In the last 20 years, the incidence of heart failure increased compared with acute myocardial infarction.1 Despite advances in medical procedures, heart failure remains a leading cause of cardiovascular morbidity and mortality worldwide. The lifetime risk of heart failure for a person aged 55 years is approximately 30%, with only 35% surviving 5 years after the first diagnosis.2
Up to now, the only curative therapy for heart failure is cardiac transplantation, but it is limited by the availability of donor organs, the side effects of immunosuppressive therapy, and the long-term failure of transplanted organs. Because of a lack of proper donor organs, only approximately 3400 heart transplantations are performed yearly worldwide.3 Thus, therapy options are limited, and new approaches are needed.
The human heart has always been defined as a postmitotic organ with a determined number of cardiomyocytes formed during the embryonic and the fetal life without the capability to regenerate.
It was assumed that if the heart loses a number of cardiomyocytes, the remaining cells have to sustain the heart function. Accordingly, cell therapy is a promising strategy to treat heart failure because it aims to regenerate the myocardium with contractile substance.
In the last years, alternative strategies in stem cell transplantation have been approved in experimental studies.
Diverse pluripotent endogenous adult stem cells were tested for their impact on myocardial regeneration.4–6 After the initial demonstration of safety use, bone marrow–derived stem cells (BMCs) especially were used in clinical trials to initiate cardiac regeneration.7–12 These revealed a modest but significant improvement in left ventricular (LV) function after BMC transplantation. Most of the studies showed a decline of this improvement over time. The effects seem to sustain for at least 2 years, but after 3 to 5 years, a significant benefit could not be detected anymore.13–15
In addition, these studies investigated the impact of only intra-arterial BMC injection after myocardial infarction; the knowledge of the exact mechanisms causing the positive effects after cell transplantation could lead to a more potent and sustained therapy in nonischemic heart failure.
Because Li et al16 showed that intra-arterial injection of BMCs leads to only minimal cell retention in the heart, we chose direct epicardial cell injection, which seems to be more efficient. In the future, this type of cell delivery could be performed during other heart surgery procedures in patients with heart failure.
Because the differentiation of a significant amount of implanted hematopoietic adult stem cells to the cardiac phenotype could not be demonstrated, it is assumed that other mechanisms are responsible for the beneficial effects after mesenchymal stem cell (MSC) transplantation.17,18
The identification of cardiac progenitor cells revealed the heart’s own capacity for regeneration.
It has been proposed that stem cells release angiogenic factors, regulate apoptotic cell death, induce proliferation of host cardiomyocytes, and may recruit cardiac progenitor cells.19–21
These results indicate the importance of paracrine effects in stem cell therapy.
To evaluate this hypothesis, we investigated the influence of the MSC injection site on cardiac performance and capillary density after epimyocardial injection of autologous MSCs in doxorubicin (Dox)–induced cardiomyopathic rabbits.
All experiments were conducted in accordance with guidelines published in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85–23, revised 1996) and under protocols approved by the Institutional Animal Care and Use Committee at Leipzig University and “Regierungspräsidium” (registration no. 24-9168.11-11/03).
Animal Experiments and Heart Failure Induction
Three-month–old male white New Zealand rabbits (Charles River, Germany) were housed individually in a temperature- and light-controlled environment with a 12-hour light/dark cycle and were provided with food and water ad libitum. Three milligrams per kilogram of doxorubicin hydrochloride (Ribodoxo-L 50) was administered once a week for 6 weeks intravenously. A 14-day interval free of doxorubicin application followed. The animals that survived 2 weeks after heart failure induction were randomized to right ventricular (RV) MSC transplantation group (RV-MSC, n = 6), LV MSC transplantation group (LV-MSC, n = 6), sham treatment (sham group, n = 6), and diseased group without therapy (Dox, n = 5). The rabbits without doxorubicin treatment served as the control group (n = 8).
Isolation, Expansion, and Labeling of Stem Cells
Four days before MSC transplantation, an aseptical femoral bone marrow aspiration was performed at the right hind limb of each rabbit. Therefore, the animals received an intramuscular ketamine (50 mg/kg) and xylazine (5 mg/kg) injection. The obtained cells were flushed with phosphate buffer saline (PBS)–EDTA (PBS, pH 7.4; EDTA, 2 mM). The MSCs were fractionated over 1.073 g/mL of Ficoll solution (Sigma, Germany), and the mononuclear cells were obtained according to Tomita et al.22 After two washing steps, MSCs were resuspended in 12 mL of Dulbecco’s Modified Eagle Medium (Sigma) by adding 10% fetal bovine serum, 100 U/mL of penicillin G, and 100 μg/mL of streptomycin (Sigma). To induce differentiation, cells were cultured with 10 μM of 5-azacytidine for 24 hours (Sigma), seeded on gelatin-coated Petri dishes and cultured at 37°C in a 5% CO2–humidified atmosphere, and tested to ensure freedom from mycoplasma contamination. The nonadherent hematopoietic cells were discarded with the medium changes, which were done every 2 days (twice). Before cell transplantation, adherent cells were detached with trypsin-EDTA, passaged and labeled with 2.5 μL/mL of Vybrant DiI cell-labeling solution (Molecular Probes) for 30 minutes, protected from light at 37°C, and washed three times with PBS. The cells passaged twice were harvested and used for cell transplantation.
The animals were premedicated with intramuscular ketamine (50 mg/kg) and xylazine (5 mg/kg), endotracheal intubated, and mechanically ventilated. Anesthesia was maintained with 2% isoflurane. A right or left thoracotomy through the intercostal space was performed, and the RV or the LV was exposed. Thereafter, the free lateral wall of the RV or the LV was identified, and either 1.5 to 2.0 × 106 cells per milliliter or media (1 mL) were injected at four locations in a circular manner with a tuberculin syringe within 2 minutes. Finally, a thoracic drainage was inserted before closing of the chest wall. The rabbits recovered from the surgical procedure and were treated with antibiotics and analgesics within 72 hours postoperatively as required.
Echocardiograms were obtained before MSC therapy/medium injection and 28 days after transplantation in triplicate by a single blinded investigator. After sedation and intubation, the rabbits were fixed supine in a left lateral position, and the thorax was shaved. Thereafter, M-mode and two-dimensional–mode images were recorded on the fourth intercostal space close to the sternum. Transthoracic echocardiography was performed with a commercially available ultrasound device (VINGMED CFM-800A, Norway) equipped with a linear 7.5-MHz transducer. Standard views were obtained in the short axis transecting the LV shortly below the mitral valve at the level of the papillary muscles, perpendicularly to the septum, in an anterior-posterior manner. Left ventricular end-diastolic (LVEDV) and LV end-systolic (LVESV) volumes were measured and averaged from three cycles. Ejection fraction (EF) was calculated using the following equation: EF (%) = (LVEDV LVESV)/LVEDV × 100.
After completion of echocardiography, the hearts were cut transversely and fixed in 4% formalin, embedded in paraffin, and stained with hematoxylin and eosin.
Quantitative and qualitative histological analyses were performed with the Axioplan2 microscope (Carl Zeiss GmbH, Jena, Germany) and the KS 300 Imaging System 3.0 (Carl Zeiss Vision GmbH, Eching, Germany).
Assessment of Regional Capillary Density
For analysis of capillary density, immunohistochemical labeling of the endothelial cells with anti-CD31 antibody was performed. Slides were predigested with 0.05% protease (Protease Type XXIV, P8038, Sigma Chemie, Deisenhofen) for 10 minutes. Monoclonal mouse anti-CD31 (dilution, 1:20; NCL-CD31-1A10, Novocastra Laboratories, United Kingdom) served as the primary antibody; rat antimouse IgG (1:100, Dianova GmbH, Hamburg) was used as the secondary antibody; and subsequently, a mouse PAP (1:500, Dianova GmbH, Hamburg) was applied. Five high-power fields (HPFs) were randomly chosen from the LV—outside the cell/medium injection zone, the RV, and the septum. The numbers of capillaries were counted in each area using a ×40 objective by two different observers without knowledge of which treatment group was analyzed. The capillaries in five different regions were averaged, with an interobserver variability of less than 5%.
All data are expressed as mean ± SD. Statistical comparison was performed by one-way analysis of variance followed by paired t test, as appropriate. Results were considered statistically significant as P < 0.05. All data analyses were performed using the SAS software, version 6.11 (SAS Institute, Cary, NC USA).
Statistical support was supplied by the Department of Biostatistics of the University of Leipzig, Leipzig, Germany.
Mortality and Body Weight
Fifteen (37%) of the doxorubicin-induced cardiomyopathic rabbits died prematurely of toxicity during the 6-week induction period. Two animals died 2 weeks after the last Dox dose, before randomization. The total mortality rate was 43%. After randomization (n = 23), no animal died within 28 days. The body weight of the MSC groups and the sham group remained nearly unchanged, and 28 days after therapy, there was no significant difference between the groups (2586 ± 79 g vs 2544 ± 83 g).
Doxorubicin treatment decreased the EF significantly compared with the control group (Dox group, 29.2% ± 2.6%, vs control group, 44.0% ± 1.6%; P < 0.05; Fig. 1) and resulted in a substantial impairment of ventricular function. There was no statistical difference between the sham and the Dox group. The hearts of the MSC-treated cardiomyopathic rabbits had a significantly increased EF 28 days after local cell injection compared with sham group (LV-MSC, 39.0% ± 1.4%, and RV-MSC, 39.2% ± 2.6%, vs sham group, 29.8% ± 3.7%; P < 0.001; Fig. 1), without significance between the MSC groups (P = 0.858). The rabbits in both MSC groups obtained partially normal EF.
Enhancement of Angiogenesis
Heart sections were examined using anti-CD31 immunostaining to evaluate the number of capillaries (Fig. 2).
The capillary density (capillaries/high-power field) decreased in all chambers of the Dox group compared with the control group (LV: 42.0 ± 2.4 vs 52.0 ± 2.0, P = 0.002; septum: 38.0 ± 1.7 vs 48.0 ± 1.7, P < 0.001; and RV: 35.0 ± 1.9 vs 41.0 ± 1.8; capillaries/HPF, P < 0.037; Fig. 3).
The RV and the LV MSC transplantation recovered the capillary density to the level of the control group, with a significant difference compared with the sham group (LV: LV-MSC, 55.0 ± 2.2, and RV-MSC, 54.0 ± 1.7, vs 42.0 ± 2.0, P < 0.05; septum: LV-MSC, 46.0 ± 1.5, and RV-MSC, 45.0 ± 2.1, vs 39.0 ± 1.7, P < 0.05; and RV: RV-MSC, 41 ± 2.2, vs 35.0 ± 1.7; capillaries/HPF, P < 0.05; Fig. 3). The capillary density in the RV of the LV-MSC group was higher compared with that of the sham group but failed to reach statistical significance (LV-MSC, 40.0 ± 2.1, vs 35.0 ± 1.7, P = 0.065; Fig. 3). There was no significant difference between the sham group and the Dox group.
We performed blood analysis in all animals after the final studies. We did not detect any signs of enhanced immune activity or inflammation. There was no significant difference in white blood cell count (P = 0.31) and C-reactive protein levels (P = 0.28) between the groups.
As far as we know, this is the first report showing that epimyocardial injection of autologous MSCs improves global heart function in nonischemic cardiomyopathy independently of the injection site.
In addition to the hemodynamic improvement, we detected an increase in capillary density in all heart chambers. The injection site did not influence this proliferation. To minimize all immunological influences of a heterologous animal model, we decided to use autologous MSCs.
Other studies using the nonischemic concept of heart failure show that injection of MSCs, embryonic stem cells, or myoblasts improves LV function.23–28 Our data support and extend these findings, showing that the beneficial effects of MSC transplantation are independent of the injection site.
Up to now, it is still highly controversial whether, and if so, how, MSC transplantation leads to an improvement of heart function.29 Different mechanisms such as proliferation and differentiation of the implanted MSCs, stimulation of resident cardiac progenitor cells, and neovascularization are discussed.
Some groups such as Orlic et al30 reported that transplanted MSCs could transdifferentiate into de novo myocardium and regenerate the heart substantially.
Behfar et al31 could improve the therapeutic benefit by guiding MSCs into a cardiac progenitor phenotype.
Other published studies could not detect more than just a rare transdifferentiation of transplanted MSCs. Instead, MSCs seem to adopt mature hematopoietic fates and form myeloid and lymphoid cell types.17,18 A study similar to ours, using a different species and technique of immunostaining, observed a small portion of transplanted MSCs forming new myocardium.23
Other studies observed spontaneous fusion of transplanted MSCs with cardiomyocytes but no formation of new cardiomyocytes. In our study, like in others, we could not detect any evidence for transdifferentiation of donor cells into cardiomyocytes 4 weeks after injection.32,33 In summary, most investigator groups agree that even if new cardiomyocytes are formed, this transdifferentiation occurs rarely and could not explain the significant recovery of cardiac function after MSC transplantation.
Consequently, other mechanisms seem to lead to the improvement of heart function and are in the focus of ongoing research.
One possibility is a direct interaction between the transplanted MSCs and resident cardiac stem cells.
Hosoda and colleagues34 reported that the transdifferentiation of human cardiac stem cells is regulated by the interaction with postmitotic cardiomyocytes. Roeske et al35 showed that MSCs influence cardiomyocytes by activating an intracellular signaling cascade and enhancing β-adrenoceptor density.
The third discussed mechanism is that the transplanted MSCs secrete paracrine mediators.
Fazel et al36 reported that c-kit–receptor activation of MSCs induces their mobilization through the bloodstream into infarct areas, where they initiate angiogenesis by secretion of angionetic substances. Another study demonstrates that murine bone marrow–derived stromal cells injected into a region of forming collaterals produce a wide array of arteriogenic cytokines. This results in improvement of perfusion and remodeling, lessens tissue damage, and enhances limb function in a mouse model of hind limb ischemia.37
Winter et al38 reported not only that different cell types can improve cardiac function but also that combining them leads to an additional improvement, probably instigated by complimentary paracrine actions. Possible ways of action could be the activation of resident cardiac stem cells or cellular network as well as direct diffusible effects.
Latest studies reported that the transplantation of MSCs and CSCs may improve heart function by stimulation of endogenous cardiac stem cells. Loffredo et al39 suggest that cell therapy with bone marrow–derived c-kit(+) cells leads to an augmentation of cardiomyocyte progenitor activity and improves cardiac function. Hatzistergos et al40 showed similar effects in an animal model. Transplantation of MSCs led to CSC proliferation into populations of adult cardioblasts and de novo vascular structures. However, in another recently published study, we analyzed the distribution of resident cardiac progenitor cells in the human heart.41 The stimulation of these cells could explain the positive effects of MSC transplantation.
A study by Gnecchi et al42 showed that Akt-MSCs exert direct salutary effects on ischemic cardiomyocytes via paracrine mediators such as VEGF, FGF, or IGF. We cannot prove that these or other secreted factors are responsible for the improvement of heart function and capillary density in our study.
However, the fact that the injection site did not influence the effect of MSC transplantation leads to the hypothesis of paracrine mechanisms. Other studies investigating the role of the injection site showed that skeletal myoblasts, regardless of whether these were injected into the infarcted or the noninfarcted myocardium, improved cardiac function and support this hypothesis.43 Two other studies demonstrated that local implantation of cells reduced ventricular remodeling of the entire heart in an animal model of global nonischemic cardiomyopathy.44,45
We are aware that RV injection of MSCs in humans is an unlikely therapy option because LV injection is easier to achieve. However, in addition to the pure scientific finding that RV injection might be as beneficial as LV injection, intraoperative injection of MSCs in both ventricles might be a promising therapeutic approach. However, it was not the aim of this study to find a new application side rather than an approach to further examine possible mechanisms of stem cell therapy.
In summary, it can be assumed that paracrine factors are at least partially involved in improving heart function after MSC transplantation. Unfortunately, it is still unknown whether the observed effects are based on angiogenesis and thus enhanced microcirculation or whether other mechanisms play a major role.
We were unable to characterize the transplanted cells in detail before injection because of unavailable or failing antibodies. Furthermore, we cannot exclude that some of the injected MSCs were accidently injected through the myocardium into the RV and then reached the LV by the bloodstream. However, we could not detect any labeled MSCs in the LV or in any remote organ such as the lung or the spleen. However, this could be due to the used labeling method. We cannot exclude the mechanism of transdifferentiation as a pathway for cardiac regeneration. The low number of animals that survived doxorubicin treatment limits the study results. One reason for the similarity of the EF after RV and LV MSC injection could be related to the limited engraftment in both ventricles, due to the thin RV and the dilated LV wall. However, in our histological sections, we observed only very few puncture channels through the RV or LV wall, which could have led to an injection inside the ventricular cavities.
Furthermore, our results refer to only MSCs. Other cell types such as endothelial progenitor cells may show similar outcomes.
Unfortunately, long-term results after MSC transplantation and cell behavior remain unclear.
In conclusion, local transplantation of autologous MSCs ameliorates global LV dysfunction and enhances remote capillary density independently of the injection site in a rabbit model of Dox-induced cardiomyopathy. The observed benefit does not seem to be the result of cell transdifferentiation but may be attributable to diffusible factors released by the transplanted cells, acting in a paracrine manner in all cardiac chambers.
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This is an interesting experimental study investigating the effect of mesenchymal stem cell transplantation in a doxorubicin-induced heart failure rabbit model. The transplantation of mesenchymal stem cells increased ejection fraction significantly and enhanced angiogenesis. Despite local application of the stem cells, the authors observed global effects, suggesting the importance of paracrine mechanisms.
The main weakness of the study is the inability of the authors to fully characterize the transplanted cells in detail before injection. They also could not exclude that some of the injected mesenchymal stem cells were injected systemically and may have reached the other parts of the heart via the bloodstream. However, the results are intriguing, and further studies are eagerly awaited, which hopefully will delineate whether paracrine mechanisms play a role in this process.