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Keywords:

  • Gene therapy;
  • Stem cells;
  • Transplantation;
  • Angiogenesis;
  • Antiapoptosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Engraftment of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for postinfarction left ventricular dysfunction. However, limited cell viability after transplantation into the myocardium has restricted its regenerative capacity. In this study, we genetically modified MSCs with an antiapoptotic Bcl-2 gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a rat left anterior descending ligation model via intracardiac injection. Rat MSCs were manipulated to overexpress the Bcl-2 gene. In vitro, the antiapoptotic and paracrine effects were assessed under hypoxic conditions. In vivo, the Bcl-2 gene-modified MSCs (Bcl-2-MSCs) were injected after myocardial infarction. The surviving cells were tracked after transplantation. Capillary density was quantified after 3 weeks. The left ventricular function was evaluated by pressure-volume loops. The Bcl-2 gene protected MSCs against apoptosis. In vitro, Bcl-2 overexpression reduced MSC apoptosis by 32% and enhanced vascular endothelial growth factor secretion by more than 60% under hypoxic conditions. Transplantation with Bcl-2-MSCs increased 2.2-fold, 1.9-fold, and 1.2-fold of the cellular survival at 4 days, 3 weeks, and 6 weeks, respectively, compared with the vector-MSC group. Capillary density in the infarct border zone was 15% higher in Bcl-2-MSC transplanted animals than in vector-MSC treated animals. Furthermore, Bcl-2-MSC transplanted animals had 17% smaller infarct size than vector-MSC treated animals and exhibited functional recovery remarkably. Our current findings support the premise that transplantation of antiapoptotic gene-modified MSCs may have values for mediating substantial functional recovery after acute myocardial infarction.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Cell transplantation utilizing fetal myocardial tissues [1], embryonic stem cells [2], skeletal myoblasts [3, 4], cardiomyocytes [5, 6], smooth muscle cells [7, 8], cardiac stem cells [9], bone marrow cells [10], and hematopoietic stem cells [11] has emerged as a promising therapeutic approach for the restoration of heart functions following myocardial infarction damage. Considerable experimental and clinical evidences have demonstrated that using different cell types to replace the necrotic tissue in the myocardium is safe and can contribute to the improvement of angiogenesis and heart functions [12, [13], [14], [15], [16]17]. Among the various cell types investigated, bone marrow mesenchymal stem cells (MSCs) are self-renewing clonal precursors of nonhematopoietic stromal tissues [18]. They can be isolated from the bone marrow or adipose tissue and expanded in culture based on their ability to adhere to culture dishes and proliferate in vitro. MSCs are multilineage potential cells [19] and can give rise to osteoblasts, chondrocytes [20], neurons [21], skeletal muscle [22], and cardiac muscle [23, 24] under appropriate conditions. Furthermore, studies on human, baboon, and murine MSCs showed that MSCs are immunosuppressive [25, 26]. Therefore, MSCs appear to be an appealing cell source for cardiac transplantation [13, 27] because of the ease of harvest and expansion ex vivo. However, the low cellular survival rate after transplantation into an infarcted heart within the first few days engenders only marginal functional improvement [24, 28]. Thus, it is necessary to reinforce MSCs against the arduous microenvironment incurred from ischemia, inflammatory response, and proapoptotic factors in order to improve the efficacy of cell therapy.

The 26-kDa Bcl-2 antiapoptotic protein belongs to the Bcl-2 family of proteins, which was originally found to be overexpressed in B-cell lymphoma [29]. It serves as a critical regulator of pathways involved in apoptosis, acting to inhibit cell death [30]. Bcl-2 gene was found to be upregulated in failing hearts [31, 32] as well as aging hearts [33]. It acts to prevent programmed cell death of ventricular myocytes [34].

Previous studies have demonstrated that Bcl-2 is the regulator of the metabolic functions of mitochondria during ischemic conditions, which can contribute to both cardiac and neuronal protection under various stresses [34, 35]. The overexpressed Bcl-2 can delay the onset of cell death and modestly augment viable cell growth in the first 48 hours of apoptosis [36]. Bcl-2 deficient mice showed abnormalities with loss of a death repressor in specific cells [37]. The protective effects of Bcl-2 for conditions of the diseased heart have been approved by previous studies [34, 38]. Interestingly, overexpressing Bcl-2 in transgenic mice improved the heart function and inhibited cardiomyocyte apoptosis [39].

In this study, adult rat bone marrow-derived MSCs were genetically modified to overexpress Bcl-2, which aimed at vivifying the stem cells and enhancing their resistance to ischemic conditions from acute myocardial infarction after transplantation into the heart. We proposed that genetic modification of stem cells with Bcl-2 would armor stem cells settling into a deteriorative ischemic microenvironment and improve stem cell viability in the early post-transplanted period, thereby enhancing cardiac functional recovery after acute myocardial infarction.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Isolation and Culturing of Rat MSCs

Male Lewis rats were obtained from Charles River Laboratories (Wilmington, MA, http://www.criver.com). All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (NIH publication number 85–23, revised 1996). Rat MSCs were isolated from the bone marrow of the femurs and tibias of rats (Charles River Laboratories). A 21-gauge needle was inserted into the shaft of the bone and flushed with 30 ml of complete α-modified Eagle's medium (α-minimal essential medium) containing 20% fetal bovine serum (lot selected for promoting rapid expansion of MSCs; HyClone, Logan, UT, http://www.hyclone.com), 2 mM l-glutamine (PAA Laboratories, Linz, Austria, http://www.paa.at), 100 U/ml penicillin (PAA), 100 μg/ml streptomycin (PAA), and 25 ng/ml amphotericin B (PAA). The harvested cells were filtered through a 70-μm nylon filter (Falcon; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) and plated into one 75-cm2 flask per rat. The cells were grown at 37°C and 5% CO2 for 3 days before the medium was replaced. The adherent cells were grown to 90% confluence to obtain samples here defined as passage 0 cells.

Fluorescence-Activated Cell Sorter Analysis

MSCs at passage 3 were analyzed for purity and epitope expression using fluorescence-activated cell sorter (FACS) analysis. The cells were blocked with Fc Block blocking reagent (BD Biosciences, San Diego, http://www.bdbiosciences.com) and incubated for 10 minutes at 4°C with the following antibodies: anti-CD29.PE (clone Ha2/5; BD Biosciences), anti-CD44.FITC (Serotec Ltd., Oxford, U.K., http://www.serotec.com), anti-CD90 (Thy1).FITC (clone MRC OX-7; Abcam, Cambridge, U.K., http://www.abcam.com), anti-CD45.PE (BD Biosciences), and anti-CD34.FITC (BD Biosciences). Isotype controls were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/index_us.shtml). After incubation, the cells were washed with 2 mM phosphate-buffered saline (PBS)/EDTA and analyzed using a FACSCalibur flow cytometer (Becton, Dickinson). Dead cells were excluded using propidium iodide (PI) staining. Data analysis was performed with CellQuest software (BD Biosciences). Histograms of cell number versus logarithmic fluorescence intensity were recorded for 10,000–20,000 cells per sample.

Genetic Modification of MSCs

A total number of 4 × 105 cells per well were plated in a 6-well plate 24 hours before transfection; jetPEI mediated transfection was performed according to the protocol given by the supplier (Polyplus-transfection, New York, http://www.polyplus-transfection.com). In brief, dilution of 3.0 μg of DNA (pEGFP-N1, pcDNA3.1/Bcl-2, or pcDNA3.1) and 6 μl of jetPEI per well was carried out in 150 mM NaCl separately. The transfection mixture was mixed immediately and added to the adherent MSCs with 1 ml of complete medium. Transfection using jetPEI was optimized according to the supplier's instructions by varying the amount of DNA (pEGFP-N1) and the volumes of transfection reagent at the number of polynitrogen (N) per DNA phosphate (P) ratios between 3:1 and 12:1. Determination of transfection efficiency was performed 24 hours after transfection by fluorescence microscopy (Leica, Heerbrugg, Switzerland, http://www.leica.com) and by FACS. For each experiment, at least three microscopic visual fields (200-fold magnification) were counted, and the ratios of enhanced green fluorescent protein-expressing cells to nonfluorescent cells were calculated. MSCs without transfection and those transfected with pcDNA3.1/Bcl-2 or pcDNA3.1 are termed “MSCs,” “Bcl-2-MSCs,” and “vector-MSCs,” respectively. Bcl-2 protein expression level was evaluated by Western blotting. Before cell injection, medium was removed from the cultures, cell layers were washed with PBS, and cells were harvested by incubation with 0.25% trypsin/EDTA. All experiments and cell number determinations were performed in triplicate.

In Vitro Functional Differentiation Assay

To induce adipogenic differentiation, MSCs after genetic modification were seeded at a density of 3 × 103 cells per cm2 and cultured for up to 3 weeks in cell culture medium supplemented with 10−8 M dexamethasone, 2.5 μg/ml insulin, and 100 μM indomethacin. To induce chondrogenic differentiation, 3 × 105 MSCs were cultured in 1 ml of chondrogenic induction medium (cell culture medium supplemented with 0.1 μM dexamethasone, 1 mM sodium pyruvate, 0.17 mM l-ascorbic acid 2-phosphate, 0.35 mM l-proline, 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenite, 5.33 μg/ml linolic acid, 1.25 mg/ml bovine serum albumin, and 0.01 μg/ml transforming growth factor-β3) in the tip of a 15-ml conical tube to allow aggregation of the cells in suspension culture. The induction of chondrogenic differentiation was performed for 4 weeks. To induce cardiomyocyte differentiation, MSCs were treated with the DNA demethylation agent 5-azacytidine following previously reported protocols. The differentiation capacity toward different cell lineages was verified by morphology changes and immunostaining for specific markers, that is, aggrecan for chondrocytes, fatty acid binding protein (FABP-4) for adipocytes, and Homeobox protein NK-2 homolog E (Nkx2.5) for cardiomyocytes.

Western Blot Analysis

The genetically modified MSCs were treated with cell lysis buffer (Promega, Madison, WI, http://www.promega.com). The protein concentration of the samples was determined by BCA protein assay (Pierce, Rockford, IL, http://www.piercenet.com). Twenty μg of total proteins were resolved by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a 0.2-mm nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany, http://www.whatman.com). The membrane was blocked in PBS buffer containing 0.2% Tween 20 and 5% skim milk overnight at 4°C. Subsequently, the blot was incubated with human Bcl-2 protein monoclonal antibody (1:200 dilution; Dako, Glostrup, Denmark, http://www.dako.com) for 3 hours. Housekeeping protein β-actin was employed as loading control. Antibody binding was detected with horseradish peroxidase conjugated anti-mouse secondary antibody (1:2,000; Pierce) and visualized by ECL kit (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). Proteins prepared from MSCs transfected with vector alone served as a control.

Apoptosis Assays

MSCs grown on glass coverslips were treated with hypoxic preconditioning of 95% nitrogen and 5% carbon dioxide. At 24 hours posthypoxic treatment, apoptotic cells were identified using the terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) method by staining the cells using the Chemicon ApopTag Assay Kit and counterstaining with PI. The coverslips were washed four times with PBS and mounted in FluorSave (Calbiochem, San Diego, http://www.emdbiosciences.com). Apoptotic nuclei were also assessed and quantified by staining with Hoechst 33342 (Molecular Probes, Eugene, OR, http://www.probes.invitrogen.com). Image acquisition was performed under phase contrast 400× on a Leica DMLB microscope. A total of 20 microscopic fields were quantitated for each coverslip. The results are representative of three independent experiments replicated with six different coverslips.

Detection of Vascular Endothelial Growth Factor Secretion

The secretion of vascular endothelial growth factor (VEGF) by rat MSCs was evaluated by the Quantikine rat VEGF immunoassay (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) according to the manufacturer's protocol.

Cell Labeling

To identify the transplanted MSCs in the hearts, the cells were labeled with bromodeoxyuridine (BrdU) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at a final concentration of 10 μmol/l in cell culture medium for 12 hours. The efficiency of labeling was 42.1% ± 3.5% (n = 3), which was evaluated by immunostaining of the cytospun cells with BrdU monoclonal antibody (Lab Vision, Fremont, CA, http://www.labvision.com). In addition, the cell engraftment was also validated with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (Vybrant CFDA SE Cell Tracer; Molecular Probes; 5 μM) labeling. The harvested cells were resuspended in culture medium at a density of 106 per 100 μl and kept on ice (less than 1 hour) until transplantation.

Myocardial Infarction and MSC Transplantation

Myocardial infarction was induced at 8–12 weeks of age (approximately 280 g of body weight). Rats were anesthetized by intraperitoneal injection with Pentobarbital (50 mg/kg of body weight; Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com), endotracheally intubated, and mechanically ventilated. The heart was exposed via a left thoracotomy. Anterior myocardial infarction was created by ligation of the left anterior descending artery with a 6–0 silk suture (myocardial infarction [MI]+) (supplemental online Fig. 1A). In the sham group, rats were sham operated (left thoracotomy without coronary artery ligation [MI−]). Successful infarction was determined by observing a pale discoloration of the left ventricular muscle and an ST elevation on electrocardiograms. Immediately after ligation, 6 × 106 vector-MSCs or Bcl-2-MSCs were injected at six injection sites into anterior and lateral aspects of the viable myocardium bordering the infarction with a 31-gauge needle (BD Biosciences) (supplemental online Fig. 1B, 1C). A further MI+ rats with Dulbecco's modified Eagle's medium injection served as medium controls. Animals were sacrificed at 4 days, 3 weeks, and 6 weeks after cell transplantation. Cell survival was examined at 4 days, 3 weeks, and 6 weeks. Angiogenesis, infarction area, and cell colocalization were evaluated at 3 weeks. The pressure-volume loop analysis was performed at 6 weeks.

Histological Analysis

At the end of the observation period, animals were sacrificed. The hearts were removed, washed with PBS, weighed, and snap frozen in liquid nitrogen. Frozen sections embedded in optimum cutting temperature medium (5 μm in thickness) were prepared and stained with Sirius red/Fast Green or hematoxylin-eosin. From every heart, three slices on mid level of the infarcted region were used for measurement of scar size. Quantification of scar area and length was performed using imaging software ESI-Vision (Soft Imaging System, Münster, Germany). The scar area was calculated as a percent of the whole area of the left ventricular section.

Quantitative Analysis of Cell Survival

Quantitative analysis of BrdU-positive cells was performed on the immunostained frozen sections. From every heart, five slices on mid level of the infarcted region were used for measurement. The myocardium extending 0.5–1.0 mm from the infarcted tissue or infarct scar was considered to represent the border zone myocardium. To avoid contamination of the remote myocardium with border zones, a myocardial area extending ∼1–2 mm from the border zone area was not included in the statistical analysis [40]. At least 20 representative microscopic fields (with oil immersion phase contrast 1,000× by a Leica DMLB) of each slide were randomly selected from the region of interest and digitally photographed. BrdU positive cells were counted in the tissue bordering infarction (4 days after infarction), in the border zones of infarct scars (3 and 6 weeks after infarction) with Leica IM 5 (V2.01) software. Cell survival was expressed as the proportion of the BrdU-positive nuclei to the total number of nuclei. The data were analyzed by two investigators who were blinded with respect to the cell treatment.

Determination of Infarct Size

Heart tissue sections were stained with hematoxylin-eosin and Sirius red F3BA (0.1% picric acid; Sigma-Aldrich). The infarcted area with an increase in collagen content was shown as Sirius red-positive and was measured by computerized planimetry. The ratio of scar length and entire circumference defined the infarct extent for the endocardial and epicardial surfaces, respectively. The final infarct size was determined as the average of endocardial and epicardial surfaces and was given in percent. Quantitative assessment of collagen deposition was performed with a multipurpose color image processor (Envision; Dako). An investigator blinded to the treatment performed the analysis.

Immunostaining

For immunohistological detection of transplanted cells, frozen tissue sections were incubated with monoclonal mouse anti-BrdU antibody (Lab Vision). After blocking in Envision blocking buffer (Dako), sections were placed in primary antibody overnight at 4°C to 8°C. On the following day, the sections were incubated with Alexa 568 (Molecular Probes) or horseradish peroxidase (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) conjugated goat anti-mouse IgG. Endothelial-like cells were stained for von Willebrand factor (vWF) (primary antibody: polyclonal anti-vWF antibody, H-300, Santa Cruz; secondary antibody: Alexa 568, Molecular Probes). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich). Cardiac-like cells were stained for Troponin T (primary antibody: monoclonal anti-Troponin T antibody, cardiac isoform, clone CT-3, Lab Vision; secondary antibody: Alexa 568, Molecular Probes). For colocalization study, BrdU-positive cells were identified (primary antibody: monoclonal mouse anti-BrdU antibody; secondary antibody: Alexa 488, Molecular Probes).

Determination of Capillary Density

The capillary density in both infarcted and normal myocardial tissue was determined as described by Weidner et al. [41]. Tissue sections were stained using anti-vWF antibody. For quantification of positively stained vessels, five sections within the infarct zone of each animal were analyzed by an investigator who was blinded with respect to the cell treatment. Capillaries were counted in 10 randomly chosen high-power fields (HPFs, 400×) in two sections per animal. The results were expressed as capillaries per high power field.

Left Ventricular Catheterization

Pressure-volume (PV) loops were recorded under isoflurane (2%) anesthesia with a conductance catheter (Millar 2 Fr catheter model SPR-838 [Millar Instruments, Houston, TX, http://www.millarinstruments.com]) connected to Aria/Power Lab data-acquisition hardware (Millar/Power Lab) by an investigator blinded to the treatment 6 weeks after cell transplantation. The cuvette volume calibration was performed according to the PVAN Pressure-Volume Analysis Software User's Guide. All data were analyzed off-line with IOX software (EMKA Technologies, Paris, http://www.emkatech.com). Maximum dP/dt (max dP/dt), minimum dP/dt (min dP/dt), end-diastolic volume (EDV), end-systolic volume (ESV), and ejection fraction (EF) were measured from the steady-state PV loops. End-systolic pressure-volume relationship (ESPVR), volume-axis intercept (V0), and maximal elastance (Emax) were obtained from PV loops measured by reducing preload through occlusion of the inferior vena cava.

Statistical Analysis

All values are presented as mean ± SD. One-way analysis of variance with Scheffe's post hoc test for unequal sample sizes was used to compare numeric data among the four experimental groups. Datasets consisting of two groups only were compared with the use of unpaired Student's t tests. A level of p < .05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

MSCs Express Markers Distinct from Hematopoietic Stem Cells

Cell isolation, expansion, characterization, and differentiation of rat MSCs have been established according to previous reports [19]. Primary culture of the marrow cells was performed according to Dexter's method [42]. Due to the presence of very little extracellular matrix in the bone marrow, gentle mechanical disruption can readily dissociate the stroma and hematopoietic cells into a single cell suspension. When plating the cells at low density, the MSCs adhere and can be easily separated from the hematopoietic stem cells by repeated washing. The morphology of rat MSCs from bone marrow displayed a homogenous spindle-shaped population and maintained a similar morphology during the subsequent passages. FACS analysis was employed to identify the surface marker expression. The MSC culture was shown to be devoid of CD34 and CD45 (Fig. 1D, 1E), which are the markers for hematopoietic cells. In contrast, a high expression of CD29, CD44, and CD90 markers were observed (Fig. 1A–1C). The results from FACS analysis were confirmed by immunocytochemistry (data not shown).

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Figure Figure 1.. Immunophenotypic profile of MSCs. Flow cytometry histograms after three passages show the expression (unshaded) of selected surface molecules; (A): CD29; (B): CD44; (C): CD90; (D): CD34; and (E): CD45. Control cells labeled without primary antibodies (shaded). The rat MSCs were positive for CD29, CD44, and CD90 but negative for CD34 and CD45 surface markers, which are commonly found on hematopoietic cells.

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Overexpression of Bcl-2 in Genetically Modified MSCs

The transfection efficiency of Polyethylenimine-Transfection Kit (Polyplus Transfection SA, Illkirch, France) for genetically modified MSCs was evaluated by pcDNA3.1/green fluorescent protein (GFP) as an internal control after 24-hour gene delivery. A representative GFP expression is shown in Figure 2A. More than 50% of MSCs were transfected based on FACS analysis (Fig. 2B). Bcl-2-MSCs and vector-MSCs have a phenotype similar to MSCs 3 days after transfection (data not shown). To evaluate the expression of Bcl-2 protein in the genetically modified MSCs in vitro, Western blot analysis was performed on cell samples at various time points (24 hours, 72 hours, 7 days, and 21 days) post-transfection. A significantly high expression of Bcl-2 was observed in Bcl-2-MSCs as early as 24 hours, which remained detectable on day 7 (Fig. 2C). In contrast, a weak expression of Bcl-2 was detected in vector-MSCs 24 hours after transfection, indicating the low level of endogenous Bcl-2 proteins in MSCs. The expression of internal housekeeping β-actin gene was at the same level. Our observation showed that the MSCs were successfully modified with Bcl-2. The upregulation of Bcl-2 was transient, and this may minimize the risk of malignant transformation or late cell failure. No apparent change of MSC phenotype after transfection was observed (data not shown).

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Figure Figure 2.. Transfection of MSCs with nonviral vector. (A): Representative photomicrograph of MSCs transfected with pcDNA3.1/GFP. (B): Fluorescence-activated cell sorter analysis showing the transfection efficiency of GFP transfected MSCs (unshaded) compared with untransfected MSCs (shaded). (C): Representative Western blots showing that Bcl-2 was overexpressed in Bcl-2 modified MSCs and lasted for 7 days after transfection. Housekeeping protein β-actin served as loading control. Abbreviations: d, days; GFP, green fluorescent protein; h, hours.

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Bcl-2 Modified MSCs Maintain Their Multidifferentiation Capacity

MSCs can differentiate into multiple lineages (such as bone, cartilage, adipose tissue, and cardiac muscle), and this ability is taken as a functional criterion defining MSC precursor cells. To verify whether the Bcl-2 modification affected the differentiation capacity, the genetically modified MSCs underwent myogenic, adipogenic, and chondrogenic differentiation using the methods previously described [23, 43] at 48 hours post-transfection. After 21 days of induction toward an adipogenic lineage, a characteristic morphological change with accumulation of lipid vacuoles was observed (Fig. 3A). Immunostaining revealed the presence of FABP-4, which is a marker protein for adipocytes. Chondrogenesis was assessed by immunostaining for aggrecan after 4 weeks of culture under chondrogenic conditions. The chondrocyte-like cells showed positive staining for aggrecan protein (Fig. 3B–3D). For cardiomyocyte differentiation, Bcl-2-MSCs were exposed to 5-azacytidine for 24 hours. After two weeks, cardiomyocyte-like cells and positive immunostaining for Nkx2.5 were noted (Fig. 3E–3G). Taken together, this evidence indicated that Bcl-2-MSCs retain their multidifferentiation potential into adipogenic, chondrogenic, and cardiomyocyte lineages.

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Figure Figure 3.. Differentiation capacity of Bcl-2-MSCs. MSCs transfected with Bcl-2 were cultured in adipogenic, chondrogenic, and myogenic medium for up to 2 months. (A): Adipogenic differentiation. Immunostaining with fatty acid binding protein-4 (brown). (B–D): Chondrogenic differentiation. Immunostaining for aggrecan ([B, D], red). Nuclei were counterstained with 4,6-diamidino-2-phenylindole ([C, D], blue). (E–G): Cardiomyocyte-like differentiation. Immunostaining with anti-Nkx2.5 antibody ([E, G], green). Nuclei were counterstained with propidium iodide ([F, G], red).

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Bcl-2 Modified MSCs Protect Against Apoptosis In Vitro

To test the capability of Bcl-2-MSCs to protect against apoptosis in vitro, the modified MSCs were treated under hypoxic conditions for 24 hours. The evaluation of apoptosis was carried out using TUNEL assay (Fig. 4A) and Hoechst 33342 staining (Fig. 4B). Under hypoxia, the rates of cell apoptosis in MSCs and vector-MSCs exceeded those of Bcl-2-MSCs by 1.5-fold. The apoptotic cell number was significantly reduced by the Bcl-2 genetic modification. Quantitative assay showed that the number of TUNEL-positive cells was decreased from 53% ± 3.5% of MSCs and 51% ± 4.7% of vector-MSCs to 36% ± 1.8% (n = 6, p < .001) of Bcl-2-MSCs. However, there was no significant difference in apoptotic cell numbers among MSCs, vector-MSCs, and Bcl-2-MSCs when evaluated under normoxia. A further confirmatory assay was based on nuclear chromatin morphology after staining with Hoechst 33342. Apoptosis was evidenced by cell shrinkage, nuclear condensation, and DNA fragmentation that occurred in cells treated with hypoxia (Fig. 4B). The quantitative assay based on Hoechst staining showed that the number of apoptotic cells was decreased from 46% ± 3.1% of MSCs and 45% ± 4.2% of vector-MSCs to 30% ± 2.6% of Bcl-2-MSCs (n = 6, p < .001). These findings demonstrated that the Bcl-2 modified MSCs could protect against apoptosis under hypoxic conditions. It is anticipated that Bcl-2 modified MSCs may retain their antiapoptotic properties in the ischemic myocardium.

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Figure Figure 4.. Antiapoptotic effect and upregulation of VEGF secretion of Bcl-2-MSCs under hypoxic conditions. (A): Representative photomicrographs of TUNEL-positive (left panels) and total (right panels) cells after 24 hours of hypoxic treatment. (B): Representative photomicrographs of apoptotic cells (arrows) with chromatin condensation by Hoechst 33342 staining after 24 hours of hypoxic treatment. (C): VEGF secretion by Bcl-2-MSCs under hypoxic conditions. After 24 hours of incubation, conditioned medium from normoxia (solid bar) and hypoxia (empty bar) treated cells (n = 6) was subjected to VEGF enzyme-linked immunosorbent assay (ELISA) assay. VEGF concentration values are mean ± SD (* p < .001). ELISA data are representative of three independent experiments. Abbreviations: PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP end labeling; VEGF, vascular endothelial growth factor.

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Bcl-2 Modified MSCs Upregulated Angiogenic Cytokine VEGF Under Hypoxia

To identify potential paracrine mechanisms responsible for the therapeutic effect of Bcl-2-MSCs, we examined the secretion of VEGF. MSCs, vector-MSCs, and Bcl-2-MSCs were cultured under either normoxic or hypoxic conditions for up to 24 hours, and VEGF secretion was quantitated by Quantikine rat VEGF immunoassay. A more than 60% increase in secretion of VEGF was detected in Bcl-2-MSCs compared with MSCs alone and vector-MSCs when the cells were cultured under hypoxic conditions (Fig. 4C). However, there was no significant difference among the groups when the cells were cultured under normoxic conditions. The angiogenic cytokine VEGF was significantly upregulated in Bcl-2-MSCs in response to hypoxic conditions. The high-level expression of VEGF from Bcl-2-MSCs may provide cardioprotective and proangiogenic effects.

Cell Engraftment with Bcl-2 Modified MSCs in the Infarcted Heart

To assess the efficacy of Bcl-2-MSC transplantation, 6 × 106 Bcl-2-MSCs or vector-MSCs from passage 3 were transplanted into the viable left ventricular (LV) myocardium bordering infarction. Medium was injected into control animals. The assessment of cell engraftment was carried out 4 days, 3 weeks, and 6 weeks after cell transplantation. BrdU-positive nuclei were identified by immunostaining (Fig. 5A–5E). Figure 5A and 5B depicts representative images 4 days following cell transplantation. Figure 5C–5E shows representative images of Bcl-2-MSC cells in the heart of a MI+ rat sacrificed 3 weeks after cell injection. The number of surviving cells in the Bcl-2-MSC group was greater than in the vector-MSCs following cell transplantation, and the differences proved to be statistically significant (Fig. 5F), with 2.2-fold enhancement of cell survival on day 4 (n = 10, p < .01), 1.9-fold enhancement on day 21 (n = 9, p < .05), and 1.2-fold enhancement after 6 weeks (n = 6, p < .05). Manipulation of MSCs with Bcl-2 gene enhanced cellular survival after myocardial infarction.

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Figure Figure 5.. Engraftment of Bcl-2-MSCs in ischemic myocardium. (A, B): Representative images from immunostaining of grafted MSCs in the infarcted rat myocardium 4 days following MSC injection. BrdU-labeled MSCs (brown) were clearly identified. (C–E): Immunofluorescence staining of BrdU-labeled cells in the Bcl-2-MSC group 3 weeks after cell treatment with high magnification. (C): BrdU (red). (D): DAPI (blue). (E): Merged image (×1,000 in all panels). Arrowheads indicate the cell engraftments. (F): Quantitative assessment of engrafted MSCs at 4 days, 3 weeks, and 6 weeks (* p < .05; ** p < .01). Abbreviations: BrdU, bromodeoxyuridine; DAPI, 4,6-diamidino-2-phenylindole.

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Furthermore, double immunofluorescence staining with BrdU and anti-vWF antibody revealed that at least some of the MSCs (3.1% ± 1.3%) appeared to display endothelial cell-like phenotype. Occasionally, blood vessels consisted of BrdU-positive cells (Fig. 6A, 6B). Three weeks after cell transplantation, we observed a very low number of MSCs colocalized with cardiac Troponin T (cTnT) (Fig. 6C–6E). The colocalization was further confirmed by the three-dimensional reconstruction of the tissue acquired by a Leica TCP2 confocal microscope (Fig. 6F–6H). The frequency of cTnT-BrdU double-positive cells from the engrafted stem cells was extremely low (Bcl-2-MSC group 0.05% ± 0.02% cTnT). There was no significant difference between Bcl-2-MSCs and the MSC group. It was not clear whether the transplanted cells had fused or differentiated into cardiomyocytes. There is no evidence for tumor formation and MSC differentiation into bone, adipose, and cartilage after 3 and 6 weeks of cell transplantation (data not shown).

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Figure Figure 6.. Bcl-2-MSCs in ischemic myocardium. (A, B): Double immunofluorescence staining revealed BrdU-labeled Bcl-2-MSCs (green) incorporated into the endothelial lining of blood vessels (red) near the infarct border zone 3 weeks after transplantation. (C–E): Three weeks after transplantation, sections near the infarct zone were double-stained for BrdU (green) and cardiac marker Troponin T (red). BrdU-positive cell colocalized with cardiac Troponin T. (F–H): Three-dimensional reconstruction of the tissue section using confocal microscopy illustrates that BrdU-positive nucleus (green) is within a cell body that expresses cardiac Troponin T. Cardiac-like cells were stained for Troponin T (primary antibody: monoclonal anti-Troponin T antibody, cardiac isoform, clone CT-3, Lab Vision; secondary antibody: Alexa 568); BrdU-positive cells were identified (primary antibody: monoclonal mouse anti-BrdU antibody; secondary antibody: Alexa 488). Abbreviations: BrdU, bromodeoxyuridine; cTnT, cardiac Troponin T.

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Capillary Density

Capillary density at the border zone of the acute myocardial infarction (AMI) was determined based on vWF immunostaining 3 weeks after cell transplantation. Representative images are shown in Figure 7A. There was a significant increase in capillary density in vector-MSC (20.5 ± 1.9 vessels per HPF) and Bcl-2-MSC (23.6 ± 1.3 vessels per HPF) groups when compared with medium-treated hearts with AMI (14.4 ± 2.1 vessels per HPF) (n = 8, p < .001) (Fig. 7B), and no obvious angiogenesis was detected in the medium group. In addition, the capillary density was 15% higher in Bcl-2-MSC treated hearts than in vector-MSC treated hearts with significant difference (n = 8, p = .002).

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Figure Figure 7.. Functional effects of Bcl-2 modified MSCs. (A, B): Bcl-2-MSCs enhanced angiogenesis in infarcted heart. (A): Representative photomicrographs of the infarct zone obtained after immunostaining for von Willebrand factor (vWF) antibody. (B): Quantitative capillary density data based on vWF immunostaining. Values are mean ± SD (* p = .002, ** p < .001). (C): Representative picrosirius red staining of transverse sections through rat hearts 3 weeks following left anterior descending ligation with medium, vector-MSC, or Bcl-2-MSC injection. Thinning of the left ventricular free wall and extensive collagen deposition (red) in scar tissue was noted in medium and vector-MSCs. (D): The scar size was significantly reduced in both the vector-MSC (* p < .001 vs. medium group) and Bcl-2-MSC groups (* p < .001 vs. medium group). The scar size was 17% smaller in the Bcl-2-MSC group than in the vector-MSC group (** p = .029). (E): Effects of MSC transplantation on left ventricular maximum dP/dt and minimum dP/dt. Values are means ± SD (* p < .001). Abbreviation: HPF, high-power field.

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Infarct Size and Cardiac Function

Left anterior descending (LAD) ligation consistently resulted in transmural myocardial infarction, exhibiting typical histologic changes including thinning of the left ventricular free wall and extensive collagen deposition 3 weeks after myocardial infarction. Representative left ventricular sections 3 weeks following LAD ligation with medium or MSC or Bcl-2-MSC injection are shown in Figure 7C. Both vector-MSC treated and Bcl-2-MSC treated MI+ animals showed smaller infarction size (34.2% ± 6.0% in vector-MSC group; 28.3% ± 5.8% in Bcl-2-MSC group) compared with the medium treated animals (47.8% ± 6.3%), and the difference was statistically significant (n = 8, p < .001 vector-MSC group vs. medium group; n = 8, p < .001 Bcl-2-MSC vs. medium group). Furthermore, the collagen content was 17% lower in Bcl-2-MSC treated hearts than in vector-MSC treated hearts (34.2% ± 6.0% of vector-MSC group vs. 28.3% ± 5.8% of Bcl-2-MSC group) (n = 8, p = .029) (Fig. 7D).

Hemodynamic parameters of left ventricular function (supplemental online Table 2) demonstrated that max dP/dt and min dP/dt were improved significantly more in the Bcl-2-MSC group than in the vector-MSC group (Fig. 7E) (n = 6, p < .001). Compared with the medium group, both Bcl-2-MSCs and the vector-MSC group had significant improvements on hemodynamic parameters (ESV, EF, max dP/dt, and min dP/dt). Furthermore, there were significant improvements in the Bcl-2-MSC group on EDV, ESPVR, and Emax compared with the medium group (Bcl-2-MSC group: 239.2 ± 35.9 μl of EDV, 0.58 ± 0.14 mmHg/μl slope ESPVR, 1.50 ± 0.34 mmHg/μl Emax vs. medium group: 292.1 ± 39.0 μl of EDV, 0.36 ± 0.16 mmHg/μl slope ESPVR, 0.93 ± 0.39 mmHg/μl Emax) (n = 6, p < .05). However, there was only a positive trend toward improvement on EDV, ESPVR, and Emax between vector-MSC group and medium group, and the difference did not reach statistical significance. No significant difference was observed on the values of V0 between the Bcl-2-MSC group (83.4 ± 14.1 μl) and the vector-MSC group (79.4 ± 13.0 μl) or between the Bcl-2-MSC group and the medium group (87.2 ± 15.8 μl).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

MSCs derived from adult bone marrow have been proposed as a promising cell source for the regeneration and restoration of infarcted heart function. However, poor survival of implanted cells hampered the therapeutic efficacy. In this study, the key roles Bcl-2 played in protecting grafted MSC survival under hypoxia-ischemia were identified in vitro and in vivo. We confirmed that Bcl-2 genetically modified MSCs retained their differentiation capacity and could give rise to different lineages in vitro. Overexpression of Bcl-2 reduces MSC death and apoptosis under hypoxic conditions. In vivo transplantation of Bcl-2-MSCs enhances the survival rate of MSCs in the LAD ligation model. It could attenuate postinfarction LV remodelling and restore LV function, which may have attributed to the enhanced antiapoptotic properties of the modified MSCs. These observations suggest that genetic modification of MSCs with Bcl-2 could be of significant value in improving the efficacy of stem cell therapy following a broad range of cardiac diseases. Our results agree with the earlier findings that transgenic mice overexpressing Bcl-2 in the heart reduced infarct size and improved recovery of cardiac function after ischemia/reperfusion injury [38, 39, 44].

There are several mechanisms contributing to the high level of stem cell death within 4 days after implantation into ischemic hearts [24, 28]. These include host inflammatory response, loss of survival signal from matrix attachments or cell-cell contact, delivery of oxygen and substrates via diffusion [28], various proapoptotic or cytotoxic factors in the ischemic myocardium [45], and ischemic/reperfusion damage to the implanted stem cells incurred from repeated bouts of ischemia [46]. Furthermore, MSCs are extremely sensitive to the hypoxic and inflammatory environment in ischemic hearts [24]. Our present studies reveal that genetic modification of MSCs with Bcl-2 effectively protects transplanted MSCs against ischemia and increases cell survival after implantation. Our investigation, consistent with previous findings from Mangi et al. [18] and Tang et al. [46], showed that the therapeutic efficacy of MSCs was closely related to the in situ survival of cells implanted in the hostile environment of hypoxia, inflammation, and scarring from myocardial infarction. Hence, the essential survival factors for MSCs, such as cytokines, chemokines, integrins, and other adhesion molecules, need to be further addressed quantitatively.

It was recently reported that persistent overexpression of VEGF in nonischemic myocardium may cause the formation of vascular tumors [47]. The present study for the first time demonstrated the high-level expression of VEGF from Bcl-2-MSCs in response to the hypoxic conditions. Therefore, transplantation of Bcl-2-MSCs could provide adequate magnitude and duration of VEGF expression in the ischemic myocardium without formation of vascular tumors. The high-level expression of VEGF from Bcl-2-MSCs transplanted to the ischemia-damaged myocardium would provide cardioprotective effects and induce functional collateral vessels, which contribute to the salvaging of ischemic myocardium and decrease the infarct area.

However, it is still not clear whether intramyocardially transplanted MSCs function as newly differentiated cardiomyocytes and endothelial cells or serve as the paracrine cells by secretion of cardioprotective proteins. Our study and those of other groups [46, 48, 49] confirmed that grafted implanted MSCs can secrete important survival factors, including VEGF, insulin-like growth factor, hepatocyte growth factor, stromal cell-derived factor-1, and basic fibroblast growth factor. Considering the low frequency of new cardiomyocytes and endothelial cells [46, 50], this paracrine effect could be even more important for the functional recovery of the infarcted heart than the transdifferentiation potential of stem cells. Therefore, a quantitative assay of the paracrine cytoprotective factors from stem cells cocultured with cardiomyocytes subjected to hypoxia deserves significant attention. The cocktail of paracrine cytoprotective factors may bring fresh therapeutic options to a number of diseases, including heart failure.

Our current work, together with many other studies, demonstrated that bone marrow MSC implantation could induce therapeutic angiogenesis in infarcted heart [11, 51, [52]53] or ischemic tissue [54]. Our data suggested that the viability of transplanted cells is one of the important factors for preservation of myocardial function, and therapeutic angiogenesis is not linearly correlated with the survival rate of the transplanted cells. The underlying mechanism of angiogenesis is very complicated. For instance, MSCs are able to differentiate into vascular endothelial cells in the ischemic myocardium and generate capillary-like structures [11, 51, 53]. Meanwhile, MSCs enhance angiogenesis partly by increasing endogenous levels of vascular endothelial growth factor and vascular endothelial growth factor type 2 receptor [54]. Our evidence supports the theory that the paracrine effect of MSCs might be one of the major reasons for therapeutic angiogenesis. Using the genetic modification approach may further enhance the cytoprotective effect and restore MSC angiogenesis effect.

In the present study, a significant increase in nucleosomal DNA fragmentation and DNA condensation by TUNEL and Hoechst 33342 nuclear staining was observed in MSCs subjected to hypoxic conditions compared with the normoxic control cells, a finding concordant with previous data verifying that hypoxia provokes apoptosis of MSCs [18]. It is known that hypoxia can induce Bcl-2 downregulation through nuclear factor-κB (NF-κB) in endothelial cells [55] and myocytes [56]. We provide the first direct evidence that Bcl-2 upregulation from exogenous delivery activates a survival pathway that is sufficient to suppress hypoxia-induced apoptosis of MSCs. The underlying mechanism by which Bcl-2 protects MSCs under hypoxia is still unknown. We speculate that NF-κB might play a pivotal role in the cytoprotective effect of Bcl-2 on MSCs under hypoxic conditions. Further studies need to be conducted in order to support the hypothesis.

Our hemodynamic data are consistent with previous reports that demonstrated cardiac functional improvement after MSC transplantation [57, 58]. We found that Bcl-2-MSC transplantation further reduced EDV and significantly enhanced the systolic functional recovery determined by slope ESPVR and Emax. These beneficial effects of Bcl-2-MSCs may, in part, attribute to VEGF secretion in response to the hypoxic environment, angiogenesis, and the improved cellular survival after pretreatment with the antiapoptotic Bcl-2 gene.

In this investigation, MSCs were genetically modified with Bcl-2 by a cationic polymer based vector with several unique advantages, such as easy surface modification with well defined structure and chemical properties [59], relatively low toxicity, high capability for carrying large therapeutic genes, and lack of immune responses. More importantly, transient overexpression of Bcl-2 modified MSCs that have benefited from the nonviral gene delivery vector may minimize the potential risk of tumorigenesis while providing safe and sufficient protection for transplanted MSCs from short-term ischemic damage, which plays a critical role in transplanted stem cell death [24]. Further work on the long-term persistence, function, and phenotype of Bcl-2 modified MSCs treated with nonviral vectors needs to be done to safely realize the full potential of MSCs for myocardial regeneration.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

In summary, we have confirmed that genetic modification with the antiapoptotic Bcl-2 gene resulted in high-level VEGF expression in response to the hypoxic conditions. It enhanced the survival of engrafted MSCs in the heart after acute myocardial infarction. The transplantation of Bcl-2 modified MSCs ameliorated LV remodeling and improved LV function. Genetically engineering cells by Bcl-2 using a nonviral vector could be an effective strategy for increasing cell survival after cell transplantation while minimizing the potential risk of tumorigenesis. Transplantation of gene-engineered MSCs may provide a novel and effective approach in the treatment of acute myocardial infarction.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

This work was supported by Helmholtz Gemeinschaft, Mecklenburg-Vorpommern (Nachwuchsgruppe Regenerative Medizin Regulation der Stammzellmigration Forderkennzeicher 0402710), BMBF, Miltenyi Biotec, START-MSC project (project 6: Kardiovaskuläre Differenzierung und Applikation definierter mesenchymaler Stammzellpopulationen), and Steinbeis Transfer Zentrum fuer Herz-Kreislaufforschung, Rostock, Germany. Authors thank Miriam Nickel, Margit Fritsche, and Daniela Kurzhals for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information
FilenameFormatSizeDescription
sc060771suppFig1.pdf42KSupplemental Figure
sc060771suppTable1.pdf41KSupplemental Table 1
sc060771suppTable2.pdf81KSupplemental Table 2

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