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Cardiovascular Research 2006 71(4):661-671; doi:10.1016/j.cardiores.2006.06.013
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Copyright © 2006, European Society of Cardiology

Bone marrow-derived cells contribute to infarct remodelling

Helge Möllmanna,*,1, Holger M. Nefa,1, Sawa Kostinb,1, Christof von Kallec, Ingo Pilzc, Michael Webera, Jutta Schaperb, Christian W. Hamma and Albrecht Elsässera

aKerckhoff Heart Center, Benekestrasse 2-8, 61231 Bad Nauheim, Germany
bMax-Planck-Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany
cUniversity Hospital, 79106 Freiburg, Germany

* Corresponding author. Tel.: +49 6032 996 0; fax: +49 6032 996 2819. Email address: h.moellmann{at}kerckhoff.mpg.de

Received 2 January 2006; revised 31 May 2006; accepted 9 June 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 
Objective The paradigm that cardiac myocytes are non-proliferating and terminally differentiated cells has recently been challenged by several studies reporting the ability of bone marrow-derived cells (BMC) to transdifferentiate into cardiomyocytes. However, these results are controversial and could not be reproduced by others. Therefore, we studied the contribution and potential transdifferentiation of BMC into different cell types during the remodelling process in mouse hearts with experimental myocardial infarction.

Methods Mice (C57BL/6J) were sublethally irradiated, and BM from enhanced green fluorescent protein (eGFP)-transgenic mice was transplanted. Coronary artery ligation was performed 3 months later. The hearts were studied 7 days (n=13) and 21 days (n=12) after infarction. Immunohistochemical staining was performed using antibodies against titin, connexin 43, vimentin, SMemb {alpha}-smooth muscle actin, CD45, CD34, F4/80, BS-1, CD31, and eGFP. Sections were analyzed using fluorescence and confocal laser microscopy.

Results Success of BM transplantation was confirmed by FACS analysis. Occlusion of the coronary artery resulted in infarct sizes of 41±6% of the left ventricle. CD45+/eGFP+ inflammatory cells were found frequently after 7 days and to a lesser degree after 21 days. In 25 examined hearts, only 3 eGFP-positive cardiomyocytes were found. However, numerous BMC-derived fibroblasts and myofibroblasts were found in the infarct area. BMC contributed to scar tissue neoangiogenesis but not to angiogenesis in the periinfarct and remote zones.

Conclusion Transdifferentiation of BMC into viable cardiomyocytes is a negligible event in normal repair processes after myocardial damage. BMC-derived fibroblasts and myofibroblasts as well as neoangiogenesis significantly contribute to post-infarction scar formation and might be important in scar tissue remodelling.

KEYWORDS Cell differentiation; Infarction; Myocytes; Remodelling; Stem cells

This article is referred to in the Editorial by I.M.C. Dixon (pages 609–611) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 References
 
Until recently, the mammalian heart was believed to be a terminally differentiated and postmitotic organ. Furthermore, it had been well established that the total number of cardiomyocytes slowly decreases during life given the lack of regenerative capacity [1,2]. Myocardial injury secondary to the sudden occlusion of a coronary artery would thus lead to an irrecoverable loss of cardiomyocytes and likewise to a loss of contractile performance. It was believed that irreversibly damaged cardiomyocytes were invariably replaced by scar tissue causing further deterioration of cardiac function [3].

However, this dogma has recently been challenged by different reports suggesting the existence of cycling ventricular myocytes in the normal and pathologic adult mammalian heart of several species including humans. In male patients undergoing cardiac transplantation and receiving a heart from a female donor, cardiomyocytes with a Y chromosome were found to a remarkable extent. This observation indicated cardiac chimerism, most probably caused by migration of undifferentiated cells from the recipient to the donor heart since stem cell factors like sca-1, c-kit, or MDR1 were positive in these cells [4]. Another report showed evidence for BM-derived cells to be part of a potential regenerative process upon cardiac injury [5]. Donor-derived cardiomyocytes were found in a mouse model of myocardial injury with a transplanted hematopoietic stem cell pool. These cells were detected primarily in the periinfarct region at a prevalence of 0.02%.

These observations prompted a number of studies in which the potential ability of bone marrow-derived cells to transdifferentiate into cardiomyocytes was used as therapeutic approach for myocardial damage. Orlic et al. injected lineage-negative (lin–), c-kit+ BMC in the contracting ventricular wall bordering on previously induced myocardial infarctions in mice and found a surprisingly high percentage of cardiomyocytes forming new myocardium in the infarct area [6]. In another study the same group could show that infarct size and cavitary dilation can be diminished and myocardial function improved when BMC are mobilized after myocardial infarction. They concluded that cytokine administration and the consequent mobilization and transdifferentiation of BMC lead to significant myocardial repair [7].

However, Murry et al. and Balsam et al. called these observations in doubt when they could not reproduce transdifferentiation of hematopoietic stem cells into cardiomyocytes on a nameable and constant basis [8,9]. Correspondingly, Nygren et al. found only transient engraftment of BM-derived cells in infarcted myocardium after their mobilization or after direct transplantation into the myocardium. Additionally, these BM-derived cells rather maintained their hematopoietic nature instead of transdifferentiating into myocytes [10]. The role of BM-derived stem cells also caused lively discussions in the clinical setting where several trials study cell-based repair after myocardial infarction [11–14]. All but one of these studies [15] reported improved myocardial function when BM-derived cells had been administered.

However, although BMC cause much excitement in both, basic and clinical research, the pathophysiological importance of these cells in repair mechanisms of myocardial damage is widely unknown until now. Therefore, we studied the contribution and potential transdifferentiation of BMC into different cell types during the healing and remodelling process in mouse hearts with experimental myocardial infarction. We used a mouse model in which the original BM was replaced by an eGFP-marked stem cell pool. These labelled cells offer the possibility to clearly identify fate and behaviour of potentially transdifferentiated offsprings of BMC.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 References
 
2.1. Bone marrow transplantation and transgenic mice
Eleven to 14 week old C57BL/6J wild type recipient mice (n=49) were sub-lethally irradiated with a dose of 11 Gy from a cobalt source. On the same day, 3–5 x 106 BM donor cells from transgenic littermates were transplanted by tail vein injection in a volume of 0.1 ml. We used the following transgenic mice as donors: The C57BL/6-TgN(ACTbEGFP)1Osb transgenic mouse line was obtained from Jackson Laboratory, Bar Harbor, Maine, USA. In this transgenic line, with an enhanced GFP cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer (cac/eGFP), all of the tissues, with the exception of erythrocytes and hair follicle cells, appear green under excitation light [16]. The BM was harvested by flushing tibias and femurs of 8- to 12-week old mice with RPMI 1640 containing 1% fetal calf serum, 100 U/ml penicillin, and 1000 U/ml streptomycin. The marrow was mechanically dissociated and filtered through a 40-µm nylon mesh to gain a single cell suspension. The cells were counted after lysis of erythrocytes. About 5 x 107 mononuclear cells were harvested from each donor.

Success of bone marrow transplantation (BMTx) was monitored by flow cytometry analysis (FACS Calibur, BD Biosciences, Heidelberg, Germany) of peripheral blood of the transplanted mice using a panel of monoclonal antibodies against CD3, CD4, CD8, CD11b, CD19, and F4/80, respectively. Additionally, FACS-analysis was used to confirm the presence of circulating CD34+sca-1+lin– progenitor cells in the blood of transplanted mice. In order to evaluate whether transplanted animals show an appropriate pool of stem and progenitor cells in BM which respond adequately to physiological stimuli, we performed colony-forming assays (CFU assay) according to the manufacturer's instructions.

2.2. Mouse model of myocardial infarction
Mice were subjected to coronary artery occlusion (n=41) or sham operation (n=6) 12 weeks after BMTx. Mice were randomized in two groups of 7 and 21 days survival after myocardial infarction, respectively. All surgical procedures were performed as described by Tarnavsky [17]. Briefly, the recipient mice were anesthetized by injection of a mixture of ketamine (100 mg/kg body weight) and xylazine (6 mg/kg body weight) intraperitoneally. Mice were endotracheally intubated and ventilated with a rodent ventilator (Harvard Apparatus). A thoracotomy was performed at the fourth intercostal space. All muscles overlying the intercostal space were dissected free and retracted with 5-0 silk threads; only the intercostal muscles were transected. A 7-0 ethilon ligature was placed around the left anterior descending artery just below the atrioventricular border. Ischemia was evident from discoloration of the ventricle. The lung was reinflated and the muscle and skin layers were closed separately. The animals were weaned from the respirator and extubated. Sham-operated animals were subjected to similar surgery, except that the ligature was not tied closely.

2.3. Perfusion fixation and tissue sampling
At days 7 and 21 after induction of myocardial infarction the mice were euthanized by cervical dislocation. The thoracic aorta was cannulated and the hearts were retrogradely perfused at a mean pressure of 100 mmHg with PBS buffer containing 0.1% adenosine (Fluka, Steinheim, Germany) and 0.05% bovine serum albumin (Sigma) for 3 min followed by fixative (3% buffered paraformaldehyde) for 4 min. Afterwards, the hearts were quickly removed. Tissue was cryoprotected in Tissue-Tek O.C.T Compound (Sakura) and stored at – 80 °C until sectioning.

2.4. Calculation of infarct size
Infarct size was evaluated using magnetic resonance imaging. Six mice were examined 7 days and 21 days after myocardial infarction, respectively. The mice were first sedated in an induction chamber by exposure to isoflurane. Anaesthesia was maintained with inhalational isoflurane. Mice were placed in a supine position and an electrocardiogram was continually registered. In vivo magnetic resonance studies were performed on a 7.05 T horizontal bore MR scanner (BRUKER, Germany) which was equipped with a 60 mm microscopy gradient system capable of 870 mT/m gradient strength and a rise time of 280 µs at complete gradient switching. For transmission and reception of the signal, a birdcage probe head with an inner diameter of 35 mm was used. MRI measurements were conducted by applying an ECG-triggered fast gradient echo cine sequence (FLASH) with the following imaging parameters: echo time, 1.6 ms; repetition time 4.3 ms; field of view, 3.0 cm2; acquisition matrix, 256 x 256; maximal in-plane resolution, 117 µm2; and slice thickness, 1.0 mm. Total ventricular volumes were calculated applying Simpson's rule [18] and infarct size was determined as the transmural infarcted portion in relation to the whole left ventricular circumference.

2.5. Histological analysis
Serial cryosections of the whole heart were cut, starting from the apex to the base (two sections every 100 µm). Slices of the whole myocardium including infarct, periinfarct and remote areas were used. Immunostaining was performed on 5-µm cryosections from myocardium, spleen, and lung of the transplanted animals. To assess incorporation of BM-derived cells into the myocardium, we ascertained the occurrence of eGFP-positive cells in all cryosections. Sections of spleen served as positive control.

In order to exclude autofluorescent effects, an anti-eGFP antibody (all antibodies are listed in Table 1) was used additionally.


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  		Table 1 List of antibodies

 
All sections were incubated for 1 h at room temperature. Incubation with the first antibody was followed by treatment with biotinylated second antibody when non-directly labelled antibodies were used. The directly labelled antibodies were conjugated to Cy3. The last incubation was carried out with fluoroisothiocyanate-linked streptavidin-Cy2 (FITC, Rockland, USA). Nuclei were stained with DRAQ-5 (Alexis). Pictures were taken with a Leica TCS SP laser scanning confocal microscope (Leica, Germany) equipped with appropriate filter blocks using a Silicon Graphics Octane workstation (Silicon Graphics, USA) and three-dimensional multichannel image processing software (Bitplane, Germany).

The number of fibroblasts was determined by immunohistochemical labelling of vimentin, the number of myofibroblasts by immunohistochemical labelling of vimentin, {alpha}-smooth muscle actin and additionally with an antibody against the embryonic form of smooth muscle myosin heavy chain (SMemb), respectively. From each heart 10 different fields of vision were randomly chosen in the infarct area and the periinfarct area and cells were counted.

HE staining was performed according to the standard protocol. Trichrome staining (Masson) (Sigma Aldrich, Steinheim, Germany) was carried out according to the manufacturer's instruction.

2.6. Statistics
Data are reported as mean±SD. All statistical analyses were performed using the GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego California USA. Statistical analyses were performed with Student's t test for unpaired samples. Cell counts were analyzed using ANOVA testing. Differences were considered to be statistically significant when p<0.05.

All investigations conform with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1996) and were approved by the appropriate authorities (Regierungspräsidium Darmstadt, Hessen, Germany).


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 References
 
3.1. Bone marrow transplantation
All but two mice survived the sub-lethal irradiation and the subsequent BMTx. Mice recovered quickly, although a reduced body weight was obvious in comparison to non-irradiated littermates (25±4 g versus 32±6 g, p<0.05).

The efficiency of BMTx into the sub-lethally irradiated mice was analyzed using FACS analysis of the peripheral blood at different time points after transplantation. Fluorescence intensity showed that 79–88% of all nucleated cells expressed eGFP 6 weeks after BMTx and 85%–92% after 12 weeks, indicating successful replacement of nearly the entire original stem-cell population (Fig. 1A). Furthermore, the proportion of leukocyte subpopulations was compared between transplanted and nontransplanted mice using flow cytometry. Significant differences between these groups were absent, asserting that the blood cell counts were within the physiological range at the time of surgery (data not shown).


Figure 1
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  		Fig. 1 A: FACS analysis of the peripheral blood of transplanted mice with 87.6% eGFP positive cells. B: Cryosection of the spleen showing the high prevalence of eGFP positive cells. C: Immunohistochemical analysis of the spleen. Nuclei blue (DRAQ-5), pan-leukocyte marker CD45 red.

 
Additionally, the success of BMTx was evaluated histologically. Fig. 1B shows a high percentage of eGFP-positive cells in the spleen. These cells were identified as leukocytes by their positive staining with the pan-leukocyte marker CD45 (Fig. 1C).

3.2. Surgery and infarct size
Survival rate after coronary artery ligation was 66% after 7 d and 61% after 21 d, respectively. Therefore, all together 13 infarcted hearts could be analyzed after seven days and 12 infarcted hearts after 21 d. All sham animals survived the scheduled interval.

Infarct size was evaluated using magnetic resonance imaging (Fig. 2A, 21 d after myocardial infarction). Ligation of the LAD resulted in an infarct size of 43±6% of the left ventricular wall after 7 d and 41±6% after 21 d (p=n.s. between 7 d and 21 d). Left ventricular enddiastolic volume showed a non-significant increase after 7 d and a significant increase after 21 d, which can be attributed to an aneurysm of the left ventricular apex. Ejection fraction and cardiac index were significantly reduced after 7 d and 21 d, respectively (Table 2). The ventricular wall showed remarkable thinning and time-dependent scar tissue formation in the infarcted regions (Fig. 2B, trichrome staining).


Figure 2
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  		Fig. 2 A: Magnetic resonance imaging was used to quantify infarct size. Example shows myocardial infarction after 21 days. B: Trichrome staining (Masson) of an infarcted heart after 21 days. C: HE staining after 7 days showing mainly an inflammatory response upon myocardial infarction. D: HE staining 21 days after myocardial infarction with a fibrotic scar tissue formation.

 

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  		Table 2 Magnetic resonance imaging data

 
3.3. Cell types of bone marrow-derived cells 7 days after infarction
Seven days after induction of myocardial infarction the HE-stained sections showed the typical accumulation of inflammatory cells and loss of myocytes and the beginning of scar tissue formation characterized by an increased amount of interstitial tissue could be demonstrated (Fig. 2C). However, the free left ventricular wall in the infarct area was still of normal thickness.

Immunohistochemical analysis showed a huge number of eGFP-positive cells in the peri-infarct zone. Frequently, the cells were present around small arteries and capillaries. The number of eGFP-positive cells was slightly lower in the infarct zone and the remote area as compared to the periinfarct region.

Most of these cells could be identified as leukocytes by their positive staining with the pan-leukocyte marker CD45. Additional characterization of these CD45-positive cells revealed that most leukocytes were F4/80 positive monocytes or macrophages (Fig. 3A/B).


Figure 3
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  		Fig. 3 A: Numerous bone marrow-derived macrophages were found in the infarct area (staining with F4/80). B: Colocalization with eGFP appears yellow. C: Specific staining for the stem cell antigen CD34 (periinfarct area). D: Colocalization with eGFP appears yellow. (* eGFP – /CD34+ cell; # eGFP+/CD34+ cell; {dagger} eGFP+/CD34 – cell).

 
Few eGFP-positive cells stained for CD34, indicating widely undifferentiated hematopoietic stem cells. These cells were predominately found in the infarct area (Fig. 3C/D).

Fibroblasts were identified by their staining with vimentin. These cells were located primarily in the infarct zone, and to a lesser degree in the periinfarct area also (p<0.05, Fig. 4A/B). 24.7±1.8% of fibroblasts showed a clear colocalization with eGFP, indicating a BM-derived origin.


Figure 4
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  		Fig. 4 A: Vimentin staining to detect fibroblasts (periinfarct area, red). B: -> Costaining with eGFP indicating that fibroblasts are in part bone marrow-derived. C: Detection of myofibroblasts by staining with {alpha}-smooth actin (periinfarct area, red). D: # Colocalization with eGFP (yellow). E: SMemb staining as a specific labelling for hypersynthetic myofibroblasts (red) in the infarct zone. F: eGFP-staining of the same section. G: Colocalization of SMemb and eGFP appears yellow. (* SMemb+/eGFP – cell, arrow: SMemb+/eGFP+ cell).

 
In the infarct zone and significantly less in the periinfarct area, a robust number of myofibroblasts was detected by specific costaining with {alpha}-smooth muscle actin and vimentin (p<0.05). 57.4±2.9% of myofibroblasts were eGFP-positive (Fig. 4C/D). This result was backed up by additional staining with SMemb in 4 mice yielding in similar results with 59.5±4.1% eGFP-positive myofibroblasts (Fig. 4E/F/G). eGFP positive fibroblasts and myofibroblasts were absent in the remote areas.

Cardiomyocytes were characterized using titin (Fig. 5A, remote zone) and connexin-43 (not shown) antibodies. In serial sections of 13 hearts 7 days after infarction, we could find only 2 cardiomyocytes which were eGFP-positive (Fig. 5C/D). These two cardiomyocytes were neither located in the infarct nor in the periinfarct area but in the remote zones of the left ventricular myocardium. All cardiomyocytes which were located in the periinfarct area were eGFP-negative excluding BM derived origin (Fig. 5B). We could not detect any viable cardiomyocytes in the infarct area.


Figure 5
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  		Fig. 5 A: Titin staining in the remote area of the MI (red). No eGFP positive cells were detected. B: Titin staining in the periinfarct region. No colocalization of eGFP positive cells and cardiomyocytes was found. C/D: An eGFP positive cardiomyocyte in the remote zone 7 d after MI.

 
Accordingly, we found no evidence for eGFP-positive cells to be involved in angiogenesis in the early post-infarction period in the periinfarct area, since no eGFP-positive smooth muscle cells or endothelial cells could be detected after serial examination of the tissue stained with {alpha}-smooth muscle actin and CD31 antibodies or BS-1 lectin by confocal microscopy (Fig. 6A/B). However neoangiogenesis was evident in the beginning of scar tissue formation in the infarct region. It involved a high number of eGFP positive vascular endothelial cells indicating newly built capillaries (Fig. 6C/D).


Figure 6
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  		Fig. 6 A/B: {alpha}-smooth actin staining in the periinfarct region showing that vascular smooth muscle cells are negative for eGFP (-> eGFP-positive cell, no colocalization to {alpha}SMA-positive cells). C/D: BS-1 staining in the infarct area (21 d) showing some eGFP+ endothelial cells (->) in capillaries.

 
Although some eGFP-positive cells were found in sham-operated animals after 7 days as well, the number was much lower. These cells were predominantly present in the area around the loosely tied thread and were characterized to be mainly CD45 positive leukocytes (data not shown). Specifically, no eGFP-positive cardiomyocytes, fibroblasts, myofibroblasts, and endothelial cells were detected.

3.4. Cell types of bone marrow-derived cells 21 days after infarction
The histological pattern changed when myocardial infarctions were examined after 21 days. The HE staining demonstrated advanced scar tissue formation with a decrease of inflammatory cells and thinning of the infarcted ventricular wall (Fig. 2D).

In immunohistochemistry, the total number of eGFP-positive cells was smaller than after seven days. Most noticeable is a decline of the number of cells staining with CD45, indicating a smaller number of leukocytes. Especially, the proportion of F4/80-positive macrophages decreased in comparison to the results after seven days. Cells with positive staining for the stem cell marker CD34 were only rarely found.

In the infarct area predominantly fibroblasts and myofibroblasts were present, which are typical of scar tissue formation. 28.6±2.2% of fibroblasts were eGFP-positive. Detection of myofibroblasts with costaining of vimentin and {alpha}-smooth muscle actin revealed 31.6±2.6% eGFP-positive cells. Again, this quantification was confirmed with an additional staining for SMemb in 4 hearts with 35.2±3.9% eGFP-positive myofibroblasts. Whereas the number of eGFP-positive fibroblasts showed no significant difference in comparison to the results after 7 days, the number of eGFP-positive myofibroblasts was significantly decreased (p<0.05).

In a total of 12 examined hearts 21 days after myocardial infarction only one eGFP-positive cardiomyocyte was identified. Again, this cardiomyocyte was located in the remote area.

In correspondence to the results after 7 days we found a remarkable degree of neoangiogenesis in the scar tissue involving BMC, i.e. the vascular endothelial cells showed distinct co-staining with eGFP and the endothelial cell markers CD31, and BS-1, respectively. Neoangiogenesis involving BMC was neither observed in the periinfarct region nor in the remote zone.

Sham-operated animals showed no obvious differences when examined after 21 days compared to the results after 7 days. However, the number of eGFP positive cells was slightly diminished (data not shown). Again, eGFP-positive cardiomyocytes, fibroblasts, myofibroblasts, or endothelial cells were absent.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
References
 
The purpose of the present study was to investigate the role of an individuum's own BMC in the healing processes after myocardial infarction.

To track and distinguish the cells of BM origin from those resident in tissue, we successfully transplanted sub-lethally irradiated mice with BM from their eGFP-transgenic littermates. BM-derived cells were traced in the infarct, periinfarct and remote areas of the entire heart. The major finding of this work is the fact that numerous eGFP-positive endothelial cells, fibroblasts, and myofibroblasts were identified in the injured hearts whereas in a total of 25 mice hearts only three eGFP labeled cardiomyocytes were found.

The conclusion was drawn that BMCs seem to be involved in reparative scar tissue formation including clearing of dead cells, replacement of cardiomyocytes by fibroblasts, myofibroblasts and excessive extracellular matrix, and angiogenesis, i.e. capillary sprouting but that BMCs are not involved in myocyte replacement in areas of myocyte loss.

In HE sections we found the typical time-dependent course of morphological and structural alterations after myocardial infarction. In the early phase after seven days the infarct area was invaded mainly by inflammatory cells most probably mediating the removal of necrotic myocytes. The infarcted area showed a nearly preserved wall thickness. After 21 days the number of inflammatory cells decreased markedly. The infarcted area was widely remodeled to a thinned scar tissue. These findings correspond excellently to the observed reduction of infarct size after 21 days compared to the results after 7 days. Similar results were reported by Virag and Murry, although they found an even more pronounced decline in infarct size after 4 weeks. However, in their study, the largest decrease of infarct size was between week 2 and 4, therefore not allowing a direct comparison to our results [19]. Lutgens et al. found only a small decrease of the infarct size after 3 weeks which is similar to our results [20]. Our results could be confirmed by immunohistochemical staining. After seven days the bulk of cells were of leukocyte nature identified by their positive staining with the pan-leukocyte marker CD45. The majority of these cells were eGFP-positive which can be explained by the near-total replacement of the BM with the transplanted stem cell pool. Further differentiation of these inflammatory cells identified primarily macrophages. After 21 days, the number of CD45 positive cells decreased suggesting an almost finished inflammatory response. Corresponding results were found by Virag and Murry in mice [19] and by Fishbein et al. in rats [21]. Therefore, our results represent the typical time course of a post-infarction inflammatory healing response, assuring that the prior BMTx did not interfere with such basic mechanisms.

Most importantly, we found in the infarct and periinfarct area fibroblasts and especially myofibroblasts which were, for a remarkable part, positive for eGFP indicating their unusual origin from BM. Interstitial fibroblasts are primarily responsible for collagen synthesis in the normal myocardium, while the phenotypically transformed fibroblasts termed myofibroblasts, are central to fibrogenesis at sites of remodelling after myocardial injury [22]. Myofibroblasts express cell-to-cell and cell-to-matrix connections that provide for a contractile assembly that contributes to scar tissue remodelling [23]. Frangogiannis et al. proposed that myofibroblasts are not only collagen-producing cells in myocardial infarction but may rather serve as a versatile cell population assuming different phenotypes depending on the physiological needs [24]. SMemb-positive myofibroblasts have been described to have an increased metalloproteinase expression [25] which may contribute to a desirable balance between collagen formation and degradation. Furthermore, they may also serve as precursor cells necessary for angiogenesis [24]. These properties make myofibroblasts essential for the capacity of the infarcted heart to heal. Moreover, the high percentage of BM derived myofibroblasts in our study may point to a particular importance of BM cells in myocardial healing processes.

Recent reports described BM derived myofibroblasts in the lung, kidney, skin, and other organs [26]. However, to our knowledge, our results show for the first time a transdifferentiation of BMC into myofibroblasts during myocardial repair. This finding are in contrast with a recent study of Yano et al. that excluded a transdifferentiation of BM derived cells into myofibroblasts [27]. These conflictive data might be explained by the use of different animal models. Yano et al. transplanted BM from eGFP transgenic mice into nude rats which functionally lack mature T-cells [28]. However, T-cells might influence the differentiation of myofibroblasts in an unpredictable manner. Virag and Murry showed the highest migration rate of myofibroblasts into the infarcted region on day 4 after myocardial infarction [19]. Thus, we might have missed the peak number of myofibroblasts when investigating the mice 7 days after myocardial infarction. Whereas the persistence of myofibroblasts in scar tissue in humans over weeks and months was described before [29], the prolonged existence of a robust number of fibroblasts in our setting is noteworthy.

Previous experimental studies aimed to establish the potential of circulating BMC to repopulate and repair the infarcted heart by transdifferentiation into cardiomyocytes. However, controversial results [5–8,30,31] have been reported. In our study we examined 25 infarcted and 6 sham hearts which were sectioned from apex to base and systematically scrutinized for eGFP positive cardiomyocytes. In total, only three eGFP-positive cardiomyocytes were detected. These cells were located in the remote area and not in the border zone or the infarction area. This result clearly excludes a cardiomyocytogenic capacity of BMC in our experimental setting. These findings are in contradiction to previous results of other groups which found robust myocardial repair of ischemically injured myocardium mediated by BMC [5–7,30,32]. Kajstura et al. reported a remarkable reduction of the infarct size (– 17%) and myocardial regeneration with as much as 2.5 to 4 million newly built cardiomyocytes per heart when BMC were injected into the periinfarct region [30]. Similarly, Kudo et al. found a reduction of infarct size from 25% to 8% and a reduced fibrotic response upon transplantation of BM stromal cells [32]. Several groups proposed that BMC might generate cardiomyocytes through cell fusion rather than through transdifferentiation [10,33,34]. However, in our study all 3 eGFP positive cardiomyocytes were mononucleated making cell fusion an unlikely explanation.

Similar to our results, Balsam et al. [9], and Murry et al. [8] found no evidence for transdifferentiation of hematopoietic stem cells into cardiomyocytes, although BMC were directly injected into myocardial tissue. All these results support our observation that BMC do not transdifferentiate into cardiomyocytes. However, most of these reports did not examine normal pathophysiological repair mechanisms, i.e. the reaction of BMC to myocardial infarction without other influences like direct injection into the infarct area or mobilizing stem cells by means of cytokines.

An explanation for the differing results might be the different experimental approach. We used a permanent occlusion of the coronary artery, thereby aggravating a migration of BMC into the infarcted area. However, since numerous inflammatory cells originating from BM were found in both, the periinfarct region and the infarct zone, it seems unlikely that the permanent coronary occlusion represents an insurmountable obstacle not allowing cellular transdifferentiation. Furthermore, we identified CD34 positive BMC within the infarct zone thereby indicating the ability of stem cells to invade the non-vascularized area. This marker is expressed mainly on pluripotent hematopoietic stem cells which are normally located exclusively in the BM and only to a negligible extent in the blood circulation [35]. However, recent reports suggest the expression of CD34 surface antigen additionally on other cell types, e.g. vascular endothelial cells [36]. Although the CD34-positive cells identified in our study did not meet the morphological criteria of endothelial cells, it cannot definitely be excluded that these BM derived cells do not have hematopoietic stem cell character.

It has been hypothesized that neoangiogenesis occurring after BMC application might cause improvement of ventricular function after myocardial infarction. In the present study, however, capillary sprouting involving eGFP positive cells was absent in the periinfarct region, which is in agreement with a recently published report by O'Neill et al., who were unable to find any involvement of BMC-derived cells in a model of hypoxia-induced angiogenesis [37]. However, our results show vascular structures containing eGFP positive endothelial and smooth muscle cells in the scar tissue indicating neoangiogenesis, i.e. the formation of microvessels ensuring viability of fibroblasts and myofibroblasts. This scar tissue neoangiogenesis might be important for positive scar tissue remodelling. However, myocardial functional improvement directly caused by BMC-derived neoangiogenesis cannot be expected, given the absence of contractile cells in the scarred tissue.

Our results indicate that BMC actively participate in post-infarction reparative processes by transforming into fibroblasts and myofibroblasts. Furthermore, there is evidence that BMC are involved in scar tissue neoangiogenesis. In our setting BMC fail to transdifferentiate into cardiomyocytes during the healing process after myocardial infarction. These data, which are in agreement with findings of several authors but in contrast to yet other reports, should prompt further research of the transdifferentiation potential of BMC in order to achieve a better understanding of basic physiological and pathophysiological mechanisms.


   Acknowledgements
 
This study was supported by grants from Max-Planck-Gesellschaft, München, Germany, for cooperation with the Kerckhoff Heart Center, Bad Nauheim, Germany (PFOR 406). We are grateful to Brigitte Matzke and Sigrun Sass for their excellent technical work.


   Notes
 
1 These authors contributed equally to this manuscript. Back

Time for primary review 31 days


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 

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