). By using TEK tyrosine-protein kinase receptor (Tie-2)/LacZ b-galactosidase (β-gal) transgenic mice, Ii et al (18) observed that EPCs, from donor mice expressing β-gal driven by the endothelial gene promoter Tie-2, are incorporated into the microvasculature following myocardial ischemia (18). This provides direct evidence that EPCs contribute to neovascularization. Furthermore, EPCs activate angiogenesis through indirect mechanisms, including through the secretion of proangiogenic factors, including VEGF, HGF and SDF-1 (5). The present study demonstrated the ability of EPCs to induce the formation of neovasculature, and the critical role of VEGF in EPC-mediated angiogenesis in infarcted rat hearts, supporting the in vitro findings.

A number of studies have reported that two types of EPCs (early and late) may be isolated from peripheral blood (19–21). The tube formation abilities of these two cell types are different. Early EPCs with a spindle shape secrete more angiogenic cytokines, including VEGF, compared with late EPCs, which have a cobblestone shape. However, late EPCs form capillary tubes more efficiently compared with early EPCs. Previous studies demonstrated that following treatment with Tβ4, late EPCs exhibit increased migration (11), and reduced senescence (12) and apoptosis (10). Considering these findings, the present study used late EPCs to assess angiogenesis under treatment with Tβ4. It was demonstrated that the Akt/eNOS pathway is a critical component in regulating the signaling of multiple biological processes (22,23). Through the activation of this pathway, the migration and anti-apoptotic ability of late EPCs increased, and finally promoted angiogenesis to aid ischemic myocardial survival.

Tβ4 is required in the development of the coronary vasculature. Knocking down Tβ4 expression in the developing heart results in congenital coronary artery anomalies, demonstrating its pivotal role in vasculogenesis (24). By using embryonic EPCs, Kupatt et al (25) revealed that Tβ4, prothymosin α and Tβ10 are among the most abundantly secreted factors of early EPCs, suggesting that Tβ4 may be involved in EPC-regulated angiogenesis. The present study provided direct evidence that Tβ4 promoted EPC-mediated angiogenesis by increasing the secretion of VEGF. In the border zone of the infarcted hearts, VEGF expression was highly induced following Tβ4-pretreated EPC transplantation. However, the mechanism underlying Tβ4-induced angiogenesis is not well defined. The upstream kinases targeting Akt include the phosphoinositide 3-kinase (PI3K), phosphorylated (p)-phosphatase and tensin homolog, p-pyruvate dehydrogenase kinase 1 and the p-serine/threonine-protein kinase mTOR pathways (26). Our previous studies demonstrated that Tβ4 predominantly activates PI3K as a principal upstream component of the Akt pathway (11,12). By using siRNAs to target Akt and eNOS, the present study demonstrated that the AKT/eNOS pathway was involved in Tβ4-mediated VEGF expression. Notably, Tβ4 and VEGF are upregulated following the induction of Akt overexpression in bone marrow-derived mesenchymal stem cells (27). This suggests that a positive feedback loop may exist between the Akt pathway and Tβ4. In addition, hypoxia-inducible factor (HIF)-1α protein stability is directly increased by the expression of VEGF induced by Tβ4 (28). It is therefore plausible that under ischemic conditions, Tβ4 induces the expression of VEGF in a HIF-1α-dependent manner.

The secretion of VEGF contributes to endothelial cell differentiation (29). However, the role of VEGF in arterial and venous specification remains unclear and further study is required.

In conclusion, the results of the present study demonstrated that the angiogenesis of EPCs was strongly enhanced following treatment with Tβ4. The Akt/eNOS pathway mediated VEGF expression, which was important for this process. The present study demonstrated a novel way to improve EPC function and may be critical for the optimization of EPC transplantation.

Acknowledgements

The authors would like to thank the Biomedical Research (Therapy) Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University (Hangzhou, China) for the use of equipment.

Funding

The present study was supported by grants from the National Natural Science Foundation of China (grant nos. 81200114 and 81570246).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

YZ contributed to the study conception and design. JS acquired, analyzed and interpreted the data. XB was involved in drafting the manuscript and interpreting the data. JG and ZS revised the article critically for important intellectual content and interpreted the data. JZ and GF gave the final approval of the version to be published and contributed to the study conception. All authors read and approved the final manuscript.

Ethics approval and consent to participate

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 all patients.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References