Optimal time for mesenchymal stem cell transplantation in rats with myocardial infarction

Optimal time for mesenchymal stem cell transplantation in rats with myocardial infarction 1 first-author †E-mail:chen_yang_jiang@yahoo.comjian_an_wang@yahoo.com †E-mail:chen_yang_jiang@yahoo.comjian_an_wang@yahoo.com chen_yang_jiang@yahoo.com 1 2 1 3 2 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:chen_yang_jiang@yahoo.comjian_an_wang@yahoo.com †E-mail:chen_yang_jiang@yahoo.comjian_an_wang@yahoo.com chen_yang_jiang@yahoo.com Department of Cardiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China Department of Cardiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China Department of Cardiology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Cardiology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Cardiology, the First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou 310006, China Department of Cardiology, the First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou 310006, China Background: 2 Bone marrow mesenchymal stem cell (MSC) transplantation is a promising strategy in the treatment of myocardial infarction (MI). However, the time for transplanting cells remains controversial. The aim of this study was to find an optimal time point for cell transplantation. Methods: 2 MSCs were isolated and cultured from Sprague-Dawley (SD) rats. MI model was set up in SD rats by permanent ligation of left anterior descending coronary artery. MSCs were directly injected into the infarct border zone at 1 h, 1 week and 2 weeks after MI, respectively. Sham-operated and MI control groups received equal volume of phosphate buffered saline (PBS). At 4 weeks after MI, cardiac function was assessed by echocardiography; vessel density was analyzed on hematoxylin-eosin stained slides by light microscopy; the apoptosis of cardiomyocytes was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay; the expressions of proteins were analyzed by Western blot. Results: 2 MSC transplantation improved cardiac function, reduced the apoptosis of cardiomyocytes and increased vessel density. These benefits were more obvious in 1-week group than in 1-h and 2-week groups. There are more obvious increases in the ratio of bcl-2/bax and the expression of vascular endothelial growth factor (VEGF) and more obvious decreases in the expression of cleaved-caspase-3 in 1-week group than those in other two groups. Conclusion: 2 MSC transplantation was beneficial for the recovery of cardiac function. MSC transplantation at 1 week post-MI exerted the best effects on increases of cardiac function, anti-apoptosis and angiogenesis. Mesenchymal stem cells (MSCs) Transplantation Myocardial infarction (MI) Apoptosis INTRODUCTION 1 Myocardial infarction (MI) leads to an irreversible loss of cardiomyocytes and scar formation in the infarct area, which are major factors in the progression of heart failure (Braunwald and Bristow, 2000 2000 ). Bone marrow mesenchymal stem cell (MSC) transplantation is considered to be an effective therapeutic approach for MI both in basic researches (Amado et al., 2005 2005 ; Tang et al., 2006 2006 ) and in clinical trials (Janssens et al., 2006 2006 ; Meyer et al., 2006 2006 ). The underlying mechanisms may involve myocardial regeneration, angiogenesis, apoptosis of myocardiocytes and inhibition of cardiac remodeling. However, the effects of cell transplantation after MI vary in different studies (Strauer et al., 2002 2002 ; Schachinger et al., 2004 2004 ; Wollert et al., 2004 2004 ; Janssens et al., 2006 2006 ), and this difference may partly result from different cell transplantation time as there is a time-dependent healing course post-MI (Koransky et al., 2002 2002 ). The infarct area appears swelling at 15 h after MI and then softening at 24~48 h, granulation tissue outlines the infarct at 3~4 d, and scar tissues mature at 2~3 months. The time of cell delivery should be at the cross over the time point when the local microenvironment is ready to receive cells at the optimum of their development and function. However, the optimal time of delivery of donor cells remains unknown. Growing evidence indicates that inhibition of cardiomyocyte apoptosis and stimulation of angiogenesis would contribute to improving cardiac function in postinfarction heart failure (Fukuda et al., 2006 2006 ; Uemura et al., 2006 2006 ). In this study, therefore, we focused on the angiogenesis and anti-apoptotic effects to investigate the optimal time for MSC transplantation in MI heart of Sprague-Dawley (SD) rats. MATERIALS AND METHODS 1 Animals 2 SD rats were obtained from the Medical Institute Animal Center of Zhejiang University, China, for conducting the proposed study. The experiments were approved by the Animal Care and Use Committee of Zhejiang Provincal 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 ( 1996 1996 ). Cell culture 2 MSCs were obtained from the femora and tibiae of male SD rats (80 g). The bones were dissected free and the proximal and distal ends were removed to reveal the marrow cavity, which was aspirated with 10 ml of Dulbecco’s modified Eagle’s medium (DMEM) through a 21G needle. The aspirant was layered over 1.073 g/ml percoll solution (GE Healthcare Bio-Sciences AB, Sweden) and centrifuged at 400× g for 25 min at room temperature. The mononuclear cells were recovered at the interface, resuspended in MSC growth medium (DMEM, 10% (v/v) fetal bovine serum, 100 U/ml penicillin G and 100 U/ml streptomycin; Gibco, USA), plated on 25 cm 2 flasks, and incubated for 24 h at room temperature. The flasks were then washed with phosphate buffered saline (PBS) to leave an adherent layer of cells containing MSCs. The cultures were maintained at 37 °C in a 5% (v/v) CO 2 incubator, and the medium was changed every 3~4 d. MI model and MSC transplantation 2 Two-month old male SD rats were intubated under general anesthesia by using 4% (w/v) 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 2~3 mm from the tip of the left auricle with a 6-0 silk suture (Min et al., 1999 1999 ). 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 5 groups (10 rats in each group): sham-operated group, PBS group (rats received PBS at 1 h post-MI), 1-h group (rats received MSCs at 1 h post-MI), 1-week group (rats received MSCs at 1 week post-MI), 2-week group (rats received MSCs at 2 weeks post-MI). The second thoracotomy was needed in 1-week and 2-week groups. The 2×10 6 cells in 150 μl PBS were directly injected into the infarct border zone according to these time points. Sham-operated and MI control rats (PBS group) received the same volume of PBS injection. Echocardiographic study 2 Echocardiography was performed blindly to assess the cardiac function 4 weeks after MI. The echocardiographic procedure was performed as previously described (Litwin et al., 1995 1995 ; 1996 1996 ). A commercially available echocardiographic system equipped with a 12-MHz probe (HP, SONOS5500) was used to obtain the measurements. Briefly, a 2D 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. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis 2 The rats were euthanized and the hearts were removed after echocardiography. The excised heart was cut into 3 transverse sections and embedded in paraffin. Sections (5-μm thick) were cut and mounted on adhered slides. After deparaffinization and dehydrated, apoptotic cardiomyocytes were evaluated by TUNEL assay with an In Situ Cell Death Detection Kit (Roche, Germany) according to the manufacturer’s instructions. In brief, the sections were incubated in proteinase K working solution (20 μg/ml in 10 mmol/L Tris/HCl, pH 7.4~8) at 37 °C in a humidified atmosphere for 15 min, and then added with 50 μl TUNEL reaction mixture each and incubated for 60 min at 37 °C. After rinsed 3 times with PBS, the sections were added with 50 μl converter-peroxidase (POD) and incubated for 30 min at 37 °C, and then rinsed 3 times with PBS again and added with 100 μl diaminobenzidine (DAB) substrate. Finally, the sections were counterstained with hematoxylin and analyzed by light microscope. Percentage of TUNEL-positive cells was assessed in 5 randomly selected fields in the border zone of the ischemic region. A total of 20 sections were analyzed for each animal from the 5 groups. Assessment of vessel density 2 The numeric density of arterioles (diameter > 20 μm) was analyzed on hematoxylin-eosin stained slides (5 slides from each of the animals in each group) by light microscopy at 400× magnification. Five high-power fields each slide in the border zone of the ischemic region were randomly selected. The number of arterioles in each was averaged and expressed as the number of arterioles per unit area (0.2 mm 2 ). Western blot analysis 2 Frozen infarcted hearts were crushed, and then homogenized by vibration in RIPA lysis buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L ethylene diamine tetraacetic acid (EDTA), 0.1% (w/v) sodium dodecyl sulfate (SDS), 1% (w/v) Nonidet P-40 (NP-40), 1 mmol/L phenylmethanesulfonyl fluoride (PMSF) and 10 mmol/L NaF at 4 °C for 30 min. The homogenate was centrifuged at 12000× g at 4 °C for 20 min. The protein content of the supernatant was determined by the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA). Protein (50~150 μg per lane) was loaded and electrophoresis was carried out in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein was transferred onto polyvinylidene difluoride (PVDF) immobilon-P membrane (Bio-Rad, CA, USA) using a transblot apparatus (Bio-Rad, CA, USA). The membranes were blocked in 10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl and 0.05% (v/v) Tween 20 (TBST) with 5% (w/v) non-fat milk at room temperature, followed by overnight incubation at 4 °C with primary antibodies (1:1000 (v/v) for bcl-2, bax, cleaved-caspase-3, and β-actin, Cell Signal, USA; 1:1000 (v/v) for vascular endothelial growth factor (VEGF), Santa Cruz, USA). After washing with TBST, the membranes were incubated for 1 h with a horseradish peroxidase (HRP)-conjugated secondary antibody, and the labled proteins were detected by using the enhanced chemiluminescence reagents and exposed to the film (Kodak, USA). Statistical analysis 2 Data were expressed as mean± SEM . Statistical significance between groups was assessed by a two-way analysis of variance (ANOVA) followed by Student-Newmen-Keuls (SNK), using SPSS 11.5. P < 0.05 was considered statistically significant. RESULTS 1 Morphology of MSCs 2 We isolated and cultured MSCs from the bone marrows of male SD rats as described above. Passage 3 MSCs were attached to culture dishes and the majority of MSCs displayed a spindle-like shape (Fig. 1 1 ). Surface molecular markers of Passage 3 MSCs were examined with flow cytometry analysis, and most adherent cells were found to express CD44 and CD90 but not CD45 (Xie et al., 2006 2006 ). Effect of MSC transplantation on cardiac function 2 Cardiac function assessment was performed 4 weeks after MI. The results showed that fractional shortening (FS) and ejection fraction (EF) were significantly decreased in all MI groups compared with sham-operated group ( P < 0.01). FS and EF were significantly increased in three MSC transplantation groups compared with PBS group ( P < 0.01). Compared with 1-h and 2-week groups, FS and EF were significantly increased in 1-week group ( P < 0.05) (Fig. 2 2 ). FS: Fractional shortening; EF: Ejection fraction; Sham: Sham-operated rats without coronary ligation; PBS: MI rats that received PBS injection; 1-h, 1-week and 2-week: Rats receiving cell transplantation at 1 h, 1 week and 2 weeks post-MI, respectively. * P < 0.01 vs Sham group; # P < 0.01 vs PBS group; † P < 0.05 vs 1-h and 2-week groups ( n =10) Effect of MSC transplantation on cardiomyocyte apoptosis 2 TUNEL labeling was performed 4 weeks after MI (Figs. 3a~3e 3a~3e ). The results show that the number of TUNEL-positive cells increased significantly in PBS group compared with sham-operated group (PBS group: (10.2±1.0)%, sham-operated group: (0.9±0.2)%; P < 0.01, Fig. 3f 3f ). Compared with PBS group, transplantation of MSCs at 1 week and 2 weeks significantly reduced the apoptosis of cardiomyocytes (1 week: (5.4±0.4)%, 2 weeks: (7.5±0.5)%; P < 0.01, Fig. 3f 3f ), and the reduction in 1-week group was more obvious than that in 2-week group ( P < 0.05, Fig. 3f 3f ). However, there was no significant difference between PBS group and 1-h group. We further examined the expression of apoptotic protein including cleaved-caspase-3, bcl-2 and bax in infarct heart. The results show that the ratio of bcl-2/bax was significantly decreased and cleaved-caspase-3 was increased in PBS group compared with sham-operated group ( P < 0.01). Compared with PBS group, the ratio of bcl-2/bax was increased in 1-week group, and cleaved-caspase-3 was decreased in 1-week and 2-week groups ( P < 0.01). However, there was no significant difference in the ratio of bcl-2/bax and cleaved-caspase-3 between PBS and 1-h groups (Fig. 4 4 ). Effect of MSC transplantation on vessel density 2 The vessel density was significantly decreased after MI compared to sham-operated group. The results also show that MSC transplantation increased vessel density, and that the vessel density was significantly greater in 1-week group than in 1-h and 2-week groups ( P < 0.05, Fig. 5a 5a ). VEGF, which could be secreted by MSCs (Uemura et al., 2006 2006 ), is an important protein for angiogenesis (Koransky et al., 2002 2002 ). We further examined its expression in infarct heart, and found that it was significantly greater in 1-week group than in other groups ( P < 0.05, Fig. 5b 5b ). However, there was no significant difference between PBS and 1-h groups. DISCUSSION 1 The present study shows: (1) MSC transplantation could improve the cardiac function, reduce the apoptosis of cardiomyocytes, increase the bcl-2/bax ratio, inhibit the activation of cleaved-caspase-3, and increase vessel density and the expression of VEGF; (2) MSC transplantation at 1 week post-MI exerted the best effect on the increase of cardiac function, anti-apoptosis and angiogenesis. Apoptosis, with a preferential localization of apoptotic cardiomyocytes at the borders of the infarcted area, may be as important as necrosis in determining the cardiomyocyte loss after acute MI (Kajstura et al., 1996 1996 ; Olivetti et al., 1996 1996 ). Apoptosis takes place at 3 to 120 h after acute MI and lasts at least 60 d (Piro et al., 2000 2000 ; Saraste et al., 1997 1997 ), and apoptosis at late post-acute MI ( > 10 d) suggests a possible relation with the progression of left ventricular dysfunction (Baldi et al., 2002 2002 ). Therefore, in this study we analyzed the apoptosis of cardiomyocytes at 4 weeks post-acute MI. MSCs have been shown to have an anti-apoptotic effect (Uemura et al., 2006 2006 ) due to their paracrine effect. MSCs could secret a great number of anti-apoptotic cytokines (Silva et al., 2003 2003 ; Wang et al., 2004 2004 ; Kemp et al., 2005 2005 ; Nagaya et al., 2005 2005 ; Uemura et al., 2006 2006 ), such as VEGF, stem cells derived factor (SDF), hetapocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), and so on. In this study, we demonstrated that MSC transplantation reduced the apoptosis of cardiomyocytes, and the best benefits were observed in 1-week transplantation group. This might be explained as followings: (1) In our study, increased VEGF expression was observed more obviously in 1-week group, so we supposed that 1 week post-MI, when scar formation has not occurred and the inflammation is reduced (Virag and Murry, 2003 2003 ), may provide a suitable environment to favor the paracrine function of the implanted MSCs; (2) We also found that the vessel density was higher in 1-week group as compared to PBS group, 1-h and 2-week groups. It might be associated with increased VEGF expression, which facilitated greater angiogenesis and further reduced the apoptosis of cardiomyocytes. However, further studies are needed to clarify this phenomenon. Our study also indicates that 1 h post-MI may not be the ideal time for cell transplantation. After 1 h post-MI, massive myocardial necrosis, leukocytes and mast cells rapidly infiltrate into the ischemic myocardium (Virag and Murry, 2003 2003 ), which may do harm for the survival of the implanted cells. By 1 week, the acute inflammatory reaction is nearly complete, and scar formation has not occurred yet, so this may be a positive stimulation for the implanted cells. By 2 weeks, scar tissues begin to form (Virag and Murry, 2003 2003 ) and adverse ventricular remodeling begins (Virag and Murry, 2003 2003 ), which may also inhibit the survival and function of the implanted cells. Similarly, Li et al.( 2001 2001 ) transplanted embryonic rat cardiomyocytes into the cryoinjuried heart and found that the transplantation at 2 weeks after cryoinjury was better than the one immediately followed or at 4 weeks post-MI. The effects of cell transplantation after MI vary in different clinical trials (Janssens et al., 2006 2006 ; Schachinger et al., 2004 2004 ; Strauer et al., 2002 2002 ; Wollert et al., 2004 2004 ), among which, cardiac function improved from approximately 2% to 9%. Different transplanted cell types (Assmus et al., 2002 2002 ; Stamm et al., 2003 2003 ; Wollert et al., 2004 2004 ), cell quantity, survival ability and delivering time may contribute to these differences. Although the healing course after MI in humans is not the same as the one in rats, this optimal time point for cell transplantation in rats may provide a suggestion for the application in human. Transplantation of stem cells after MI leads to an engraftment of the implanted cells in the peri-infarct area (Uemura et al., 2006 2006 ). These transplanted cells can differentiate into multiple phenotypes of myocardium, and thereby contribute to myocardial regeneration (Shim et al., 2004 2004 ). However, other studies (Nygren et al., 2004 2004 ; Garbade et al., 2005 2005 ) have suggested that phenotypic changes may occur as a consequence of cell fusion rather than transdifferentiation. The contribution of fusion in cardiac regeneration was still unknown (Yoon et al., 2005 2005 ). Further studies will be done to illuminate whether there is an optimal time point for differentiation or fusion. In conclusion, MSC transplantation was beneficial for the recovery of cardiac function in experimental rats. 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Myocardial regeneration with bone-marrow-derived stem cells We thank Xing Zhang and Jiang Cao (Center Lab, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China) for technical support. 10.1631/jzus.B0820004 Project (No. 2004QN018) supported by the Health Bureau of Zhejiang Province, China 2008-01-04 2008-04-24 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 09