We investigated the impact of bone marrow-derived mesenchymal stem cells (BM-MSCs) alone or in combination with hepatocyte growth factor (HGF) transplantation via noninfarct-relative artery in a swine myocardial infarction (MI) model. Donor BM-MSCs were derived in vitro from swine auto-bone marrow cultures labeled by bromodeoxyuridine (BrdU) incorporation. Host MI swine model was created by ligating the distal left anterior descending artery. After 4 weeks, age-matched male MI swines were used for the transplantation. Male MI swines were transfused via noninfarct-relative artery with vehicle (control, n=6) or BrdU-labeled BM-MSCs (5 × 106) alone (MSCs, n=6) or BrdU-labeled BM-MSCs (5 × 106) combined with HGF (4 × 109 PFU) (MSCs+HGF, n=6). To evaluate the collateral artery growth (Rentrop) and cardiac perfusion in these animals, gate cardiac perfusion imaging and coronary angiography were performed before and 4 weeks after transplantation, respectively. To assess the contribution of donor-originated cells in stimulation of cardiomyocyte regeneration and angiogenesis, immunohistochemistry for BrdU and α-smooth muscle actin (α-SMA) and quantitative image analysis were performed at 4 weeks after transplantation. The results are as follows: (1) BrdU-positive cells were detected in host myocardium in both MSCs and MSCs+HGF groups, but not in the vehicle group. Most BrdU-positive cells expressed myosin heavy chain β. (2) α-SMA-positive arteriole densities in the infarcted border area and infarcted area were increased significantly in both transplantation groups compared with the vehicle group. (3) Gate cardiac perfusion imaging demonstrated that the cardiac perfusion was significantly improved in transplantation groups compared with the vehicle group. (4) Ejection fraction and α-SMA-positive arteriole densities were increased significantly in both transplantation groups compared with the vehicle group. However, there was no difference in ejection fraction and α-SMA-positive arteriole densities between the MSCs group and the MSCs+HGF group. Growth of collateral arteries was not detected by coronary angiography in all three groups. In conclusion, the current study indicates that BM-MSCs transplantation via noninfarct-relative artery stimulates cardiomyocyte regeneration and angiogenesis and improves cardiac function, but does not stimulate collateral artery growth. BM-MSCs transplantation combined with HGF therapy is not superior to BM-MSCs alone transplantation. BM-MSCs transplantation via noninfarct-relative artery may be an alternative for those patients who cannot be transplanted via infarct-relative artery in clinical practice.
After myocardial infarction (MI), some heart cells are lost and others hibernate because they are underperfused. The previous reperfusion therapy could not reverse the progress in deterioration and subsequently heart failure became inevitable. Bone marrow stem cells have myogenic potential and offer the promise to perfuse the hibernating cells (angiogenesis).1, 2 Therefore, they are promising therapies for myocardial disease. Recently, new therapeutic strategies based on gene and protein are proposed. For example, vascular endothelial growth factor and angiopoietin-1 have been demonstrated to stimulate angiogenesis in acute MI. Hepatocyte growth factor (HGF), which has the potential angiogenic, anti-apoptotic, anti-fibrotic and anti-inflammatory benefits, has been given increasing attention in ischemic heart disease lately.3, 4 In our current study, to investigate the therapeutic impact of stem cells and gene therapy in acute MI, we employed mesenchymal stem cells (MSCs) alone or in combination with HGF.
A critical step for the clinical success of transplantation for myocardial repair depends on an efficient approach. In clinical practice, in some patients, the situation did not allow transplantation via infarct-relative artery, so we proposed a new approach to deliver MSCs, either alone or in combination with HGF via noninfarct-related artery.
Characterization of donor BM-MSCs
Mononuclear cells were prepared by Percoll gradient centrifugation technique with two major types of cells, MSCs and hematopoietic stem cells. The MSCs that attached to the bottom of the culture dish within 2–3 days proliferated in culture.1, 2 The medium was changed every 2 days, and hematopoietic stem cells that did not attach to the dish were washed from the culture with each medium change. Four days after bone marrow-derived stem cell seeding in culture plates, a contrast phased microscope revealed adherent cells in small colonies with fibroblast-shaped morphology. The fibroblast-like morphology was also maintained after cell passages and throughout the culture period.
At the end of cultures, cells were stained immunocytochemically for BrdU, vimentin and β-MHC. Results showed that almost all cells were positive for BrdU following 24 h of BrdU incorporation (Figure 1a). In the view of the fact that vimentin is a marker for MSCs, we examined the expression of vimentin in cultured cells. All fibroblastic cells were immunopositive for vimentin (Figure 1b). However, these cells were negative for β-MHC, as demonstrated by immunostaining (Figure 1c). These results revealed that cells for transplantation were proliferating MSCs, but did not express the specific protein for cardiomyocytes.
To determine whether MSCs alone or BM-MSCs+HFG transplantation stimulated collateral artery growth, the growth of collateral artery was examined by coronary angiography 4 weeks after ligating the left anterior descending coronary artery. Our results showed that growth of collateral arteries was not detected in three groups at 4 weeks after transplantation. The grades of collateral arteries were not altered significantly at 4 weeks after the transplantation compared to that before transplantation (Table 1).5
To evaluate whether MSCs alone or BM-MSCs+HFG transplantation improved cardiac perfusion, cardiac perfusions were determined by gate cardiac perfusion imaging before transplantation and at 4 weeks after transplantation. Cardiac perfusions were not altered in the vehicle group before and after the transplantation, but improved significantly in MSCs alone and BM-MSCs+HFG transplantation groups at 4 weeks after the transplantation compared with that before transplantation or when compared to the vehicle group (Figure 2).
To determine whether MSCs alone or BM-MSCs+HFG transplantation improved heart function, the ejection fractions were examined by gate cardiac perfusion imaging. Results showed that the left ventricular ejection fractions (LVEFs) were not altered in the vehicle group before and after the transplantation and improved significantly in MSCs and BM-MSCs+HFG transplantation groups at 4 weeks after the transplantation compared with that before transplantation and when compared to the control group. However, there were no further improvement in BM-MSCs+HFG transplantation groups compared with the MSCs transplantation group (Table 2).
To evaluate the specific colonization site of the labeled BM-MSCs, histological studies were performed on tissue sections at 4 weeks after infusion. We were able to confirm that the infarcted border area contained the donor cells, which were prelabeled with BrdU (Figure 3), but there were no donor cells in the remote intact myocardium. In addition, we were able to identify cells that were stained positively for myosin heavy chain β (β-MHC) in the infarcted border area.
Blood vessels in the infarcted area and infarcted border area stained positively for SMA (α-SMA+) (Figure 4). Vessel densities were higher in the infarcted area and infarcted border area in transplantation groups than in the control group (52.1±4.1 and 50.1±4.0 vs 16.4±3.5; 48.0±4.7 and 48.5±3.2 vs 15.7±3.2, P<0.05) (Table 3).
Ischemic heart disease accounts for 50% of all cardiovascular deaths and is the leading cause of congestive heart failure as well as premature permanent disability in workers. MI is, by nature, an irreversible injury.6, 7 After an MI, some heart cells are lost and others hibernate because they are underperfused. Cell transplantation and gene therapy offer the promise to replace the lost cells (myogenesis) and to perfuse the hibernating cells (angiogenesis). Recent reports have suggested that the MSCs and HGF could prevent heart failure after a myocardial injury.
As the physiological condition of swine is very similar to human beings, and growth of collateral arteries is almost complete at 4 weeks after acute MI, and hence the drugs (stem cells or HGF) could reach the target site through the collateral arteries, we employed the swine MI model in the present study. On the other hand, the spontaneous formation of collateral artery in the swine is poor after myocardial ischemia.8 In our study, macroscopic collateral artery growth (blood vessel diameter >200 μm) was not found by coronary angiography at 4 weeks after ligation. Thus, at the time of intervention, some animals in the treatment group were infused with the drugs (HGF and/or stem cells) through RCA and others were infused through left main circumflex.
So far, stem cell delivery and gene therapy for cardiac repair have been carried out by three approaches: via an intracoronary arterial route or by injection of the ventricular wall via a percutaneous endocardium or surgical epicardial approach. The advantage of intracoronary infusion – using standard balloon catheters – is that cells or genes can travel directly into myocardial regions, and also nutrient blood flow and oxygen supply are preserved. Therefore, this approach ensures a favorable environment for cell survival, a prerequisite for stable engraftment also.9 Wang et al.10 demonstrated that the transplanted bone marrow stem cells via coronary artery formed cardiocytes and endotheliocytes. However, in clinical practice, some patients who had complete occlusion in coronary artery were not allowed to be transplanted with stem cells or HGF via infarct-relative artery. For some reasons, for these patients. coronary stunting and coronary artery bypass grafting surgery did not suit. In the present study, we delivered the BM-MSCs alone or BM-MSCs with HGF via noninfarct-relative artery. Although the intracoronary delivery of BM-MSC may induce arteriolar embolism in coronary circulation system and result in a mild MI as described previously, we prepared BM-MSC into a single cell suspension achieved by forcefully expelling the cells through a 22-gauge syringe needle for the transplantation, and hence our study did not find similar damages as reported previously.
In our studies, cells prelabeled with BrdU were detected in the infarcted border area in both MSCs alone and BM-MSCs+HFG transplantation groups, but there were no donor cells in the remote, intact myocardium. β-MHC was also confirmed. The BrdU-positive cardiomyocytes contained organized sarcomeres, and most of the BrdU-positive myogenic cells located in the periphery of the infarct region. No junctions were seen between the BrdU-labeled cells and the host cardiomyocytes in the groups above. No gap junctions were present between the BrdU-labeled cells either. Similar to the findings of Orlic et al.,11 we showed that transplanted stromal cells formed cardiomyocytes in the two stem cell transplantation groups.
In the transplantation groups, we found that arterioles were stained positively for α-SMA in the infarcted area and infarcted border area. Arteriole densities were higher in the infarcted area and infarcted border area than in the control group. But between the stem cell group and the stem cells with HGF group, there were no changes in arteriole densities. Coronary angiography showed that there were no differences in the degree of collateral artery growth in the cell transplantation group and the control group at 4 weeks after infarction and cell transplantation (P>0.05). These results indicate that bone MSCs and HGF can induce angiogenesis, but do not contribute to small arteries. Gate cardiac perfusion imaging showed that cardiac function was improved in cell transplantation groups than in the control group (P<0.05), but there is no difference between the two groups. In our additional study, we discovered that HGF could improve cardiac function (LVEF) and there were no differences in LVEF between the HGF group and the BM-MSCs group (data not shown). However, in the present study, we found that HGF does not increase the effects of stem cells in cardiac function and angiogenesis in MI.
In summary, BM-MSCs combined with HGF transplantation via noninfarct-relative artery can stimulate cardiomyocyte regeneration and angiogenesis (arterioles) and improve cardiac function. But, there is no synergetic effect between BM-MSCs and HGF, and BM-MSCs combined with HGF transplantation cannot stimulate collateral artery growth.
Materials and methods
Preparation of donor cells
A 10 ml portion of bone marrow was aspirated from sterna of 3-month-old swines with a bone marrow aspirate needle under general anesthesia. After a homogenous cell suspension was achieved, the cells were centrifuged at 1400 r.p.m. for 8 min and then resuspended in complete culture medium (Iscove's modified Dulbecco's medium (IMDM)). Then, they were cultured in IMDM+20% FBS+1% antibiotics of benzylpenicillin and streptomycin (ABS) at 37°C in a humidified atmosphere with 5% CO2 for 3 days before the first medium change. After this, the medium was changed every 2 days. The mesenchymal population was isolated on the basis of its ability to adhere to the culture plate,12, 13 and hematopoietic stem cells that did not attach to the dish were washed from the culture with each medium.
To monitor cell and detect distribution in vivo, or discriminate the transplanted cells in the recipient myocardium, representative cultures were incorporated with bromodeoxyuridine (BrdU). BrdU solution (10 μmol/ml) was added into cultures before cell harvest for 24 h. Almost all adherent cells from these cultures were positive for BrdU detected by immunocytochemistry (Figure 1a). The stromal cells were detached from the culture dish with 0.05% trypsin digestion, collected and centrifuged. The cell pellets were resuspended with culture medium at a concentration of 5 × 106 cells/ml. A single cell suspension was achieved by forcefully expelling the cells through a 22-gauge syringe needle for the transplantation. These cells were used for transplantation back to the same swine.
MI animal model
Three-month-old (weighing 30±5 kg) male swine were anesthetized with a combination of ketamine (50 mg/kg) and xylazine (10 mg/kg). MI model was created by ligating at the distal one-third of the left anterior descending coronary artery and at the origin of the second diagonal artery. The animals were intubated with a cuffed endotracheal tube and ventilated with 100% oxygen to maintain PaCO2 between 35 and 45 mm Hg. Electrocardiography was used to monitor heart rate, rhythm and ST-segment changes during the surgical procedure.
Cell (HGF) transfer and cardiac function measurements
Eighteen male MI swines were divided randomly into three groups, vehicle, MSCs and MSCs+HFG, respectively, at 4 weeks after the MI. These animals were anesthetized with a combination of ketamine (50 mg/kg) and xylazine (10 mg/kg). The vehicle group, the MSCs group and MSCs+HFG group were infused via noninfarct-relative artery (right coronary artery or left circumflex artery) with IMDM culture medium, bone marrow-derived MSCs (BM-MSCs) (5 × 106) and BM-MSCs (5 × 106) with HGF (4 × 109 pfu), respectively. All the infusions were carried out through cardiac catheterization. The cardiac function was evaluated by gate cardiac perfusion imaging before and 4 weeks after cell (HGF) transplantation. Simultaneously, cardiac perfusion was evaluated by gate cardiac perfusion imaging.
Evaluation of the degree of collateral artery growth by Rentorp
At the end of 4 and 8 weeks after ligation, the grades of collateral artery growth were evaluated through coronary angiography (Rentorp). Grades of collateral filling from the contralateral vessel were as follows: 0=none; 1=filling of side branches of the artery to be dilated via collateral channels without visualization of the epicardial segment; 2=partial filling of the epicardial segment via collateral channels; 3=complete filling of the epicardial segment of the artery being dilated via collateral channels.14
Histology and immunohistochemistry
To assess the contribution of donor-originated cells in stimulation of cardiomyocyte regeneration and angiogenesis, immunohistochemistry for BrdU and α-smooth muscle actin (α-SMA) was performed at 4 weeks after transplantation. Tissue pieces (0.5 cm3) from the transplant and control sites were removed from the fixed heart and embedded in paraffin after which 10 μm sections were cut on a rotary microtome. The sections were stained with hematoxylin and eosin for cell and blood vessel identification. Immunohistochemical detection of specific antigens was performed for Brdu, β-MHC and α-SMA. Stained sections were observed under optical microscope and images were recorded using computer picture analysis apparatus.
Quantitative analysis for α-SMA+ arterioles
Following immunostaining for α-SMA, α-SMA+ arterioles were counted in each tissue section at magnification × 400 in the infarcted region and the border area of the infarction. Five high-power fields in each section were randomly selected and their blood vessel densities, which were expressed as α-SMA+ arterioles/mm2 (an area of high-power field is equal to 0.152 mm2), were calculated. Averages of five fields of three samples from each animal were used for comparison.
Data are expressed as mean±s.e. Analysis system software SPSS 11.0 was used for all analysis. A level of P<0.05 was considered as significant difference. Differences between groups were compared by analysis of variance.
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This work was supported by a grant from the Natural Science Foundation of Jiangsu Province (no. RC2002043).
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