Adult Sprague-Dawley rats, weighing 200±20 g were obtained from the Experimental Animal Center, Shanghai Medical College of Fudan University. Rats were bred and kept in special-pathogen-free (SPF) condition, with free access of food and water. All animal experiments were approved by the Ethics Committee of Shanghai Medical College of Fudan University and performed in accordance with institutional guidelines for animal experiments.

The male rats were sacrificed by cervical dislocation and immersed in 75% alcohol for 8 min for sterilization. Subsequently, the skin and fascia were cut with eye scissors and the muscles were stripped. Then the femurs and tibias were isolated and placed in pre-cooled Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA). Afterwards, the bone marrow was flushed from femurs and tibias of the rats using a syringe with DMEM, collected in a 15-ml centrifuge tube and dissociated into a single-cell suspension by pipetting. Then the single-cell suspension was slowly transferred to a new centrifuge tube containing 10 ml lymphocyte separation medium and centrifuged for 20 min at 2,000 rpm. The liquid in the middle layer was separated and resuspended with DMEM. After centrifugation at 1,000 rpm for 10 min, EPCs were obtained and suspended with Endothelial Basal Medium-2 (EBM-2; Lonza, Basel, Switzerland), and inoculated in a 6-cm culture dish pre-coated with 0.1% fibronectin at 5×10⁵ cells/ml. Cells were incubated in a 37°C and 5% CO₂ incubator. When cells were grown to 70–80% confluence, they were used for further analysis.

The cultured EPCs (passage 0) were digested with 0.25% trypsin-EDTA solution for 2 min at room temperature after washing with phosphate-buffered saline (PBS). Then the cells were collected, counted and resuspended with PBS containing 0.5% bovine serum albumin (BSA) at 1×10⁸ cells/ml. Afterwards, the cells were incubated with FITC-conjugated antibodies against CD34, PE-conjugated antibodies against CD133 and VEGFR-2 at 4°C for 10 min. After centrifugation, the harvested cells were smeared onto glass slides and observed under confocal microscopy.

The identified EPCs (passage 0) were seeded into 24-well plates at 1×10⁵ cells/ml and treated with DMEM (control) and Tβ4 at different concentrations (0.05, 0.1 and 0.2 µM), respectively.

Twenty-four hours after Tβ4 treatment, 0.5% (w/v) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS was added to each well and the mixture was incubated at 37°C for 4 h. Afterwards, dimethyl-sulfoxide (DMSO) was added to the mixture and the mixture was incubated with gentle shaking for 10 min. Cell viability was determined by the absorbance at 570 nm using a micro-plate reader. Three replicate trials were conducted for each concentration of Tβ4.

Twenty-four hours before the experiment, DMEM was placed in a hypoxic chamber (1% O₂, 94% N₂ and 5% CO₂) for pretreatment. After cells were treated with Tβ4 at different concentrations, they were incubated in pretreated DMEM without fetal bovine serum (FBS) in the hypoxic chamber (1% O₂, 94% N₂ and 5% CO₂) for 12 h. Subsequently, a portion of the cells was added together with 0.5% w/v MTT solution for detection of cell viability. The other portion of cells was stained with ethidium bromide and acridine orange (EB/AO) and observed under a fluorescence microscope. Five random fields in each well were analyzed for calculating surviving and apoptotic cells.

The changes in EPC migration after Tβ4 treatment were measured in Transwell cell culture chambers with 6.5-mm diameter polycarbonate membrane filters (8-µm pore). The Transwell chambers were placed in 24-well plates, and the membrane filters were coated with 40 ml of Matrigel solution [diluted with FBS-free DMEM solution]. After drying, EPCs were seeded into the chamber at a density of 5×10⁵/chamber and then treated with Tβ4 at different concentrations. FBS-free EBM-2 was added into the upper chamber of the devise, whereas the lower chamber was filled with EBM-2 containing 5% FBS. After 12 h of incubation at 37°C, non-migrated cells were removed from the upper surface of the membrane using a cotton swab. Then the filters were fixed in 90% alcohol for 30 min and stained with crystal violet for 10 min. The migrated cells were observed under a microscope and determined by the absorbance at 600 nm using a microplate reader after addition of 10% acetic acid.

Firstly, the 96-well plates were filled with pre-cooled Matrigel solution (50 µl in each well) and placed in a cell culture chamber with 5% CO₂ at 37°C for 30 min. Then EPCs were inoculated into the 96-well plates at a density of 1×10⁵/well and treated with Tβ4 at different concentrations. In addition, 5% FBS was added to each well and EPCs were incubated in 5% CO₂ at 37°C for 6 h. Three different images from each well were captured, and the total tubule number was calculated by two independent investigators.

EPCs after treatment of Tβ4 at different concentrations were lysed with RIPA lysis buffer containing 10 mM phenylmethylsulfonyl fluoride (PMSF). Then the lysates were isolated by centrifugation and total protein was quantified using a standard bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). After quantification, the proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene difluoride membrane. The membranes were incubated in 5% milk in 0.1% Tris-buffered saline (TBS) at room temperature for 1 h. Afterwards, the membranes were incubated with anti-Akt and anti-p-AKT antibodies (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, Cambridge, MA, USA) at a dilution of 1:5,000. Finally, the membranes were washed and visualized using an enhanced chemiluminescence system (GE Healthcare, Piscataway, NJ, USA).

MI was induced by surgical ligation of the left anterior descending coronary artery in 24 female rats. Firstly, the animals were anesthetized with ketamine (80 mg/kg) and placed in a supine position. Then they were intubated and mechanically ventilated with a rodent ventilator with a respiratory rate of 80 beats/min, and tidal volume of 7 ml. A left-side thoracotomy was performed to expose the heart. After removing the pericardium, the left arterial descending coronary artery was permanently ligated. After establishment of the MI model, rats were randomized into three groups, with 8 rats in each group: control group, EPC group and Tβ4-EPC group. Rats in the control group received an intramyocardial injection of 100 µl PBS; rats in the EPC group received an intramyocardial injection of 100 µl PBS containing 2×10⁶ EPCs; and rats in the Tβ4-EPC group were administered 2×10⁶ EPCs pretreated with Tβ4 (2 µM). Another 8 mice underwent a sham operation including every step except coronary ligation and were assigned into the sham group. Surgery was finally completed by chest closure and muscle and skin suture. Rats in each group were treated with penicillin after surgery to prevent infection.

Four weeks after EPC injection, the cardiac function of all rats was monitored using an ultrasound system with a 15 MHz probe, sample volume of 0.6 mm and scanning speed of 100 mm/sec. M-mode images were obtained by drawing an anatomical M-mode line perpendicular to the interventricular septum and left ventricular posterior wall. The measurements for left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) were carried out according to the recommendations of the American Society of Echocardiography. The ejection fraction of the left ventricle (EF) and fractional shortening (FS) were calculated using the Teichholz formula: EF ( % ) = [ ( LVEDV − LVESV ) / LVEDV ] × 100 % FS ( % ) = [ ( LVEDD − LVESD ) / LVEDD ] × 100 % .

The value of each parameter was obtained from three different cardiac cycles.

After echocardiography, the rats were anesthetized with ketamine and the heart was removed from the open chest of the rats. Then the hearts were washed with normal saline, fixed in 4% paraformaldehyde for 24 h, immersed in 15 and 30% sucrose in PBS for dehydration and embedded in OCT compound. Then the tissue samples were cut into 5-µm sections and stained with Masson's trichrome. Myocardial tissue and scar tissue in the infarcted area were observed under light microscopy and the epicardial perimeters of the infarcted area and the whole ventriculus sinister were assessed using Image-Pro Plus 6.0 software. The infarcted size was determined by the following formula: Infarcted size = epicardial perimeters of the infarcted area / epicardial perimeters of the whole ventriculus sinister × 100 % .

For immunohistochemistry, tissue sections were blocked in goat serum. Then the sections were incubated with mouse specific primary antibodies [anti-CD31 and smooth muscle actin-α (α-SMA)] at a dilution of 1:100 at 4°C overnight, followed by incubated with DyLight 594-conjugated and FITC-conjugated goat anti-mouse secondary antibodies at room temperature for 1 h. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Vascular structures positive for the expression of both CD31 and α-SMA were observed under fluorescence microscopy at ×200 magnification. Five random fields from each slide were used to calculate the microvessel density and three slides selected from the upper, middle and lower region of the infarcted area of the heart were used for analysis.

Tissue sections were first immersed in PBS containing 0.2% Triton X-100. After washing with PBS, the sections were treated with 0.2 N HCl solution for 20 min at room temperature and digested with 0.1 mg/ml proteinase K solution in a humidified atmosphere. Then probe buffer was added onto the slides at 37°C for 30 min to block non-specific binding. Afterwards, the sections were incubated with cDNA probe buffer containing 1 ng/µl Y-chromosome probe at 95°C for 10 min and at 37°C overnight. On the next day, the slides were washed with pre-warmed 2× SSC solution [17.53 g of sodium chloride (0.29 M), 8.82 g of sodium citrate and distilled water in a final volume of 1,000 ml, pH 7.0], 2× SSC solution and 0.1× SSC solution, respectively. After blocking with 5% goat serum for 5 min, the slides were treated with FITC-labeled streptavidin and then incubated for 1 h at room temperature. Finally, the sections were immunostained with the anti-CD31 antibody at a dilution of 1:100. DAPI was used to stain cell nuclei. The distribution and endothelial differentiation of cells positive for Y-chromosome were observed under a microscope.

All statistical analysis was conducted with SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation (SD) and independent t-test was utilized to perform comparison between two groups. A value of P<0.05 was considered to indicate a statistically significantly difference.

EPCs were isolated from bone marrow and cultured in a 37°C and 5% CO₂ incubator. Three days after inoculation, colony growth occurred on adherent cells. After additional culture of 2 days, the cells were grown to 80% confluence. The cells were spindle-shaped and covered with projections. The results from the immunofluorescent staining revealed that >90% of the cells expressed CD34, CD133 and VEGFR-2 (Fig. 1). The results indicate that the EPCs were successfully prepared.

As shown in Fig. 2, there was a significant increase in cell viability in the EPCs following treatment with Tβ4 compared to the viability of EPCs without Tβ4 treatment (P<0.05). The effects of Tβ4 in promoting EPC viability were in a dose-dependent manner (0.05 vs. 0.1 µM, 0.05 vs. 0.2 µM and 0.1 vs. 0.2 µM: 113.2±6.23 vs. 120.9±8.73%, 113.2±6.23 vs. 135.7±8.71% and 120.9±8.73 vs. 135.7±8.71%, P<0.05).

Based on the results from the MTT assay (Fig. 3A), compared to the EPCs without Tβ4 treatment, there was an obvious increase in cell viability in the EPCs after Tβ4 treatment at different concentrations (P<0.05) under hypoxia and serum deprivation and the growth-promoting effects were also in a dose-dependent manner. In addition, after staining with EB/AO, the apoptotic cells in the Tβ4 treatment groups were less than those without Tβ4 treatment (Fig. 3B).

As shown in Fig. 4A–D, the number of migrated cells in the Tβ4 treatment group was increased compared to that in the control group. After staining with crystal violet, the numbers of migrated cells from each group were quantified. A significant increase in the number of migrated cells was observed in the Tβ4 treatment group (P<0.05) and the migration-promoting effects of Tβ4 was in a dose-dependent manner (Fig. 4E).

In each group, 6 h after inoculation of EPCs into Matrigel, most of the cells were covered with projections. These projections connected with each other, leading to the formation of tubular structure. Moreover, Tβ4 promoted the formation of EPC-based vascular structure. As the concentration of Tβ4 was increased, more tubular structures were observed (Fig. 5A–D). After quantification analysis, compared to the number of tubular structures in the control group, there was a significant increase in EPCs treated with Tβ4 at different concentrations (P<0.05) (Fig. 5E). In addition, the promoting-effect of Tβ4 on EPC-based neovascularization was in a dose-dependent manner.

Following the results from the western blot analysis (Fig. 6), an obviously upregulation of the expression of phosphorylated Akt was noted in the EPCs treated with Tβ4 at different concentrations compared to the EPCs treated with DMEM (P<0.05). Additionally, as the dose of Tβ4 increased, the promoting effects of Tβ4 on p-Akt expression were also elevated (0.05 vs. 0.1 µM, 0.05 vs. 0.2 µM and 0.1 vs. 0.2 µM, P<0.05).

Based on the results of echocardiography, transplantation of the EPCs or Tβ4-EPCs both reduced left ventricular chamber and increased the left ventricular wall thickness in rats with MI (Fig. 7A). After statistical analysis (Fig. 7B–G), compared to the rats in the control group, both EF and FS were significantly increased in the rats transplanted with EPCs and Tβ4-EPCs (P<0.05). Moreover, both parameters were also significantly higher in the rats transplanted with Tβ4-EPCs than those with EPCs (P<0.05). Furthermore, both LVESD and LVESV were significantly decreased in the rats transplanted with Tβ4-EPCs compared to the rats in the control group (P<0.05).

Masson's trichrome staining showed an obvious expansion of the left ventricular chamber and thinning of the left ventricular wall in rats that underwent surgical ligation of the left anterior descending coronary artery. Moreover, most of the myocardium in the infarcted area was replaced by scar tissue. Whereas, in the MI-induced rats transplanted with EPCs or Tβ4-EPCs, the left ventricular chamber was smaller, the left ventricular wall was thicker and there was much less scar tissue and more myocardial cells compared to the rats in the control group (Fig. 8A). Statistical results indicated that in rats transplanted with Tβ4-EPCs the left ventricular wall thickness was significantly larger and the infarcted size was significantly smaller than these parameters in the control and EPC groups (P<0.05) (Fig. 8B and C).

Angiogenesis was observed in the marginal zone of the infarcted area as confirmed by immunofluorescence. Transplantation with EPCs promoted angiogenesis as confirmed by the increase in CD31- and αSMA-positive cells in the infarcted area (Fig. 9A). In addition, transplantation with Tβ4-EPCs showed more promoting effects on angiogenesis. Based on the statistical results, the microvessel density was significantly higher in rats transplanted with Tβ4-EPCs than that in rats in the EPC and control groups (P<0.05) (Fig. 9B).

Furthermore, based on the results from FISH, Y-chromosome-positive cells were observed in the marginal zone of the infarcted area, which constitutes the endothelial tissue. There were a higher number of Y-chromosome-positive cells in rats transplanted with Tβ4-EPCs than the number in rats transplanted with EPCs (P<0.05) (Fig. 9C).