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European Journal of Heart Failure 2008 10(5):454-462; doi:10.1016/j.ejheart.2008.03.017
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© 2008 European Society of Cardiology

Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction

Manuel Mazoa,1, Valérie Planat-Bénardb,1, Gloria Abizandaa, Beatriz Pelachoa, Bertrand Léobonb, Juan José Gavirac, Iván Peñuelasd, Arantxa Cemboraina, Luc Pénicaudb, Patrick Laharragueb, Carine Joffreb, Marie Boissonb, Margarita Ecayd, Maria Collantesd, Joaquin Barbac, Louis Casteillab,* and Felipe Próspera,*

a Hematology and Cell Therapy and Foundation for Applied Medical Research, Division of Cancer, University of Navarra Pamplona, Spain
b UMR 5241 CNRS UPS, Institut Louis Bugnard Toulouse, France
c Department of Cardiology and Cardiovascular Surgery, Clínica Universitaria Pamplona, Spain
d Department of Nuclear Medicine and MicroPET Research Unit CIMA-CUN, Clínica Universitaria Pamplona, Spain

* Corresponding authors. Casteilla is to be contacted at UMR-CNRS 5241 IFR31, CHU Rangueil 1 avenue Jean Poulhès, 31432 Toulouse cedex 4, France. Tel.: +33 5 62 17 08 91; fax: +33 5 62 17 09 05. Prósper, Hematology and Cell Therapy Area, Clínica Universitaria, Av Pío XII 36, 31008 Pamplona, Spain. Tel.: +34 948 255400; fax: +34 948 296500. E-mail addresses: casteil{at}toulouse.inserm.fr (L. Casteilla), fprosper{at}unav.es (F. Prósper).


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
 References
 
Aims: To determine the effect of transplantation of undifferentiated and cardiac pre-differentiated adipose stem cells compared with bone marrow mononuclear cells (BM-MNC) in a chronic model of myocardial infarction.

Methods: Ninety-five Sprague–Dawley rats underwent left coronary artery ligation and after 1month received by direct intramyocardial injection either adipose derived stem cells (ADSC), cardiomyogenic cells (AD-CMG) or BM-MNC from enhanced-Green Fluorescent Protein (eGFP) mice. The control group was treated with culture medium. Heart function was assessed by echocardiography and 18F-FDG microPET. Cell engraftment, differentiation, angiogenesis and fibrosis in the scar tissue were also evaluated by (immuno)histochemistry and immunofluorescence.

Results: One month after cell transplantation, ADSC induced a significant improvement in heart function (LVEF 46.3±9.6% versus 27.7±8% pre-transplant) and tissue viability (64.78±7.2% versus 55.89±6.3% pre-transplant). An increase in the degree of angiogenesis and a decrease in fibrosis were also detected. Although transplantation of AD-CMG or BM-MNC also had a positive, albeit smaller, effect on angiogenesis and fibrosis in the infarcted hearts, this benefit did not translate into a significant improvement in heart function or tissue viability.

Conclusion: These results indicate that transplantation of adipose derived cells in chronic infarct provides a superior benefit to cardiac pre-differentiated ADSC and BM-MNC.

Key Words: Cell therapy • Stem cells • Chronic ischaemic heart failure • angiogenesis

Received December 11, 2007; Revised February 26, 2008; Accepted March 26, 2008


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
 References
 
Heart failure is a leading cause of hospitalisation and death in industrialized countries [1] and is reaching epidemic proportions. Ischaemia is regarded as the main mechanism that induces myocardial injury and subsequent cardiomyocyte (CM) loss. The low regenerative capacity of CMs results in scar formation and a decrease in pump force [2,3]. Over the past decade, cell therapy has emerged as a new approach to repairing the ischaemic heart by repopulating the fibrotic tissue (reviewed in [4,5]). While the ideal cell type should be capable of differentiating into a functional cardiomyocyte and electrically coupling with the remaining myocardium, the potential to form new vessels to nourish the damaged area could also stimulate tissue regeneration. Cell types from several different sources have already been tested in animal models [6-10], and bone marrow derived cells and skeletal myoblasts have been used in clinical trials [11,12]. Although results have shown an improvement in cardiac function due mostly to paracrine effects [13], formation of a new myocardial mass has only been established for embryonic stem cells [14].

Among the different putative source of stem cells, the initial enthusiasm over the use of bone marrow mononuclear cells (BM-MNC) in patients with acute MI has been dampened by the results of several recent clinical trials [15-17] (reviewed in 5). Increasing evidence suggests that adipose tissue may be a suitable source of regenerative cells, as it can be easily sampled and contains multipotent cells [18,19]. Adipose derived stromal cells (ADSC) seem to have a great regenerative angiogenic potential due to their capability to differentiate into endothelial lineages [20,21] and because their ability to secrete angiogenic and antiapoptotic factors [22] could play a therapeutic role. Moreover, some rare cells from adipose tissue can differentiate into spontaneous contractile cardiomyocytes in specific culture conditions [23]. Although some groups including our own have previously determined the beneficial effect of ADSC injection in ischaemia models [20,22], the effect of engraftment of adipose derived cardiomyoblasts (AD-CMG) was only recently tested in a murine model of acute myocardial infarction [24,25].

In the current study, we compared the potential of mouse derived ADSC, AD-CMG and BM MNC for improving cardiac function after engraftment into a rat model of chronic myocardial infarction.


   2. Materials and methods
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Source of funding
 References
 
2.1. Isolation, characterization and preparation of injected cells
All enhanced-Green Fluorescent Protein (eGFP) cells (from bone marrow and adipose tissue) were isolated from eGFP-mice kindly provided by Dr Okabe [26]. Bone marrow mononuclear cells (BM-MNCs) were obtained from 3-week old C57B1/6N male mice. Briefly, bone marrow cells were harvested from the mouse femurs and MNCs were isolated by Ficoll-Paque density gradient centrifugation. Cells were washed and resuspended in culture media (DMEM, Invitrogen, Leiden, The Netherlands) at a concentration of 106 cells per 70 {varepsilon}l. GFP adipose tissue cells were isolated from mice inguinal and interscapular adipose tissues. Inguinal fat pads were digested at 37 °C in DMEM-F12 (Invitrogen) containing 2% BSA and 2 mg/ml collagenase (Roche, Barcelona, Spain) for 30 min. After filtration (25 {varepsilon}m filters), mature adipocytes were separated from the stromal fraction by centrifugation (600x g for 10 min). The pellet was re-suspended in culture medium and cells were then seeded (10,000 cells/cm2) in DMEM-F12 with 10% Fetal Calf Serum (FCS) medium and maintained in 5% CO2 and humidified atmosphere. Six hours after plating, all non-adherent cells were removed by washing. Sub-confluent ADSCs were obtained after 6 days. AD-CMG cells were purified and amplified as described previously [23]. Freshly prepared adipose stromal cells (30,000 cells/ml) were plated and maintained for 2 weeks in methylcellulose (Methocult MG3534, StemCell Technologies). Early contracting clones were dissected under an inverted phase-contrast microscope and plated (1500 cells/cm2) into 30-mm culture dishes (Greiner Bio-one) coated with 0.1% gelatin (Sigma) in BHK21 medium containing 10% FCS (StemCell Technologies) supplemented with β-mercapto-ethanol 10–4 M (Sigma), Glutamine 2 mM (Gibco BRL), Pyruvate 1 mM (Gibco BRL), non-essential amino acid 0.1 mM (Gibco BRL), and a solution of amphotericin 0.25 {varepsilon}g/ml, penicillin G 100 U/ml and streptomycin 100 {varepsilon}g/ml (Sigma). AD-CMG cells were collected 2 to 3 weeks later.

2.2. Experimental model
A total of 95 female Sprague-Dawley rats (Harlan IBERICA S.L. Barcelona, Spain) underwent coronary artery ligation of the left coronary artery as previously described [27]. Only those animals whose LVEF fell below 40% at 1 month post-MI were included in the study (n=80). Four weeks after induction of myocardial infarction, rats underwent sternotomy and received 106 cells per heart by direct intramyocardial injection (total volume: 70 µl) of AD-CMG (n=20), ADSCs (n=20), BM-MNC (n=20) obtained from male eGFP-mice or vehicle medium (n=20), injected at 2 points of the infarct region border. All animals were immune-suppressed daily with cyclosporin A (20 mg/kg/d i.p. Sandimmun, Novartis) from 2 days pre-transplantation until sacrifice. All experiments were performed in accordance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources, Commission on Life Science, National Research Council, and published by the National Academy Press, revised 1996. All animal procedures were approved by the University of Navarra Institutional Committee on Care and Use of Laboratory Animals.

2.3. Tissue processing and immunostaining
After sacrifice, rat hearts were excised, fixed in 4% paraformaldehyde for 4 h at 4 °C, and cut in 3 equal size blocks (apical, mid-ventricular and basal). Hearts were either dehydrated in ethanol 70% (4 °C o/n) and embedded in paraffin, or incubated in glucose 25% (4 °C o/n) and embedded in O.C.T. compound and snap-frozen at –80 °C. For histological analysis, 3 {varepsilon}m sections were obtained. Cell detection was based on the presence of GFP-positive signals by immunohistochemical methods (anti-GFP polyclonal Ab (pAb), diluted 1:500 in TBS, Invitrogen) or immunofluoresecence. For immunofluorescence, a tyramide amplifying kit (Invitrogen) was used following manufacturer's instructions. Immunolabelling was performed with antibodies against {alpha}-Smooth Muscle Actin ({alpha}-SMA) (mouse monoclonal antibody (mAb) diluted 1:1000 in TBS, Sigma, Madrid, Spain), cardiac troponin T (cTT) (mAb, diluted 1:100 in TBS; Labvision), ventricular myosin (mAb diluted 1:2 in TBS; BioCytex), Ki-67 (mAb, diluted 1:25 in TBS; DAKO, Barcelona, Spain), PCNA (mAb, diluted 1:100 in PBS; Santa Cruz), CD14 (pAb, diluted 1:50 in PBS; Santa Cruz, Heidelberg, Germany), CD68 (mAb, diluted 1:200 in TBS; Serotec, Oxford, UK) and BSL-I lectin (diluted 1:100 in PBS; Vector, Barcelona, Spain). Secondary antibodies labelled with AlexaFluor-594 were purchased from Molecular Probes (Invitrogen) if needed. EnVisionTM-HRP conjugated system (Dako) was used as secondary reagent for immunohistochemistry.

Sirius Red staining and haematoxylin-Eosin staining were performed as detailed elsewhere [27,28].

2.4. Morphometric analysis
Quantification of vascular density was performed in animals sacrificed 4 weeks post-transplantation. For capillary density (capillaries/mm2), a total of 15 serial sections, 30 {varepsilon}m apart, were stained with BSL-I lectin-Cy3 and a total of 4 intra-infarction images were analyzed. Arteriolar density (arterioles/mm2) and arteriolar area (µm2) were quantified in the same way after staining with anti-alpha smooth muscle actin-Cy3 in the following sections. Pictures were taken using a Nikon Eclipse E800 microscope equipped with epifluorescence optics and digital images were analyzed using imaging software (Jay Image). The degree of fibrosis and the infarct size were determined as previously described [28,29]. Briefly, a mean of 15 serial sections were stained, each 30 {varepsilon}m apart. Infarct size was assessed as the mean percentage of infarcted area vs. total LV area, whereas fibrosis was measured in high power photographs within the infarct border as the percentage of collagen area (red) vs. total tissue area, using AnalySISR software.

2.5. PCR analysis
DNA was extracted from paraffin-embedded tissue. Briefly, 15-20 serial slides per heart were deparaffinised and hydrated. Tissue was carefully separated from glass and DNA extracted with DNAeasy Mini Kit (Quiagen, Venlo, The Netherlands), following the manufacturer's instructions. GFP cell expression was detected by PCR (GFP-upstream primer: 5'-GCA CCA TCT TCT TCA AGG AC-3'; GFP-downtream primer: 5'-ACT TGT ACA GCT CGT CCA TG-3'). Amplification conditions were as follows: 95 °C for 2 min followed by 40 cycles consisting on 94 °C for 30 s, 59 °C for 30 s and 72 °C for 30 s, plus an extension phase of 72 °C for 10 min. All samples were also amplified for GAPDH (GAPDH-upstream primer: 5'-CATGGCCTTCCGTGTTCCTA-3'; GAPDH-downstream primer: 5'-CTGGTCCTCAGTGTAGCCCAA-3') as control for the presence of amplified DNA. Also, the presence of transplanted cells was tested using PCR for the Y-chromosome gene sry (sry-upstream primer: 5'-AGGCACAGAGATTGAAGATCCTAC-3'; sry-downstream primer: 5'-ATATCAACAGGCTGCCAATAAAAG-3'), with the same GAPDH-internal control.

2.6. Echocardiographic studies
Animals were lightly anesthetized prior to the study with 2% isoflurane (Forane®. Lab. ABBOTT S.A, Madrid, Spain) in 100% O2 gas and placed in the supine or lateral position on a warming pad for transthoracic two-dimensional echocardiography, M-mode recordings, and Doppler ultrasound measurements as previously described [27]. Echocardiography was performed using a Sonos 4500 ultrasound system (Philips) with a 12 MHz linear array transducer and Doppler measurement. Left ventricular remodelling was assessed by measuring end systolic and diastolic volumes and diameters, according to the American Society of Echocardiography and the left ventricular ejection fraction was determined according to Teichholz in parasternal short axes [30]. Diastolic function was assessed by measuring E and A waves of the mitral filling pattern by pulsed echo-Doppler technique in four-chamber apical views. Echocardiographic studies were performed at baseline (before infarct), before cell transplantation and at day 30 post-transplant by 2 different investigators blinded to the type of therapy. Measurements were done in three cycles and the mean value was obtained.

2.7. 18F-FDG PET imaging protocols, image reconstruction and semi-quantitative evaluation
Immediately before injection of cells and 30 days later, animals were subjected to PET analysis. Rats were anesthetized with 2% isoflurane in 100% O2 gas for 18F-FDG injection (75 MBq in 100-200 {varepsilon}L) in a tail vein, and were immediately awakened afterwards and placed back in the cage. Two hours after administration of the tracer injection, animals were anesthetized with isoflurane, placed prone on the PET scanner cradle and were maintained under continuous anaesthesia for the duration of the study. A static 60-min study (sinogram) was acquired using a Mosaic (Philips) small animal dedicated imaging tomograph. No transmission scan was performed. The scanner has an 11.9 cm axial and 12.8 cm transaxial field of view (FOV) allowing a rat whole body study without moving the animal. Sinograms were reconstructed using the 3D Ramla algorithm (a true 3D reconstruction [31]) with 2 iterations and a relaxation parameter of 0.024 into a 128x128 matrix with a 1 mm voxel size. Polar maps were generated and divided into 17 different ROIs and individual quantification of the 18F-FDG uptake in each of them was calculated. The total number of counts obtained for each of the ROIs was divided by its corresponding area to obtain counts per area unit. For each PET study, the maximum value of the 17 ROIs was considered as 100% and the remaining data transformed into percentage values. All further calculations and statistical analysis were performed on these sets of re-scaled numerical data.

2.8. Statistical analysis
All data are expressed as mean±SD and comparisons for repeated measurements were performed by one way ANOVA. Kolmogorov-Smironov and Shapiro-Wilk normality tests were used to verify that the data had a Gaussian distribution, which justifies the use of parametric tests. Statistical analysis was performed with SPSS 11.0 software and differences were considered statistically significant when p<0.05.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Source of funding
 References
 
ADSC and AD-CGM cells were negative for expression of CD45 and CD31 and positive for CD44 and CD81. ADSC were negative for troponin T while AD-CMG expressed Troponin T (data not shown), which is consistent with our previous results, and demonstrates that AD-CGM differentiate into functional cardiomyocytes in vitro [23].

3.1. Adipose tissue derived cells improve cardiac function and tissue metabolism
In order to assess the effect of cell transplant on cardiac function and tissue metabolism we performed both cardiac echocardiography and MicroPET imaging of myocardial 18F-FDG metabolism at baseline, pre-transplantation and 1 month post-transplantation. As expected, there was a marked reduction in LVEF from baseline (70.87±6.61% mean±SD) to pre-transplant which did not differ significantly between the 4 groups (29.71±7.72% mean±SD), suggesting that initial ischaemic injuries were similar in the four groups. One month after cell transplantation, LVEF was unchanged in the control group. Injection of BM-MNC and AD-CMG did not induce a statistically significant change in LVEF compared to the control group. In contrast, transplantation of ADSCs was associated with a significant improvement in LVEF compared to the pre-injection value (P<0.05, Fig. 1). There were no differences in other echocardiographic parameters related to heart remodelling and diastolic function (data not shown). Semi-quantitative analysis of polar maps including all 17 cardiac segments also indicated that transplantation of ADSCs was associated with a significant increase in FDG uptake (P<0.01) (Fig. 2) which suggested that transplantation of adipose derived cells induced an increase in tissue metabolism.


Figure 01
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  		Fig. 1 Echocardiographic assessment of LVEF before transplant and 1 month after cell injection (mean±SD; *P<0.05).

 


Figure 02
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  		Fig. 2 MicroPET evaluation of 18F-FDG uptake. A: Representative images obtained in one animal in each group of treatment pre-injection (upper row) and 1 month after injection of cells (lower row). B: Comparison of 18F-FDG uptake for each group of animals before and 1 month after transplant. Results are expressed as the percentage (mean±SD) of 18F-FDG uptake in the 17 segments calculated as described in the materials and methods. **P<0.01.

 
3.2. Cell engraftment and in vivo differentiation
Cell fate was analyzed at 1 and 4 weeks after transplantation by immunohistochemistry against GFP. BM-MNC and ADSC but not AD-CMG GFP-positive cells were detected 1 week post-transplantation (BM-MNC 0.10±0.02%; mean±SD of injected cells at 1 week) and ADSC (0.25±0.04%; mean±SD at 1 week) versus total transplanted cells (Fig. 3a-f). We analyzed the degree of proliferation as well as the differentiation of the engrafted cells. No specific differentiation to smooth muscle (SM-actin), endothelium (BSL-I lectin), cardiomyocytes (MCL2V) or haematopoietic cells (CD14) could be detected at this time-point (Fig. 4a-d). Although proliferating cells were detected in the infarct area, none of the GFP positive cells co-stained with Ki67 or PCNA (Fig. 4e,f). However, proliferating cells were detected surrounding the graft area, which could indicate a putative trophic effect of the transplanted cells. As no GFP-positive signals were detected 4 weeks after transplantation in any of the animals, we performed PCR of genomic DNA in order to detect the presence of GFP and sry gene to detect the Y chromosome as male cells from mice were engrafted in female recipient rats. Both genes were detected in the groups transplanted with either BM-MNC or ADSCs at 1 week but not at 4 weeks (not shown), which is consistent with the immunohistochemistry data.


Figure 03
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  		Fig. 3 Analysis of cell engraftment by immunohistochemistry against GFP. BM-MNC (A,B), ADSCs (C,D) and AD-CMG (E,F) cells from male mice were injected in the myocardium of sex mismatched rats 4 weeks after inducing an infarct. Representative areas are shown. Scale bars: 50 {varepsilon}m.

 


Figure 04
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  		Fig. 4 In vivo cell differentiation. No differentiation of transplanted cells to cardiomyocytes, smooth muscle, endothelial or haematopoietic cells was detected 1 or 2 weeks after transplant as shown by the lack of co-staining between GFP and markers for cardiomyocytes (MCL2V, A), smooth muscle (alpha-SM actin, B), endothelial cells (BSL-I, C) or haematopoietic cells (CD14, D). Immunofluorescence against PCNA (E) and Ki67 (F) shows no eGFP positive proliferating cells. A representative study is depicted in the figure. Scale bars: A, C, D, E: 8 {varepsilon}m; B: 16 {varepsilon}m; F: 3.2 {varepsilon}m.

 
3.3. Adipose derived cells and BM-MNC have a positive effect on angiogenesis and fibrosis
To determine the potential mechanism(s) involved in the improvement in cardiac function and myocardial viability, we analyzed the angiogenic and vasculogenic effect of cell transplant in the injured tissue. Vascular density was determined 1 month after transplantation by quantification of the lectin-positive and {alpha}-SMA-positive vessels in the scar area. As shown in Fig. 5, the number of capillaries per mm2 was significantly increased (P<0.05) in all three cell treated groups when compared with the medium-injected controls. This increase was even more pronounced in animals treated with ADSC cells (P<0.01). No statistically significant increase in vasculogenesis was observed either in {alpha}-SM actin-positive vessel density or in the area occupied by {alpha}-SM actin-positive vessels 1 month after transplantation (not shown). Finally, transplantation of all cell types was associated with a reduced infarct size from 19.66±1.21% in the control group to 13.29±2.67% in animals receiving BM-MNC (P<0.05), 11.98±3.04 in AD-CMG (P<0.05) and 11.3±3.36% in ADSC (P<0.01) (mean±SD) (Fig. 6). The reduced infarct size was also associated with a decrease in the percentage of collagen volume fraction (CVF) from 61.05±3.98% in the control group to 50.49±7.31 (BM-MNC), 53±5.07 (AD-CMG) and 48.4±7.58 (ADSC) (mean±SD).


Figure 05
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  		Fig. 5 Angiogenesis and vasculogenesis after transplantation of BM-MNC, ADSC and AD-CMG. A: Angiogenesis as determined by the number of lectin-positive vessel per mm2 in the infarct area in the different groups of animals. Results represent the mean±SD. *P<0.05; **P<0.01 among control group and cell treated groups. B: Representative images of lectin-stained sections. Scale bars: 25 µm.

 


Figure 06
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  		Fig. 6 Infarct size. A: Infarct size was measured by Sirius red staining in the different groups of animals and quantified as the area occupied by collagen. Data represent the mean±SD. *P<0.05; **P<0.01 among control group and cell treated groups B: Representative images of Sirius Red-stained sections.

 

   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Source of funding
 References
 
Although cardiac regeneration with stem cells has become a major goal of modern cardiology, the evidence is far from conclusive and only a modest improvement in cardiac function has been demonstrated to date [5]. A number of cell types have been used in both animal models and patients. Clinically, BM-MNC have been the most commonly used source of stem cells for cardiac regeneration, probably due to the fact that they are easy to obtain, and because this cell source contains a combination of stem cell populations (haematopoietic, endothelial and mesenchymal stem cells) [32]. Unfortunately, the positive initial results of phase I/II studies have not been fully supported by more recent randomised studies [15-17,33,34]. Among other sources of stem cells, adipose tissue has been found to nurse attractive populations with regard to their differentiation capacities [35,36]. Stem cells present in the adipose tissue show a strong angiogenic potential but also contain a different and rare subset of cells which are able to spontaneously differentiate into cardiomyocytes [20,23]. The ability to obtain numerous adipose-derived cells using the easy and non-invasive procurement method from adipose tissue in adults, has made these cells a good candidate for cell therapy in ischaemic diseases [37].

In our study we compared the effect of BM-MNC with 2 populations of adipose derived cells endowed with either angiogenic potential (ADSCs) or cardiomyogenic potential (AD-CMG) in a chronic model of MI. This pathophysiological situation is characterized by the presence of scar tissue which does not lend itself to the engraftment of viable cells. Despite this disadvantageous context, we demonstrated that adipose derived cells improved heart function by increasing angiogenesis and decreasing the degree of fibrosis in the infarcted tissue. Importantly, functional evaluations as well as 18FDG-PET studies revealed the benefit induced by ADSC transplantation. On the other hand, this study also showed that little benefit is derived from transplantation of BM-MNC. Our data are consistent with the results of recent randomised clinical trials in which transplantation of BM-MNC was performed in the setting of chronic as well as acute MI [15-17].

It is reasonable to speculate that angiogenic events are involved in increasing heart viability and function. We hypothesize that the mechanism of this improvement may be due, at least in part, to paracrine induction of the neovascularization processes. It has been previously demonstrated that ADSCs secrete high levels of angiogenic factors such as vascular endothelial growth factor (VEGF), the transforming growth factor (TGF-β) and the basic growth fibroblast growth factor (bFGF), which induce endothelial cells (EC) and smooth muscle cells (SMC) growth and/or differentiation [22]. Overall, we consider that induction of vascularization is one possible explanation for the reduction of the fibrotic area and, in turn, the increase in tissue viability. We further speculate that the increased blood influx associated with cell engraftment may improve the rescue of hibernating cardiomyocytes or may even stimulate cardiac stem cells to divide and replace, to a limited extent, the lost contractile tissue. All of these effects result in an improvement of cardiac performance. On the other hand, besides a putative difference in paracrine effects, the ability of ADSCs to remodel scar tissue could also be due to their macrophage properties, as we have previously demonstrated that preadipocytes develop phagocytic properties and can function as macrophage cells thus participating in the inflammatory reaction that develops during the healing process after MI [38], which may help in the healing process by helping to reduce remodelling [39].

Another issue is the fate of the transplanted cells with different level of engraftment and persistence and the mechanism of the positive effect observed after transplant. The presence of ADSC and BM-MNC was detected 1 week post-injection, both by immunohistochemical and genomic DNA-PCR methods. Even with such a transitory survival, it is possible that these cells may have a paracrine effect in the surrounding tissue cells which further supports the paracrine hypothesis.

There are certainly some caveats to this study. First, 1 month follow up is not sufficient to detect a more robust effect of the transplanted cells on cardiac function. Furthermore, it is possible that expression of GFP may be a potential handicap for cardiac performance, as has previously been described [40]. It should also be noted that AD-CMG cells failed to improve heart function, this may perhaps be due to the scar tissue which may provide too harsh an environment for these cardiac-like cells to develop. Additional experiments where combinations of undifferentiated and cardiac pre-differentiated cells are transplanted together in the myocardium may help to improve the results. Another issue that needs to be resolved is the low level of cell engraftment even at one week after transplantation. Possible reasons for this low engraftment may include a combination of high cell leakage following injection into the myocardium together with high rates of cell-death in the first few hours [41]. The persistence of the beneficial effect at one month, despite the lack of engraftment, points to a paracrine effect as the mechanism of improvement [42], also suggesting that an increase in cell engraftment may result in more substantial benefits. We are currently undertaking longer term studies in which cells are transplanted into a matrix support, which it is hoped will favour their retention and survival. Results will certainly help us to obtain clearer results regarding the differentiation capability of these cells. Finally, the fact that we are using a xenogeneic (mouse cells into rat) model, despite immune-suppression, can also affect the degree of engraftment and in vivo differentiation. New strategies to achieve more efficient engraftment and to prevent apoptosis [43-46] are now required.

In conclusion, in spite of the limitations outlined above, which certainly need to be addressed, our results indicate that transplantation of adipose-derived cells contribute to improved LV function and tissue viability in a chronic model of MI in rats. The promising features of these progenitor cells require further evaluation in larger animal models prior to consideration for new clinical trials in patients with myocardial infarction.


   Source of funding
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
References
 
Supported in part by grants from the Fondo de Investigaciones Sanitarias PI042125, ISCIII-RETIC RD06/0010, FEDER (INTERREG IIIA), the National Research Program on cardiovascular diseases (n° PNC0402), and the Cell and Gene therapy program from the Région Midi-Pyrénées (# 03011999). This project was funded in part through the "UTE project CIMA".


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
 References
 
1 Contributed equally to this study and should be considered equal first authors. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
 References
 

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