Published online before print August 7, 2007, doi: 10.1161/CIRCULATIONAHA.106.668178
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(Circulation. 2007;116:917-927.)
© 2007 American Heart Association, Inc.
Molecular Cardiology |
Preservation of Left Ventricular Function and Attenuation of Remodeling After Transplantation of Human Epicardium-Derived Cells Into the Infarcted Mouse Heart
From the Departments of Anatomy & Embryology (E.M.W., B.H., H.L.-V., R.V.S., S.M., M.C.D., R.E.P., A.C.G.-d.G.), Cardiology (R.W.G., J.v.T., P.S., A.v.d.L., M.J.S., D.E.A.), Molecular Cell Biology (J.v.T., A.A.F.d.V.), and Radiology (R.v.d.G.), Leiden University Medical Center, Leiden, The Netherlands; and Department of Cardiology, University Medical Center Utrecht (P.A.D.), Utrecht, The Netherlands.
Correspondence to Professor Dr A.C. Gittenberger-de Groot, Leiden University Medical Center, Department of Anatomy and Embryology, Einthovenweg 20, PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail acgitten{at}lumc.nl
Received October 4, 2006; accepted June 20, 2007.
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Abstract |
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Background— Proper development of compact myocardium, coronary vessels, and Purkinje fibers depends on the presence of epicardium-derived cells (EPDCs) in embryonic myocardium. We hypothesized that adult human EPDCs might partly reactivate their embryonic program when transplanted into ischemic myocardium and improve cardiac performance after myocardial infarction.
Methods and Results— EPDCs were isolated from human adult atrial tissue. Myocardial infarction was created in immunodeficient mice, followed by intramyocardial injection of 4x105 enhanced green fluorescent protein–labeled EPDCs (2-week survival, n=22; 6-week survival, n=15) or culture medium (n=24 and n=18, respectively). Left ventricular function was assessed with a 9.4T animal MRI unit. Ejection fraction was similar between groups on day 2 but was significantly higher in the EPDC-injected group at 2 weeks (short term), as well as after long-term survival at 6 weeks. End-systolic and end-diastolic volumes were significantly smaller in the EPDC-injected group than in the medium-injected group at all ages evaluated. At 2 weeks, vascularization was significantly increased in the EPDC-treated group, as was wall thickness, a development that might be explained by augmented DNA-damage repair activity in the infarcted area. Immunohistochemical analysis showed massive engraftment of injected EPDCs at 2 weeks, with expression of 
-smooth muscle actin, von Willebrand factor, sarcoplasmic reticulum Ca2+-ATPase, and voltage-gated sodium channel (-subunit; SCN5a). EPDCs were negative for cardiomyocyte markers. At 6-weeks survival, wall thickness was still increased, but only a few EPDCs could be detected.
Conclusions— After transplantation into ischemic myocardium, adult human EPDCs preserve cardiac function and attenuate ventricular remodeling. Autologous human EPDCs are promising candidates for clinical application in infarcted hearts.
Key Words: myocardial infarction • stem cells • remodeling • magnetic resonance imaging • angiogenesis
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Introduction |
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Current therapy aimed at alleviating the sequelae of sustained myocardial infarction (MI) is not able to restore the function of the scarred area. Stem cell therapy poses a promising alternative therapy. Because therapeutic use of embryonic stem cells is an ethically intricate issue and is technically difficult in relation to the possible rejection of the cells and tumor formation, use of adult stem cells appears to be a more feasible option. Many different types of adult cells have been demonstrated to improve cardiac function after a MI, although the underlying mechanism has only been partially unraveled (for review, see Murry et al1). Most of the cell types used are not known to be of importance during normal cardiogenesis. We chose to transplant epicardium-derived cells (EPDCs) because these cells are known to be crucial for cardiac development, because of both their physical contribution2 and their modulatory role.3,4
Clinical Perspective p 927
During embryogenesis, epicardium migrates from the extracardiac proepicardium to cover the premature heart, which by that time consists of only myocardium and endocardium, with cardiac jelly in between.5 A subset of the epicardial cells undergoes epithelial-mesenchymal transformation (EMT). These cells are called EPDCs.2 EPDCs migrate into the myocardium to differentiate into interstitial cardiac fibroblasts, subendocardial and atrioventricular cushion mesenchymal cells, and coronary smooth muscle cells and adventitial fibroblasts.2,6–8 In addition to this physical contribution of EPDCs to the coronary vessels and the fibrous heart skeleton, EPDCs have a modulatory role in cardiogenesis.9,10 Formation of the compact myocardium,3,4 coronary vessel formation,11–13 and Purkinje fiber cell differentiation2,14 are dependent on EPDC regulation. If epicardial outgrowth is inhibited completely, the myocardium remains thin, and normal septation does not take place, which leads to early embryonic death.3 In addition to hampered formation of the compact myocardium, vascular development is severely disturbed in partial epicardial abrogation.11,15
The role of EPDCs during postnatal cardiac growth has never been elucidated. It is unknown whether new EPDCs are generated continuously through EMT, contributing to the growing structures of the heart and regulating developmental processes. It has been shown, however, that adult rat epicardial cells are at least still able to undergo EMT and differentiate into smooth muscle cells in vitro.16 We recently demonstrated that adult human cells derived from epicardial explants (hEPDCs) can undergo EMT spontaneously, as observed by a transformation from cobblestone into spindle-shaped morphology, while losing their ß-catenin expression.17 In the present study, we also used Wilms’ tumor 1 suppressor protein (WT1) expression to investigate this aspect. During cardiogenesis, expression of WT1 is observed in the proepicardium and the epicardial cells but is lost in the EPDCs soon after they have undergone EMT.18,19 Furthermore, it has been shown that adult epicardial cells positively modify cardiomyocyte phenotype and function20 and that WT1 is switched on de novo in adult coronary vessels in ischemic myocardium.21
Being relatively undifferentiated cells that can give rise to differentiated progeny of at least smooth muscle cells and fibroblasts, embryonic EPDCs have been referred to as stem cells.22 Given that adult hEPDCs can still undergo EMT and can give rise to the above-mentioned cell types,17 we consider adult hEPDCs as progenitor cells. We hypothesized that adult hEPDCs could recapitulate part of their embryonic program when transplanted into diseased myocardium, thereby positively modifying the surrounding myocardium.
In the present study, we investigated in a mouse model whether adult hEPDCs could improve cardiac performance after MI. Adult spindle-shaped hEPDCs were transplanted into ischemic murine myocardium. Evaluation of this population revealed that these cells showed no expression pattern indicative of endothelial cells or cardiomyocytes.17
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Methods |
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See the online Data Supplement for an expanded Methods section.
Primary Cultured hEPDCs
EPDCs were cultured from human adult epicardium, which was separated mechanically from atrial appendages. Spindle-shaped cells (see Data Supplement Figure I) from passages 2 to 4 were used for transplantation experiments after transduction with a human vector expressing the enhanced green fluorescent protein (eGFP) gene, which enabled cell tracing. An adenoviral and a lentiviral vector were used for short-term (2-week survival) and long-term (6-week survival) experiments, respectively.
Creation of the MI and Cell Transplantation
To avoid rejection of transplanted human cells, nonobese diabetic–severe combined immunodeficient (NOD/scid) mice were used.23 All animal procedures were approved by the Animal Ethics Committee of Leiden University and conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 85-23, revised 1996). For short-term experiments, the main left anterior descending coronary artery was permanently ligated. For long-term experiments, only the branch supplying the ventral wall of the left ventricle (LV) was ligated, because extended survival until 6 weeks is not possible with the entire LV infarcted. Immediately after ligation, transplantation of 4x105 hEPDCs suspended in M199 (hEPDC group; 2-week survival, n=22; 6-week survival, n=15) or cell-free M199 (medium group; 2-week survival, n=24; 6-week survival, n=18) was performed into the ischemic myocardium. Sham-operated animals (2-week survival, n=16; 6-week survival, n=3) were operated on similarly but without ligation of the main left anterior descending coronary artery and without fluid injection. Animals were randomized to treatment.
Short-Term Experiments
Magnetic Resonance Imaging
Infarct size (2 days after surgery) and cardiac function (2 and 14 days after surgery) were assessed with contrast-enhanced and cine MRI images (9.4T). Images were analyzed by manual delineation of endocardial and epicardial borders (hEPDC group, n=17; medium group, n=14; sham group, n=13) with dedicated software (see Data Supplement for detailed information). LV infarcted area, end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and ejection fraction (EF) were computed automatically.24 Infarct area measurements were used to correct functional parameters for potential differences in initial infarct size.
Immunohistochemical Assessment
Animals were euthanized 15 days after surgery. Paraffin sections of the hearts (n=5 per group) were used for immunohistochemical analysis. To investigate host tissue properties, serial sections were stained for CD31, /
-muscle actin, -smooth muscle actin, proliferating cell nuclear antigen (PCNA), phosphohistone H3, and phosphohistone H2AX. Nonfluorescent anti-eGFP staining was performed to visualize the injected hEPDCs because the strong and irregular autofluorescence of the heart disturbs assessment of spontaneous eGFP fluorescence to detect engrafted cells.
To identify which proteins were expressed by the injected hEPDCs, double stainings were performed for eGFP and other proteins, including -smooth muscle actin, von Willebrand factor (vWF), sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA2a), voltage-gated sodium channel (-subunit; SCN5a), cardiac troponin I, atrial natriuretic peptide, /-muscle actin (clone HHF35), and sarcomeric myosin (clone MF20). A red Qdot conjugated goat anti-rabbit antibody was used to visualize eGFP staining. An appropriate biotinylated secondary antibody in combination with yellow Qdot conjugated to streptavidin was used to visualize the other proteins. For details, see the Data Supplement.
LV Vascular Profile and Wall Thickness
To evaluate the angiogenic effect of hEPDC transplantation, the cumulative area of CD31-stained vessel lining per total LV area was determined in the hEPDC and medium group (n=5 for each group). LV wall thickness was measured in the hearts of the hEPDC and medium group (n=5 for each group) by an observer blinded to treatment. Wall thickness was measured in both border zones of the infarcted wall, in the mid-infarcted area between the border zones, and in the middle of the interventricular septum.
General Health and Survival
Body weight was determined just before surgery at day 0 and before euthanasia at day 15. Pulmonary water content was estimated after euthanasia by subtracting dry weight from wet weight of the lungs. To correct for differences in body mass, the amount of lung fluid was expressed relative to body weight. Survival proportions were assessed.
Long-Term Experiments
Magnetic Resonance Imaging
The effect of hEPDC transplantation in the long term was evaluated by cine MRI images 42 days after surgery only (hEPDC group, n=15; medium group, n=14; sham group, n=3) because of the high risk of mortality during repeated imaging procedures.
Immunohistochemical Assessment
Animals were euthanized 43 days after surgery. Excised hearts were treated as described for short-term experiments, except for 3 hearts in the medium and hEPDC groups that were frozen. To identify injected cells, nonfluorescent stainings were performed in paraffin sections for eGFP, human-specific CD31, and human-specific vWF and in frozen sections for human nucleus.
LV Vascular Profile and Wall Thickness
Anti-CD31 and Sirius red staining and wall thickness measurements were used for host tissue investigation.
Survival
Survival proportions were assessed.
Statistical Analysis
Data are presented as mean±SEM. Data were analyzed with SPSS software (SPSS Inc, Chicago, Ill). For comparisons of more than 2 groups, a 1-way-ANOVA was performed (or the nonparametric Kruskal-Wallis test for the dependent variable lung weight). If the omnibus tests among groups were significantly different, post hoc tests between groups with t tests (and Mann-Whitney test for lung weight) were performed. Infarct size was used as a covariate in an ANOVA/ANCOVA of the functional data to correct for baseline differences in infarct size among groups. Differences in mortality were evaluated with the Breslow test. A level of P<0.05 was considered to represent a significant difference.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Cultured Adult hEPDCs
Within a few days, hEPDCs migrated from the epicardium dissected from the atrial appendages. In the first passage, mainly cobblestone and a few spindle-shaped cells were observed, whereas after 2 passages, all cells had adopted a spindle-shaped morphology (with loss of WT1 expression; Data Supplement Figure I). Cells could easily be kept in culture, with a constant doubling time between day 10 and day 50, after which they went into senescence (Figure 1). For a more extensive characterization of in vitro cultured hEPDCs, see Van Tuyn et al.17
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Short-Term Experiments
LV Function
LV EF and SV decreased significantly after onset of MI. LV EF and SV were similar in the medium- and hEPDC-treated groups 2 days after surgery. However, at 14 days after surgery, significantly greater LV EF and SV were observed in the hEPDC-treated group than in the medium-treated group (Figure 2a and 2b). LV EDV and LV ESV were significantly smaller in the hEPDC-treated group than in the medium-treated group, both at 2 and 14 days after surgery (Figure 2c and 2d). Both medium- and hEPDC-treated mice showed heart-function parameters that differed significantly from the sham-operated animals. However, with respect to EDV on day 2 and SV on day 14, the hEPDC-treated group resembled the sham-operated group (Figure 2a through 2d).
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P<0.05 vs sham group.
LV Wall Structure
Histological evaluation showed that at day 15, transplanted hEPDCs had engrafted mainly in the ischemic LV wall and formed layers of cells (Figure 3a and 3b). Transplanted hEPDCs were not found in the lining of the coronary vessel wall (Figure 4a and 4b).
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The infarcted host tissue only showed the remaining cardiomyocytes in the subendocardial and, to a lesser extent, subepicardial position, which was not different between the hEPDC and medium groups (Figure 3c and 3d). The cardiomyocytes and the intermediate fibrous wall tissue were negative for -smooth muscle actin (Figure 3e and 3f). Nuclear PCNA expression was increased in the infarcted LV wall (Figure 3g) and the border zone (Figure 3h) of the hEPDC group compared with the medium group (Figure 3i and 3j). DNA damage was present in these areas of both the medium and hEPDC groups, as demonstrated by phosphohistone H2AX staining (not shown). Phosphohistone H3 staining revealed that the low number of mitotic figures was not different between groups (not shown).
Hearts from the hEPDC group showed a complex vascular network that consisted of capillaries, veins, and arterioles with a high vascular density throughout the ischemic area (Figure 4c) but that lacked the distribution of fine capillaries observed in healthy myocardium (Figure 4e). Medium-injected infarcted hearts were poorly vascularized, with small capillaries unequally dispersed throughout the ventricular wall (Figure 4d). When determined quantitatively, free-wall endothelial density, expressed relative to septal endothelial density, was significantly higher in all parts of the LV of the hEPDC group than in the medium group (Figure 4f and 4g). Wall thickness of the infarcted LV wall, the border zone, and the interventricular septum was significantly greater in the hEPDC-treated group than in the medium-treated group (Figure 4h).
By double-staining techniques, we were able to investigate a number of differentiation markers in the engrafted hEPDCs. Almost all hEPDCs expressed -smooth muscle actin (Figure 5a through 5c) and vWF (Figure 5d through 5f). A number of hEPDCs were positive for SERCA2a and SCN5a (Figure 5g through 5l). The transplanted cells did not express the (cardio)myocyte markers atrial natriuretic peptide, cardiac troponin I, sarcomeric myosin, or /-muscle actin (not shown).
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General Health and Survival
Only a minor decrease in body weight between the day of surgery and the day of euthanasia (day 15) was observed in the hEPDC-treated group (–0.5±0.6 g), which was not statistically different from that in the sham-operated group (0.1±0.3 g). In contrast, the decrease in body weight was significantly larger in the medium-treated group (–2.7±1.0 g) than in the sham-operated group or the hEPDC-treated group (Figure 6a). The decrease in body mass relative to the original weight amounted to 10±4% in the medium group, 2±2% in the hEPDC group, and 0±1% in the sham group. Similarly, no significant difference at day 15 in the amount of lung fluid, corrected for body weight, was observed between the sham-operated and hEPDC-treated group, whereas lung edema was present in the medium-treated group (Figure 6b).
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P<0.05 vs hEPDC group; Cumulative survival of medium-treated mice over the 2-week time period was significantly lower than that of the sham-operated mice. Survival of hEPDC-treated mice was not different from survival of sham-operated animals (Figure 7).
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Long-Term Effect of hEPDC Transplantation
The effect of hEPDC transplantation on cardiac function 6 weeks after MI was investigated in separate experiments. LV EF and SV were significantly greater 6 weeks after creation of MI in the hEPDC group than in the medium-treated group (Figure 8a and 8b). Similarly, LV EDV and ESV were significantly smaller in the hEPDC group than in the medium group (Figure 8c and 8d). Sham-operated animals showed values for these functional parameters that were all significantly different from those of the medium- and hEPDC-treated animals, except for SV (Figure 8a through 8d). No deaths were observed in the hEPDC or sham group, in contrast to 4 deaths in the medium group (Figure 8e).
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Anti-eGFP staining only demonstrated a few engrafted hEPDCs, which were not embedded in the vessel lining (Figure 8f and 8g). No cells positive for human-specific CD31 or vWF were observed in sections consecutive to the eGFP-stained sections. In the frozen sections of the hEPDC group, no cells were detected that stained positive for human nucleus (not shown).
Wall thickness of infarcted area and border zone was increased in the hEPDC group compared with the medium group (Figure 8h). Properties of the scar itself were not significantly different between hEPDC- and medium-treated animals. The remaining cardiomyocytes observed in the scar area at week 2 had disappeared. The infarcted area in both groups consisted mainly of fibrous tissue (Figure 8i), with many vessels situated in the border zone (Figure 8j) and few vessels in the scar area (Figure 8k).
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Discussion |
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The main findings of the present study are that adult human EPDCs (1) can be isolated and cultured, during which they undergo EMT; (2) engraft and survive in ischemic murine myocardium for at least 2 weeks, whereas only a few cells can be detected at 6 weeks; and (3) preserve LV function and attenuate postischemic remodeling until 6 weeks after MI. Embryonic EPDCs are known to be of crucial importance during cardiogenesis. They are essential for proper myocardial architecture and coronary vessel formation, both through their physical contribution and through regulation of these developmental processes.2,3,10 Little is known about the role of adult EPDCs in the normal and diseased adult heart. We describe for the first time the use of adult hEPDCs, cells grown from human adult epicardial explants after EMT,17 in cardiac regeneration therapy, applying them to possibly trigger embryological developmental processes that might restore or preserve cardiac function.
Immunologic Characterization of hEPDCs In Vivo
After 2 weeks, many hEPDCs were observed in the LV wall. They expressed the smooth muscle cell marker -smooth muscle actin. This is in line with the fact that embryonic EPDCs express this protein when they differentiate into coronary smooth muscle cells, in addition to interstitial and adventitial coronary fibroblasts.2 The -smooth muscle actin–positive hEPDCs were not observed in the vessel wall but as isolated cells located in the scar tissue, having a shape similar to that of the surrounding fibroblasts. Part of the engrafted hEPDCs also expressed the marker SERCA2a, which is expressed in smooth muscle cells, skeletal muscle cells, and cardiomyocytes.25 Staining for /-muscle actin (clone HHF35), normally expressed by almost all muscle cells,26 was negative in transplanted hEPDCs, which suggests that engrafted cells did not fully differentiate into a smooth muscle cell phenotype.
The injected hEPDCs did not acquire a cardiomyocyte phenotype, because the engrafted hEPDCs did not express the cardiomyocyte markers atrial natriuretic peptide, sarcomeric myosin, or cardiac troponin I. This is consistent with the finding that EPDCs do not differentiate into cardiomyocytes during embryonic heart development.2,6 Remarkably, immunostaining for SCN5a was positive in some engrafted hEPDCs. SCN5a is mainly expressed in cardiomyocytes and has recently also been described in human gastrointestinal smooth muscle.27 Therefore, we consider the expression pattern of the transplanted hEPDCs as that of a smooth muscle cell with extraordinary features, such as SCN5a expression.
Although the engrafted cells did not integrate into the vessel wall, a large portion of them were positive for the endothelial cell marker vWF at 2 weeks after MI. It is interesting that the engrafted adult hEPDCs expressed vWF, because the possible contribution of embryonic EPDCs to coronary endothelium is still a subject of debate.13,28,29 Relevant to these findings are reports describing that endothelial markers can also be expressed by nonendothelial cells, such as certain skeletal muscle cells.30
The expression profile of cultured hEPDCs in vitro is different from that of engrafted hEPDCs in vivo. Whereas engrafted hEPDCs in vivo stained positive for -smooth muscle actin, vWF, SERCA2a, and SCN5a proteins, hEPDCs in vitro contained only -smooth muscle actin mRNA, and mRNA for SCN5a and SERCA2a was not observed. Moreover, vWF staining was negative in cultured hEPDCs in vitro.17 This implies that the expression pattern of hEPDCs changes in reaction to the surrounding ischemic tissue, which results in relatively undifferentiated engrafted cells with a myoendothelial phenotype.30
The engraftment of the hEPDCs is temporary, because only a few hEPDCs could be detected at week 6. These cells did not express vWF, which indicates that the myoendothelial phenotype is at least partly transitional.
Histological Characteristics of the Surrounding Host Tissue
High vascular densities were observed in all parts of the LV wall of hEPDC-injected hearts 2 weeks after MI. The highly organized vascular network in the host tissue of the hEPDC group consisted of variably sized but mainly large-diameter vessels, whereas the medium group contained only a few vessels that were spread irregularly throughout the ischemic area. However, the high density of capillaries observed in healthy myocardium was lacking in the hEPDC group. The vessels must have been of murine origin, because no hEPDCs were observed to be integrated in the vessel lining, a finding confirmed at 6 weeks’ survival. It remains to be investigated whether more vessels had survived or new murine (host) vessels were formed after hEPDC injection. An indirect paracrine stimulatory effect of hEPDCs on vessel survival or angiogenesis is suggested, because (1) hEPDCs were not found in vessel linings, and (2) vessels were found throughout the entire LV wall of the hEPDC group, not just in areas with a high density of engrafted hEPDCs. At week 6, differences in vascular profiling had disappeared, which suggests a transitional effect of hEPDCs on the vessels.
The increased PCNA expression in the infarcted area and border zone of the hEPDC group compared with the medium group might explain in part the significantly increased wall thickness in these areas of the hEPDC group, both early and late after MI. PCNA is a central protein in both replication and DNA-damage repair.31–33 Because phosphohistone H3 staining revealed only a few mitotic figures, which was not different between groups, it is likely that the PCNA-positive cells represent cells with DNA-damage repair rather than proliferating cells. This was supported by the fact that DNA damage was indeed present in both groups. Increased cellular survival34 due to augmented repair might then have contributed to a thicker wall. Further research, however, is needed to unravel the processes that underlie the increased PCNA activity and to determine whether the PCNA-positive cells are indeed repairing DNA damage or whether they represent activated and proliferating cells as well.
On the other hand, diminished ventricular dilatation itself,35 as well as increased proliferation of cardiac stem cells36 and other host tissue cells immediately after MI (before day 14), might have affected wall thickness.34,37–39 It seems unlikely that new cardiomyocyte formation40 contributed to the increment in wall thickness in the hEPDC group, because the cardiomyocytes observed in the subendocardial and subepicardial regions of the infarcted area of the hEPDC and medium group were negative for -smooth muscle actin, which is normally expressed by primitive but not by adult cardiomyocytes.41 Moreover, whereas wall thickness at week 6 was still increased in the hEPDC-treated hearts, hardly any cardiomyocytes were observed in the ischemic area.
Functional Improvement
Main left anterior descending coronary artery occlusion results in MIs that vary in size, with associated variability in ventricular volumes.42 To discern possible treatment effects, it appears mandatory to determine the initial infarct size and to correct functional data for any differences in infarct size. We performed this step by assessing infarct size with contrast-enhanced MRI images and subsequent covariance analysis of functional parameters.43 Functional data were acquired by MRI, which is considered to be the "gold standard" for ventricular function assessment in small animals,44 creating high-resolution images, especially at 9.4T. In contrast to 1D or 2D echocardiography and conductance catheter measurements, computation of ventricular volumes from MRI images is not based on specific geometric assumptions but on real data, which makes it a reliable method for determination of infarcted, abnormally shaped hearts.
We showed that hEPDC transplantation in the acutely infarcted myocardium improved cardiac function 2 weeks after induction of MI. This improvement was represented by a higher EF, larger SV, and less lung edema in the hEPDC group than in medium-treated animals. Moreover, a smaller EDV in the hEPDC group demonstrated that ventricular remodeling was reduced by hEPDC transplantation. However, EDV was still 2- to 3-fold higher in the hEPDC group than in the sham group, which illustrates the fact that hEPDC transplantation does result in less deterioration or preservation of cardiac function, not in restoration of normal function. An early protective effect of the hEPDCs was indicated by the fact that a smaller LV EDV and ESV were observed as soon as 2 days after the onset of MI. It cannot be determined whether this early effect is responsible for the observed positive influence of hEPDC transplantation after several weeks. Studies in which hEPDCs are injected a few days after MI might reveal this contribution.
The increased survival proportions in the hEPDC group might be explained by the improved cardiac function and reduced ventricular remodeling, because LV dysfunction and mortality are highly correlated.45 Animals in the hEPDC group did not show cardiac cachexia 2 weeks after MI, defined as weight loss >7.5% of the original weight,46 which is known to be a severe complication of chronic heart failure and which is associated with a poor prognosis. Cardiac cachexia, however, was observed in the medium-treated group, which had a relative weight loss of 10%. Mice in the hEPDC group showed less lung edema than mice in the medium group, which illustrates that the absence of weight loss in the hEPDC group was not obscured by edema.
To investigate whether the beneficial effect of hEPDC transplantation on the infarcted heart remained until a definitive scar had been formed, an additional set of experiments was performed with analysis 6 weeks after MI. We demonstrated that EF and SV were still significantly higher in the hEPDC group than in the medium group. Moreover, ESV and EDV were again significantly smaller in the hEPDC-injected group, which demonstrates decreased remodeling 6 weeks after MI. The survival benefit for the hEPDC-treated group after 6 weeks confirmed the beneficial influence of hEPDC transplantation in the long term. These data suggest that the effect of hEPDCs on cardiac function is stable, although we still cannot exclude an early paracrine effect, as has been described for other stem cells.34,47
The exact mechanism underlying the improvement in cardiac function caused by hEPDC transplantation remains to be investigated. We suggest from the present data that the injected hEPDCs protect host tissue cells through augmented DNA-damage repair, which results in augmented cellular survival34 and subsequent prevention of extreme wall thinning, which will contribute to preservation of LV function and reduced functional remodeling in both the short and long term. A paracrine effect of the transplanted cells on the host tissue is indicated, because functional data demonstrate an effect of the hEPDCs as early as day 2, and because histological data show differences between groups in host tissue properties rather than newly formed donor-derived tissue in the hEPDC group.
Clinical Relevance
We showed that adult hEPDCs grow easily in culture during several passages and acquire spindle-shaped morphology, similar to embryonic EPDCs that have undergone EMT, which enables migration into the myocardium.2 Because they preserved cardiac function and reduced remodeling both early and late after the onset of MI, autologous EPDCs appear promising for use in cardiac regeneration therapy. Preferably, these cells should be injected intramyocardially, by use of the catheter-based methods described previously.48 In the present study, atrial appendages were harvested during CABG procedures. For broader clinical applications, a minimally invasive cardiac surgical technique is to be preferred, such as endoscopic surgery. Atrial appendages are removed by this technique as therapy in atrial fibrillation.49 Transplantation of autologous EPDCs will not generate ethical problems, and there will be little risk of rejection. This makes them ideal candidates for cell therapy. Spontaneous tumor formation is not an issue, because we demonstrated that adult hEPDCs do not divide indefinitely, nor did we observe any tumor formation in the ischemic mouse heart up to 6 weeks after transplantation. In a clinical setting, EPDCs can probably only be transplanted in the chronically infarcted, reperfused heart. We are currently studying this aspect.
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Acknowledgments |
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The authors are indebted to Pieter Voigt and Robert Klautz for kindly supplying the leftover surgical atrial appendages, to Jan Lens for excellent preparation of the figures, and to the animal facility.
Source of Funding
This research was funded by grant 53.345 from the Interuniversity Cardiology Institute of the Netherlands.
Disclosures
None.
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References |
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1. Murry CE, Reinecke H, Pabon LM. Regeneration gaps: observations on stem cells and cardiac repair. J Am Coll Cardiol. 2006; 47: 1777–1785.
2. Gittenberger-de Groot AC, Vrancken Peeters MP, Mentink MM, Gourdie RG, Poelmann RE. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ Res. 1998; 82: 1043–1052.
3. Gittenberger-de Groot AC, Vrancken Peeters MP, Bergwerff M, Mentink MM, Poelmann RE. Epicardial outgrowth inhibition leads to compensatory mesothelial outflow tract collar and abnormal cardiac septation and coronary formation. Circ Res. 2000; 87: 969–971.
4. Kang JO, Sucov HM. Convergent proliferative response and divergent morphogenic pathways induced by epicardial and endocardial signaling in fetal heart development. Mech Dev. 2005; 122: 57–65.[CrossRef][Medline] [Order article via Infotrieve]
5. Viragh S, Gittenberger-de Groot AC, Poelmann RE, Kalman F. Early development of quail heart epicardium and associated vascular and glandular structures. Anat Embryol (Berl). 1993; 188: 381–393.[Medline] [Order article via Infotrieve]
6. Männer J. Does the subepicardial mesenchyme contribute myocardioblasts to the myocardium of the chick embryo heart? A quail-chick chimera study tracing the fate of the epicardial primordium. Anat Rec. 1999; 255: 212–226.[CrossRef][Medline] [Order article via Infotrieve]
7. Vrancken Peeters MP, Gittenberger-de Groot AC, Mentink MM, Poelmann RE. Smooth muscle cells and fibroblasts of the coronary arteries derive from epithelial-mesenchymal transformation of the epicardium. Anat Embryol (Berl). 1999; 199: 367–378.[CrossRef][Medline] [Order article via Infotrieve]
8. Dettman RW, Denetclaw WJ, Ordahl CP, Bristow J. Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev Biol. 1998; 193: 169–181.[CrossRef][Medline] [Order article via Infotrieve]
9. Männer J, Perez-Pomares JM, Macias D, Munoz-Chapuli R. The origin, formation and developmental significance of the epicardium: a review. Cells Tissues Organs. 2001; 169: 89–103.[CrossRef][Medline] [Order article via Infotrieve]
10. Winter EM, Gittenberger-de Groot AC. Cardiovascular development: towards biomedical applicability: epicardium-derived cells in cardiogenesis and cardiac regeneration. Cell Mol Life Sci. 2007; 64: 692–703.[CrossRef][Medline] [Order article via Infotrieve]
11. Eralp I, Lie-Venema H, DeRuiter MC, Van Den Akker NM, Bogers AJ, Mentink MM, Poelmann RE, Gittenberger-de Groot AC. Coronary artery and orifice development is associated with proper timing of epicardial outgrowth and correlated Fas-ligand-associated apoptosis patterns. Circ Res. 2005; 96: 526–534.
12. Perez-Pomares JM, Phelps A, Munoz-Chapuli R, Wessels A. The contribution of the proepicardium to avian cardiovascular development. Int J Dev Biol. 2001; 45: S155–S156.
13. Guadix JA, Carmona R, Munoz-Chapuli R, Perez-Pomares JM. In vivo and in vitro analysis of the vasculogenic potential of avian proepicardial and epicardial cells. Dev Dyn. 2006; 235: 1014–1026.[CrossRef][Medline] [Order article via Infotrieve]
14. Eralp I, Lie-Venema H, Bax NA, Wijffels MC, van der LA, DeRuiter MC, Bogers AJ, Van Den Akker NM, Gourdie RG, Schalij MJ, Poelmann RE, Gittenberger-de Groot AC. Epicardium-derived cells are important for correct development of the Purkinje fibers in the avian heart. Anat Rec A Discov Mol Cell Evol Biol. 2006; 288: 1272–1280.[Medline] [Order article via Infotrieve]
15. Lie-Venema H, Gittenberger-de Groot AC, van Empel LJ, Boot MJ, Kerkdijk H, de Kant E, DeRuiter MC. Ets-1 and Ets-2 transcription factors are essential for normal coronary and myocardial development in chicken embryos. Circ Res. 2003; 92: 749–756.
16. Wada AM, Smith TK, Osler ME, Reese DE, Bader DM. Epicardial/mesothelial cell line retains vasculogenic potential of embryonic epicardium. Circ Res. 2003; 92: 525–531.
17. van Tuyn J, Atsma DE, Winter EM, van der Velde-van Dijke I, Pijnappels DA, Bax NAM, Knaan-Shanzer S, Gittenberger-de Groot AC, Poelmann RE, van der Laarse A, van der Wall EE, Schalij MJ, de Vries AAF. Epicardial cells of human adults can undergo an epithelial-to-mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells. 2007; 25: 271–278.[CrossRef][Medline] [Order article via Infotrieve]
18. Moore AW, McInnes L, Kreidberg J, Hastie ND, Schedl A. YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. Development. 1999; 126: 1845–1857.[Abstract]
19. Perez-Pomares JM, Phelps A, Sedmerova M, Carmona R, Gonzalez-Iriarte M, Munoz-Chapuli R, Wessels A. Experimental studies on the spatiotemporal expression of WT1 and RALDH2 in the embryonic avian heart: a model for the regulation of myocardial and valvuloseptal development by epicardially derived cells (EPDCs). Dev Biol. 2002; 247: 307–326.[CrossRef][Medline] [Order article via Infotrieve]
20. Eid H, Larson DM, Springhorn JP, Attawia MA, Nayak RC, Smith TW, Kelly RA. Role of epicardial mesothelial cells in the modification of phenotype and function of adult rat ventricular myocytes in primary coculture. Circ Res. 1992; 71: 40–50.
21. Wagner KD, Wagner N, Bondke A, Nafz B, Flemming B, Theres H, Scholz H. The Wilms’ tumor suppressor Wt1 is expressed in the coronary vasculature after myocardial infarction. FASEB J. 2002; 16: 1117–1119.
22. Wessels A, Perez-Pomares JM. The epicardium and epicardially derived cells (EPDCs) as cardiac stem cells. Anat Rec A Discov Mol Cell Evol Biol. 2004; 276: 43–57.[CrossRef][Medline] [Order article via Infotrieve]
23. Meyerrose TE, Herrbrich P, Hess DA, Nolta JA. Immune-deficient mouse models for analysis of human stem cells. Biotechniques. 2003; 35: 1262–1272.[Medline] [Order article via Infotrieve]
24. van der Geest RJ, Buller VG, Jansen E, Lamb HJ, Baur LH, van der Wall EE, de Roos A, Reiber JH. Comparison between manual and semiautomated analysis of left ventricular volume parameters from short-axis MR images. J Comput Assist Tomogr. 1997; 21: 756–765.[CrossRef][Medline] [Order article via Infotrieve]
25. Le Jemtel TH, Lambert F, Levitsky DO, Clergue M, Anger M, Gabbiani G, Lompre AM. Age-related changes in sarcoplasmic reticulum Ca2+-ATPase and alpha-smooth muscle actin gene expression in aortas of normotensive and spontaneously hypertensive rats. Circ Res. 1993; 72: 341–348.
26. Tsukada T, Tippens D, Gordon D, Ross R, Gown AM. HHF35, a muscle-actin-specific monoclonal antibody, I: immunocytochemical and biochemical characterization. Am J Pathol. 1987; 126: 51–60.[Abstract]
27. Ou Y, Gibbons SJ, Miller SM, Strege PR, Rich A, Distad MA, Ackerman MJ, Rae JL, Szurszewski JH, Farrugia G. SCN5A is expressed in human jejunal circular smooth muscle cells. Neurogastroenterol Motil. 2002; 14: 477–486.[CrossRef][Medline] [Order article via Infotrieve]
28. Poelmann RE, Gittenberger-de Groot AC, Mentink MM, Bokenkamp R, Hogers B. Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras. Circ Res. 1993; 73: 559–568.
29. Merki E, Zamora M, Raya A, Kawakami Y, Wang J, Zhang X, Burch J, Kubalak SW, Kaliman P, Belmonte JC, Chien KR, Ruiz-Lozano P. Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation. Proc Natl Acad Sci U S A. 2005; 102: 18455–18460.
30. Tavian M, Zheng B, Oberlin E, Crisan M, Sun B, Huard J, Peault B. The vascular wall as a source of stem cells. Ann N Y Acad Sci. 2005; 1044: 41–50.[CrossRef][Medline] [Order article via Infotrieve]
31. Jonsson ZO, Hubscher U. Proliferating cell nuclear antigen: more than a clamp for DNA polymerases. Bioessays. 1997; 19: 967–975.[CrossRef][Medline] [Order article via Infotrieve]
32. Holmes AM, Haber JE. Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases. Cell. 1999; 96: 415–424.[CrossRef][Medline] [Order article via Infotrieve]
33. Essers J, Theil AF, Baldeyron C, van Cappellen WA, Houtsmuller AB, Kanaar R, Vermeulen W. Nuclear dynamics of PCNA in DNA replication and repair. Mol Cell Biol. 2005; 25: 9350–9359.
34. Uemura R, Xu M, Ahmad N, Ashraf M. Bone marrow stem cells prevent left ventricular remodeling of ischemic heart through paracrine signaling. Circ Res. 2006; 98: 1414–1421.
35. Gerdes AM, Capasso JM. Structural remodeling and mechanical dysfunction of cardiac myocytes in heart failure. J Mol Cell Cardiol. 1995; 27: 849–856.[CrossRef][Medline] [Order article via Infotrieve]
36. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003; 114: 763–776.[CrossRef][Medline] [Order article via Infotrieve]
37. Fazel S, Cimini M, Chen L, Li S, Angoulvant D, Fedak P, Verma S, Weisel RD, Keating A, Li RK. Cardioprotective c-kit+ cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines. J Clin Invest. 2006; 116: 1865–1877.[CrossRef][Medline] [Order article via Infotrieve]
38. Yoon YS, Wecker A, Heyd L, Park JS, Tkebuchava T, Kusano K, Hanley A, Scadova H, Qin G, Cha DH, Johnson KL, Aikawa R, Asahara T, Losordo DW. Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction. J Clin Invest. 2005; 115: 326–338.[CrossRef][Medline] [Order article via Infotrieve]
39. Messina E, De Angelis L, Frati G, Morrone S, Chimenti S, Fiordaliso F, Salio M, Battaglia M, Latronico MV, Coletta M, Vivarelli E, Frati L, Cossu G, Giacomello A. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res. 2004; 95: 911–921.
40. Beltrami AP, Urbanek K, Kajstura J, Yan SM, Finato N, Bussani R, Nadal-Ginard B, Silvestri F, Leri A, Beltrami CA, Anversa P. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 2001; 344: 1750–1757.
41. Leor J, Patterson M, Quinones MJ, Kedes LH, Kloner RA. Transplantation of fetal myocardial tissue into the infarcted myocardium of rat: a potential method for repair of infarcted myocardium? Circulation. 1996; 94 (suppl II): II-332–II-336.[Medline] [Order article via Infotrieve]
42. Degabriele NM, Griesenbach U, Sato K, Post MJ, Zhu J, Williams J, Jeffery PK, Geddes DM, Alton EW. Critical appraisal of the mouse model of myocardial infarction. Exp Physiol. 2004; 89: 497–505.
43. Yang Z, Berr SS, Gilson WD, Toufektsian MC, French BA. Simultaneous evaluation of infarct size and cardiac function in intact mice by contrast-enhanced cardiac magnetic resonance imaging reveals contractile dysfunction in noninfarcted regions early after myocardial infarction. Circulation. 2004; 109: 1161–1167.
44. Jacoby C, Molojavyi A, Flogel U, Merx MW, Ding Z, Schrader J. Direct comparison of magnetic resonance imaging and conductance microcatheter in the evaluation of left ventricular function in mice. Basic Res Cardiol. 2006; 101: 87–95.[CrossRef][Medline] [Order article via Infotrieve]
45. Gottlieb SH, Ouyang P, Gottlieb SO. Death after acute myocardial infarction: interrelation between left ventricular dysfunction, arrhythmias and ischemia. Am J Cardiol. 1988; 61: 7B–12B.[Medline] [Order article via Infotrieve]
46. Anker SD, Sharma R. The syndrome of cardiac cachexia. Int J Cardiol. 2002; 85: 51–66.[CrossRef][Medline] [Order article via Infotrieve]
47. Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, Mu H, Melo LG, Pratt RE, Ingwall JS, Dzau VJ. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J. 2006; 20: 661–669.
48. Beeres SL, Bax JJ, Kaandorp TA, Zeppenfeld K, Lamb HJ, Dibbets-Schneider P, Stokkel MP, Fibbe WE, de Roos A, van der Wall EE, Schalij MJ, Atsma DE. Usefulness of intramyocardial injection of autologous bone marrow-derived mononuclear cells in patients with severe angina pectoris and stress-induced myocardial ischemia. Am J Cardiol. 2006; 97: 1326–1331.[CrossRef][Medline] [Order article via Infotrieve]
49. Mack MJ. Minimally invasive cardiac surgery. Surg Endosc. 2006; 20 (suppl 2): S488–S492.[CrossRef][Medline] [Order article via Infotrieve]
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The online-only Data Supplement, consisting of Methods and a figure, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.668178/DC1.
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