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TRANSLATIONAL AND CLINICAL RESEARCH: MESENCHYMAL STEM CELLS SERIES |
Departments of aCardiology,
bMolecular Cell Biology,
cAnatomy and Embryology and
dDivision of Image Processing, Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands
Key Words. Mesenchymal stem cells • Cellular therapy • Transgene expression • NOD/scid mouse
Correspondence: Correspondence: M.J. Schalij, Ph.D., M.D., Department of Cardiology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Telephone: 31-71-526-2020; Fax: 31-71-526-6809; e-mail: M.J.Schalij{at}lumc.nl
Received on July 9, 2007;
accepted for publication on January 9, 2008.
First published online in STEM CELLS EXPRESS January 17, 2008.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Myocardin is a pivotal cardiomyogenic transcription factor that transactivates the ubiquitous transcription factor serum response factor [15]. Myocardin regulates the expression of many growth-related and muscle-restricted genes [16]. Overexpression of the longest splice variant of the human myocardin gene in human mesenchymal stem cells (hMSCs) [17] and human myocardial scar fibroblasts [18] induced the expression of cardiac and smooth muscle cell genes in vitro, without expression of skeletal muscle genes [17]. Therefore, it was hypothesized that forced myocardin expression in hMSCs may enhance their propensity to differentiate into cardiomyocytes in vivo, thereby increasing the potential therapeutic effect of mesenchymal stem cell therapy in patients with ischemic heart disease (IHD). In this study, the effects of forced myocardin expression in hMSC from IHD patients on the in vivo engraftment rate, differentiation, and preservation of LV function were evaluated in an immune-compromised mouse model of AMI.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Purification and Expansion of hMSC
Human cellular material was obtained after written informed consent, and experiments were approved by the institutional medical ethics committee. BM samples were obtained from four adult IHD patients who were enrolled in ongoing clinical stem cell trials [2]. hMSCs were purified as described previously [17], expanded by serial passage, and used from passage 3 to passage 8 in culture conditions. The hMSC surface antigen profile was characterized as previously described [17]. hMSCs abundantly expressed hyaluronate receptor (CD44), major T-cell antigen (Thy-1; CD90), endoglin (CD105), vascular cell adhesion molecule-1 (CD106), and human leukocyte class I (HLA-ABC) antigens. These cells also expressed low levels of transferrin receptor (CD71), P-selectin (CD62P), β3 integrin (CD61), neural cell adhesion molecule (CD56), and membrane cofactor protein of the complement system (CD46) at their surface. hMSCs differentiated into adipocytes and osteoblasts after proper stimulation, confirming their multipotent nature (data not shown).
Adenoviruses and Gene Transfer
The generation of the fiber-modified human adenovirus serotype 5 vectors encoding the longest splice variant of the human myocardin gene (myocL) (hAd5/F50.CMV.myocL), the enhanced green fluorescent protein (eGFP) gene (hAd5/F50.CMV.eGFP), and the control vector (hAd5/F50.empty) is described elsewhere [17]. Before transduction, hMSC were seeded at a density of 2 x 104 hMSCs per cm2 in 10-cm2 dishes and cultured overnight. Next, the culture medium (Dulbecco's modified Eagle's medium + 10% fetal bovine serum) was replaced by 1 ml of fresh culture medium per dish and supplemented with 5 mM sodium butyrate and 100 infectious units per cell of hAd5/F50.CMV.eGFP and hAd5/F50.CMV.myocL or hAd5/F50.CMV.eGFP and hAd5/F50.empty. After overnight incubation, the cells were washed with phosphate-buffered saline (PBS) and cultured for 24 hours in normal culture medium.
Myocardial Infarction and Cell Implantation
Animals were preanesthetized with 5% isoflurane in a mixture of oxygen and nitrogen. After endotracheal intubation and ventilation using a Harvard Rodent Ventilator (model 845; Harvard Apparatus, Holliston, MA, http://www.harvardapparatus.com) (200 breaths per minute, with a stroke volume of 200 µl), animals were kept anesthetized with 0.5%–1.5% isoflurane for the remainder of the surgical procedure. After a left thoracotomy, the left anterior descending (LAD) coronary artery was ligated using a 7-0 prolene suture (Johnson and Johnson, New Brunswick, NJ, http://www.jnj.com).
After 10–20 minutes post-AMI, animals were grouped to receive injections of 2 x 105 hMSCs transduced with eGFP and myocardin (hMSCmyoc) in 20 µl of culture medium (MI+hMSCmyoc group; n = 10), hMSCs transfected with eGFP and empty vector (MI+hMSC group; n = 10) or medium only (MI+medium group; n = 12). Sham-operated animals were used to determine baseline characteristics (sham group; n = 12). The intramyocardial injections were performed at five sites within infarcted area and the border zone using a 20-µl Hamilton syringe with a 33-gauge needle. The chest was closed in layers, and animals were allowed to recover.
Cardiac Magnetic Resonance Imaging
LV anatomy and function were serially assessed 2 and 14 days after surgery by magnetic resonance imaging (MRI) as previously described [19].
Infarct Size. To determine the extent of the infarcted area at day 2, contrast-enhanced MRI was used after injection of a 150-µl (0.05 mmol/ml) bolus of gadolinium (Gd)-DPTA (Dotarem; Guerbet, Gorinchem, The Netherlands, http://www.guerbet.nl) via the tail vein.
Left Ventricular Anatomy and Dimensions. At days 2 and 14, a high-resolution two-dimensional FLASH cine sequence was used to acquire a set of contiguous 1-mm slices in the short-axis orientation covering the entire long axis of the heart.
Image Analysis. The percentages of MI volume, LV end-diastolic volume (EDV), LV end-systolic volume (ESV), and ejection fraction (EF) were computed as previously described [19].
Left Ventricular Function by Pressure-Volume Loops
Instrumentation. A 1.4-F pressure-conductance catheter (SPR-719; Millar Instruments, Houston, TX, http://www.millarinstruments.com) was introduced via the right carotid artery, positioned into the LV, and connected to a Sigma-SA signal processor (CD Leycom, Zoetermeer, The Netherlands, http://www.cdleycom.com). Calibration was performed as previously described [20]. The abdomen was opened to enable temporary preload reductions by directly compressing the inferior vena cava. All data were acquired using Conduct-NT software (CD Leycom) at a sample rate of 2,000 Hz and analyzed off-line with custom-made software.
Pressure-Volume Relationships.
LV pressure-volume signals were acquired in steady state to quantify general hemodynamic conditions; heart rate, stroke volume, cardiac output, EDV, ESV, EF, end-diastolic pressure, and end-systolic pressure (ESP) were determined. Stroke work (SW) was determined as the area of the pressure-volume loop, and the maximal and minimal rates of LV pressure change, dP/dtMAX and dP/dtMIN, and isovolumic relaxation time constant 
were calculated. Load-independent indices of systolic and diastolic LV function were determined from pressure-volume relationships obtained during preload reductions. To quantify systolic function, we used the end-systolic pressure-volume relationship (ESPVR), the relationship between dP/dtMAX and EDV, and the preload recruitable stroke-work relationship (PRSWR; SW vs. EDV). The slopes of these relationships, end-systolic elastance (EES), slope of dP/dtMAX-EDV relationship, and slope of PRSWR, respectively, are sensitive measures of intrinsic systolic LV function. In addition, the positions of the pressure-volume relationships were quantified by their intercepts at ESP = 82 mm, SW = 1,059 mmHg·µl, and dP/dtMAX = 6,090, respectively. (These levels were selected retrospectively as the overall mean values of ESP, SW, and dP/dtMAX for all groups.) For diastolic function, the end-diastolic stiffness (EED) was determined from a linear fit to the end-diastolic pressure-volume points.
Histological Examination
At day 15, mice were sacrificed, and hearts and lungs were removed. The body weight and wet lung weight were measured from all animals, and lungs were then freeze-dried. The difference between wet and dry lung weight was used as a measure of pulmonary congestion.
From each group, four hearts were immersion-fixed in 4% paraformaldehyde, dehydrated in graded ethanol and xylene, and subsequently embedded in paraffin. Serial sections of 5 µm were cut along the entire long axis of the LV for immunohistochemical analysis. Sections were deparaffinated and dehydrated in xylene and graded alcohol. Antigen retrieval was performed by heating in a microwave oven (97°C) in 0.01 M citric buffer, pH 6.0, for 10 minutes. Sections were incubated overnight at room temperature with the primary antibodies diluted in PBS with 1% bovine serum albumin and 0.05% Tween.
Engraftment Rate of Transplanted hMSCs
hMSC engraftment was detected by immunostaining with a rabbit anti-green fluorescent protein (anti-GFP) antibody (A11122
[GenBank]
; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) followed by a biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). For visualization of the eGFP-specific antibody, the signal was amplified with the ABC staining kit (PK-6100; Vector Laboratories). 3,3'-Diamino-benzidine tetrahydrochloride (D5637; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) was used as substrate for horseradish peroxidase. Sections were counterstained with Mayer's hematoxylin. The number of engrafted hMSCs was determined by counting the GFP-positive cells with a magnification of x20 in every 10th serial section of the entire long axis of the heart. Only elongated cells with a length >20 µm were counted. The number of GFP-positive cells was multiplied by 10 to estimate the total number of engrafted cells in the heart. The hMSC engraftment rate was calculated by dividing this number by the number of injected hMSCs (2 x 105 cells).
Assessment of Cell Differentiation
Double immunostainings were performed with mouse monoclonal antibodies against 
-sarcomeric actin (SA; clone 5C5, A2172; Sigma-Aldrich); atrial natriuretic factor (ANF; CBL66; Chemicon, Temecula, CA, http://www.chemicon.com); anti-myosin, cardiac ventricular heavy chain /β (cMHCβ, clone F26.2D11); anti-cardiac troponin T (cTnT; AB33589; Abcam, Cambridge, MA, http://www.abcam.com); cardiac troponin I (cTnI; clone 19C7; HyTest, Turku, Finland, http://www.hytest.fi); a polyclonal rabbit anti-myosin light chain 2, atrial isoform (MLC2a, gift from S.W. Kubalek); and goat anti-myosin light chain 2, ventricular isoform (MLC2v; C-17; sc-34490; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). Primary antibodies (Abs) were visualized with appropriate secondary biotinylated Abs followed by Qdot 655 streptavidin-conjugated (Q10121MP; Invitrogen) Abs. The eGFP-labeled cells were detected by immunostaining with monoclonal rabbit anti-GFP or polyclonal goat anti-GFP (AB6673; Abcam) antibodies followed by an appropriate donkey Alexa Fluor 488 antiserum (A21206
[GenBank]
and A11055
[GenBank]
). Colocalization of eGFP and differentiation markers was examined using a Nikon eclipse E800 fluorescence microscope (Nikon, Melville, NY, http://www.nikon.com) equipped with dedicated Qdot 655- and Alexa 488-compatible filter sets.
Differentiation In Vitro
To compare the protein expression of in vitro-cultured and in vivo-injected hMSCs, a small fraction of hMSCs and hMSCsmyoc from the same cell batch that was injected in the mice was propagated ex vivo. The cells were maintained in vitro for 15 days and evaluated for cardiomyocyte-specific protein expression by immunofluorescence staining using the antibodies described above.
Analysis of Vascular Density
To determine the effect of hMSC transplantation on vascular density, vascular endothelial cells were stained with a platelet cell adhesion molecule (PECAM)-1 antibody (CD31; clone MEC13.3; BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) followed by a biotinylated goat anti-rat IgG (BD Pharmingen). Visualization was enhanced by the CSA system (K1500; Dako, Glostrup, Denmark, http://www.dako.com). 3,3'-Diamino-benzidine tetrahydrochloride was used as substrate for horseradish peroxidase. Sections were counterstained with Mayer's hematoxylin. Morphometric measurements were performed on three equidistant slices (at the midpoint between LAD ligation and the apex, between the midpoint and the LAD ligation, and between the midpoint and the apex) from four animals per group. Per slice, four areas of interest equally distributed in the infarcted anterolateral wall of the LV were photographed at a magnification of x20. Vascular density was then determined by the cumulative area of PECAM-1-stained vessel lining per total left ventricular area. Endocardial endothelium staining was excluded from the quantitative analysis. The percentage of PECAM-1 staining was measured using the Image-Pro Plus software package (Media Cybernetics, Carlsbad, CA, http://www.mediacy.com). The value was expressed as the ratio of the sum of all areas containing PECAM divided by the total area of the image. The measurements were performed by two independent examiners who were blinded to the treatment assignment.
Statistical Analysis
Numerical values are expressed as mean ± SEM. Comparisons of parameters among the sham, medium, hMSC, and hMSCmyoc groups were made using one-way analysis of variance. If the omnibus tests among groups were significantly different, post hoc tests between groups using unpaired t tests were used. A p < .05 was considered significant.
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RESULTS |
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Hemodynamic Measurements
The functional pressure-volume-derived data for all four groups are presented in Table 1. Summarized schematic pressure-volume loops (based on mean end-systolic and end-diastolic pressures and volumes) and mean end-systolic pressure-volume relationships for all four groups are shown in Figure 2E. Consistent with the MRI data, the pressure-volume loops revealed substantial cardiac dilatation and a decreased stroke volume post-MI; it is of interest that these effects were less pronounced after cell treatment, particularly in the hMSCmyoc group. As expected, MI caused a marked deterioration in LV function, demonstrated by significant differences for almost all conductance catheter-derived indices between the sham group and the MI+medium group (Table 1). In both hMSC and hMSCmyoc groups, an improvement in dP/dtMAX, SW, ESVINT, and EED was observed compared with the MI+medium group. In addition, in the hMSCmyoc group but not in the hMSC group, ESP, , –dP/dtMIN, EES, EDVINT, and the slope and intercept of the dP/dtMAX-EDV relationship were also improved. Comparison of the hMSC group with the hMSCmyoc group showed a significant difference for and ESVINT, whereas the intercepts of PRSWR and the dP/dtMAX-EDV relationship displayed a nonsignificant trend.
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hMSC Engraftment and Differentiation
Fifteen days after cell transplantation, substantial engraftment was observed in both cell transplant groups (Fig. 3A, 3B). However, quantitative assessment revealed a significantly higher engraftment rate in the hMSCmyoc group (5.8% ± 0.5%) compared with the hMSC group (4.2% ± 0.3%; p < .05) (Fig. 3C). Sections were examined to assess differentiation of hMSCs and hMSCsmyoc into a cardiomyocyte-like phenotype. None of the injected hMSCs expressed SA, MLC2a, ANF, MLC2v, cMHCβ, cTnT, or cTnI (Fig. 4, left panel). In contrast, overexpression of myocardin in hMSCs resulted in expression of sarcomeric -actin, ANF, MLC2a, MLC2v, cMHCβ, and cTnT (Fig. 4, right panel). The differentiation rate of the engrafted cells was approximately 90%–100% for the various differentiation markers. The staining pattern for these proteins, however, was diffuse, and we did not observe cross-striations in the hMSCsmyoc. Furthermore, none of the hMSCsmyoc expressed cardiac troponin I (Fig. 4, right panel).
After 15 days of in vitro culture of cells from the same cell batches that were injected in the mice, immunohistochemistry revealed that hMSCs transduced with hAd5/F50.CMV.eGFP and hAd5/F50.empty did not express SA, Mlc2a, ANF, MLC2v, cMHCβ, cTnT, or cTnI (Fig. 5A–5G). Transduction of hMSCs with both hAd5/F50.CMV.myocL and hAd5/F50.CMV.eGFP, however, resulted in the expression of all these proteins (Fig. 5H–5M), with the exception of cTnI (Fig. 5N). However, the hMSCs did not exhibit sarcomeric organization or spontaneous beating within the 15-day period of observation.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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MSCs are able to differentiate into a number of organ-specific cell types [4]. Therefore, the MSC population is potentially an attractive therapeutic cell type for patients with IHD, with reported beneficial effects on LV function in animal studies [6, 7, 21] and a initial clinical trial [22]. However, the underlying mechanism is still not fully understood, as the ability of BM-derived stem cells, including MSCs, to differentiate into cardiomyocytes has been questioned [10, 23]. This lack of cardiomyogenic differentiation potential prompted the development of techniques to stimulate cardiomyogenic differentiation [12] after in vivo transplantation. Stimulation of MSCs with 5'-azacytidine [3, 9]; with a combination of several growth factors, including fibroblast growth factor, IGF-1, and BMP-2 [13]; or with TGF-β1 [14] resulted in increased cardiomyocyte differentiation and improved functional recovery after myocardial infarction in animal models [3, 13, 14].
However, pharmacological stimulation either did not result in cardiomyogenic differentiation of all treated cells [3, 13, 14] or resulted in incomplete differentiation [3, 14]. To achieve effective cardiomyogenic differentiation, it is therefore mandatory to find a more physiological way of improving the in vivo differentiation of transplanted MSCs.
Genetic modification of stem cells can be another approach to improving the cardiomyogenic differentiation potential of these cells. Transduction of mouse MSCs with a hypoxia-regulated heme oxygenase-1 vector improved cell survival in the ischemic mouse myocardium [24]. Also, transplantation of rat MSCs overexpressing the prosurvival gene Akt-1 in a rat MI model resulted in the prevention of remodeling and improvement of cardiac function [25]. In addition, rat MSCs co-overexpressing angiopoietin, a mediator of angiogenesis, and Akt-1 restored cardiac function after permanent coronary artery occlusion [26]. However, to our knowledge, genetic modification to stimulate cardiomyogenic differentiation of MSCs has not been used.
We recently demonstrated that transduction of hMSCs and human myocardial scar fibroblasts with human adenovirus vectors expressing the longest splice variant of the human myocardin gene in vitro induced the expression of a panel of genes involved in the development of cardiomyocytes, as well as smooth muscle cells, but not skeletal muscle cells [17, 18]. Forced myocardin expression in hMSCs before transplantation in a mouse model of AMI may therefore enhance their propensity to differentiate into cardiomyocyte-like cells in vivo.
Effects of hMSC and hMSCmyoc Transplantation on Infarct Size and Left Ventricular Function
Delayed-enhancement 9.4-T MRI and LV conductance measurements in small animals are relatively new techniques allowing detailed assessment of LV anatomy and function in different pathophysiological states [27]. MRI revealed no differences in infarct size at day 2 among the different groups, and all three AMI groups showed typical changes indicative of left ventricular failure, as reflected by an increase in LV volume and a decrease in LVEF. This indicates that hMSC or hMSCmyoc transplantation had no protective effect on early LV remodeling and function. However, 14 days after AMI, progressive deterioration of LVEF was observed in the medium group only, whereas deterioration of LVEF was significantly attenuated in the hMSC and hMSCmyoc groups. Despite the preservation of LVEF, hMSC transplantation did not prevent LV remodeling at 2 weeks. In contrast, hMSCmyoc transplantation resulted in attenuation of LV remodeling, indicating an additional beneficial effect of forced myocardin expression. Pressure volume loop measurements at day 15 were consistent with the MRI data at day 14. In addition, LV systolic function parameters, such as maximum rate of LV pressure (dP/dtMAX) and total amount of external work performed by the LV (stroke work), also improved after hMSC and hMSCmyoc transplantation compared with the medium-only group. Furthermore, after hMSCmyoc and, to a lesser extent, hMSC transplantation, the ESPVR (a load-independent parameter of LV systolic function), shifted toward smaller volumes, indicating improved systolic function. Moreover, hMSCmyoc transplantation improved the other intrinsic LV function parameter, such as PRSWR and the relationship between dP/dtMAX and EDV (dP/dtMAX-EDV), effects not observed after hMSC transplantation alone. Regarding the LV diastolic function, the slope (EED) of the end-diastolic pressure-volume relationship significantly decreased after both hMSC and hMSCmyoc transplantation, indicating a decreased diastolic stiffness (i.e., improved diastolic function). Importantly, transplantation of hMSCmyoc but not transplantation of hMSC resulted in a significant increase in the peak rate of pressure decline (dP/dtMIN) and decrease of the relaxation time constant , indicating a faster isovolumic relaxation. From these results, it can be concluded that transplantation of hMSCsmyoc and, to a lesser extent, of hMSCs had beneficial effects on infarct size and systolic and diastolic LV function 15 days after myocardial infarction compared with the medium-only group. The decline in cardiac function in the MI+medium group was associated with a significant weight loss (qualifying as cardiac cachexia [28]). This weight loss was significantly attenuated after hMSCmyoc transplantation but not after hMSC transplantation. Furthermore, in the medium group, MI resulted in an increase in lung fluid, indicative of pulmonary congestion, which was not observed after hMSC and hMSCmyoc transplantation.
Possible Mechanisms of Benefit After hMSC and hMSCmyoc Transplantation
In this study, it was demonstrated that forced expression of myocardin in hMSCs confers an additional beneficial effect on LV function after injection into the acutely infarcted myocardium of NOD/scid mice compared with non-myocardin-transfected hMSCs. Although the improved preservation of LV function in the hMSCmyoc group could be caused by differentiation of hMSCmyoc into a cardiomyogenic phenotype, no fully developed cardiomyocytes with sarcomeric striations were observed, making a contractile contribution of transplanted cells unlikely. Although hMSCmyoc transplantation in the immune-compromised NOD/scid mouse model of acute MI resulted in the expression of -sarcomeric actin, ANF, atrial and ventricular myosin light chain 2 (Mlc2a and Mlc2v), cMHCβ, and cardiac troponin T, the staining patterns for muscle-specific proteins remained diffuse. This was consistent with the hMSCs that were kept in in vitro culture.
Furthermore, none of the myocardin-transduced hMSCs expressed cTnI, which is in accordance with our previous reverse transcription-polymerase chain reaction (PCR) results in myocardin-transduced hMSC, where, also, no cTnI but only conversely slow-twitch skeletal troponin I (ssTnI)-specific transcripts were found [17]. ssTnI is active not only in skeletal muscle but also in the embryonic, fetal, and neonatal myocardium [29]. These results are in line with the expression of the more atrial proteins ANF and MLC2a, as well as that of the ventricular proteins cTnT, MLC2v, and cMHCβ. In the human embryonic heart, ANF has also been demonstrated in the trabeculated part of myocardium of the ventricles [30], and MLC2a mRNA expression has been demonstrated until 15 weeks in the ventricles by in situ hybridization techniques [30, 31]. The expression of ssTnI, ANF, and MLC2a therefore suggests that myocardin expression activates an early developmental gene program but does not result in complete ventricular cardiomyocyte differentiation of hMSCs.
The observed cardiomyocyte-specific protein expression, together with the lack of cTnI expression, is in agreement with our previously published in vitro PCR data. These results indicate that in vivo, also, only the myocardin-inducible genes are expressed and that the influence of the ischemic environment did not result in complete differentiation.
The relative preservation of LV function observed in the hMSC group and the amelioration of remodeling and contractility observed in the hMSCmyoc group can be explained by paracrine effects mediated by cytokines and/or other signaling molecules secreted by engrafted hMSCs and hMSCmyoc. Nagaya et al. demonstrated that rat MSCs increased capillary density in a rat model of dilated cardiomyopathy by secretion of the angiogenic factors vascular endothelial growth factor (VEGF) and hepatocyte growth factor [8]. Furthermore, Tang et al. demonstrated that rat MSCs engrafted in ischemic myocardium secrete angiogenic factors, including stromal cell-derived factor-1 (SDF-1), VEGF, and bFGF [5]. In line with these results, we observed that hMSC and hMSCmyoc injections resulted in a higher vessel density in the infarcted area compared with medium-treated animals. As no eGFP-positive cells were found incorporated into the vascular structures, this indicates that the vessels were derived from the host tissue. However, although LV function was better in the hMSCmyoc-treated group, vessel density in the scar area was the same in both groups. This is comparable to results from Li et al. showing that pretreatment of mouse MSCs with TGF-β1 to enhance myogenic differentiation after AMI did not result in a higher microvessel density compared with nontreated MSCs [14]. Also, in vitro pretreatment of rat BMC with 5-aza did not result in an increased capillary density compared with normal BMCs in a rat model of chronic ischemia [3]. Therefore, the additional beneficial effects of hMSCmyoc transplantation cannot be fully attributed to possible paracrine angiogenic effects.
Substantial engraftment of both hMSCs and hMSCsmyoc after injection in the ischemic myocardium was observed. However, quantitative assessment of engrafted cells revealed a significantly higher engraftment rate in the hMSCmyoc group. Interestingly, Madonna et al. recently demonstrated that expression of myocardin A in adipose tissue-derived murine MSCs promoted telomerase activity by enhancing the telomere points [32]. The antiapoptotic function of telomerase could therefore have contributed to the improved survival of hMSCmyoc compared with hMSC, resulting in a more robust therapeutic effect. This is comparable to the results from Mangi et al., who demonstrated that transduction of rat MSCs with Akt, a serine-threonine kinase and powerful survival signal, protects MSCs against apoptosis and as a result prevented remodeling and preserved LV function [25]. In addition, this beneficial effect was also dose (cell number)-dependent, where a 20-fold increase in the number of transplanted cells resulted in a significant increase in LV function [25]. Interestingly, Berry et al. demonstrated by atomic force microscopy that hMSC engraftment attenuates postinfarction remodeling by softening of the border zone area, thereby improving the elastic moduli, resulting in a more compliant infarct scar [33]. Furthermore, our group recently demonstrated that myocardin transduction also leads to the functional expression of cellular components involved in electrical conduction [18]. Therefore, transplantation of forced myocardin-expressing hMSCs might result in improved electrical coupling between areas of surviving myocardium, leading to a more efficient contraction of the scarred myocardium.
Other beneficial effects of MSCs have been attributed to the secretion of substances acting on spared host cardiomyocytes to reduce apoptosis [34] or reduce scarring [33]. In addition, MSCs also secrete IGF-1, which plays an important role in myocardial muscle growth and can exert positive inotropic effects [8]. However, the exact mechanisms responsible for the observed additional beneficial effects of forced myocardin expression in injected hMSCs remain unclear at present and need to be defined in future studies.
Limitations
Despite the clinical relevance (as most patients will be IHD patients), one of the limitations of the present study is the lack of a control group with hMSCs derived from healthy subjects, as the differentiation potential of IHD-derived hMSCs may be less than the that of hMSCs of healthy subjects. Second, we used a model of acute MI with permanent ligation of the LAD coronary artery, which does not reflect contemporary medical practice, where patients with a MI undergo early reperfusion of the culprit artery. Furthermore, human adenovirus serotype 5 vectors were used; these vectors do not integrate into the genome, resulting in transient expression of eGFP and myocardin genes, thereby allowing only short-term experiments. Although the effects of forced myocardin expression were studied over a 15-day period, the stability of the observed beneficial effects should be studied over a longer period. As lentiviral vectors are more suitable for stable long-term transgene expression, these vector systems may prove to be necessary for future experiments.
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CONCLUSION |
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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