Published online before print August 13, 2007, doi: 10.1161/CIRCULATIONAHA.106.650903
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2007;116:1176-1184.)
© 2007 American Heart Association, Inc.
Molecular Cardiology |
Bone Marrow–Derived Cells Are Involved in the Pathogenesis of Cardiac Hypertrophy in Response to Pressure Overload
From the Department of Regenerative Medicine and Advanced Cardiac Therapeutics (J.E., M.S., J.F., K.H., S.Y., S.M., K.F.), Cardiology Division (J.E., J.F., K.H., S.O.), Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan, and Advanced Medical Research Institute of Fertility (N.A., Y.T., O.K.), Kato Lady’s Clinic, Tokyo, Japan.
Correspondence to Dr Keiichi Fukuda, Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan. E-mail kfukuda{at}sc.itc.keio.ac.jp
Received July 10, 2006; accepted June 15, 2007.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— Bone marrow (BM) cells possess broad differentiation potential and can form various cell lineages in response to pathophysiological cues. The present study investigated whether BM-derived cells contribute to the pathogenesis of cardiac hypertrophy, as well as the possible cellular mechanisms involved in such a role.
Methods and Results— Lethally irradiated wild-type mice were transplanted with BM cells from enhanced green fluorescent protein–transgenic mice. The chimeric mice were subjected to either prolonged hypoxia or transverse aortic constriction. BM-derived enhanced green fluorescent protein–expressing cardiomyocytes increased in number over time, emerging predominantly in the pressure-overloaded ventricular myocardium, although they constituted <0.01% of recipient cardiomyocytes. To determine whether BM-derived cardiomyocytes were derived from cell fusion or transdifferentiation at the single-cell level, lethally irradiated Cre mice were transplanted with BM cells from the double-conditional Cre reporter mouse line Z/EG. BM-derived cardiomyocytes were shown to arise from both cell fusion and transdifferentiation. Interestingly, BM-derived myofibroblasts expressing both vimentin and 
-smooth muscle actin were concentrated in the perivascular fibrotic area. These cells initially expressed MAC-1/CD14 but lost expression of these markers during the chronic phase, which suggests that they were derived from monocytes. A similar phenomenon occurred in cultured human monocytes, most of which ultimately expressed vimentin and -smooth muscle actin.
Conclusions— We found that BM-derived cells were involved in the pathogenesis of cardiac hypertrophy via the dual mechanisms of cell fusion and transdifferentiation. Moreover, the present results suggest that BM-derived monocytes accumulating in the perivascular space might play an important role in the formation of perivascular fibrosis via direct differentiation into myofibroblasts.
Key Words: hypertrophy • cell fusion • bone marrow • fibroblasts
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cardiac hypertrophy initially allows an overloaded heart to maintain normal function and cardiac output by ameliorating ventricular wall stress; however, progressive ventricular dilatation and heart failure ensue if the causative stress persists. Recent developments in molecular biology and genetics have increased our understanding of the signaling pathways that distinguish and regulate compensatory versus maladaptive features of cardiac hypertrophy.1 The cellular basis of cardiac hypertrophy involves all cells of the myocardium, and despite a variety of causes, it shares common preceding cell events with heart failure, including cardiomyocyte hypertrophy and interstitial (mainly perivascular) fibrosis. The current model to explain the rapid increase in wall volume after acute pressure overload is cardiomyocyte hypertrophy and proliferation of cardiac fibroblasts. A possible role for other noncardiac cells in cardiac hypertrophy remains unknown.
Clinical Perspective p 1184
Recent advances in stem cell biology have revealed that many types of stem cells can be induced to differentiate into cardiomyocytes.2–4 Two different sources of such cardiomyogenic stem cells have been well characterized: bone-marrow (BM)–derived mesenchymal stem cells5 and cardiac-resident stem cells.6–9 We and others have studied the potential of BM-derived cells to regenerate cardiac muscle after myocardial infarction.10,11 We showed recently that BM-derived mesenchymal stem cells were prominent in the regeneration of myocardial tissue after acute myocardial infarction using a single-cell transplantation technique. Clonally purified BM-derived mesenchymal stem cells5 mobilized in significant numbers to the peri-infarct region and differentiated into cardiomyocytes, whereas single-cell–derived hematopoietic stem cells contributed little to the cardiomyocyte regeneration. Despite these findings, it remains unclear whether BM-derived cells make any contribution to the pathogenesis of pathological hypertrophy and heart remodeling.
BM-derived stem cells show a broad differentiation potential both in vivo and in vitro, referred to as plasticity. Stem cell plasticity was initially ascribed to transdifferentiation, in which cells differentiate cell-autonomously into fully functional cells. Subsequently, several studies demonstrated that stem cells can spontaneously generate hybrid cells with differentiated cells in vitro, and therefore, cell fusion provides an alternative explanation for many cases of stem cell plasticity.12 The relative contribution of each mechanism to cardiac regeneration remains unknown.13–15
Cardiac hypertrophy is also accompanied by perivascular inflammation and reactive fibroblast proliferation. The accumulation of inflammatory hematopoietic cells surrounding intermuscular coronary vessels precedes the onset of perivascular fibrosis.16 The resulting interstitial fibrosis is detrimental for the diastolic and systolic stiffness of the myocardium and thus contributes to the transition from compensated cardiac hypertrophy to heart failure. Thus, it is crucial to understand how the inflammatory cell accumulation that occurs in response to pressure overload inevitably causes an increase in perivascular fibroblasts.
The present study examined whether BM-derived cells could be mobilized to hypertrophied myocardium and differentiated into cardiomyocytes by acute pressure overload induced via either prolonged hypoxia or transverse aortic constriction (TAC), and if so, the cellular mechanisms involved (cell fusion versus transdifferentiation). We further characterized the phenotype of BM-derived cells that infiltrated into the perivascular space and examined the role of these cells in the accompanying fibrosis. Illumination of these events and the underlying mechanisms might lead to more effective treatments of cardiac hypertrophy and heart failure.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Wild-type C57BL/6 mice were purchased from Japan CLEA (Tokyo, Japan). Enhanced green fluorescent protein (EGFP)–transgenic mice (C57BL/6 background) were a gift from Professor M. Okabe (Osaka, Japan).17 CAG-Cre mice (C57BL/6 background), which express Cre recombinase ubiquitously under the control of the chicken β-actin promoter, were gifted by Professor Jun-ichi Miyazaki (Osaka, Japan). The double-conditional Cre reporter line Z/EG mice (C57BL/6 background) were purchased from Jackson Laboratories (Bar Harbor, Me). All experimental procedures and protocols were approved by the Animal Care and Use Committee of Keio University, Japan.
BM Transplantation
BM cells were harvested from 8-week-old EGFP-transgenic mice. After irradiation with a single dose of 11.5 Gy, the unfractionated EGFP+ BM cells (1x106 cells) were injected via the tail vein into C57/Bl6 mice (n=60). To assess chimerism, peripheral blood cells were collected from the recipient mice 8 weeks after BM transplantation, and the frequency of EGFP+ cells among peripheral nucleated blood cells was determined with a fluorescence-activated cell sorter (Becton Dickinson, San Jose, Calif) after hemolysis by ammonium chloride to eliminate erythrocytes.
Determination of Transdifferentiation and Cell Fusion
To distinguish cell fusion and transdifferentiation, we used a Cre-lox recombination system and performed BM transplantation (n=30). For the present study, we used CAG-Cre mice as recipients and Z/EG mice as donors and performed BM transplantation. In Z/EG mice, the LacZ reporter gene is ubiquitously expressed under the control of a hybrid cytomegalovirus-enhancer β-actin promoter. When Cre-expressing cells fuse with Z/EG cells, Cre recombinase excises the loxP-flanked LacZ reporter/stop cassette in the Z/EG nuclei, which results in expression of EGFP in the fused cells. Hematopoietic reconstitution was confirmed by fluorescence-activated cell sorter analysis based on the frequency of LacZ+ cells in peripheral blood cells with the FluoReporter lacZ flow cytometry kit (Molecular Probes, Eugene, Ore). Chimeric mice were then subjected to either hypoxia-induced pulmonary hypertension (n=10) or TAC (n=10), and an additional 10 mice were used as controls. Hearts from these mice were serially sectioned and stained for the presence of either EGFP+ or lacZ+ cells. For this study, we used anti-EGFP antibody (Medical & Biological Laboratories, Nagoya, Japan) as a primary antibody and anti-mouse IgG antibodies conjugated with Alexa Fluor488 (Molecular Probes) as a secondary antibody, because the native fluorescence signal from EGFP that resulted from Cre-mediated recombination was not sufficient to detect it on the section. LacZ+ cells were detected by 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) staining.
Statistical Analyses
Values are presented as mean±SEM. Statistical significance was evaluated with the unpaired Student t test for comparisons between 2 mean values. Comparisons between >3 groups were performed with ANOVA. A value of P<0.05 was considered significant. All other experimental procedures are described in the online-only Data Supplement.
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.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
BM-Derived Cardiomyocytes Integrated Into Hypertrophic Right Ventricles After Induction of Pulmonary Hypertension
Accumulating evidence suggests that BM-derived cells have the potential to migrate and differentiate into a variety of cell lineages during tissue remodeling. The role of endogenous BM-derived cells in the pathogenesis of cardiac hypertrophy, however, remains elusive.
To visualize BM-derived cells in a population, we developed chimeric mice by grafting in EGFP-transgenic BM cells (EGFP-BM transplanted mice). Eight weeks after transplantation, the frequency of EGFP+ cells in the peripheral nucleated cells was determined by fluorescence-activated cell sorter analysis. We selected high-efficiency EGFP-BM transplanted mice for subsequent experiments; the mean frequency of EGFP+ cells in the peripheral nucleated cells was 98±3% (n=10; Figure 1A). Initially, the EGFP-BM transplanted mice were subjected to prolonged hypoxia to induce pulmonary hypertension. The right ventricular (RV) systolic pressure was elevated significantly over time in hypoxia-exposed mice (39.6±4.6 mm Hg at 4 weeks, 45.0±6.7 mm Hg at 8 weeks; P<0.05) compared with normoxia-exposed control mice (22.3±4.4 mm Hg; Figure 1B). In accordance with the progression of pulmonary hypertension, RV hypertrophy became evident (Figure 1D); the ratios of RV weight to the weight of the left ventricle (LV) plus the septum were increased significantly (31.4±4.6% at 4 weeks and 34.4±4.9% at 8 weeks; P<0.05) relative to control mice (23.4±2.7%; Figure 1C). Radiation did not affect RV systolic pressure or the ratio of RV weight to LV-plus-septum weight (data not shown).
|
EGFP+/-actinin+ cells, which indicate BM-derived cardiomyocytes, were observed in hypoxia-exposed mice after 4 and 8 weeks (Figure 2A). Mapping of the EGFP+/-actinin+ cells along the short-axis view demonstrated that these cells existed predominantly in the RV free wall, although a few cells were also observed in the LV free wall (Figure 2B and 2C). Using the 100 short-axis sections (each 8 µm) obtained from the midventricular level of each heart, we counted and mapped the EGFP+ cardiomyocytes. Figure 2B shows the map that resulted from a representative sample. Each mark (+) represents 1 EGFP+ cardiomyocyte, which indicates that positive cells were relatively scarce in the sections. The number of EGFP+/-actinin+ cells increased with the progression of RV hypertrophy (Figure 2D). In contrast, EGFP+/-actinin+ cells were not observed in normoxia-exposed control mice at any time point. The cells that were integrated into the myocardial wall were aligned correctly and were morphologically indistinguishable from the surrounding cardiac muscle fibers. Metaspectrometer measurements confirmed that the green fluorescence was emitted by EGFP (Figure 2E). Radiation did not mobilize EGFP+ cells to the heart before hypoxia (Figure 2F). We also assessed the expression of connexin43, which was stained in punctate between BM-derived EGFP+ and resident cardiomyocytes, indicating a functional connection between the endogenous and BM-derived cells (Figure 2G).
|
BM-Derived Cardiomyocytes Integrated Into Hypertrophic LVs After TAC
Strong accumulation of EGFP+ cardiomyocytes in hypertrophied RVs in pulmonary hypertensive mice tempted us to speculate that pressure overload might trigger the homing and differentiation of BM-derived cells into cardiomyocyte populations. To address this hypothesis, we next induced LV hypertrophy in the EGFP-BM transplanted mice by TAC. Gross morphological analysis demonstrated the success of this approach (Figure 3A). M-mode echocardiographic studies at 4 weeks after TAC revealed increased LV wall thickness (sham 0.7±0.1 mm versus TAC 1.1±0.1 mm, P<0.05), increased LV end-diastolic dimension (sham 1.4±0.2 mm versus TAC 2.4±0.2 mm, P<0.05), and reduced fractional shortening (sham 60.0±3.5% versus TAC 32.2±5.2%, P<0.05; Figure 3B and 3C). Radiation did not affect the LV wall thickness, LV end-diastolic dimension, or fractional shortening (data not shown).
|
EGFP+ cardiomyocytes were detected only when the chimeric mice were subjected to TAC (Figure 3D). EGFP+ cardiomyocytes at 4 weeks after TAC were found almost exclusively in either LV free wall or septum, and no EGFP+ cardiomyocytes were evident in RV free wall (Figure 3E and 3F). The map shown in Figure 3E was generated as for Figure 2B, with each mark (+) representing 1 EGFP+ cardiomyocyte. These were first observed at 2 weeks after TAC and increased in number with time (Figure 3G). Together, these findings indicate that the BM-derived cells mobilized and differentiated into cardiomyocytes in a "stressed" chamber–specific manner during the pathogenesis of cardiac hypertrophy.
BM-Derived Cells Differentiated Into Cardiomyocytes via Both Cell Fusion and Transdifferentiation Mechanisms
Two mechanisms have been suggested for the contribution of BM-derived cells to adult tissue regeneration: the de novo generation of tissue-specific cells from BM-derived cells (transdifferentiation)14,18 and the fusion of BM-derived cells with preexisting cells.19–21 It remains unresolved whether 1 of these mechanisms dominates over the other in their contribution to cardiac regeneration by different stems cells. To address this question in the present model, we used genetic tracing with Cre-loxP technology to discriminate cell fusion and transdifferentiation at the single-cell level. Lethally irradiated Cre mice were transplanted with BM cells from the double-conditional Z/EG Cre reporter mouse line.22,23 Cells originating from Z/EG mice constitutively express lacZ, and when Cre-expressing cells fuse with Z/EG cells, Cre recombinase excises the loxP-flanked LacZ reporter cassette in Z/EG nuclei, which results in expression of EGFP in the fused cells (Figure 4A). Chimeric rates were obtained by LacZ staining of red blood cell–lysed peripheral blood, and chimeric rates of >90% were used in the present study. Eight weeks after high levels of donor-derived lacZ+ cells were established in the circulation, these mice were subjected to hypoxia before immunohistological analysis of their hearts.
|
EGFP+ fused cardiomyocytes were detected only when the BM-reconstituted Cre mice were subjected to hypoxia-induced pulmonary hypertension (Figure 4B). Some of these cardiomyocytes contained 2 nuclei, which supports the idea of cell fusion. Next, we stained the heart for β-galactosidase to test for transdifferentiation of BM-derived cells into cardiomyocytes. LacZ+ cardiomyocytes were found in the hypertrophic RV (Figure 4C), although in lower percentages than the EGFP+ fused cardiomyocytes (Figure 4D). Interestingly, the LacZ+ cardiomyocytes tended to be smaller in diameter than preexisting cardiomyocytes (Figure 4E).
Metabolic cooperation or nanotubule formation between Cre-positive recipient cells and adjacent myocytes carrying the loxP sequences can result in translocation of Cre recombinase to the cells and green fluorescent protein expression that is independent of cell fusion.24 To exclude this possibility, we performed fluorescence in situ hybridization analysis for the Y chromosome (Y-FISH) on the immunofluorescently stained serial sections of mouse heart for (1) the male donor–female recipient combination and (2) the male donor–male recipient combination. The Y-FISH analysis for the male donor–female recipient combination revealed that EGFP+ cardiomyocytes were positive for the Y chromosome (Figure 4F), which indicates that they were derived from donor cells. However, although Y-FISH analysis of the male donor–male recipient combination (n=10, EGFP+ cardiomyocytes; data not shown) was able to detect single Y chromosomes in single EGFP+ cardiomyocytes, technical difficulties that arose from the use of thin sections of these large cells precluded detection of 2 Y chromosomes in single EGFP+ cardiomyocytes. Thus, we could neither directly show that cell fusion had occurred nor exclude the possibility that Cre recombinase had been translocated independently of cell fusion.
To investigate whether BM-derived cells reprogram to express cardiac-specific genes when they fuse with donor cardiomyocytes,25 we co-immunostained 20 EGFP+ cardiomyocytes with anti-Nkx2.5 antibodies (Figure 4G). In all of the EGFP+ cardiomyocytes, the nuclei were immunoreactive for Nkx2.5. Although not a direct indicator of reprogramming, this suggested the likelihood of some promotion or upregulation of expression of cardiac-specific genes. Together, these findings indicated that cardiomyocytes were regenerated from BM-derived cells via both cell fusion and transdifferentiation, with fusion possibly being the principal mechanism in response to pressure overload.
BM-Derived Cells Contributed to the Formation of Perivascular Fibrosis
In parallel with cardiomyocyte hypertrophy, pressure overload induces monocyte accumulation and fibrosis in the perivascular space.16 The proliferation of fibroblasts extends progressively from the perivascular space to the adjacent intermuscular interstitial space. Although monocyte infiltration shares a similar spatial and temporal distribution to the fibrosis, its role in the pathogenesis of myocardial fibrosis remains unknown. To address this issue, we first performed co-immunostaining for CD31 (an endothelial marker), smooth muscle myosin-1, and cardiac actinin. The labeled sections were used to quantify the endothelial cells, smooth muscle cells, and cardiomyocytes (Figure 5A). Interestingly, the majority of EGFP+ cells were negative for these markers, which suggests that they were other cell types. BM-derived EGFP+ cells and fibrosis surrounding the vasculature were both evident at 4 weeks after TAC (Figure 5B). Radiation did not affect the induction of perivascular fibrosis (data not shown). EGFP+ cells were clustered predominantly in the perivascular fibrotic region, showing a 12-fold higher abundance in this area than in nonfibrotic areas (Figure 5C). They decreased in proportion to the distance from the vascular wall (Figure 5D). Immunostaining revealed that these EGFP+ cells expressed -smooth muscle actin and showed a fibroblast-like elongated form (Figure 5E). Most of the EGFP+–-smooth muscle actin+ cells were negative for smooth muscle myosin-1 (Figure 5A), which indicates that they were not mature smooth muscle cells but myofibroblasts in the perivascular area. Some of these cells showed faint expression of MAC-1 and CD14 (monocyte and macrophage markers), which were detected only by use of the Tyramide Signal Amplification Kit (Perkin Elmer, Boston, Mass) (Figure 5F and 5G). Most of these did not express MAC-1 or CD14 and had developed a fibroblast-like phenotype.
|
To confirm that monocytes can develop a myofibroblast-like phenotype, primary cultures of isolated human monocytes were maintained on fibronectin-coated dishes for 14 days and examined by immunofluorescent staining (Figure 6). The isolated monocytes strongly expressed CD45 (a leukocyte marker) and CD11b (a monocyte marker; human MAC-1) after 24 hours in culture; however, at day 14, they had a flattened, fibroblast-like appearance. Expression of CD45 and CD11b was gradually decreased during the time in culture, and at 14 days, >90% of the monocytes did not express these markers. In contrast, some isolated monocytes weakly expressed vimentin and -smooth muscle actin at 24 hours, but expression of these markers was strong in most cells at day 14. To rule out the possibility of overgrowth by contaminating mesenchymal stem cell–like cells, we selected CD45-negative cells from the initial cells and performed experiments using the same protocols; however, we did not obtain any plate-attached cells from this fraction. Together, these findings showed that the monocytes developed myofibroblast-like phenotypes during long-term culture in vitro. These results indicated that in response to pressure overload, BM-derived cells, most likely monocytes, directly differentiated into myofibroblasts in the perivascular region to participate in reactive perivascular fibrosis.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
It is generally believed that cardiac hypertrophy arises from hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts, and the contribution of BM-derived cells in this process remains unknown. To address the role of BM-derived cells in the present study, we generated BM-transplanted mice from EGFP-transgenic mice and subjected them to either prolonged hypoxia or TAC to impose pressure overload on the RV or LV myocardium, respectively. Consistent with previous studies, negligible numbers of BM-derived cardiomyocytes were present under normoxia or unstressed conditions. However, on exposure to stress, BM-derived cardiomyocytes accumulated in the mouse hearts in a time-dependent manner, localized primarily in the pressure-overloaded ventricular myocardium in both the pulmonary hypertension and TAC models. Together, these findings suggested that pressure-induced stress triggers the mobilization and homing of BM-derived cells into the heart for differentiation into cardiomyocytes.
BM-derived cells contributed significantly to cardiomyocyte repopulation in the chronic pressure-overload models. Although only a restricted number of BM-derived cardiomyocytes were detected in these models, we suggest that the contribution of these cells could be much higher in situ. As we have demonstrated previously, BM mesenchymal stem cells but not hematopoietic stem cells contributed to cardiomyocyte regeneration.15 The chimeric rate of the peripheral blood was >90% in the present study, but BM mesenchymal stem cells are relatively resistant to irradiation and therefore more difficult than BM hematopoietic stem cells to exchange completely. Thus, the number of BM-derived cardiomyocytes might be underestimated. Moreover, turnover of the cardiomyocytes is considered to be a very slow process, whereas the present results were obtained in a very short time period. In future studies, long-term observations will be required. We assumed that BM-derived cells might play a compensatory role for the loss of nonrenewable, terminally differentiated cardiomyocytes. The functional significance of myocyte loss in the progression to heart failure has been well established through both pharmacological and genetic rescue experiments.26,27 Nonetheless, if cardiomyocytes could be differentiated from BM-derived precursors to compensate for the sporadic myocyte loss in chronically remodeling hearts, the progression to heart failure would be delayed or prevented.
It was recently demonstrated that cell fusion–derived cardiomyocytes appeared in the surviving myocardium remote from an infarcted region.21 Somatic nuclei also have the potential to acquire totipotency through fusion events.12,28,29 BM-derived cells have also been shown to rescue a fetal degenerative metabolic disease in mice by fusing with genetically deficient hepatocytes.30,31 Using Cre-lox recombination, we showed here that BM-derived cardiomyocytes arose from both cell fusion and transdifferentiation and that cell fusion might be the major mechanism of this phenomenon. However, these findings require cautious interpretation. It has recently been suggested that metabolic cooperation between Cre-positive cells and adjacent myocytes carrying loxP sequences can result in the translocation of Cre recombinase, with EGFP expression thereby arising independently of cell fusion.24 In addition, the formation of nanotubules between donor and recipient cells can result in the transfer of Cre recombinase, with the resulting EGFP expression mimicking a fusion event in the absence of the generation of heterokaryons. Unfortunately, we could not directly show, using the present Y-FISH experiments, that EGFP+ cardiomyocytes were a result of cell fusion. It was technically difficult to show the simultaneous presence of 2 Y chromosomes in a single EGFP+ cardiomyocyte with the use of thin sections of large cells (in contrast to the use of cultured cells). Further studies will be required to resolve this issue.
The frequency of cell fusion and transdifferentiation between BM cells and preexisting cardiomyocytes was increased by mechanical stress but was still limited. Thus, these phenomena might play a restricted physiological role in the maintenance of cardiac function. In particular, it is unclear whether new cardiomyocytes are generated in chronic hypertrophic myocardium by cell fusion. Unfortunately, we failed to detect any mitotic nuclei in the BM-derived fused cardiomyocytes in >200 hypertrophied hearts. It therefore remains unknown whether new cardiomyocytes are generated by this mechanism.
In addition to BM-derived stem cells, cardiac-resident stem cells might also contribute to cell regeneration in remodeling hearts. Resident myocardial progenitors can be identified by the expression of distinct cell surface markers, such as c-kit or Sca-1.6,7 We postulate that these cardiac stem cells also might contribute to cardiomyocyte regeneration in pressure-overloaded myocardium.
Previous work demonstrated the infiltration of inflammatory cells, such as monocytes and macrophages, into the perivascular space in response to pressure overload. This accumulation was observed predominantly in the perivascular space adjacent to intramuscular coronary arteries expressing intercellular adhesion molecule-1 and monocyte chemotactic protein-1.32,33 The inflammatory process also plays a causative role in reactive fibrosis, because blocking of either intercellular adhesion molecule-1 or monocyte chemotactic protein-1 function significantly attenuated both accumulation of inflammatory cells and subsequent fibroblast proliferation.16 However, the cellular mechanism underlying the progression of inflammatory accumulation to perivascular fibrosis remains elusive. It has been proposed that resident cardiac fibroblasts are stimulated by proinflammatory cytokines secreted from the infiltrating immune cells, which then acquire the myofibroblast phenotype and proliferate. Here, we presented new evidence for the direct differentiation of BM-derived MAC-1+/CD14+ monocytes into vimentin-positive and -smooth muscle actin–positive myofibroblasts and show that this process contributed to the perivascular fibrosis. Both cardiac-resident EGFP– and BM-derived EGFP+ myofibroblasts were detected in the perivascular space. Some but not all BM-derived myofibroblasts coexpressed MAC-1 or CD14, which suggests that the monocyte-derived myofibroblasts initially expressed both monocyte (MAC-1 and CD14) and myofibroblast (vimentin and -smooth muscle actin) cell markers, subsequently losing the monocytic character as they terminally differentiated into mature myofibroblasts. Results with the primary cultured human monocytes strongly supported this idea.
Circulating monocytes derived from hematopoietic stem cells are precursors to a variety of phagocytes, such as macrophages, dendritic cells, osteoclasts, Kupffer cells, and microglia. However, recent evidence from several groups suggested that monocytes also have the potential to differentiate into nonphagocytic cells, including endothelial-like cells and osteoblast-like cells, in vitro.34,35 Consistent with our observation in the present study, BM-derived myofibroblasts have been found in a number of organs, such as gut, kidney, skin, and liver.36,37 Finally, perivascular fibrosis can adversely affect LV systolic and diastolic function via increased tissue stiffness. Thus, the present findings suggest that any therapeutic effort to enhance the mobilization of BM cells in cases of heart failure might have both a useful and an adverse effect and would require a cautious approach. Regeneration of cardiomyocytes and vascular cells through enhanced mobilization of BM-derived cells is seemingly beneficial; however, the enhanced differentiation of BM-derived cells into myofibroblasts in the interstitial space may damage cardiac contractile function.
In conclusion, we found that BM-derived cardiomyocytes contributed to the pathogenesis of cardiac hypertrophy. Cardiomyocytes were regenerated via both cell fusion and transdifferentiation, together with perivascular fibrosis in response to the pressure overload. Cell fusion appeared to be the dominant cellular mechanism for the BM-derived cardiomyocyte regeneration. BM-derived monocytes infiltrating into the perivascular space might also play an important role in perivascular fibrosis by directly differentiating into myofibroblasts.
|
Acknowledgments |
|---|
Sources of Funding
This study was supported by the program for Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation, Osaka, Japan, and research grants from the Ministry of Education, Science and Culture, Tokyo, Japan.
Disclosures
None.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Olson EN, Schneider MD. Sizing up the heart: development redux in disease. Genes Dev. 2003; 17: 1937–1956.
[Free Full Text] - Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol. 2005; 23: 845–856.[CrossRef][Medline]
[Order article via Infotrieve]
- Rubart M, Field LJ. Cardiac regeneration: repopulating the heart. Annu Rev Physiol. 2006; 68: 29–49.[CrossRef][Medline]
[Order article via Infotrieve]
- Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J Clin Invest. 2005; 115: 572–583.[CrossRef][Medline]
[Order article via Infotrieve]
- Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, Pan J, Sano M, Takahashi T, Hori S, Abe H, Hata J, Umezawa A, Ogawa S. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest. 1999; 103: 697–705.[Medline]
[Order article via Infotrieve]
- 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]
- Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y, Pocius J, Michael LH, Behringer RR, Garry DJ, Entman ML, Schneider MD. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A. 2003; 100: 12313–12318.
[Abstract/Free Full Text] - Cai CL, Liang X, Shi Y, Chu PH, Pfaff SL, Chen J, Evans S. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell. 2003; 5: 877–889.[CrossRef][Medline]
[Order article via Infotrieve]
- Tomita Y, Matsumura K, Wakamatsu Y, Matsuzaki Y, Shibuya I, Kawaguchi H, Ieda M, Kanakubo S, Shimazaki T, Ogawa S, Osumi N, Okano H, Fukuda K. Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart. J Cell Biol. 2005; 170: 1135–1146.
[Abstract/Free Full Text] - Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature. 2001; 410: 701–705.[CrossRef][Medline]
[Order article via Infotrieve]
- Kawada H, Fujita J, Kinjo K, Matsuzaki Y, Tsuma M, Miyatake H, Muguruma Y, Tsuboi K, Itabashi Y, Ikeda Y, Ogawa S, Okano H, Hotta T, Ando K, Fukuda K. Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood. 2004; 104: 3581–3587.
[Abstract/Free Full Text] - Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature. 2002; 416: 542–545.[CrossRef][Medline]
[Order article via Infotrieve]
- Vassilopoulos G, Russell DW. Cell fusion: an alternative to stem cell plasticity and its therapeutic implications. Curr Opin Genet Dev. 2003; 13: 480–485.[CrossRef][Medline]
[Order article via Infotrieve]
- Kajstura J, Rota M, Whang B, Cascapera S, Hosoda T, Bearzi C, Nurzynska D, Kasahara H, Zias E, Bonafe M, Nadal-Ginard B, Torella D, Nascimbene A, Quaini F, Urbanek K, Leri A, Anversa P. Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fusion. Circ Res. 2005; 96: 127–137.
[Abstract/Free Full Text] - Zhang S, Wang D, Estrov Z, Raj S, Willerson JT, Yeh ET. Both cell fusion and transdifferentiation account for the transformation of human peripheral blood CD34-positive cells into cardiomyocytes in vivo. Circulation. 2004; 110: 3803–3807.[CrossRef][Medline]
[Order article via Infotrieve]
- Kai H, Kuwahara F, Tokuda K, Imaizumi T. Diastolic dysfunction in hypertensive hearts: roles of perivascular inflammation and reactive myocardial fibrosis. Hypertens Res. 2005; 28: 483–490.[CrossRef][Medline]
[Order article via Infotrieve]
- Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. "Green mice" as a source of ubiquitous green cells. FEBS Lett. 1997; 407: 313–319.[CrossRef][Medline]
[Order article via Infotrieve]
- Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001; 98: 10344–10349.
[Abstract/Free Full Text] - Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 2004; 428: 668–673.[CrossRef][Medline]
[Order article via Infotrieve]
- Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO, Rubart M, Pasumarthi KB, Virag JI, Bartelmez SH, Poppa V, Bradford G, Dowell JD, Williams DA, Field LJ. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature. 2004; 428: 664–668.[CrossRef][Medline]
[Order article via Infotrieve]
- Nygren JM, Jovinge S, Breitbach M, Sawen P, Roll W, Hescheler J, Taneera J, Fleischmann BK, Jacobsen SE. Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation. Nat Med. 2004; 10: 494–501.[CrossRef][Medline]
[Order article via Infotrieve]
- Harris RG, Herzog EL, Bruscia EM, Grove JE, Van Arnam JS, Krause DS. Lack of a fusion requirement for development of bone marrow-derived epithelia. Science. 2004; 305: 90–93.
[Abstract/Free Full Text] - Novak A, Guo C, Yang W, Nagy A, Lobe CG. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis. 2000; 28: 147–155.[CrossRef][Medline]
[Order article via Infotrieve]
- Will E, Klump H, Heffner N, Schwieger M, Schiedlmeier B, Ostertag W, Baum C, Stocking C. Unmodified Cre recombinase crosses the membrane. Nucleic Acids Res. 2002; 30: e59.
[Abstract/Free Full Text] - Lapidos KA, Chen YE, Earley JU, Heydemann A, Huber JM, Chien M, Ma A, McNally EM. Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle. J Clin Invest. 2004; 114: 1577–1585.[CrossRef][Medline]
[Order article via Infotrieve]
- Imahashi K, Schneider MD, Steenbergen C, Murphy E. Transgenic expression of Bcl-2 modulates energy metabolism, prevents cytosolic acidification during ischemia, and reduces ischemia/reperfusion injury. Circ Res. 2004; 95: 734–741.
[Abstract/Free Full Text] - Chandrashekhar Y. Role of apoptosis in ventricular remodeling. Curr Heart Fail Rep. 2005; 2: 18–22.[Medline]
[Order article via Infotrieve]
- Ying QL, Nichols J, Evans EP, Smith AG. Changing potency by spontaneous fusion. Nature. 2002; 416: 545–548.[CrossRef][Medline]
[Order article via Infotrieve]
- Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, Alvarez-Buylla A. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature. 2003; 425: 968–973.[CrossRef][Medline]
[Order article via Infotrieve]
- Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature. 2003; 422: 901–904.[CrossRef][Medline]
[Order article via Infotrieve]
- Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S, Grompe M. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature. 2003; 422: 897–901.[CrossRef][Medline]
[Order article via Infotrieve]
- Kuwahara F, Kai H, Tokuda K, Niiyama H, Tahara N, Kusaba K, Takemiya K, Jalalidin A, Koga M, Nagata T, Shibata R, Imaizumi T. Roles of intercellular adhesion molecule-1 in hypertensive cardiac remodeling. Hypertension. 2003; 41: 819–823.
[Abstract/Free Full Text] - Egashira K, Koyanagi M, Kitamoto S, Ni W, Kataoka C, Morishita R, Kaneda Y, Akiyama C, Nishida KI, Sueishi K, Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy inhibits vascular remodeling in rats: blockade of MCP-1 activity after intramuscular transfer of a mutant gene inhibits vascular remodeling induced by chronic blockade of NO synthesis. FASEB J. 2000; 14: 1974–1978.
[Abstract/Free Full Text] - Schmeisser A, Garlichs CD, Zhang H, Eskafi S, Graffy C, Ludwig J, Strasser RH, Daniel WG. Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions. Cardiovasc Res. 2001; 49: 671–680.
[Abstract/Free Full Text] - Zhao Y, Glesne D, Huberman E. A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc Natl Acad Sci U S A. 2003; 100: 2426–2431.
[Abstract/Free Full Text] - Direkze NC, Forbes SJ, Brittan M, Hunt T, Jeffery R, Preston SL, Poulsom R, Hodivala-Dilke K, Alison MR, Wright NA. Multiple organ engraftment by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrow-transplanted mice. Stem Cells. 2003; 21: 514–520.
[Abstract/Free Full Text] - Forbes SJ, Russo FP, Rey V, Burra P, Rugge M, Wright NA, Alison MR. A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis. Gastroenterology. 2004; 126: 955–963.[CrossRef][Medline]
[Order article via Infotrieve]
CLINICAL PERSPECTIVE
Bone marrow (BM)–derived cells are known to have a broad potential to differentiate into various cells in response to physiological cues. It is generally thought that cardiac hypertrophy arises from hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts, but it remains unknown whether BM-derived cells are involved in this process. We found that BM-derived cells are mobilized and differentiated into cardiomyocytes in the hypertrophied heart of both hypoxia-induced pulmonary hypertension and transverse aortic constriction models. BM-derived cardiomyocytes are observed in a "stressed" chamber–specific manner during the pathogenesis of cardiac hypertrophy. Both cell fusion and transdifferentiation mechanisms are involved in this regeneration process, with fusion possibly being the principal mechanism in response to pressure overload. Moreover, the present results suggest that BM-derived monocytes might play an important role in the formation of perivascular fibrosis via direct differentiation into myofibroblasts, although it is conventionally believed that only resident myofibroblasts contribute to perivascular fibrosis in hypertrophied heart. Our findings suggest that regeneration of cardiomyocytes through enhanced mobilization of BM-derived cells is seemingly beneficial; however, the enhanced differentiation of BM-derived cells into myofibroblasts in the interstitial space may damage cardiac contractile function. Any therapeutic effort to enhance the mobilization of BM cells in cases of heart failure might have both a useful and an adverse effect and would require a cautious approach.
|
Footnotes |
|---|
The online-only Data Supplement, consisting of an expanded Methods section, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.650903/DC1.
Related Article:
Circulation 2007 116: 1107.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||