Eur J Cardiothorac Surg 2004;25:188-195
© 2004 Elsevier Science NL
Enhanced cytoprotection and angiogenesis by bone marrow cell transplantation may contribute to improved ischemic myocardial function
Shaoheng Zhanga,b,
Ping Zhanga,b,
Jingxuan Guob,
Zhuqing Jiaa,c,
Kangtao Mac,
Yonggang Liub,
Chunyan Zhoua,c*,
Linsong Lia
a Stem Cell Research Center, Peking University, 38 Xue Yuan Road, Hai Dian District, Beijing 100083, China
b Department of Cardiology, The Third Hospital of Peking University, 49 North Garden Road, Hai Dian District, Beijing 100083, China
c Department of Biochemistry and Molecular Biology, The School of Basic Medical Sciences, Peking University, 38 Xue Yuan Road, Hai Dian District, Beijing 100083, China
Received 13 June 2003;
received in revised form 15 October 2003;
accepted 20 October 2003.
* Corresponding author. Tel.: +86-10-8280-2417; fax: +86-10-6201-5582
e-mail: chunyanzhou{at}bjmu.edu.cn
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Abstract
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Objective: Heat shock proteins (HSPs) are cytoprotective proteins. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor. This study aimed to elucidate the possible role of cytoprotection and angiogenesis on cardiac function after bone marrow cell transplantation (BMT). Methods: Myocardial infarction was induced in inbred Lewis rats by left anterior descending artery ligation. A total of 5x106 bone marrow-mononuclear cells were transplanted into the ischemic zone by direct injection. At 1, 3, 7, 14 and 28 days post-transplantation, cardiac function was evaluated by echocardiography. The expressions of HSP32, HSP70 and VEGF were assessed by immunofluorescence and RT-PCR. The number of vessels was examined by immunohistochemistry. The differentiation of the transplanted cells was determined by immunofluorescence. Results: Echocardiography showed BMT led to sustained improvement in cardiac function, as assessed by left ventricle ejection fraction and fraction of shortening. Immunofluorescence revealed that the expressions of HSP32, HSP70 and VEGF were promoted in both transplanted bone marrow cells and recipient cardiomyocytes. RT-PCR showed that the mRNA expression levels of HSP32, HSP70 and VEGF in the BMT group were markedly higher in comparison with injection of peripheral blood cells or saline (P<0.01) by day 7. Seven days later, the vessel count showed that angiogenesis had been induced to a significantly greater degree in the BMT groups. Fourteen days later, specific markers for myocardial or vascular endothelial cells were detected in the transplanted bone marrow cells. Conclusions: BMT upregulated the expressions of HSP32, HSP70 and VEGF in both transplanted bone marrow cells and recipient endogenous cardiomyocytes in the early phase post-transplantation. This enhanced cytoprotection and angiogenesis, and contributed to the functional recovery following cardiac infarction. In the late phase, the transplanted bone marrow cells might differentiate into both myocardial and vascular endothelial cells that enhanced the ischemic cardiac function further.
Key Words: Cytoprotection • Angiogenesis • Ischemia • Stem cells • Transplantation
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1. Introduction
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Despite advances in the treatment of myocardial infarction (MI), congestive heart failure secondary to infarction continues to be a major complication. The cardiomyocytes lost during MI cannot be regenerated, and the extent of the loss is directly related to the reduced cardiac output [1]. Cell therapy has previously been used in the treatment of this disease in which terminally differentiated cells were irreparably damaged [2]. Recently, it was suggested that bone marrow cell transplantation (BMT) might be effective in the treatment of MI [3]. There is now compelling evidence that BMT reduces infarction area and improves cardiac function via differentiation and angiogenesis in an ischemic heart model in the late phase post-transplantation (>7 days) [4]. However, the mechanism via which BMT improves cardiac function in the early phase post-transplantation (
7 days), generally before cell differentiation, remains largely a matter of speculation. Most researchers have suggested that transplantation of bone marrow cells into ischemic myocardium enhanced collateral perfusion and regional function via supply of angioblasts and angiogenic ligands such as VEGF [5], but others disagree [6]. Thus, there are probably other mechanisms. Heat shock proteins (HSPs) are correlated with enhanced recovery of myocardial contractility after acute MI [7,8]. HSPs can be induced by brief episodes of several stresses including ischemia, heat shock or injury. HSP32 and HSP70, two members of the cytoprotective proteins family, have been shown to protect cells from ischemic injury [9,10]. In this study, we used a rat myocardial ischemia model to investigate the change of cardiac function following acute MI, and attempted to elucidate the role of myocardial protection and angiogenesis on cardiac function after BMT.
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2. Materials and methods
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2.1. Animals
Male inbred Lewis rats (200–250 g) were obtained from Beijing Animal Administration Center at 8 weeks of age as donors and recipients. All animal experiments were approved by the Animal Care and Use Committee of Peking University and were in compliance with the European Convention on Animal Care.
2.2. Preparation and labeling of mononuclear cells
Bone marrow and peripheral blood were collected from the tibia and the hearts of donor animals, respectively. Bone marrow-mononuclear cells (BM-MNCs) and peripheral blood-mononuclear cells (PB-MNCs) were prepared by Ficoll-Hypaque gradient centrifugation (Lymphoprep, Nycomed) and then stained with Hoechest-33342 (Sigma-Aldrich, St Louis, Missouri) [11]. Cells were suspended in cold phosphate-buffered saline (PBS) at a density of 5x107 cells/ml and kept on ice for up to 4 h before being transplanted to recipient rats.
2.3. Animal models and cell transplantation
Recipient animals with the same body weight and cardiac function (assessed by echocardiography) were randomly assigned to experimental or non-experimental groups. Normal animals receiving neither infarction nor cell transplantation served as non-experimental group (NOR group). MI was induced in the experimental animals by ligation of the left anterior descending coronary artery as previously described [12]. And then, the experimental animals were randomly chosen to receive either a total of 100 µl of PBS (PBS group), or PB-MNCs (PBT group) or BM-MNCs (BMT group) cell suspension, by direct injection into the infarct and peri-infarct regions of the recipients (5x106 cells, three sites, 1–2 cm apart). In each group, the animals were further divided into five subgroups according to five time points: 1, 3, 7, 14 and 28 days post-transplantation. Ten animals were used in each subgroup.
2.4. Cardiac function
Echocardiography was performed on 7.5-MHz phased-array transducer (Acuson Sequoia 256) at time endpoints of each subgroup [12]. Left ventricular internal dimensions (LVID) were measured as: FS(%)=[(LVIDd-LVIDs)/LVIDd]x100, EF(%)=(EDV-ESV)/EDVx100%, where d stands for diastole, s for systole; EDV and ESV stand for end-diastolic and end-systolic LVID, respectively. All measurements were averaged for three consecutive cardiac cycles and were carried out by three experienced technicians who were unaware of the identity of the experimental groups.
2.5. Immunohistochemistry
The hearts were removed from animals after echocardiography Immunohistochemistry was performed on a series of cryostat left ventricular tissue sections. The primary antibodies used in this study included: HSP70 (Sc-24, Santa Cruz Biotech, Inc., CA), heme oxygenase-1 (HSP32, OSA-111, Stressgen Biotech, Inc., San Diego, CA) and VEGF (JH121, NeoMarkers, Inc., Fremont, CA) monoclonal antibodies; goat anti-myosin heavy chain (MHC) (Y-20, Santa Crunz Biotech, Inc., CA) and rabbit anti-factor VIII related antigen (10665638, Zymed Lab, South San Francisco, CA) polyclonal antibodies. The secondary antibodies included Rhodamine-conjugated (TRITC) goat anti-mouse IgG, TRITC-conjugated goat anti-rabbit IgG and FITC-conjugated rabbit anti-goat IgG (Jackson ImmunoResearch Laboratories, Inc., Pennsylvania, PA).
In order to quantify capillary density, additional slides were prepared from animals in each subgroup. The sections were stained with rabbit anti-factor VIII antibody and counterstained with an immunoperoxidase kit (Vector Labs, Burlingame, CA). Ten fields from each slide were randomly chosen from infarct portions that were bordered by non-infarct portions along the LAD. Capillary density was expressed as the number of factor VIII+endothelial cells per square millimeter [5]. The vessels were defined as round or ellipse structures with a central lumen lined by cells staining positively to factor VIII. A pathologist who was blinded to the groups' identities evaluated the capillary density by counting vessels in the chosen areas. Appropriate immunohistological controls were performed to assess specificity, including exclusion of primary antibody and use of mouse, goat and rabbit sera isotype in place of the antibodies.
2.6. RT-PCR
Total RNA was isolated from the infarct and peri-infarct myocardial tissues using TRIzol Reagent (15596-018, Gibco BRL). The expressions of VEGF, HSP70 and HSP32 at the mRNA level were evaluated with RT-PCR. PCR conditions for HSP32 were: 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min, and extension at 72 °C for 3 min. Amplification conditions for HSP70 and VEGF were: 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 60 s, and extension at 72 °C for 30 s. The primers and the size of the expected products were as follows (forward and reverse, respectively): HSP32, ACT TTC AGA AGG GTC AGG TGT CC/TTG AGC AGG AAG GCG GTC TTA G (514 bp); HSP70, AAC GTG CTG CGG ATC ATC AA/TCG GAT GGA CGT GTA GAA GT (393 bp); VEGF, ACT GGA CCC TGG CTT TAC TG/ACG CAC TCC AGG GCT TCA TC (256 bp). GAPDH served as a control: GGA AAG CTG TGG CGT GAT GG/GTA GGC CAT GAG GTC CAC CA (393 bp). The PCR products were subject to electrophoresis on 1.5% agarose gels, scanned, and semi-quantitated using Image-Quant software (Kodak 1D V3.53, 4 Science Park, New Haven, CT065//USA).
2.7. Statistical evaluation
Statistical analyses were performed with one-way ANOVA followed by SPSS software (SPSS Science, Chicago, IL) 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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3.1. Cardiac function
Echocardiographic assessments of LV function were shown in Table 1. Results from infarcted rats in the PBT and the PBS groups showed significant reduction of cardiac function in comparison with the NOR group. In the BMT group, cardiac function exhibited a gradual improvement. In comparison with the PBS group, EF and FS in the BMT group were improved significantly, by 17 and 29%, respectively (P<0.01 and <0.05, respectively), at day 7 post-transplantation. At day 28, EF and FS in the BMT group were 30 and 56% higher than those in the PBS group (P<0.01). EF and FS in the PBT group did not show significant change.
3.2. HSP32, HSP70 and VEGF expressions in the ischemic myocardium
The expressions of HSP32, HSP70 and VEGF were examined with immunofluorescence and RT-PCR. As shown in Fig. 1
, HSP32, HSP70 and VEGF were more abundantly expressed in the recipient myocardial and endothelial cytoplasm in the BMT group (Fig. 1a, d and g) than in the PBT group (Fig. 1b, e and h) or the PBS group (Fig. 1c, f and i) on day 3 post-transplantation. We also observed positive double-staining of Hoechest33342 and HSP32, HSP70 or VEGF in the cytoplasm of the transplanted BM-MNCs (indicated by arrowheads in Fig. 1a, d and g) whereas no cells were double-stained in the PBS groups. RT-PCR in Fig. 2a
revealed that HSP32, HSP70 and VEGF in the BMT group expressed more remarkably than in the PBT and PBS groups. Semi-quantitative analysis showed after day 1 post-transplantation, the mRNA expressions of HSP32 (Fig. 2b), HSP70 (Fig. 2c) and VEGF (Fig. 2d) were significantly higher in the BMT group than in the PBT and PBS groups, peaked at day 3 (4.7-, 5.2-and 1.6-fold more than the PBS group, respectively, P<0.01), and then decreased significantly. No significant difference was observed between the PBT and the PBS groups.
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Fig. 1. Immunofluorescence micrographs of the ventricular sections around the infarction zone on day 3 post-transplantation. The nuclei of transplanted BM-MNCs and PB-MNCs, which were pre-labeled with Hoechest33342, are stained blue. (a–c) and (d–f) show HSP32 and HSP70 expressions (stained red), respectively, in the myocardial, transplanted BM-MNCs and endothelial cytoplasm. HSP32 and HSP70 expressions in the BMT group (a and d) were significantly stronger than those in the PBT (b and e) and the PBS groups (c and f). Some Hoechest-33342 positive BM-MNCs in the BMT groups were double-stained with HSP32 or HSP70 (arrowheads), whereas no cells were double-stained in the PBT and the PBS groups. (g–i) show that VEGF expression (red) in the vascular endothelial, transplanted BM-MNCs and myocardial cytoplasm in the BMT group (g) was significantly stronger than those in the PBT (h) and the PBS groups (i). Some Hoechest-33342 positive BM-MNCs in the BMT group were double-stained with VEGF, but no cells were double-stained in the PBT and the PBS groups (each bar stands for 50 µm).
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Fig. 2. mRNA expressions of HSP32, HSP70 and VEGF in the BMT, PBT and PBS groups. (a) shows the electrophoresis results of RT-PCR products for HSP32, HSP70 and VEGF in these three groups. (b–d), respectively, show the semi-quantitative analysis of mRNA expression of HSP32, HSP70 and VEGF in these groups. #P<0.01 vs day 1 post-transplantation in the same group; *P<0.01 vs PBS group (X+S, n=9. RT-PCR experiments were performed in triplet. The results were the average of nine experiments on three animals in every subgroup).
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3.3. Vessel density in the infarcted myocardium
A significant difference in vessel density was observed between the BMT group and the control groups. Fig. 3
showed the representative immunohistochemical staining of factor VIII in the infarcted myocardium of the BMT group (Fig. 3a), the PBT group (Fig. 3b) and the PBS group (Fig. 3c) on day 28 post-transplantation. The density of capillaries in the infarcted area was significantly higher in the BMT group than in the PBT or PBS groups on day 7, 14 and 28, being 45, 78 and 125% (P<0.01), respectively (Fig. 3d). There was no significant difference between the PBS and the PBT groups at any time point after the transplantation.
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Fig. 3. Effects of BMT on neovascularization. Vascular endothelial cells were stained with anti-factor VIII antibody. Light photomicrograph of a cross-section of the infarcted myocardium in the BMT group (a), the PBT group (b), and the PBS group (c) at day 28 post-transplantation. The capillaries were stained with DAB (original magnification, x40). (d) shows that the number of vessels in the BMT group was significantly greater than those in the other groups 7, 14 and 28 days after BMT, respectively. Neither the PBS group nor the PBT group showed any significant changes. *P<0.01 vs the PBS group (X+S, n=25. The number of vessels in each slide was calculated based on 10 microscopic fields. The results were the average of 25 slides equally from five animals in every subgroup).
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3.4. Incorporation of BM-MNCs into myocardium and endothelium
In order to assess whether BM-MNCs were incorporated into myocardium or newly formed capillary walls, we examined Hoechest33342-labeled cells in combination with specific markers for myocardial or vascular endothelial cells, MHC and factor VIII using fluorescence microscopy. We observed that on day 14, 8% of transplanted Hoechest-33342 positive BM-MNCs began to express MHC, and 9% began to express factor VIII; on day 28, 17% of transplanted Hoechest-33342 positive BM-MNCs expressed MHC (as shown in Fig. 4a–c)
, and 12% expressed factor VIII (Fig. 4d–f). In contrast, transplanted PB-MNCs did not express MHC (Fig. 4g) or factor VIII (Fig. 4h) at any time points observed. To exclude the possibility that the differentiated cells resulted from infiltrating hematopoietic cells, some sections were double-stained with antibodies against CD45. The Hoechest-positive cells that stained positive with antibodies against MHC or factor VIII were not positively stained for CD45 (data not shown).
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Fig. 4. Differentiation of bone marrow-mononuclear cells (BM-MNCs) into myocardial and endothelial cells. BM-MNCs were pre-labeled with Hoechest33342 (a and d), which stained the nuclei blue. Myocardial cytoplasm and vascular endothelial cytoplasm were stained green and red with antibodies against MHC and factor VIII (b and e), respectively. Some of the transplanted BM-MNCs double-stained with Hoechest33342 and MHC or factor VIII (c and f, arrowheads). Transplanted PB-MNCs did not express MHC and factor VIII (g and h) (each bar stands for 50 µm).
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4. Discussion
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MI is a leading cause of heart failure and death in many countries. Although post-infarction survival rates have been improved in recent years, reduced cardiac function due to excessive loss of cardiomyocyes remains a major problem. The lack of resident stem cells in the heart has led to an intensive search for alternative sources of cardiomyocyte progenitors. Embryonic stem cells have shown to differentiate into cardiomyocytes that can form stable intracardiac grafts. Skeletal myoblasts or cardiomyocytes from fetal or neonatal mice have also shown to take up residence in cardiac tissue after injury. However, bone marrow cells, especially autologous bone marrow cells, have been identified as a progenitor population readily accessible from an adult patient's own tissue that can be adapted to clinical therapy [13–15]. In the present study, we investigated the role of cytoprotection and angiogenesis in the improvement of ischemic heart function followed BMT.
To our knowledge, we were the first to observe that endogenous expression of HSP32 and HSP70 was strongly promoted within cardiomyocytes in the infarction zone and the peri-infarct zone, especially on day 3 after transplantation. We also found the expression of HSP32 and HSP70 within some transplanted bone marrow cells. Suzuki et al. [16] demonstrated that HSPs enhanced graft cell survival in skeletal myoblast transplantation to the heart. Katori et al. [17] found that prior induction of HSPs by nitric oxide donor attenuated cardiac ischemia/reperfusion injury in the rat. Mestril et al. [10] showed that adenovirus-mediated gene transfer of HSP70 protected hearts against simulated ischemia. Our study demonstrated the overexpression of HSP32 and HSP70 within transplanted bone marrow cells and recipient endogenous myocytes, suggesting that the upregulation of HSP32 and HSP70 contributed to myocytes protection against ischemic injury, enhanced post-ischemic myocardial salvage, and therefore improved cardiac function. Although this action was not long-lived, it is useful for animals to survive the acute phase of MI and the perioperative period. This also suggested that the conventional treatments of coronary artery disease, percutaneous transluminal coronary angioplasty and coronary artery bypass grafting might work more beneficially in combination with stem cell transplantation.
Usually, HSP70 is expressed in normal, unstressed mammalian cells and can be detected in the samples from normal rat, rabbit and pig heart [18]. The ischemia injury can stimulate HSPs expression but it lasts for no more than 4 days [9,19,20]. In our study, we observed the expression of HSP70 and HSP32 in all three groups. However, the expression levels in the BMT group were much higher than those in the other two groups. The expression of HSPs, especially HSP70, in the BMT group lasted for 7 days. It indicated that BMT enhanced and prolonged the expression of HSPs in the ischemia myocardium. The biological mechanisms, via which BMT upregulates the expression of HSPs remain to be understood by further investigation. On the basis of our study, we speculate that several factors may contribute to the increase in the expression of HSPs. First, HSPs activity was enhanced during bone marrow cell differentiation [18], which contributed to upregulation of HSPs expression. Second, transplanted BM-MNCs autologously produced large amount of HSPs in vivo at ischemic microenvironment and enhanced the expression of HSPs in the host ischemic myocardium. Third, allograft stimulated the expression of HSPs [21]. Fourth, myocardial ischemia upregulated the expression of HSPs. Finally, it is possible that the mild transient inflammatory effect after operation may have contributed to the initiation of the HSPs expression. However, the latter three factors might not be major influent ones, because we did not find that the expression of HSP32 and HSP70 in the PBT group kept pace with overexpression of HSP32 and HSP70 in the BMT group. It should be noted that the expression levels were analyzed by semi-quantitative RT-PCR rather than real-time RT-PCR. Further quantitative analysis is required in future studies.
In our study, at the early phase of cell transplantation, the cardiac function in the BMT group gradually recovered, whereas neither the PBT group nor the PBS group demonstrated such effects. In a parallel time frame with the observed enhancement of cardiac function in the BMT group, the amount of angiogenesis gradually increased. This suggests that angiogenesis played an important role in the enhancement of cardiac function. A variety of growth factors in bone marrow cells are known to induce angiogenesis. VEGF is the most potent growth factor. We also found that the cardiac expression of VEGF was upregulated by BMT but not PB-MNCs or PBS injection. Kamihata et al. [5] reported that in a swine MI model, the mRNA expression of VEGF assessed by Northern blots in the BMT group was most abundant after 1 day and reverted to the baseline level after 14 days. In the present rat model, RT-PCR results showed in detail that VEGF in the BMT group was highly expressed after 1 day, peaked at 3 days, decreased after 7 days, and then reverted to the baseline after 14 days. VEGF expressions in the PBT and the PBS groups were abundant between 1 and 3 days, and reverted to the baseline level after 7 days, which were in consistence with those from the pig study by Heba et al. [22] which indicated that the expression of VEGF was upregulated between 1 and 4 days after MI. This suggests that intramyocardial injection of BM-MNCs, but not injection of PB-MNCs or injury/ischemia, prolonged upregulation of VEGF expression. Upregulation of VEGF may contribute to further enhancement of angiogenesis.
Several groups have demonstrated that transplanted bone marrow cells could differentiate into myocardial and vascular endothelial cells [2,3,23]. We also found the expression of specific markers for myocardial or vascular endothelial cells, MHC and factor VIII, in transplanted BM-MNCs on day 14 post-transplantation. On day 28, these expressions were enhanced further. In contrast, transplanted PB-MNCs did not express MHC or factor VIII. This suggests that exogenous BM-MNCs were able to differentiate into new myocytes and to form new vessels (vasculogenesis). In parallel with differentiation of transplanted BM-MNCs, cardiac function in the BMT group was further improved 14 days after operation. It suggested that differentiation of BM-MNCs contributed to the recovery of cardiac function in the late phase of post-infarction. Because BM-MNCs contain various cell lineages, such as mesenchymal cells, hematopoietic cells and endothelial progenitor cells, it is not clear which populations of BM-MNCs work beneficially in ischemic myocardium. Further investigation on BMT treatment is required to clarify the optimal populations in whole bone marrow cells that have the most potency for differentiation.
In consistence with continuous improvement of cardiac function, BMT could sustain enhancement of angiogenesis in the ischemic myocardium. Vessel counting showed that the density of capillaries in the infarcted area was 45, 78 and 125% higher in the BMT group than in the PBS groups on days 7, 14 and 28, respectively. It indicated that angiogenesis contributed to sustained improvement of infarcted heart function. Kocher et al. [4] considered that vessel density enhancement caused by BMT included vasculogenesis and angiogenesis, which enhanced the blood supply of ischemic myocardium and post-ischemic myocardial salvage. Whether this vasculogenesis alone or in combination with angiogenesis contributes to improvement of cardiac function remains to be precisely determined.
The main findings of this study include (1) at the early phase (1–7 days post-transplantation), BMT upregulated the expression of HSP32, HSP70 and VEGF in both transplanted BM-MNCs and recipient endogenous cardiomyocytes, and enhanced cytoprotection and angiogenesis, which may contribute to recovery of infarcted heart function; and (2) at the late phase, the transplanted bone marrow cells might differentiate into both myocardial and vascular endothelial cells that enhance ischemic cardiac function further. Further research would be required for quantitative analysis of changes in VEGF and HSPs after BMT by using Northern blots, Real-time PCR or Western blots. The relationship between upregulation of HSPs and improvement of cardiac function also needs to be clarified.
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Acknowledgments
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This work was supported by the National High Technology Research and Development Program (2001AA2160313), the National Natural Sciences Foundation of China (30170382), Beijing Science and Technology Commission (H020220010490) and 985 Project Foundation of Peking University. The authors thank Professors Youyi Zhang and Chaoshu Tang, for their useful discussion during the course of this study; Professors Jiang Gu, Shulin Liu and Dr Jason Wong for the help in preparation of this manuscript.
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Abstract
1. Introduction
