Eur J Cardiothorac Surg 2007;31:438-443. doi:10.1016/j.ejcts.2006.11.057
Copyright © 2007, European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved
Optimal temporal delivery of bone marrow mesenchymal stem cells in rats with myocardial infarction
Xinyang Hua,
Jianan Wangb,*,
Jie Chenc,
Ronghua Luoa,
Aina Heb,
Xiaojie Xiea,
Jiahui Lia
a Department of Cardiology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, 3 East Qing Chun Road, Hangzhou 310016, China
b Department of Cardiology, Second Affiliated Hospital, College of Medicine, Zhejiang University, 88 Jie Fang Road, Hangzhou 310009, China
c Department of Cardiology, First Affiliated Hospital, Zhejiang Chinese Medical University, 54 You Dian Road, Hangzhou 310006, China
Received 28 September 2006;
received in revised form 21 November 2006;
accepted 24 November 2006.
* Corresponding author. Tel.: +86 571 87784677. (Email: wang_jian_an{at}tom.com).
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Abstract
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Objective: This study was designed to determine the optimal time point for bone marrow mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI). Methods: MSCs from donor rats were labeled with DAPI before transplantation. The animals underwent MI by ligation of left anterior descending coronary artery, and received intramyocardial injection of MSCs at 1 h, 1 week and 2 weeks after MI, respectively. Sham-operated and MI control groups received equal volume phosphate buffered saline. Cardiac function, histological analysis and immunoblot for troponin T were performed 4 weeks after cell transplantation. Results: MSC transplantation attenuated left ventricular chamber dilation, reduced infarct size, and improved cardiac function in rats after MI. The greatest benefit was achieved in rats that received cells 1 week after MI, engrafted MSC survival, angiogenesis and functional cardiomyocytes in the injured hearts were more abundant in these rats than that in other transplantation groups. Conclusions: The optimal functional benefit of MSC transplantation was observed in 1-week transplantation group. At this time point scar formation has not occurred and the inflammation is reduced, which should facilitate integration of transplanted cells and functional recovery.
Key Words: Cell transplantation • Time point • Myocardial infarction
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1. Introduction
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Myocardial infarction (MI), leading to irreversible loss of cardiomyocytes and scar formation, is the leading cause of congestive heart failure [1]. It has been shown that bone marrow stem cells are able to differentiate into cardiomyocytes, endothelial cells, and vascular smooth muscle cells when they are stimulated appropriately [2]. Animal and clinical studies show that bone marrow mesenchymal stem cell transplantation can repair cardiac infarcts and improve cardiac function [3,4]. Potential mechanisms may include angiogenesis and myogenesis within the ischemic heart [5]. The local environment, such as inflammatory cytokines released from infarcted hearts, may also play an important role in these processes, particularly during stem cell homing, survival, engraftment, and differentiation [6]. On one hand, inflammatory cytokines may favor stem cell differentiation into endothelial cells, smooth muscle cells, and cardiomyocytes. However, the adverse inflammatory environment, with its excessive oxidative load, may be deleterious to implanted cells.
It remains unclear whether engrafted stem cells could generate different effects if cells are transplanted at different time points after MI. Previous studies [7] have shown that cardiac function improves by approximately 6% when the MSCs are administrated during the early stages of an acute MI. The present study was designed to test if there is an optimal time point for delivering bone marrow MSCs.
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2. Materials and methods
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Two-month-old adult male Sprague-Dawley rats were obtained from the Medical Institute Animal Center of Zhejiang University for conducting the proposed study. The experiments were approved by the Animal Care and Use Committee of Zhejiang Province Medical Institute and were in compliance with the ‘Guide for the Care and Use of Laboratory Animals’ as published by the US National institutes of Health (National Institutes of Health publication no. 85-23, revised 1996).
2.1 MSC preparation and labeling
MSCs were isolated and harvested as previously described [8]. Briefly, MSCs were acquired from the tibia and femur of donor adult rats. Cells were isolated by gradient centrifugation, and then cultured in DMEM with 20% fetal bovine serum to reach a final concentration of 1 x 105 nucleated cells per ml. The cells were incubated with 95% room air and 5% carbon dioxide at 37 °C for 24 h and then washed with phosphate-buffered saline solution (PBS). The medium was changed twice a week for 28 days and cells were observed by inverted microscopy every day. Hematopoietic stem cells were washed away after changing medium. The primary cultures were passaged to two new flasks once the MSCs reached 80% confluence.
The transplanted cells were thereafter labeled with DAPI as previously described [9] to track engrafted cells. Briefly, sterile DAPI was added to culture medium at the day of transplantation at a final concentration of 50 µg/ml for 2 h. The cells were rinsed 6 times in PBS to remove unbounded DAPI, detached with 0.25% (w/v) trypsin EDTA and suspended in PBS for cell transplantation.
2.2 Myocardial infarction model and stem cell transplantation
SD rats were intubated under general anesthesia using 4% chloral hydrate (4 mg/kg, administered intraperitoneally) and ventilated with room air by using a small animal ventilator (Zhejiang University Apparatus). MI was induced by ligation of the left anterior descending coronary artery (LAD) 2–3 mm from the tip of the left auricle with a 6-0 silk suture [10]. Successful performance of coronary occlusion was verified by blanching of the myocardium distal to the coronary ligation. The sham-operated group received the same procedure of thoracotomy without coronary ligation. The rats were divided randomly into five groups (10 rats in each group): sham-operation group, PBS group (rats received PBS), 1 h group (rats received MSCs 1 h after MI), 1 week group, 2 week group. Next, 2 x 106 cells in 150 µl PBS were directly injected into the infarct border zone according these time points. Sham and MI control rats received the same volume of PBS injection as transplant subjects.
2.3 Echocardiographic study
Four weeks after cell transplantation, echocardiography was performed blindly to assess the cardiac function. The echocardiographic procedure was performed as previously described [11,12]. A commercially available echocardiographic system equipped with a 12-MHz probe (HP, SONOS5500) was used to obtain the measurements. Briefly, a two-dimensional short-axis view of the left ventricle was obtained at the level of the papillary muscles, and M-mode tracings were recorded and analyzed to evaluate cardiac function after cell transplantation.
2.4 Morphology and histology of infarcted myocardium
Subsets of animals were sacrificed after assessment of echocardiography. The hearts were quickly harvested, and tissues from the free wall of the left ventricle including the infarct and peri-infarct regions were then embedded in OCT tissue freezing medium. Frozen sections (6 µm in thickness) of left ventricular samples were made for identification of implanted cells. Another subsets of hearts were fixed in 10% formaldehyde, embedded in paraffin and sectioned at 4 µm thickness for hematoxylin and eosin staining. Images were digitized using a computerized image analysis system (NIH image). Infarct size was calculated by dividing the sum of the planimetered endocardial and epicardial circumferences of the infarcted area by the sum of the total epicardial and endocardial circumferences of the left ventricular (LV) [13].
2.5 Assessments of engrafted cells and arteriole density
The survival of engrafted cells was identified by the presence of DAPI-positive cells in frozen sections made from hearts with MI. The number of DAPI-positive cells was evaluated by counting five randomly chosen high-power (200x) fields from each of five sections taken from each animal.
The numeric density of arterioles (diameter >20 µm) was analyzed on hematoxylin and eosin-stained slides (five slides from each of the animals in each group) by light microscopy at 400x magnification. Five high-power fields in the transplanted area were randomly selected. The number of arterioles in each was averaged and expressed as the number of arterioles per unit area (0.2 mm2) [14].
2.6 Western blot analysis
Troponin T (TnT) expression was detected by western blot. Total protein was prepared from the infarcted myocardial tissues, and the corresponding tissues were obtained from the same area in the sham group. Forty micrograms of protein were electophoresed on a 12% SDS-polyacrylamide gel and transferred to PVDF membrane. After blocking with 0.2% Tween in Tris-buffered saline (TBS-T) containing 5% milk at room temperature for 2 h, membranes were incubated for 2 h at room temperature with rabbit polyclonal anti-TnT (1:500, Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was washed three times with TBS-T and incubated with AP-conjugated secondary antibody for 2 h at room temperature. The signal was detected by the addition of ECL solution.
2.7 Statistical analysis
All data are expressed as mean ± SD. Differences between groups were evaluated by one-way ANOVA using SPSS 11.0 statistical software. Data were considered statistically significant at a value of P
< 0.05.
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3. Results
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3.1 Characterization of MSCs
The majority of cultured bone marrow cells were spindle-like mesenchymal stem cells, which attached to the culture dish tightly (Fig. 1A), while hematopoietic stem cells were rounded in shape and failed to attach to the culture dish, and were washed away by changing the culture medium. The surface markers of MSC were determined by flow cytometry analyses as previous reported [15], MSC expressed CD44 and CD90 at moderate to high level, while CD45 was negative. Fig. 1B shows DAPI-labeled mesenchymal stem cells.
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Fig. 1. Characterization of MSC and DAPI labeled MSCs. (A). Mesenchymal stem cells in culture. The majority of cultured bone marrow cells were spindle-like MSCs. (B). MSCs were labeled with DAPI. (Original magnification: 100x).
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3.2 Effect of MSC transplantation on cardiac function
Cardiac function measurements are summarized in Table 1
. Four weeks after MI, fractional shortening (FS) and ejection fraction (EF) were significantly decreased; left ventricular diastolic dimension (LVDd) and left ventricular systolic dimension (LVDs) were increased in MI rats (P
< 0.01 vs the sham-operation group, Table 1). Intramyocardial transplantation of MSCs improved cardiac function and attenuated heart size enlargement significantly, and the effects peaked in the 1-week group.
3.3 Infarct size and identification of engrafted MSCs
Hematoxylin-eosin staining of cardiac tissue obtained 4 weeks after MI reveals fibrosis in the infarct region 4 weeks after MI (Fig. 2
). Noticeable scar formation was evident in hearts injected with PBS. However, the scar size was reduced in the groups that received MSC transplantation compared to the PBS group. In the 1 h group, 1 week group and 2 week group, the scar size was 41.4 ± 9.3% (P
< 0.05 vs PBS group), 32.8 ± 7.3% (P
< 0.01 vs PBS group), 37.1 ± 6.7% (P
< 0.01 vs PBS group), respectively, there was trend of smaller scar size in 1 week group as compared with the other transplantation groups. Fig. 3
shows engrafted cells in ischemic myocardium, which demonstrates that implanted cells do survive in the peri-infarct region for at least 4 weeks post-transplantation. A greater number of DAPI-positive cells were present in the myocardium in the 1 week group than that in the other transplantation groups.
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Fig. 2. Hematoxylin-eosin staining of transverse sections from rat left ventricles at the level of the posterior papillary muscle. (A) From a sham rat, shows normal histological structure of myocardium. (B) From a rat 4 weeks after MI that received PBS as transplantation control. Parts (C–E) are from rats 4 weeks after MI that received MSC transplantation at 1 h, 1 week and 2 weeks post-MI, respectively. Part (F) is the measurement of infarct size in experimental groups. *
P
< 0.05, **
P
< 0.01 versus MI plus PBS group.
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Fig. 3. The number of MSCs engrafted in the recipient myocardium 4 weeks after MI and MSC transplantation. Parts (A–C) demonstrate immunofluroscence micrographs of DAPI positive cells around the infarct border zone in rats that received cells at 1 h, 1 week and 2 weeks after MI, respectively (original magnification: 200x). (D) Analysis of DAPI positive cell counts within the infarct border from the experimental groups. *
P
< 0.05 versus both 1 h and 2 week group.
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3.4 TnT expression in heart
To further verify the survival of functional cardiomyocytes, we examined TnT expression in the cardiac tissues. Fig. 4
reveals that TnT expression is noticeably decreased in MI hearts compared to sham (P
< 0.01). TnT expression was significantly greater in the 1 week group than that in PBS group (P
< 0.05). However, there was no significant difference between PBS group and 1 h or 2 week group.
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Fig. 4. Protein expression of TnT (n
= 5 for each group) determined by Western blot of tissue from left ventricles from sham rats, MI rats with PBS injection, MI rats with MSC transplantation at 1 h after MI, MI rats with MSC transplantation at 1 week after MI, and MI rats with MSC transplantation at 2 weeks after MI. (A) Western blot; (B) densitometric analysis of TnT/ß-actin. *
P
< 0.01 versus Sham group; **
P
< 0.01 versus PBS group; #
P
< 0.05 versus PBS group.
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3.5 Assessment of vessel density
The vessel density was markedly decreased after MI compared to sham controls. Fig. 5
shows MSC transplantation increased vessel density. The vessel density was significantly greater in the 1 week MSC transplantation group than that in the 1 h and 2 week MSC transplantation groups (P
< 0.01). The vessel density value in the 1-week group was close to normal level so that there was no significant difference compared to that of the sham group.
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Fig. 5. Numeric density of vessels in myocardium from experimental groups at 4 weeks after MI. MSC transplantation significantly increased vessel density compared to the PBS group, especially in the 1 week group. *
P
< 0.01 versus Sham group; #
P
< 0.01 versus PBS group; 
P
< 0.01 versus 1 h transplantation group or versus 2 week transplantation group.
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4. Discussion
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The main findings of this study are (1) MSC transplantation improved heart function and reduced infarct size after MI, and the optimal benefits were observed in 1 week group; (2) the arteriole density in 1 week transplantation rats was greater than that in the other MSC transplantation groups; (3) morphological study indicated that a greater number of transplanted MSCs survived and engrafted in the myocardium in the 1 week group.
The present study reveals that transplantation of MSCs at 1-week post-MI produced the optimal therapeutic benefit, reflected by minimal scar formation and maximal heart function at 4 weeks after MI and cell transplantation therapy. In this study, although the time from the coronary ligation to echo analysis in each group is not the same (between 4 and 6 weeks after MI), the time from transplantation to echo analysis is the same (all the groups received echo analysis 4 weeks after transplantation). In addition, it was reported that, in rat MI model, over 6 weeks after MI, LVEF did not change significantly [16], and the collagen content at 4 week was the same compared to that at 13 weeks [17]. This observation suggests that fair comparisons may be performed among our experimental groups. Similarly, Li et al. [18] reported that cardiomyocyte transplantation at 2 weeks post-MI resulted in better heart function and greater developed pressure compared to transplantation immediately following or 4 weeks post-MI. The optimal therapeutic benefit in 1 week transplantation group might be explained by several mechanisms: (1) the implanted cells survived better in this group; (2) there was greater angiogenesis within the injured hearts in the 1 week group; (3) enhanced survival implanted cells had the increase paracrine effect that protected cardiomyocyte from apoptosis or stimulated the angiogenesis. It has been reported that the paracrine effect of MSC also is a possible mechanism for heart function recovery [19].
Surviving cell grafts play an important role in enhancing functional improvement, and most implanted cells died within 4 days after transplantation, which limits the positive effects of cell transplantation [20,21]. Multiple mechanisms could contribute to the death of grafted cells, including hypoxic and inflammatory environment after myocardial infarction, loss of survival signal from cell to cell contact. In addition, direct intramyocardial injection also causes high mortality of implanted cell because of mechanical damage and subsequently provokes an acute inflammatory response in the ischemic heart [22]. So it is important to investigate whether there is the optimal time point to deliver stem cell to improve grafted cell survival. Our work indicates that 1 week after MI appears to be the optimal timepoint for cell homing and survival.
It has been demonstrated that stem cells can differentiate into endothelial cells, and angiogenesis has been one of the putative mechanisms in cardiac functional recovery after MI and stem cell transplantation [23]. Our study shows that the vessel density is higher in 1-week group as compared to the other groups that received cells at either 1 h or 2 weeks after MI. The underlying mechanism might be associated with increased MSC differentiation into endothelial cells, which facilitates greater angiogenesis. In turn, this process may further attenuate left ventricular remodeling and contribute to the improvement of cardiac function.
In addition, we also report increased TnT expression in rat myocardium from 1-week transplantation rats compared to other transplantation groups. The greater expression of TnT indicates there are greater numbers of functional cardiomyocytes within the injured hearts, which might be explained by two mechanisms: (1) the increased TnT expression suggests that there are more newly regenerated functional cardiomyocytes; (2) paracrine action from engrafted MSCs prevents cardiomyocytes from apoptosis and restores more functional cardiomyoctes [19]. Whether MSCs differentiate into cardiomyocytes remains controversial. Although several investigators have reported that MSCs differentiate into cardiomyocytes, recent in vitro studies have shown that stem cells may fuse with the host cells. Our experiments were not designed to address whether or not stem cells differentiated into cardiomyocytes. Further experiments need to be conducted to clarify this question.
The myocardial substrate, course of infarct healing, and tissue remodeling determine the optimal time point for delivery of donor cells. Acute and chronic inflammatory reactions occur following an acute MI. Following massive myocardial necrosis, leukocytes and mast cells rapidly infiltrate into the ischemic myocardium and peak between 24 and 72 h after MI. By 1 week, the majority of the infarct zone is composed of granulation tissue and necrosis, and the acute inflammatory reaction is nearly complete. By 2 weeks, scar tissues begin to form [24] and ventricular remodeling begins. Corresponding with our present study, the niche of the local myocardium at 1 week after MI appears to be the optimal microenvironment for stem cell homing, survival, and differentiation. More experiments need to be conducted to provide direct evidence for these mechanisms.
For clinical application of stem cell transplantation, most clinical trials have reported positive results [3], but a few recent trials show that there is no significant difference in global LVEF between transplantation group and control group [25]. The different results may relate to the different transplanted cell and different patient population. It should be noticed that the healing course after MI in patients can be different from rat, thus the optimal time point for cell transplantation in rat may not be extended into clinical application directly.
Taken together, transplantation of MSCs could improve cardiac function in MI rat. The optimal functional benefit was achieved in the rats that received cells at 1 week after MI. At this time point scar formation has not occurred and the inflammation is reduced, which should facilitate integration of transplanted cells and functional recovery.
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Acknowledgments
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The study was supported in part by a grant from the Chinese National Natural Science Foundation.
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Abstract
1. Introduction
