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Eur J Cardiothorac Surg 2006;30:353-361
© 2006 Elsevier Science NL
a Department of Physiology, XiangYa School of Medicine, Central South University, Changsha, Hunan 410078, People's Republic of China
b Institute Of Clinical Medicine, Renmin Hospital, YunYang Medical College, Shiyan, Hubei 442000, People's Republic of China
c Department of Physiology, YunYang Medical College, Shiyan, Hubei 442000, People's Republic of China
d Stem Cells Research Institute of Hainan Province, Haikou 570102, Hainan, People's Republic of China
Received 6 October 2005; received in revised form 11 February 2006; accepted 20 February 2006.
* Corresponding author. Address: Department of Physiology, XiangYa School of Medicine, Central South University, Changsha, Hunan 410078, People's Republic of China. Tel.: +86 731 8905361; fax: +86 731 8905361. (Email: tangjm416{at}163.com; qxie18{at}hotmail.com; qxie{at}xysm.net; panther75{at}163.com; rywjn{at}vip.163.com; slwangmingjiang{at}sina.com).
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Key Words: Myocardial infarction • Mesenchymal stem cell • Angiogenesis • Transplantation • Rats
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Mesenchymal stem cells (MSCs) are pluripotent, adult stem cells and mostly reside within the bone marrow. In contrast to their hematopoietic cells, MSCs are adherent and can be expanded in culture. MSCs can differentiate not only into osteoblasts, neurons, and skeletal muscle cells but also into vascular endothelial cells and cardiomyocytes [2,3]. In vitro, MSCs can be induced to differentiate into cardiomyocytes under special culture conditions (e.g., when treated with 5-azacytidine) [4]. In vivo, MSCs directly injected into an infarcted heart have been shown to induce myocardial regeneration and improve cardiac function [5]. It is interesting that the cytokine vascular endothelial growth factor (VEGF) is a major inducer of angiogenesis [6]. With myocardial ischemia, VEGF expression is increased in response to ischemia to promote vascular repair. And VEGF is widely recognized as a potential therapeutic target for regulating angiogenesis [7]. In addition, MSC transplantation induces therapeutic angiogenesis in a rat model of hindlimb ischemia through VEGF production by MSCs [8]. These findings raise the possibility that transplanted MSCs have the effects on myocardial structure and function via myogenesis and angiogenesis. However, little information is available about the therapeutic potential of MSCs for myocardial ischemia with reperfusion (MI).
A model of MI in the rat has been created by release of left anterior descending artery (LAD) ligation for 1 h, which results in severe heart failure characterized by increased cardiac fibrosis and left ventricular (LV) dilation. The purpose of this study was to investigate the following topics: (1) whether transplantation of MSCs induces myogenesis and angiogenesis, and thereby changes myocardial histomorphology and improves cardiac function in a rat model of MI and (2) whether the beneficial effects of MSCs are mediated by their differentiation into vascular cells by VEGF.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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2.2 Isolation and culture of MSCs and multi-differentiation of MSCs
Isolation and primary culture of MSCs were performed with the method described by Chedrawy et al. [2,9]. Briefly, rat MSCs were isolated from bone marrow with density gradient centrifugation and cultured in low-glucose DMEM (Gibco Co.) supplemented with 10% fetal calf serum (FCS, Hyclone Co.), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. The culture medium was removed and replaced with fresh medium two times weekly. At confluence, the cells were harvested for passage with 0.25% trypsin (Solon, OH, USA) containing 0.02% EDTA (Sigma, St. Louis, MO, USA). All experiments were performed using cells from the second passage.
For osteogenic differentiation, a 70% subconfluent culture of MSCs from passage two was used. The medium was replaced by osteogenic medium with 10–7 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM ß-glycerophosphate (all Sigma). After day 15 in osteogenic medium, cell colonies displayed bone-like nodular aggregates of matrix mineralization. Von Kossa staining for calcium could visualize the mineral deposition. For adipogenic differentiation, the medium was replaced by adipogenic medium with 1 µM dexamethasone, 0.2 mM metacen, and 0.5 mM IBMX, 10 µg/ml insulin (all Sigma). The medium was replaced every 3–4 days for 21 days. Fat cells could be visualized by oil red staining for fatty drop. For endothelial cell differentiation, MSCs were cultivated in the presence of 10% FCS and 20 ng/ml VEGF (Sigma) for 7 days. Medium was changed every 3 days.
2.3 Labeling of MSCs
After passage, two cells became nearly confluent. Sterile 4,6-diamidino-2-phenylindole (DAPI) solution was added to the culture medium on the day of implantation at final concentration of 50 µg/ml for 30 min [9]. The MSCs were rinsed six times in Hanks balanced salt solution to remove all excess unbound DAPI. The MSCs were harvested (approximately 1 x 107 cells for each implantation) and resuspended in minimal volume of serum-free DMEM.
2.4 Myocardial ischemia model and cell implantation
MI was performed by the ligation of the left coronary artery as described previously [10]. Briefly, male Wistar inbred rats were anesthetized with ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and tracheally ventilated with room air using a Colombus ventilator (HX-300, Taimeng Instruments, and Chendu, China). After left lateral thoracotomy in the fourth intercostal space, the LAD was ligated for 1 h with reperfusion. MI was assessed by electrocardiograph and histology. Experimental animals were randomized for each group. One week after MI induction, the DAPI-labeled MSCs in the serum-free medium (1 x 107 in 0.5 ml) were separately injected into three different sites (0.16 ml per site) for each MI heart in the MSC-transplanted group with a 30-gauge tuberculin syringe. Two injection sites were in the myocardium bordering the ischemic area and one within the ischemic area. Control group received the same MI operation but was only injected with an equivalent volume of the cell-free medium. The sham group underwent the identical surgery with neither ligation of the coronary artery nor MSC transplantation. Penicillin (150 000 U/ml) was given intravenously before each operation and buprenorphine hydrochloride (0.05 mg/kg) was administered subcutaneously twice a day for the first 48 h after operation.
2.5 Measurement of hemodynamics
Four weeks after transplantation, hemodynamic measurements in vivo were performed with the methods as following. Rats were anesthetized with pentobarbital sodium (60 mg/kg, i.p.). A carotid artery and femoral artery were isolated. The two catheters filled with heparinized (10 U/ml) saline solution were connected to Statham pressure transducer (Gould, Saddle Brook, NJ, USA). The carotid arterial catheter was advanced into the left ventricle to record ventricular pressure for a brief period of time. And the femoral artery catheter was inserted into an isolated femoral artery to monitor mean arterial pressure (BP), and heart rate (HR). These hemodynamic parameters were monitored simultaneously and recorded on a thermal pen-writing recorder (RJG-4122, Nihon Kohden, Japan) and on an FM magnetic tape recorder (RM-7000, Sony, Tokyo, Japan). Tapes were later played back. After the measurements, the heart was rapidly removed from the killed rats (each group, n
= 10) [11].
2.6 Histology and morphometric measurement
After the measurement of hemodynamics, the hearts were quickly removed and was cut into six transverse slices from apex to base and processed for histology. Subsequently, partial 8 µm transverse slices from each section were prepared for hematoxylin and eosin staining, and the images were digitized. 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 LV. The LV-wall thickness was measured at three widely spaced locations within the scar segment as well as the non-infarcted region for each slide and the average of these multiple measurements were calculated. To estimate an overall degree of ventricular remodeling, expansion index was computed. The analyses of LV-wall thickness and expansion index were performed upon two middle slides and the averages were calculated for each heart (each group, n
= 10) [12,13].
2.7 Immunohistochemistry analysis
The subsets of animals were killed 1, 2, and 4 weeks after MSC transplantation (each time point, n
= 5). After quick removal of the hearts, the free wall of the LV including the infarcted and peri-infarcted regions was embedded in tissue-frozen medium (Fisher Scientific, Fair Lawn). Frozen tissue was sectioned into 8 µm slides and stained with hematoxylin and eosin. Survival of engrafted cells was confirmed by identification of DAPI-positive spots under fluorescent microscopy. Potential transdifferentiation of myocardial-like cells from implanted MSCs was verified by antibody immunostaining for troponin T (cTnT). Briefly, frozen tissue sections were fixed in acetone at 4 °C for 10 min and incubated separately with a mouse anti-rat cTnT (NeoMarkers Co.) for 60 min at room temperature. After a washing with PBS solution, sections were incubated with a goat anti-mouse-conjugated FITC IgG for cTnT. Neovascular transformation of implanted MSCs was verified by antibody immunostaining for a-smooth muscle actin (SM-actin) and CD31. Frozen sections of the left ventricle were processed as described above using the following antibody sets: primary-rabbit anti-rat SM-actin (NeoMarkers Co.) and mouse anti-rat CD31 (Serotec), secondary-goat anti-rabbit, or goat anti-mouse-conjugated FITC IgG (Acris Antibodies GmbH Co.) [14].
2.8 VEGF expression analysis
To identify the protein expression of VEGF, Western blotting was performed with rabbit polyclonal antibody raised against VEGF (Zymed Laboratories Co.). The LV obtained from individual rats was used for comparison among all three groups (each group, n
= 5). These samples were homogenized on ice in 0.1% Tween 20 homogenization buffer with a protease inhibitor. Then, 50 µg of protein was transferred into sample buffer, loaded on a 7.5% sodium dodecyl sulfate–polyacrylamide gel, and blotted onto a polyvinylidene fluoride membrane (Millipore Co.). After being blocked for 120 min, the membrane was incubated with primary antibody at a dilution of 1:200. The membrane was incubated with horseradish peroxidase labeled with secondary antibody (Beijing Zhongshan Biotechnology Co., China) at a dilution of 1:1000. Positive protein bands were visualized with diaminobenzidine (Lab Vision Co.) and measured by densitometry. And quantitative analysis of cardiac tissue contents of VEGF was measured by enzyme immunoassay (R&D System Inc.). Similarly, the level of VEGF was detected 4 days after transplanted MSCs were cultured [15].
2.9 Measurement of capillary density
The hearts were quickly removed and cut into six transverse slices from apex to base and processed for histology 4 weeks after treatment (each group, n
= 10). Partial 8 µm transverse slices from each section were prepared for hematoxylin and eosin staining (three transverse slices each section). Subsequently, the effect of MSC transplantation on angiogenesis was evaluated by counting the number of capillary vessels within the infarcted zone from frozen sections with hematoxylin and eosin staining under light microscopy. A capillary vessel was defined as a vessel with a diameter less than 20 µm. The number of capillaries was counted under microscopy (magnification, 400 x ) for five random fields in the infarcted area or myocardial wall of each transverse slices in the all three groups and presented as the mean of blood vessels per unit area (0.2 mm2), respectively. The capillary count of these sections was compared with the control and the sham group [16].
2.10 Data analysis
Statistical analyses were performed with one-way ANOVA followed by SPSS software (SPSS Science, Chicago, IL, USA) analysis. Data (mean ± SD) were considered statistically significant at a value of P
< 0.05.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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3.2 Multi-differentiation of MSCs
The ability of MSCs to differentiate into osteocytes and adipocytes was tested in all cultures from various donors. When cultured in osteogenic medium for 15 days, the morphology changed on: (A) day 1, a nearly confluent spindle-shaped layer; (B) days 5–7, cells form nodular aggregates, and (C) days 12–15, cells began to mineralize their matrix and were positive for Kossa staining. They were also able to differentiate into adipocytes, and cells accumulated different amounts of lipid vacuoles after cultivation in adipogenic medium.
We introduced differentiation into endothelial-like cells by cultivating confluent MSCs in the presence of 10% FCS and 20 ng/ml VEGF for 15 days. The morphology changed on: (A) day 1, a nearly confluent spindle-shaped layer; (B) day 4, a few MSCs could alter spindle-shape into round or oval-shape; (C) day 8, some MSCs showed round or oval-shape; and (D) day 12, paving stone-shape. Immunohistochemical staining for CD31 was chosen for the basal characterization of endothelial-like cells. Undifferentiated MSCs showed almost no specific staining for CD31, but after 7 days of cultivation CD31 expression of the differentiated MSCs was markedly enhanced.
3.3 Identification of DAPI-labeled MSCs in vivo after transplantation
The DAPI-labeled MSCs showed clear nuclear and faint cytoplasmic blue fluorescence when viewed under an epifluorescence microscope. The labeling efficiency of cultured MSCs with DAPI approached 100%. One, two, and four weeks after MSC transplantation, freeze sections of the implant sites of the hearts were made to evaluate the morphology and phenotype changes of implanted MSCs. DAPI-labeled cells could be identified in all specimens 1, 2, and 4 weeks after transplantation (five rats with transplanted MSCs at random). One and two weeks after implantation numerous scattered DAPI-labeled cells (blue fluorescence) were found in the specimen. One and two weeks after transplantation the consecutive section showed concordance between dense host myocardium and presence of DAPI fluorescence. The MSC-derived donor cells showed an immature appearance with a large nucleus-to-cytoplasm ratio. There was no obvious inflammatory response at this time. Four weeks after transplantation, a few clearly DAPI-labeled cells were found to incorporate into the host myocardium. They had abundant cytoplasm and had aligned themselves with non-labeled cells (host cardiomyocytes).
3.4 Measurement of hemodynamics
To test whether transplantation of MSCs into injured myocardium would even further improve cardiac function, we carried out measurement of hemodynamics four weeks after transplantation. Measurement of hemodynamics in vivo showed that LV function was significantly lower in MSC group and control group than in the sham operation group. However, improvement of LV function was significantly greater in the MSC group than in the control group. The differences of LVSP (n
= 10, P
< 0.05, ±dp/dt
max and LVEDP (n
= 10, P
< 0.05 were statistically significant between the two groups (Fig. 1
).
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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MSCs are not only in vitro capable of transdifferentiating into functional cardiomyocytes under special culture conditions (e.g., when treated with 5-azacytidine) [4] but also in vivo MSCs differentiate cardiomyocyte-like cells in the acute ischemia model or in a myocardial environment, which express desmin, troponin T, adrenoceptor, and sarcomeric MHC and produce a concomitant benefit [3,5]. In this study, colocalization of MSCs with cardiac muscle-specific troponin T protein was observed. And MSCs can differentiate into endothelial-like cells in vitro. Moreover, engrafted MSCs were present obviously in the luminal face of the endothelium of several vessels and expressed CD31, suggesting their transdifferentiation into endothelial cells, and MSCs present in vessel walls were positive for a-smooth muscle actin, suggesting their transdifferentiation into smooth muscle cells (SMCs). These transdifferentiation contributed to the significantly higher capillary density and cardiomyocyte-like cell numbers in the infarcted areas of MSC-treated animals. These changes could lead to decrease scar sizes, reduce remodeling and improve cardiac function, which is a consequence of blood supply improvement in damaged heart sites.
In our study, we evaluated the functional improvement seen after MSC transplantation by cardiac catheterization to detect ventricular pressure. MSC group and control group showed the expected decline in ±dp/dt max and LVSP, and markedly increase in LVEDP. In contrast, and as expected, the MSC transplantation group showed a lower decline in ±dp/dt max and LVSP, and a lower increase in LVEDP. MSCs contributed to preservation of ±dp/dt max, LVSP, and LVEDP after intramyocardial injections. It is interesting that our study has showed the change of cardiac function was related with dimension of infarct areas after different occlusion time course of LAD in rats. (These data will be published in Acta Laboratorium Animalis Scientia Sinica.) In this study, qualitative histomorphometric examinations showed that new myocardium was located in the middle of the scar or adjacent to the border zone, which was not seen in any of the controls. In contrast, the infarcted area in medium-injected scars showed transmural and thin scar tissue composed of collagen. Quantitative histomorphometric examinations showed a significantly lower infract size, a greater LV-wall thickness, and a lower Hochman-Choo expansion index in the MSC-treated group compared with the control group. It is interesting that the non-infarct LV wall was thicker than the infarct LV wall in the control and MSC groups. And thickness of infarct, non-infarct, and average LV wall were greater in the MSC group than in the control group. And MSC-transplanted hearts were less dilated and a combination of less dilatation and a trend toward less paradoxical systolic bulging probably led to the improved ventricular pressure. Furthermore, MSC transplantation could inhibit LV remodeling, augment or preserve the myocardial elasticity, improve heart function and reduce the expression of extracellular matrix genes (e.g., TIMP-1, TGF-beta (1), collagen, etc.) [15]. Recently, Chedrawy et al. [9] reported that colocalization of MSCs with adherens and gap junction proteins (connexin-43 is the major gap junction protein1 responsible for electrical coupling, N-cadherin is the major protein responsible for mechanical coupling) make it likely that the transplanted MSC-derived cardiomyocyte cells were directly contributing to meaningful systolic function within the infarct zone. It is more likely that they played an active role by thickening the scar, reducing paradoxical bulging, and limiting LV dilatation and remodeling [12]. This is further suggested by the lower expansion index in rats treated with MSC implantation versus those not transplanted. Although this study does not provide direct evidence that regional MSC injection does actually improve regional myocardial function, angiogenesis may contribute to the maintenance of cardiac function by preserving residual, viable cardiomyocytes, and neovascularization might also restore contractility. For example, Kamihata et al. [19] reported that implanted BM-MNCs enhanced collateral perfusion and regional function via side supply of angioblast, angiogenic ligands, and cytokines in porcine models of MI. Therefore, these above parameters in the MSC group were better than the control group, the capillary density and the transdifferentiated smooth muscle and endothelial cells and cardiomyocyte-like cells seen in the MSC group might have contributed to preservation of ±dp/dt max, LVSP, and LVEDP, indicating improvement of heart function after myocardial ischemia.
It is interesting that our study have showed MSC differentiation into endothelial cells was related with the presence of VEGF. Recently, Kinnaird et al. [20,21] reported that MSCs produce a wide array of arteriogenic cytokines and improve perfusion and remodeling in mouse models of hindlimb ischemia, and these effects appear to be mediated through paracrine mechanisms associated with local release of the arteriogenic cytokines. In our study, VEGF expression level in the MSC group obviously increased. Increased VEGF could involve in or trigger angiogenic progress of MSCs, even might contribute to postnatal neovascularization by mobilizing bone marrow-derived EPCs [22]. Of course, besides VEGF, released cytokines after MI include fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and so on [23]. These cytokines could contribute to stem cell-mediated repair of infarcted myocardium. For example, Rosenblatt-Velin et al. [24] recently reported that FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes. At the same time, cell-to-cell direct contact between resident cells and MSCs play an important role in MSCs’ differentiation into cardiomyocytes, endothelial cells, or SMCs [25]. These suggest that ischemia environment may drive MSCs differentiate into vascular cells and cardiomyocyte-like cells through the symphysis effect of paracrine mechanisms and cell-to-cell direct contact.
In this study, the technique of cell labeling used is of paramount importance, because it is crucial for differentiating cells derived from the implanted MSCs from host myocardium. DAPI, a fluorescent dye, has been used to visualize DNA in living and fixed cells. The fluorescence quantum yield of the free dye is low, but binding to DNA results in a highly fluorescent complex. DAPI is relatively non-toxic to living cells and does not alter the ultrastructure of cell organelles. An advantage of DAPI is the high labeling efficiency via simple procedure. In our observation, the labeled cells were clearly detected, and the initial labeling efficiency was 100%. However, disadvantages of DAPI include the loss of labeling intensity due to cell division and proliferation [9]. We did not attempt quantitative analysis of our date, which was a limitation of this study.
In conclusion, this study suggests that transplantation of MSCs into ischemic myocardium with reperfusion is safe and effective. MSC differentiation into endothelial cells may be related with the presence of VEGF in vivo and in vitro. Implanted MSCs improved cardiac structure and function through the symphysial effect of myogenesis and angiogenesis. The finding suggesting that MSC transplantation might one day be an alternative therapy for ischemic heart failure.
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Acknowledgments |
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We thank Professor Qiru Wang and Mengqun Tan for their helpful suggestions in study design and invaluable discussion during the preparation of the manuscript. We are grateful to Dr Jing Yang for his help with the measurement of infarct size, left ventricle wall thickness, and vessel density, and Dr Yuanchong Xu and Guang Qiu with immunofluorescence.
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References |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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