Male Sprague-Dawley (SD) rats were purchased from the Experimental Animal Center of Anhui Province (Anhui, China). The rats received a 12 h light and dark cycle everyday, and were kept at 20–25°C and 40–70% humidity. In addition, the rats were fed standard laboratory rodents feed ad libitum. All animals used in the present study received the appropriate care according to the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996). The present study was approved by the ethics committee of the Experimental Animal Center of Anhui Province.

MSCs were isolated from bone marrow of male SD rats (4–6 weeks old) following the standard procedure with certain modifications (22). In brief, bone marrow cells were collected from the bilateral femurs and tibias by removing the epiphyses, flushing the cavity with Dulbecco’s modified Eagle’s medium (DMEM) and centrifuging the suspension for 10 min at 300 × g. The cell pellet was then resuspended and cultured in 25 cm² culture flasks with complete media containing 10% fetal bovine serum (HyClone, Logan, UT, USA), at 37°C, 90% humidity and 5% CO₂. Non-adherent cells in the suspension were discarded following 48 h and fresh complete medium was added and replaced every 3–4 days thereafter. At 90% confluence, the cells were trypsinized (0.25% trypsin) and passaged at 1:3 ratios. Cells were identified by flow cytometry as described previously (23). MSCs between passages three and four were used for the following experiments.

MSCs were stimulated for 24 h with IL-1β (PeproTech, Rocky Hill, NJ, USA; 10 or 20 ng/ml), TNF-α (PeproTech; 10 or 20 ng/ml) or IL-1β (10 ng/ml) combined with TNF-α (10 ng/ml). In the meantime, cells in the control group were incubated in parallel without stimulation. Control and treated cells were used for subsequent experiments. Each experiment was repeated at least three times.

The control and treated MSCs were harvested and adjusted to a cell density of 10⁶/ml, then resuspended in 100 μl phosphate-buffered saline (PBS; 1×10⁶ cells). The cells were then incubated with phycoerythrin (PE)-VCAM-1 (BD Biosciences, Franklin Lakes, NJ, USA) antibody at room temperature for 30 min in the dark. Following this, the cells were washed twice with PBS and dispersed to make a single cell suspension in 400 μl PBS. Labeled cells were assayed using a flow cytometer (BD FACSCalibur; BD Biosciences) and analyzed with FCS4 software (version 1.2.4.1; De Novo Software, Los Angeles, CA, USA). At least 10,000 events were analyzed for each sample.

To analyze the MSC adhesion capacity, MSCs (5×10⁴cells/well) were seeded in collagen-coated 24-well plates in 250 μl complete medium and incubated for 20 min as previously described (8,24). The wells were gently washed twice with PBS to remove the non-adherent cells and the adherent cells were counted in six random fields per well under a microscope (magnification, ×100). The quantity of cells adhered to the plate reflected the relative adhesion ability of the MSCs.

Total RNA was extracted by using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), cDNA was obtained using a RevertAid First Strand cDNA Synthesis kit (Thermo Fischer Scientific) and then an amplification reaction was performed according to the manufacturer’s instructions. The gene-specific primers were designed using Primer Premier 5 software (Premier, Canada) based on cDNA sequences from Genebank and they were as follows: VCAM-1 forward, 5′-CCA GCG AGG GTC TAC CA-3′ and reverse, 5′-ACA GGG CTC AGC GTC AG-3′; β-actin forward, 5′-GTG GGC GGC CCT AGG CAC CA-3′ and reverse, 5′-CTC TTT AAT GTC ACG CAC GAT-3′. A Biometra T-Gradient thermal cycler (Biometra, Göttingen, Germany) was used for PCR. The PCR conditions were as follows: denaturation at 94°C for 30 sec, annealing at 60°C (VCAM-1)/51°C (β-actin) for 30 sec and extension at 72°C for 40 sec for 35 cycles. The PCR products were electrophoresed on a 1.0% agarose gel stained with 0.5 μg/ml ethidium bromide. The electrophoresis gel containing the PCR products was scanned using the UVP gel imaging system (JD-801; Jieda, Nanjing, Jiangsu, China). The expression of VCAM-1 mRNA was normalized to the expression of β-actin mRNA.

Western blot analysis of cell lysates was performed as previously described (25), proteins were denatured in Laemmli sample buffer (Beyotime Institute of Biotechnology, Shanghai, China) for 5 min at 95°C, samples were separated on 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in 5% non-fat dried milk in Tris-buffered saline containing 0.05% Tween-20 (TBST; Sigma, St. Louis, MO, USA) for 2 h prior to incubation with anti-VCAM-1 (1:1,000; Bioworld Technology, Inc., Minneapolis, MN, USA) overnight at 4°C and then conjugated with a secondary antibody, anti-rabbit Immunoglobulin G-horseradish peroxidase (Beyotime Institute of Biotechnology), for 1 h at room temperature. Membranes were washed three times in TBST and positive bands were detected by the enhanced chemiluminescence kit (Thermo Fischer Scientific). All the protein bands were scanned using Chemi Imager 5500 V2.03 software (Alpha Innotech, San Leandro, CA, USA). Protein band intensities were then analyzed by computerized image analysis system (Gel-Pro analyzer 4 software; Media Cybernetics, Rockville, MD, USA) and equal protein was normalized to β-actin.

Myocardial infarction was produced in male SD rats (weighing, 180–220 g) as previously described (26,27). First, a left thoracotomy was performed through the fourth intercostal space to expose the rat heart. Then, the left anterior descending coronary artery (LAD) was ligated with a 6-0 polyester suture. Successful ligation was confirmed by the typical myocardial infarction waves in electrocardiography recordings. The cells were harvested for 1 h prior to transplantation. The infarcted hearts (n=8) received intramyocardial injections of 100 μl control or treated MSCs (1×10⁶ cells). The injections were performed at four different sites in the free wall of the left ventricles.

Left ventricular (LV) function was assessed in anesthetized animals four weeks following transplantation of MSCs using two-dimensional echocardiography equipped with a 12-MHz probe (Philips Healthcare, Woerden, Netherlands). The animals were placed on a warming pad in the supine or lateral position. The greatest LV diameter of the internal end-diastole (LVID,d) and internal end-systole (LVID,s) was measured from the long axis view. The LV ejection fraction (LVEF,%) was calculated as [(LVID,d)3-(LVID,s)3]/(LVID,d)3 × 100. All measurements were averaged on at least three consecutive cardiac cycles and analyzed by an observer blinded to the treatments received by the animals.

Animals were euthanized prior to isolation and sectioning of their hearts into two transverse slices through the infarct area. The hearts were fixed in 10% formaldehyde prior to being embedded in paraffin. Sections (3 μm) were stained with Masson’s trichrome according to the manufacturer’s instructions (Maixin, Fuzhou, Fujian, China). Images of each slide were captured with an Olympus BX41 microscope (Tokyo, Japan). Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) was used to evaluate the percentage of myocardial infarction area which exhibited collagen deposition. The percentage of collagen deposition area was calculated as: (Fibrotic area/total LV area) × 100.

All values are expressed as the mean ± standard deviation (SD). Statistical analysis of the results between two groups was performed using a Student’s t-test. Differences among the groups were determined by a one-way analysis of variance. The analysis was performed using Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference between values.

Following the collection and immunostaining of the cells with antibodies, fluorescence was measured by flow cytometry. The results demonstrated the expression of cell surface markers, and that the majority of the cells were positive for CD29 and CD90, while they were negative for CD34. PE-VCAM-1 fluorescence intensities of treated MSCs were markedly increased and had statistical significance when compared with the control group (P<0.01). The expression of the VCAM-1 was moderately elevated with increasing concentration of the cytokines, but this difference was not statistically significant (P>0.05). However, co-treatment with the two factors markedly increased the VCAM-1 expression of MSCs as compared with the cells treated with one factor only (P<0.01; Fig. 1).

Pre-treatment with IL-1β and TNF-α significantly increased the MSC adhesion ability in vitro (P<0.01) with responses similar to those of the VCAM-1 expression. Incubation in 20 ng/ml cytokine moderately increased the number of adhered cells as compared with the 10 ng/ml treatment group; however, this difference was not statistically significant (P>0.05). There were only 8.4±2.3 cells adhered to the plate in the control group, but when treated with IL-1β, the number of MSCs adhered to the plate increased to 28.0±5.2 in the 10 ng/ml group and 30.4±3.4 in 20 ng/ml group. Similarly, in the TNF-α stimulation groups, the number of MSCs increased from 47.1±4.3 in the 10 ng/ml group to 49.7±6.2 in the 20 ng/ml group. By contrast, the number of adherent cells in the combined cytokine treatment group was significantly increased compared with the single cytokine groups (P<0.01). In the combined cytokine group, the number of MSCs adhered to the plates increased to 68.8±5.8 when treated with TNF-α (10 ng/ml) and IL-1β (10 ng/ml) (Fig. 2).

qPCR was used to detect VCAM-1 mRNA levels of MSCs. Following subtraction of the background, VCAM-1 mRNA levels were compared among the different groups relative to the β-actin mRNA levels. Incubation with IL-1β (10 or 20 ng/ml) induced the mRNA expression of VCAM-1 to 0.27±0.03 and 0.29±0.03, respectively, compared with 0.09±0.01 in the untreated control group. Stimulation with TNF-α (10 or 20 ng/ml) induced the transcription of VCAM-1 to ~0.33±0.03 and 0.36±0.04, respectively. By contrast, exposure of MSCs to IL-1β (10 ng/ml) and TNF-α (10 ng/ml) resulted in a marked increase in mRNA synthesis with a value of 0.52±0.05 relative to the level of β-actin mRNA (Fig. 3).

To further confirm the above results, the protein expression of VCAM-1 was quantified by measuring protein bands which were transferred to a PVDF membrane. In concordance with the flow cytometry results, adhesion experiments and mRNA data, the western blot analysis demonstrated that stimulation with IL-1β and TNF-α induced an evident increase in the VCAM-1 protein expression levels. IL-1β alone (10 ng/ml, 2.4±0.2-fold; 20 ng/ml, 2.7±0.3-fold), TNF-α alone (10 ng/ml, 3.1±0.2-fold; 20 ng/ml, 3.2±0.4-fold) and combination of the two cytokines (4.8±0.6-fold) markedly increased the protein expression of VCAM-1 in MSCs in vitro (Fig. 4).

To examine the therapeutic efficacy of treated MSCs in MI in vivo, the cells were transplanted into the border region between the infarcted and normal area of rat hearts following coronary ligation. At the end of the fourth week following surgery, nine of the rats had not survived the experiment, three of them in the control group, two in the IL-1β (10 ng/ml) group and the other four were one for each group. The LVID,d and LVID,s of the heart was measured and then the LVEF was calculated (Table I). The LVEFs of the stimulation groups were evidently improved as compared with the control group (28.6±1.5%) and the specific measurement results were as follows: The LVEFs in the 10 and 20 ng/ml IL-1β groups were 33.7±2.1 and 34.8±1.7%, respectively, in the 10 and 20 ng/ml TNF-α groups they were 40.9±2.2 and 43.0±2.1%, respectively, and in the co-treatment group, the LVEF was 49.9±2.4%.

To further verify the myocardial protection effect in vivo, the cardiac slices were stained with Masson’s trichrome. The non-infarcted left ventricular appeared red, while the infarcted myocardium replaced with fibroblasts and collagen appeared blue. The measurements revealed that the myocardial infarct size in both the IL-1β (10 ng/ml, 23.11±1.64; 20 ng/ml, 21.61±1.94%) and TNF-α (10 ng/ml, 17.71±1.85%; 20 ng/ml, 16.23±1.85%) groups was notably reduced compared with the control group (32.44±2.74%; P<0.01). Furthermore, the infarct size was even more reduced in the cytokine combination treatment group (8.37±1.60%). However, there was no significant difference between the infarct size in the hearts of the 20 ng/ml and 10 ng/ml cytokine stimulation groups (P>0.05; Fig. 5).