CLINICAL RESEARCH: PRECLINICAL STUDY
A novel application of myocardial contrast echocardiography to evaluate angiogenesis by autologous bone marrow cell transplantation in chronic ischemic pig model
Hiroko Fujii, MD, PhD* ,
Shinji Tomita, MD, PhD* ,2,
Takeshi Nakatani, MD, PhD ,
Shinya Fukuhara, MD||,
Akihisa Hanatani, MD, PhD,
Yoshinori Ohtsu, MD*,
Michiko Ishida, MD,
Chikao Yutani, MD, PhD||,
Kunio Miyatake, MD, PhD, FACC and
Soichiro Kitamura, MD, PhD, FACC,1
* Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center, Osaka, Japan
Department of Cardiovascular Medicine, National Cardiovascular Center, Osaka, Japan
Department of Cardiovascular Surgery, National Cardiovascular Center, Osaka, Japan
Department of Organ Transplantation, National Cardiovascular Center, Osaka, Japan
|| Department of Pathology, National Cardiovascular Center, Osaka, Japan
Manuscript received July 11, 2003;
revised manuscript received September 1, 2003,
accepted September 16, 2003.
2 Correspondence: Dr. Shinji Tomita, Cardiothoracic Surgical Unit, Auckland City Hospital, Private Bag 92024, Auckland, New Zealand.
shinjitomita{at}hotmail.com 1 Reprint requests: Dr. Soichiro Kitamura, President of National Cardiovascular Center, Chairman of Department of Cardiovascular Surgery, 5-7-1 Fujishirodai, Suita, Osaka, 565-8565 Japan.
skitamura{at}hsp.ncvc.go.jp
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Abstract
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OBJECTIVES: We investigated the feasibility of myocardial contrast echocardiography (MCE) to evaluate regional perfusion after bone marrow cell transplantation.
BACKGROUND: The myocardial microvessels improved by cell transplantation are too small to visualize with conventional angiography.
METHODS: Fourteen mini-pigs from the Nippon Institute for Biological Science were used. The proximal left anterior descending coronary artery was ligated. One month later, nine pigs survived. Six pigs received autologous cell transplantation into the left ventricular anterior wall: bone marrow mononuclear cells (BMMNCs) (n = 3) and bone marrow stromal cells (BMSCs) (n = 3). The other three pigs received saline (control group, n = 3). The pigs were sacrificed one month later. Myocardial contrast intensity (MCI) with a contrast agent was measured using the SONOS 5500 system (Philips). Capillary density (CD) and MCI were measured at four areas: anteroseptum (nontransplanted infarct area), anterior wall (transplanted infarct area), septum (border zone), and lateral wall (normal). We compared the anteroseptum with the anterior wall by MCI and CD.
RESULTS: In the BMMNC and BMSC subsets, the CD of the anterior wall was higher than that of the anteroseptum (p < 0.001). There was a linear relation between MCI and CD (acoustic unit [AU2] = 0.234 CD + 0.010, r = 0.92, p < 0.001). At one month after cell transplantation, MCI of the anterior wall increased in the BMMNC and BMSC subsets (p < 0.05), although it did not change in the control group. The ratio of wall thickness (systole/diastole) in the transplanted infarct area was larger than that in the nontransplanted infarct area (p < 0.01).
CONCLUSIONS: Myocardial contrast echocardiography is useful to evaluate regional perfusion, which was enhanced by bone marrow cell transplantation.
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Abbreviations and Acronyms
| | AU | = acoustic unit | | BMMNC | = bone marrow mononuclear cell | | BMSC | = bone marrow stromal cell | | CD | = capillary density | | MCE | = myocardial contrast echocardiography | | MCI | = myocardial contrast intensity | | MI | = myocardial infarction |
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Several researches reported that bone marrow cell transplantation into the heart induced angiogenesis (1–4). Assessment of myocardial perfusion after therapy is important. For a long time, vascular density on histologic study has been used to evaluate the therapeutic effectiveness of cell transplantation on myocardial infarction (MI) in experimental studies (5). However, it is invasive and difficult to perform a biopsy of the targeted area in clinical examinations.
Microvessels induced by bone marrow cell transplantation are too small (<50 µm) to visualize using conventional angiography (2,6). Although previous studies have found bone marrow cell transplantation to be effective, concrete and objective evidence for this therapy in daily clinical examinations has not been demonstrated.
Myocardial contrast echocardiography (MCE) is a noninvasive and inexpensive tool to evaluate myocardial perfusion in the microvasculature (7,8), and it can be performed even at the bedside. The purposes of this study were to verify the feasibility of MCE and to evaluate angiogenesis after bone marrow cell transplantation in the chronic ischemic heart of pigs.
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Methods
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Animal model.
All animals received humane care in compliance with the "Principles of Laboratory Animal Care," formulated by the National Society for Medical Research, and the "Guide for the Care and Use of Laboratory Animals," prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). All procedures were approved by the Animal Care Committee of the National Cardiovascular Center, Osaka, Japan.
Fourteen mini-pigs (weight 25 to 30 kg) from the Nippon Institute for Biological Science (NIBS; Nosan Corp., Japan) were used. The pigs were premedicated with ketamine hydrochloride (600 mg intramuscularly), stressnyl (2 ml), and atropine (1.5 mg) (9). Anesthesia was induced using a ventilation mask with 1% to 3% isoflurane, oxygen at 10 l/min, and ketamine hydrochloride (12 to 15 mg/kg intravenously). The animals were intubated using a cuffed endotracheal tube and were ventilated with 50% oxygen to maintain end-tidal CO2 between 30 and 35 mm Hg. Anesthesia was maintained with 0.5% to 1.0% isoflurane in oxygen at a flow rate of 3 to 4 l/min and fraction of inspired oxygen (FIO2) at 50%. Electrocardiography was used to monitor the heart rate, rhythm, and ST-segment changes during the surgical procedure.
Under general anesthesia, a left lateral thoracotomy was performed. The left anterior descending coronary artery was double-ligated with 2-0 silk (2). Ventricular fibrillation and tachycardia were treated with xylocaine and a defibrillator. After the lungs were adequately inflated, the pericardium was closed using 4-0 prolene sutures, and the chest was closed with #1 prolene. A 10-ml volume of sternal bone marrow was aspirated for culture. The pigs were weaned from anesthetics, extubated, and kept warm in a pen. The surgical team administrated analgesic to the pigs, if needed.
By one month after MI, 5 of the 14 pigs had died. The nine pigs that survived were randomized as follows. Six of the nine pigs were assigned to a cell transplantation group whose subsets were bone marrow mononuclear cells (BMMNCs) (n = 3) and bone marrow stromal cells (BMSCs) (n = 3). The other three pigs received saline (control group, n = 3) (9).
Cell preparation.
Bone marrow mononuclear cells
In the BMMNC subset, at one month after MI, immediately before cell transplantation, 50 to 100 ml of fresh bone marrow was aspirated and loaded onto Lymphoprep (AXIS-SHIELD PoC AS, Oslo, Norway) (2). The solution was centrifuged at 800g at 4°C for 20 min. A white layer was aspirated and washed with phosphate-buffered saline twice, and 1 x 108 cells/ml was created.
Bone marrow stromal cells
A 10-ml volume of bone marrow was aspirated from the sternum on the same day as MI and then cultured in cell culture medium: Iscove's modified Dulbecco's medium with 10% fetal bovine serum, penicillin G (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.2 µg/ml) (9). The cells were incubated with 95% air and 5% CO2 at 37°C for one month.
The cultured BMSCs were dissociated from the culture dish with 0.05% trypsin in phosphate-buffered saline, collected, and centrifuged at 570g. The cells were then suspended to obtain a concentration of 1 x 108 cells in 1.0 ml. This suspension was injected in the same pig as registered in this BMSC subset.
Under general anesthesia, we performed bone marrow cell transplantation after a median sternotomy. We longitudinally opened the pericardium, exposing the heart to visualize the left anterior descending and diagonal arteries, so that we could recognize where the anterior wall was. Bone marrow cells were autologously injected using a 27-gauge needle with a 1-ml syringe. We made sure that the tip of the needle was not ">intracavitary with negative pressure when using the syringe and then injected each 0.1-ml cell solution of 108 cells/ml at each of the 10 sites on the left ventricular anterior free wall at the mid-ventricle.
Myocardial contrast echocardiography.
A specialist in MCE performed MCE at two stages: one month after infarction (before cell transplantation) and one month after cell transplantation. A SONOS 5500 system (Philips, Andover, Massachusetts) was used (2,10). All imaging was performed at the mid-papillary muscle level of the left ventricle, under a closed-chest condition. The MCE imaging was performed in the second harmonic mode, in which ultrasound was transmitted at 1.8 MHz and received at 3.6 MHz. The mechanical index was set at 1.4 to 1.6. The maximal dynamic range was 60 dB. As a venous contrast agent, we used the second-generation microbubbles called YM-454 (Definity, Bristol-Myers Squibb Medical Imaging Inc., North Billerica, Massachusetts) (8). They are composed of a bilayer phospholipid shell and perfluoropropane. The mean size of gas-filled liposomes is about 2.5 µm. This agent was injected intravenously at a rate of 100 µl/min. We used the intermittent mode and the multiframe trigger method in MCE, and we set the trigger point at the end-systolic phase every 4 beats in this study.
Data were recorded on 1.25-cm videotape with an S-VHS recorder and on a 5-inch magnetic optical disk (11,12) and analyzed off-line using QuantiCon (Echotech 3D Imaging Systems, GmbH, Hallbergmoos, Germany). We put the region of interest on targeted areas and calculated their myocardial intensities. We subtracted the intensity of phase B from that of phase A at a target. Phase A occurred when the microbubbles sufficiently filled the myocardium, and phase B occurred when about 80% of microbubbles were destroyed by a series of three frames of the ultrasound beam within the end-systolic phase. We regarded this subtracted intensity as myocardial contrast intensity (MCI) for analysis. The acoustic unit (AU) was determined using the Acoustic densitometry software package (Philips), which displays intensities in dB (13). Acoustic densitometry provides an integrated on-line capability to measure, display, and analyze the average acoustic image intensity. Off-line densitometric systems that offer a "digital" solution for data storage using magnetic/optimal disk media are also plagued with the problem of nonlinearity in the image data (13). We calculated AU2 from dB using the equation:  and we assessed myocardial intensity using AU2. We put the region of interest on four different target areas for measurement of intensity: anteroseptum (nontransplanted infarct area), anterior wall (transplanted infarct area), septum (border area), and lateral wall (normal area). At these four target areas, MCI and capillary density (CD) were measured and compared.
Wall thickness.
To evaluate wall motion, we measured the wall thickness of the systolic and diastolic phases at the anteroseptum (nontransplanted infarct area), anterior wall (transplanted infarct area), and lateral wall (normal area). We calculated the wall thickness (systole/diastole) ratio and assessed the regional wall motion.
Histologic studies (CD).
At one month after cell transplantation, we performed MCE to assess MCI, after which the heart was arrested with potassium chloride and excised for histologic study. The coronary arteries were then perfused with 10% formaldehyde (100 ml), and the heart was immersed in the formaldehyde for 20 days.
After fixation, a cube (5 x 10 x 10 mm) of tissue from each site, where MCE was evaluated, was embedded in paraffin and cut into 6-µm sections for staining with hematoxylin-eosin, as described in the manufacturer's specifications (Sigma Chemical Co., St. Louis, Missouri), and von Willebrand factor-related antigen (14). A pathologist and an orthopedist investigated bone formation and tumorigenic formation. An observer blinded to the treatment groups and subsets determined the CD of the anteroseptum (nontransplanted infarct area), anterior wall (transplanted infarct area), septum (border area), and lateral wall (normal area). Five fields of each section were randomly selected, and the CD was averaged and expressed as the number of blood vessels per mm2 (15).
Statistical methods.
Data are expressed as the mean value ± SD. Interstage comparisons in each group and intrastage differences between two groups were confirmed using the Mann-Whitney U test. Analysis System software (Statcel, OMS Publishing Company, Tokorozawa, Saitama, Japan) was used for these two analyses. Correlations were performed using logarithmic or linear regression analysis. Differences were considered significant at p < 0.05.
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Results
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There was no critical arrhythmia before, during, or after cell transplantation. Heart rate and blood pressure were stable during transplantation. The nine pigs that survived for one month after MI completed the whole procedure.
Capillary density and MCI of normal, nontransplanted infarct, and transplanted infarct areas.
The CD of the normal area was 2.12 ± 0.58 (x103/mm2) and that of the nontransplanted infarct area was 0.46 ± 0.19 (x103/mm2) (Table 1). The MCI of the normal area was 0.54 ± 0.19 and that of the nontransplanted infarct area was 0.13 ± 0.02 (Table 2). Both the CD and MCI of the normal area were significantly higher than those of the nontransplanted infarct area (p = 0.00035 and p = 0.00035, respectively).
On the transplanted infarct area, the CD of the transplant group (including the BMMNC and BMSC subsets) was 1.53 ± 0.63 (x103/mm2) and that of the control group was 0.27 ± 0.07 (x103/mm2) (Table 1). On the same area, the MCI of the transplant group was 0.38 ± 0.20 and that of the control group was 0.12 ± 0.02 (Table 2).
The CD and MCI of the transplanted infarct area in the transplant group were significantly higher than those of the same area in the control group (p = 0.020 and p = 0.028, respectively).
Wall thickness ratio (systole/diastole) of normal, nontransplanted infarct, and transplanted infarct areas.
The wall thickness systolic/diastolic ratio at the normal area was significantly larger than that at the nontransplanted infarct area (1.50 ± 0.18 and 1.07 ± 0.09, respectively; p = 0.0003) (Table 3). In the cell transplant group, the wall thickness systolic/diastolic ratio at the transplanted infarct area was significantly larger than that at the nontransplanted infarct area (1.48 ± 0.15 and 1.08 ± 0.1, respectively; p = 0.004), although there was no difference in the control group. Further, when we compared the systolic/diastolic ratio between the cell transplant group and control group at the transplanted infarct area, the ratio in the transplant group was significantly larger than that in control group (1.48 ± 0.15 and 1 ± 0, respectively; p = 0.02) (Table 3).
Comparison of CD in nontransplanted and transplanted infarct areas.
The histologic findings of the section stained for von Willebrand factor are shown in Figure 1. In both the BMMNC and BMSC subsets, the anteroseptum (nontransplanted infarct area) contained very few capillaries (Fig. 1 [1a and 2a]). In contrast, the anterior wall (transplanted infarct area) contained more capillaries than the anteroseptum (Figs. 1 [1b and 2b]). The size of the capillaries at the transplanted infarct area ranged from 5 to 20 µm, which varied more than that of the normal area (10 to 20 µm). Bone formation or tumorigenic formation was not observed at the transplanted area in any pig.
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Figure 1 Capillary formation at the anteroseptum and anterior wall. Number 1 represents the bone marrow mononuclear cell (BMMNC) subset; number 2 represents the bone marrow stromal cell (BMSC) subset; and number 3 represents the control group (magnification x200). The top row (1a, 2a, 3a) shows the findings of the anteroseptum (nontransplanted infarct area), and bottom row (1b, 2b, 3b) shows the findings of the anterior wall (transplanted infarct area). In the BMMNC and BMSC subsets, the anterior wall (1b, 2b) contained more capillaries than the anteroseptum (1a, 2a). In contrast, in the control group, both the anteroseptum (3a) and anterior wall (3b) contained very few capillaries.
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In the cell transplant group, the CD of the anterior wall was significantly larger than that of the anteroseptum (p = 0.0081) (Fig. 2A) (BMMNC subset, p = 0.025; BMSC subset, p = 0.025) (Fig. 2B). In the control group, the CD of both the anterior wall and anteroseptum was low, and there was no significant difference between the two areas (p = NS) (Fig. 2A). The CD of the anteroseptum in the BMMNC subset was larger than that of the other subset or group (p = 0.027).
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Figure 2 The capillary density (CD) of the anteroseptum (open bars) and anterior wall (solid bars). The CD of the anterior wall was higher than that of the anteroseptum in the cell transplant group (**p = 0.0081; A) (*p = 0.025 and *p = 0.025 for bone marrow mononuclear cell (BMMNC) and bone marrow stromal cell (BMSC) subsets, respectively; B), although there was no difference between the two areas in the control group (A).
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Relationship between MCI and CD.
Figure 3 shows typical graphs of the four target areas at one month after BMMNC transplantation. The MCI of the anterior wall (transplanted infarct area) was higher than that of the anteroseptum (nontransplanted infarct area). The MCI and CD of four target areas at the same stage were plotted (Fig. 4).
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Figure 3 The myocardial contrast intensity (MCI) of the bone marrow mononuclear cell subset at one month after cell transplantation. The four graphs enable us to quantify MCI and to recognize the differences in the anteroseptum, anterior wall, septum, and lateral wall. The MCI in the anterior wall was higher than that in the anteroseptum.
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Figure 4 Relationship between myocardial contrast intensity (MCI) and capillary density (CD). (A) When MCI was evaluated by dB, a logarithmic relation was seen between MCI and CD. (B) When MCI was evaluated by AU2, a linear relation was seen between them.
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The MCI and histologic CD correlated well based on data derived from four areas of all pigs. When MCI was analyzed by decibel, MCI and CD were correlated logarithmically (dB = 7.32Log[CD] + 5.89; r = 0.90, p < 0.001) (Fig. 4A). There was a significant linear relation between MCI analyzed by AU2 and CD (MCI [AU2] = 0.23 CD + 0.01; r = 0.92, p < 0.001) (Fig. 4B).
Time course of MCI.
In the control and cell transplant groups, there was the same time trend of MCI at the anteroseptum (nontransplanted infarct area), which slightly decreased (p = NS) (Fig. 5A). In contrast, the MCI of the anterior wall (transplanted infarct area) significantly increased in the cell transplant group (p = 0.018), although it did not change in the control group (p = NS) (Fig. 5B).
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Figure 5 Time course of myocardial contrast intensity (MCI) in the anteroseptum and anterior wall before and after transplantation. The straight line indicates the cell transplant group (bone marrow mononuclear cell and bone marrow stromal cell subsets), and the dashed line indicates the control group. (A) At the anteroseptum (nontransplanted infarct area), there was no significant change between the groups or stages. (B) At the anterior wall (transplanted infarct area), the MCI of the cell transplant group significantly increased after transplantation (*p = 0.018), although that of the control group did not change.
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Discussion
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The importance of this study is to establish a method to follow the effect of bone marrow cell transplantation not only in experimental studies but also in clinical examinations. Therefore, we performed MCE using Definity as the contrast agent and a commercially available ultrasound machine, based on the hypothesis that MCE would be a useful tool to assess the microcirculation after cell transplantation.
This study demonstrated that: 1) MCI measured by MCE was closely correlated with CD; 2) bone marrow cell transplantation increased blood perfusion at the infarct area; and 3) bone marrow cell transplantation into the heart was performed safely.
Relationship between MCI and CD for assessment of myocardial perfusion.
The relationship between MCI and CD showed a strong correlation. This result indicates that MCE is as useful as CD for evaluating angiogenesis. In previous experimental studies, CD or microsphere analysis was generally used to assess angiogenesis (8,16). However, in clinical examinations, we cannot use these two methods. In contrast, MCE is a noninvasive and repeatable method to evaluate angiogenesis induced by bone marrow cell transplantation to the heart for clinical application.
The microsphere method reflects functional myocardial perfusion. There are several reports stating that MCI using MCE reflects myocardial perfusion using the microsphere method (11,17). Therefore, MCI reflects functional myocardial perfusion.
Recently, it was reported that MCI evaluated by AU2 is proportional to the concentration of microbubbles (13). In this study, we proved that MCI evaluated by AU2 is proportional to CD. This result indicates that the new microvessels created by bone marrow cell transplantation were functional.
Importance of angiogenesis by bone marrow cell transplantation.
Bone marrow cell transplantation (BMMNCs and BMSCs) induced angiogenesis in the transplanted area, as proved by CD and MCI. The angiogenesis induced by bone marrow cell transplantation may make the residual cardiac muscle and transplant cells survive, which may contribute to improvement of contraction (1).
The BMMNC subset.
The BMMNCs contain various kinds of cells, such as hematopoietic cells, fibroblasts, myogenic cells, and endothelial cells, and they can work beneficially in the ischemic myocardium (12). This study was consistent with our previous result (1). We observed angiogenesis, as described earlier, and did not see any bone formation. The BMMNCs have several advantages. Special facilities are not required because we can process them fresh by a clinically established protocol. This procedure is simple and can be performed safely.
The BMSC subset.
The BMSCs were cultured by Caplan's method, as previously reported (9,18). The BMSCs can be isolated from the other cells in marrow by their tendency to adhere to the culture dish. Stromal cells were a heterogeneous population (1). Our study suggested that the BMSCs could include stem cells differentiating into endothelial cells due to an increase in CD and improvement of myocardial perfusion.
The potential mechanisms inducing angiogenesis after endothelial cell transplantation are formation of blood vessels by transplanted endothelial cells and stimulation of angiogenesis by growth factors such as vascular endothelial growth factor, beta-fibroblast growth factor, and insulin-like growth factor-1 (2), expressed or stimulated by transplanted endothelial cells (15).
The BMSCs can be cultured from only 10 ml of bone marrow; however, it takes time to expand and requires culture facilities satisfying good manufacturing practice for clinical applications.
Effects of BMMNC and BMSC transplantation.
The increases in CD and MCI after cell transplantation were recognized in both the BMMNCs and BMSCs. The BMSCs stimulated angiogenesis as fresh bone marrow cells (BMMNCs), as in our previous rat study (1). The significant increases in CD and MCI at the transplanted infarct area indicated that bone marrow cell transplantation improved regional perfusion where cells were injected in both BMMNC and BMSC subsets.
Wall motion after cell transplantation.
In the cell transplant group, the regional wall motion of the transplanted infarct area was significantly improved after cell transplantation. This result indicates that improvement of perfusion after bone marrow cell transplantation is reflected in regional wall motion where bone marrow cells are transplanted.
Follow-up MCE after cell transplantation.
The MCI using MCE enabled us to assess the effects of this therapy not only by comparing the transplanted infarct area with the nontransplanted infarct area, but also by comparing the transplanted infarct area before and after cell transplantation; therefore, it is useful for follow-up after this therapy.
Bone marrow cell transplantation for clinical application.
Bone marrow cell transplantation was performed safely in our study. During the acute phase (perioperative period), there were no detrimental effects such as hypotension, arrhythmia, or hypoxemia. During the subchronic phase, there was no critical arrhythmia. There was no ectopic differentiation (bone formation) or tumorigenic formation at the transplant sites. From the point of view of clinical application, we regarded both the BMMNC and BMSC transplantations as feasible.
Study limitations.
The sample size of this study is low at the subset level. The bone marrow cell transplantation group was composed of BMMNC and BMSC subsets, and not only was their wall motion statistically improved after cell transplantation, but also their CD and MCI were statistically higher than those in the nontransplanted area. This project is adequately powered by statistical analysis.
We measured CD and MCI only at one month after cell transplantation. It will be necessary to evaluate myocardial perfusion and especially cardiac function in the long term.
Conclusions.
We proved that bone marrow cell transplantation improved the regional perfusion in the chronic ischemic heart. Because angiogenesis after cell transplantation is the most important factor that improves myocardial condition, MCI using noninvasive MCE could be a promising tool to evaluate functional perfusion after bone marrow cell transplantation in the clinical setting.
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Acknowledgments
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We thank Mr. K. Masuda, Ms. E. Takeda, and Dr. H. Ishibashi-Ueda for their technical assistance in the histologic study, as well as Ms. K. Hattori for her help in performing surgical procedures. We also thank Dr. H. Ohgushi (Tissue Engineering Research Center, National Institute of Advanced Industrial Science of Technology, Japan) for the investigation of bone formation and tumorigenic formation.
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Footnotes
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This research was supported in part by a Health Science Research Grant from the Ministry of Health, Labor, and Welfare (Research for Cardiovascular Diseases [13C-1] and Research on the Human Genome, Tissue Engineering Food Biotechnology [12-007]) and by Grants-in-Aid for Scientific Research (B) and for Exploratory Research from the Japan Society for the Promotion of Science.
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Abstract
Methods