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European Journal of Heart Failure 2007 9(6-7):558-567; doi:10.1016/j.ejheart.2007.02.003
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© 2007 European Society of Cardiology

Cellular cardiomyoplasty in large myocardial infarction: Can the beneficial effect be enhanced by ACE-inhibitor therapy?

Emerson L. Olivaresa,b,*, Ricardo H. Costa-e-Sousaa,b, João P.S. Werneck-de-Castroa,f, Vanessa Pinho-Ribeiroa, Márcia G. Silvac, Karla C. Ribeiroa, Elisabete C. Mattosc, Regina C.S. Goldenberga, Antonio C. Campos de Carvalhoa,d and Masako Oya Masudaa,e

a Instituto de Biofísica Carlos Chagas Filho UFRJ, Rio de Janeiro, CEP 21949-9000, Brazil
b Departamento de Ciências Fisiológicas, Instituto de Biologia, UFRRJ, Seropédica, CEP 23890-000, Brazil
c Ecodata Exames Médicos LTDA, Rio de Janeiro, CEP 22020-120, Brazil
d Albert Einstein College of Medicine, Bronx, New York 10461, USA
e Fundação CECIERJ, Rio de Janeiro, CEP 20943-001, Brazil
f Departamento de Biociências da Atividade Física, UFRJ, Rio de Janeiro, CEP 21949-9000, Brazil

* Corresponding author. Departamento de Ciências Fisiológicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro BR 465, Km 7, 23890-000, Seropédica, Rio de Janeiro, Brazil. Tel.: +55 21 26821210. E-mail address: elopes{at}ufrrj.br


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background: Cellular cardiomyoplasty with bone marrow derived stromal (MSC) and mononuclear (BMNC) cells has been shown to improve performance of infarcted hearts. We performed a comparative study with MSC and BMNC and tested the hypothesis that captopril treatment could enhance the beneficial effect of cell therapy in large myocardial infarctions.

Methods: Male syngeneic Wistar rats underwent experimental infarction and were randomized to receive 1–3x106 MSC, 108 BMNC or vehicle (BSS group). Two additional groups were treated with captopril and received 1–3x106 MSC (Cap.MSC) or vehicle (Cap).

Results: The ejection fraction (EF%) of MSC and BMNC-treated rats was higher than in the BSS rats, eight weeks after transplantation (33.0±4.0, 34.0±2.0 and 20.0±2.0% respectively, P<0.01). Both captopril-treated groups improved EF% similarly. But only captopril plus MSC treatment almost restored cardiac function to control levels, 8 weeks after injection (60.50±5.40% vs. 41.00±4.50% in Cap.MSC and Cap respectively, P<0.05). Many DAPI-labelled cells were found in the scar tissue of the left ventricle only in the Cap.MSC group.

Conclusions: Cell transplantation with both MSC and BMNC produced a similar stabilisation of heart function, but the success of the cell engraftment and the recovery of cardiac performance were dependent on concomitant treatment with captopril.

Key Words: Cell therapy • Stem cells • Myocardial infarction • Captopril

Received July 21, 2006; Revised October 6, 2006; Accepted February 6, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Bone marrow cell transplantation, a relatively new approach based on implantation of donor cells into damaged myocardium, has been identified as a promising strategy to restore the functional competence of the damaged heart [1-3]. This is an interesting therapeutic alternative considering the limitations of currently available therapies in patients with congestive heart failure and the limited capacity of the mammalian heart to replace damaged myocardium. Several cellular types have already been tested, but the optimal choice still needs to be determined. In recent years, bone marrow derived progenitor cells (BMPC) have become one of the most promising cellular candidates. These undifferentiated cells have been reported to transdifferentiate into cellular components of the heart, as cardiomyocytes and vascular cells, and ultimately recover heart function [1-3]. Moreover, from a clinical standpoint, the use of BMPC is very attractive because of the possibility of using the patient's own cells, thereby avoiding the need for immunosuppressive therapy.

Although the therapeutic effects of these cells have already been recognized, it is still not known whether a specific sub-population in the bone marrow cell pool is actually responsible for these effects. Many groups have isolated the so called "mesenchymal stem cells" (MSC) from the bone marrow, using different protocols in vitro [2,4,5], but the question of whether culture procedure is critical to cell therapy is controversial. From a clinical point of view, because unfractionated mononuclear bone marrow cells (BMNC) contain both haematopoietic and non-haematopoietic cells and can be easily obtained, avoiding cell culture manipulation and therefore minimizing contamination or cell death, it has been suggested that perhaps the BMNC may be more appropriate for cell therapy [6].

Although some studies observed either no improvement [7] or even a decrease [8] in heart function after cell therapy in chronic infarction models, most have suggested safety with diverse levels of improvement in cardiac performance after transplantation of BMNC, both in experimental infarction [9,10] and in patients with chronic myocardial infarction [11].

In line with this, our group has demonstrated the safety and therapeutic potential of BMNC therapy in post-ischemic patients with severe ventricular dysfunction [12,13]. In addition, we verified that cell therapy with MSC improved myocardial function in healed infarcted rat hearts [2]. However, a comparative study between the therapeutic potential of MSC and BMNC in large healed myocardium infarction (more than 40% of scar tissue in left ventricle) is still lacking.

Despite several approaches to enhance the beneficial effects of cell therapies, such as gene transfer of angiogenic [14] and anti-apoptotic [4] factors, studies associating cell therapy with classical pharmacological approaches with recognized beneficial effects on the remodelling process and heart function, such as the use of angiotensin converting enzyme inhibitors (ACEi) [15], have not been systematically tested.

Therefore, we used a rat model of heart failure, obtained by permanent coronary occlusion resulting in large myocardial infarctions, to compare the therapeutic potential of MSC and BMNC, and also to test the possible synergistic effect of adding an ACEi.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the institutional animal welfare committee.

2.1. Animals
Male inbred Wistar rats (200 to 250 g) were obtained from Centro de Pesquisa Gonçalo Muniz (Fiocruz-Bahia/Brazil). Animals were housed at controlled temperature (23 °C) with daily exposure to 12 h light-dark cycles and free access to water and standard rat chow. These isogenic rats were used as both donors and recipients of the bone marrow cells to simulate clinical autologous implantation.

2.2. Cell isolation and culture procedures
The bone marrow derived mononuclear cells (BMNC) were obtained from donor isogenic rats (80-100 g). The animals were anesthetized with ethyl ether (Reagen) and killed by cervical dislocation. Bone marrow was aspirated from femoral and tibial bones with a syringe containing Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Grand Island, NY) with an 18-gauge needle. The marrow cells were homogenized and centrifuged at 450 xg for 10 min. Afterwards this cell suspension was loaded on a gradient of Ficoll-Hypaque (Sigma, St. Louis, MO) and centrifuged at 800 xg for 30 min. Cells retained on the Ficoll-medium interface were washed 3 times with buffered salt solution (BSS).

The isolation and primary culture of marrow stromal cells (MSC) was performed according to Caplan [16]. Briefly, BMNC were harvested from bone marrow as described before and cultured in DMEM supplemented with 20% fetal bovine serum (GIBCO BRL), 2 mM L-glutamine (Sigma), antibiotics (100 U/ml Penicillin G; 100 µg/ml Streptomycin, GIBCO BRL) and maintained at 37 °C in a 5% CO2 incubator. The cells were maintained for one week and medium was changed at least twice during this period, which washed away all floating haematopoietic cells. At approximately 80 to 90% confluence, the cells were detached from the culture flasks with 0.25% trypsin-EDTA (sodium ethylenediaminetetraacetic acid, Sigma), resuspended in DMEM and labelled with 4', 6-diamidino-2-phenylindole (DAPI, Sigma) for 30 min (37 °C). The cells were rinsed 3 times in BSS to remove unbound DAPI and kept in warm BSS for a few minutes before transplantation. This labelling protocol was also used for BMNC and in both cases was very efficient, ensuring close to 100% labelling of cell nuclei.

2.3. Myocardial infarction and cell transplantation
Left ventricular myocardial infarction (MI) was induced following the procedure described by Johns and Olson [17]. Briefly, male Wistar rats (200 to 250 g) were anesthetized with Halothane PA (Merck, Deutschland) and a 2 cm incision was made on the left side of the thorax, parallel to the sternum. The fifth and sixth ribs were separated, exposing the heart, and the left anterior descending coronary artery was permanently occluded just underneath the left atrial appendage by ligation with a 6-0 silk thread. The chest was then closed with continuous silk stitch and the rats were allowed to recover.

For the injection procedure, the animals were anesthetized with Ketamine (50 mg/kg) and Xylazyne (5 mg/kg) administered intra-peritoneally and maintained under positive pressure ventilation with room air supplemented with oxygen (2 l/min, Harvard Apparatus Rodent Respirator — model 680) at 60 to 80 cycles/min, with a tidal volume of 1 ml/100 g of body weight. The heart was exposed, the macroscopic scar area was visualized and 200 µl of a 108 BMNC or 1-3x106 MSC suspension was injected with a tuberculin syringe into two to three sites along the borders of the left ventricular wall scar tissue. The same volume of BSS was injected into two to three points on corresponding areas in the hearts of the control animals. After treatment with cells or BSS, the chest was stitched closed and the animals were carefully monitored for 4 h postoperatively.

The experimental mortality rate was 30% within 24 h following infarction surgery and 26% within 24 h following cellular or saline-injection.

2.4. Functional assessment
2.4.1. Electrocardiographic study
The electrocardiogram (ECG) was performed as described before [2,18]. In brief, rats were anesthetized with Ketamine and Xylazyne and placed in supine position for ECG recording (Cardimax FX-2111 — Fukuda Denshi) in six limb leads (L1, L2, L3, aVR, aVL, aVF) and two chest leads (Vant recorded half-way from the sternal manubrium and the xyphoid process and Vlat, recorded at the mid-axillary line, at the same height as the first chest lead). In order to obtain reproducible ECG recordings, the anesthetized animals were fixed to a board with their anterior paws in orthogonal direction with respect to the body and posterior limbs were kept free. Additionally, all animals were carefully examined at the time of medium or cell-injection (in the infarcted group) and post mortem (all groups) in order to detect alterations in the anatomical relations of the heart in the thoracic cage that might lead to misleading results in the evaluation of the ventricular depolarization vector angle (ÂQRS). ECG was recorded for 3 min after which the anesthetized animals were subject to echocardiographic examination. During the 5-15 min duration of the echo exam we continuously recorded a L2 lead ECG.

The ECG parameters studied were: heart rate, presence of a P wave larger than 0.1 mV, PR interval, QRS duration, QT interval, frontal QRS axis (ÂQRS), QRS amplitude index (I-QRS, the sum of QRS complex voltage in L1, L2 and L3) and presence of Q wave in L1, L2, L3, aVF, Vant and Vlat. In all infarcted animals (n=34) the presence of an ÂQRS larger than 90° (right deviation) and the presence of Q wave in L1 were used as evidence of an extensive infarction as demonstrated by post-mortem pathological analysis that confirmed the presence of transmural scar tissue larger than 40% of the left ventricle. Thus, the data for ÂQRS were represented as frequency of animals with ÂQRS larger than 90° (Right) or between 0 and 90° (Left).

2.4.2. Echocardiographic study
The echocardiogram (ECHO) was performed as described before [2]. In this study we used an echocardiographic colour-system (Megas/Esaote) equipped with a 10 MHz electronic-phased-array transducer. Under Ketamine and Xylazyne anaesthesia, the chests of the animals were shaved and they were maintained either in left lateral decubitus or supine position. All ECHO analyses were performed blind by the same echocardiographer and included morphologic and functional parameters. Images were obtained from the left parasternal and apical windows. Short-axis 2-dimensional views of the left ventricle (LV) were taken at the level of the papillary muscles to obtain the M-mode recordings. Anterior and posterior end-diastolic and end-systolic wall thickness, LV, left atrium and aorta internal dimensions (LA/Ao) ratio, and relative wall thickness (RWT, 2x posterior end-diastolic wall thickness/LV internal end-diastolic dimension, LVEDd) were measured following the American Society of Echocardiography (ASE) leading-edge method. Systolic function was expressed as ejection fraction (EF%), calculated by Simpson's method, after left ventricular (LV) volume calculation: systolic and diastolic LV long axis were measured on the long-axis view and systolic and diastolic LV short axis, traced at papillary muscle level, were measured on transverse view. The pulsed-wave Doppler spectra of mitral inflow were recorded from the apical four-chamber view with the guidance of the colour Doppler. All Doppler spectra (mitral flow velocity pattern: peak early diastolic filling velocity, E velocity; peak filling velocity at atrial contraction, A velocity; and their ratio: E/A) were recorded and morphologic parameter values were measured during the echocardiographic exam.

2.5. Post-mortem study
The rats were anesthetized with ether and killed by cervical dislocation 13 weeks post-surgery. The pleural effusion was collected by needle aspiration and its volume was measured by gravimetric method [19]. This procedure was performed blind by the same researcher and the data are shown as the number of animals with pleural effusion volume >0.1 ml. The heart, lung and liver were removed and weighed corrected according to the body weight of the animal, then expressed as heart, lung and liver index respectively.

2.5.1. Histopathology and immunohistochemistry
After macroscopic analysis, the heart was perfused with 4% paraformaldehyde in phosphate buffer. The percentage of scar tissue in LV was calculated as described by Spadaro et al. [20]. Briefly, the left ventricle was cut into four slices from apex to base. The slices were approximately the same thickness (1-2 mm) and were labelled slices A (at the apex), B, C and D, respectively. Histological analysis with hematoxilin-eosin and Picrosirius staining was performed in representative sections obtained from slice C, described as the most representative [20] and confirmed by a previous study from our group (unpublished data) of the total infarcted length, using an Axiovert 100 microscope (Zeiss Inc. Germany). Sections stained with Picrosirius were recorded with a digital camera and stored for later analysis. The digital files were analyzed with the ImageJ software (version 1.27 z, National Institutes of Health, USA) which allowed quantification of the relative infarct size of the left ventricle. The length of the infarcted endocardium is measured, as well as the total perimeter of the endocardial surface. From the ratio of these values, the percentage of infarcted endocardium was calculated. The same procedure was used for the epicardial surface, to obtain the value for the percentage of infarcted epicardium. From these two values the average percentage infarct size was estimated.

The remaining heart sections were cryoprotected, cooled in liquid nitrogen and 10 µm consecutive cryostat sections were obtained. Thereafter, the BMNC and MSC were identified by DAPI-labelled nuclei using an Axiovert 135 microscope coupled to a high resolution Axiocam HR CCD video camera (Zeiss, Germany). The sections were processed for immunofluorescence using a monoclonal anti-{alpha}-smooth muscle actin antibody (clone 1A4, Dako A/S, Denmark) and a monoclonal anti-{alpha}-sarcomeric actin (clone 5C5, Sigma). Both antibodies were used at 1:100 dilutions. Goat secondary antibodies from Sigma were used: anti-mouse IgG FITC conjugate (F-2012), and anti-mouse IgM FITC conjugate (F-9259) both at 1:50 dilutions. Fluorescence images were obtained from regions of the infarcted left ventricle displaying both nuclear and cytoskeletal markers using an Axiovert 135 microscope coupled to a high resolution Axiocam HR CCD video camera (Zeiss, Germany). Images were overlaid and processed using Photoshop 7 software (Adobe, USA).

2.6. Experimental protocols
This study was conducted in two parts. In the first part, the infarcted rats (n=22) were randomized to injection with 1-3x106 MSC (MSC group, n=7), 108 BMNC (BMNC group, n=9) or vehicle, BSS, (BSS group, n=6). The first functional assessment (baseline) was performed four weeks after surgery, immediately prior to cell transplantation or BSS injection. Two additional recordings were made four and eight weeks after treatment with cell or vehicle.

In the second part, twelve infarcted rats received captopril (Extracto Vital®, 2 g/l administered in the drinking water) [15] immediately after surgery and continuously up to the end of the experiment (thirteen weeks after surgery). Four weeks after infarction surgery, these 12 animals were randomly selected to receive either 1-3x106 MSC (Cap.MSC, n=6) or BSS injection alone (Cap, n=6). The serial functional assessments were performed at the same time points as in the first part of the study. Histopathology was done at the end of the study (nine weeks after any treatment) in both parts of the study.

2.7. Statistical analysis
All values are expressed as mean±SEM. Differences between electrocardiographic and echocardiographic measurements at baseline and 4 or 8 weeks after vehicle or cell-treatment and among groups were evaluated by two-way analysis of variance. The results obtained from the post-mortem study were analyzed by one-way analysis of variance. In both tests Graphpad Prism software (version 4, Graphpad Software, Inc., San Diego, USA) was used. Values were considered different when P<0.05 after Bonferroni's correction. When frequency data were compared, the non-parametric Fischer exact test with a 0.05 level of probability was used.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Functional assessment
3.1.1. Electrocardiographic study
The ECGs recorded in the first part of the study were not significantly different between the BMNC, MSC and BSS groups at any time of observation and resembled a large myocardial infarction. There was a significant rightward deviation of the frontal QRS axis (ÂQRS), the Q wave was observed in L1 and Vlat and the I-QRS decreased in all 22 animals compared with data obtained in 33 sham-operated and 34 normal rats (data not shown).

In the second part of the study, the ECG tracings in the 12 captopril-treated animals showed improvement in some parameters compared to the infarcted untreated rats (BSS group). The most outstanding effect was the attenuation of the rightward deviation of the QRS, in all captopril-treated rats, four weeks after infarction (baseline). However, the beneficial effect of captopril treatment on ÂQRS did not persist in all animals. Only those animals treated with captopril plus MSC (Cap.MSC) maintained normal ÂQRS 4 and 8 weeks after cell transplantation (Fig. 1). The animals treated solely with captopril (Cap) displayed a rightward deviation comparable to the BSS group in the two subsequent ECG evaluations (4 and 8 weeks later). All other ECG parameters did not differ significantly between the three groups (data not shown).


Figure 01
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  		Fig. 1 Frontal QRS axis (ÂQRS) assessed by ECG in BSS, Cap and Cap.MSC groups before (baseline), and four and eight weeks after cell therapy or BSS injection. Dotted line represents the limit for normal maximal value of ÂQRS in rats. *P<0.05 vs. Control and #P<0.05 vs. BSS and Cap.

 
3.1.2. Echocardiographic study
Heart rate was continuously monitored during echocardiography and was not statistically different among the groups studied, ranging from 250 to 300 beats per minute under anaesthesia in both parts (data not shown).

All groups (BSS, BMNC and MSC) had similar baseline echocardiographic values (four weeks after myocardial infarction) and clear signs of severe heart failure, in the first part (Table 1). A significant increase in LA/Ao ratio, LVEDd and E/A ratio (restrictive mitral flow) as well as a significant decrease in both RWT (relative wall thickness) and EF% was observed in all animals in the infarcted group when compared to the sham (n=33) and normal (n=34) values described in our previous study [2]. No major changes were detected in other ECHO parameters (data not shown).


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  		Table 1 Echocardiographic data for BSS, BMNC and MSC groups obtained in Part 1

 
As shown in Table 1, cell transplantation with BMNC or MSC did not reverse the enlargement of the left atrial and ventricular chambers induced by the infarction, 4 and 8 weeks after treatment. However, while LA/Ao ratio progressively increased at weeks 4 and 8 in the BSS group, this parameter remained stable in both the BMNC and MSC groups. The relative wall thickness was not different among groups after myocardial infarction.

In addition, whereas the BSS group showed a progressive decrease in EF% after BSS injection, systolic function in the BMNC and MSC-treated rats stabilized after cell transplantation (Table 1). The difference between the EF% values attained 8 weeks after BSS injection and cell injection (BMNC or MSC) was highly significant (P<0.01).

Doppler analysis did not show any differences in mitral flow pattern between cell-treated (BMNC or MSC) and BSS-injected rats. E/A velocity ratio>3.0, described as "restrictive flow pattern" [2,19,21,22], was observed in all groups independent of the period of observation (data not shown).

In Part 2, the captopril-treated groups showed a significant decrease in LVEDd (Fig. 2B) and posterior wall thickness in diastole (PW — Fig. 2C), compared to the BSS group, four weeks after myocardial infarction (baseline). The RWT (Fig. 2D) and LA/Ao ratio (Fig. 2A) did not differ among the groups in the same period. However, while the captopril plus MSC treatment (Cap.MSC) decreased LA/Ao ratio to normal values early (4 weeks after treatment), the animals treated solely with captopril normalized this parameter only 8 weeks after BSS injection. Regarding LV geometry, both captopril-treated groups showed a clear decrease in PW and LVEDd, but only Cap.MSC increased RWT significantly 4 and 8 weeks after cell transplantation.


Figure 02
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  		Fig. 2 Left atrium-to-aorta diameter (LA/Ao) ratio (A), left ventricular end-diastolic diameter (LVEDd, B), posterior wall thickness in diastole (PW, C) and relative wall thickness (RWT, D) assessed by echocardiography in BSS, Cap and Cap.MSC groups before (baseline), and four and eight weeks after cell therapy or BSS injection (control). Although Cap and Cap.MSC prevented an increase in LA/Ao ratio, LVEDd and PW, only Cap.MSC showed normal values of RWT four and eight weeks after cell transplantation. Dotted line represents the normal values of the respective parameter in rats. *P<0.05 and **P<0.01 vs. BSS and #P<0.05 vs. BSS and Cap.

 
As shown in Fig. 3A, systolic function (represented by ejection fraction, EF%) of the captopril-treated groups was slightly improved at baseline confirming a previous study [22]. Despite this increase in cardiac performance shown by the rats treated solely with captopril, only captopril-MSC (Cap.MSC) was effective in fully restoring heart function 4 and 8 weeks after cell transplantation. Fig. 3B shows representative M-mode tracings of the infarcted rats treated with captopril before and eight weeks after BSS (Cap) or MSC (Cap.MSC) injection. Note a clear improvement in left ventricular contractility in Cap.MSC, which, in this and in most animals studied, was particularly striking in the anterior wall. One echocardiogram from an infarcted non-treated rat (representative of the BSS group) is shown for comparative analysis.


Figure 03
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  		Fig. 3 Ejection fraction by Simpson's method (A) and representative images from M-Mode recordings (B) assessed by echocardiography in BSS, Cap and Cap.MSC groups. The captopril-treated groups showed a significant increase in EF% throughout the period of observation compared to BSS, but ejection fraction only fully recovered in the group treated additionally with cell therapy. Dotted line represents the normal values of the EF% in rats (A). Note in B, a decrease of left ventricle inner dimension (clear during the systole) in Cap.MSC compared to BSS. *P<0.05 and **P<0.01 vs. BSS within the same week and #P<0.05 vs. Cap at 4 and 8 weeks.

 
Doppler analysis performed in the Cap and Cap.MSC groups showed a low frequency of rats with E/A velocity ratio >3.0 compared to the BSS group at all observation times (data not shown). The normalization of the E/A ratio after treatment with angiotensin converting enzyme inhibitors alone has been described before [22].

3.2. Post-mortem study
The hearts from all animals were carefully examined post mortem and none were found to present either tissue adherence or abnormal heart position in the thoracic cage that might lead to misleading interpretation of electrocardiographic parameters, mainly in the ÂQRS axis.

Table 2 shows the macroscopic study undertaken in both Parts of this study. Data from a sham-operated group (animals subjected to the same surgical procedure as the infarcted group but the left coronary artery was not ligated) are also represented for comparative analysis. As expected, after the severe ventricular damage in this model, heart and lung indexes were increased in all rats from the BSS group, which also showed pleural effusion during the necropsy procedure. In contrast, the BMNC and MSC groups exhibited a decrease in lung index despite no changes in heart index compared to the BSS group. Captopril treatment seems to be effective in decreasing both heart and lung indexes and in eliminating the pleural effusion, since in all animals from the Cap and Cap.MSC groups these alterations were not present. No changes were observed in liver index.


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  		Table 2 Post-mortem parameters in all groups nine weeks after cell transplantation or BSS injection

 
3.2.1. Histopathology and immunohistochemistry
In Part 1, gross examination showed a large transmural post-infarct scar in all hearts from all groups, nine weeks after cell transplantation or BSS injection (Fig. 4A). Curiously, despite the same level of LV dilatation, the thickness of the scar tissue was increased in the BMNC and MSC groups compared to the BSS group (Fig. 4B). Few cells (less than 0.01% of the injected cells) were engrafted outside and within the scar tissue as detected by DAPI labelled nuclei, and there was no evidence of differentiation into cardiomyocytes or blood vessels either in BMNC or MSC treated animals (data not shown).


Figure 04
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  		Fig. 4 Percentage (A) and thickness (B) of scar tissue in left ventricle and cross sections of heart slices stained with picrosirius-red (C) in BSS, BMNC, MSC, Cap and Cap.MSC groups. One slice from a representative sham-operated animal is shown for comparison. The infarct (stained red) contains regions of cardiomyocytes (stained yellow) in all cell-treated groups. Note the thickening of the scar in BMNC and MSC. Although all captopril-treated rats showed a small scar in the left ventricle, only captopril plus cell therapy increased thickness of scar tissue. *P<0.05 vs. BSS and #P<0.05 vs. BMNC, MSC and Cap.MS.

 
In Part 2, infarct size was decreased in both the captopril-treated groups compared to the BSS group (Fig. 5A). However, only Cap.MSC increased the thickness of scar tissue nine weeks after cell transplantation (Fig. 4B). The effectiveness of the captopril plus MSC-treatment in increasing the scar thickness resembles that of the BMNC and MSC-treated animals and contrasts with the results obtained in the infarcted group treated with captopril plus BSS (Fig. 4B). Fig. 4C shows representative cross sections of heart slices taken at papillary muscle level, obtained from BSS, BMNC, MSC, Cap and Cap.MSC rats nine weeks after cell treatment or BSS injection. An additional image from a sham-operated animal is also represented for comparative analysis of cardiac morphology.


Figure 05
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  		Fig. 5 Representative cross section of a heart slice from a Cap.MSC treated animal stained with picrosirius-red (A) and fluorescence images (B, C and D) obtained from regions of the infarcted left ventricle. Note many DAPI-labelled cells (blue) in the antero-lateral left ventricular wall scar tissue nine weeks after cellular cardiomyoplasty with MSC. Scale bar, 84.37 µm.

 
In contrast to the BMNC and MSC groups, DAPI-labelled transplanted cells could be easily identified in consecutive sections obtained from the different portions of the left ventricle of the Cap.MSC animals. We determined an average of 5x104 DAPI-labelled nuclei in a 6-mm2 area around the scar (which should account for the localization of most of the injected cells), suggesting that 1.5-5% of the injected cells were still present in the heart nine weeks after cell injection.

Fig. 5 shows a representative image of many DAPI-labelled nuclei in the left ventricular wall from an infarcted rat heart treated with captopril plus MSC nine weeks after cell transplantation.

Positive immunostaining for smooth muscle co-localized with DAPI labelled nuclei in individual smooth muscle cells from the tunica media of coronary vessels. However, it was a rare event, and not all the cells presenting with both nuclear and cytoskeletal fluorescent labels seemed to have achieved a fully differentiated state. When this was the case, cell shape was round and the cells were frequently found in groups or distributed along the cardiomyocytes.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The present investigation demonstrates that direct transplantation of bone marrow derived mononuclear (BMNC) or stromal (MSC) cells into the ventricular wall of rats with large myocardial infarcts and chronic heart dysfunction, stabilizes cardiac performance at a time when a fibrous scar was already present and severe heart failure had developed. Cell transplantation avoids the further progression in functional degeneration observed in vehicle treated infarcted rats. Treatment with captopril, starting in the early phase of cardiac remodelling and given continuously thereafter, in addition to a single dose of 1-3x106 MSC, fully restored the functional competence of the infarcted rat hearts.

In a previous report [2] we showed that cellular cardiomyoplasty using 1-3x105 MSC alone was sufficient to improve cardiac performance. ECG and ECHO parameters in MSC-treated animals improved significantly when compared to vehicle-treated animals, suggesting the involvement of MSC in the chain of events that ultimately led to the improvement in cardiac function. What then is the reason for the difference between the results observed in our previous investigation [2] and in this study, which did not show a full improvement in heart function after MSC treatment alone? One possibility is the difference in infarct size: scar tissue represented 30% of the left ventricle in our previous report, while in the present study, it corresponded to 47%. As a consequence, it is possible that cardiac dysfunction, haemodynamic alterations and cardiac remodelling may have developed at different levels at the time of cell transplantation. Indeed, classical studies, such as that by Pfeffer et al. [23] have demonstrated significant differences in geometry and heart function between small and large infarctions induced by left coronary occlusion in rats. Therefore, we suggest that the enhanced left chamber deformity (remodelling) observed in the present report might account for the limitations in cardiac improvement after BMNC or MSC transplantation. This hypothesis is supported by Chiu [24], who suggested difficulties in engraftment of any cellular type in large myocardial infarctions.

Overall, the functional stability elicited by BMNC or MSC transplantation in this "inhospitable" scenario, contrasting with the progressive decrease in cardiac performance presented in infarcted non-treated rats, can still be considered advantageous from the clinical point of view. In this case, albeit beyond the scope of this study, we agree with other reports which propose a paracrine action of bone marrow cells in large myocardial infarctions. The paracrine release of growth factors, such as vascular endothelial (VEGF) or basic fibroblast (bFGF) growth factors could be more important than the transdifferentiation into muscle or blood vessel [5,25,26]. Supporting this idea, Kinnaird et al. [25] working with a murine hind limb ischemia model, demonstrated that injection of 1x106 MSC 24 h after ischemic insult enhanced collateral flow recovery and improved limb function. Labelled MSC were seen dispersed between muscle fibres but were not incorporated into mature collaterals. Recently, Uemora et al. [5] demonstrated that bone marrow stem cells protect ischemic murine myocardium through paracrine effects, i.e., release of survival factors, such as Akt and eNO, which prevent apoptosis in cardiomyocytes adjacent to the infarcted area. These data indicate that paracrine signalling is an important mediator of bone marrow cell therapy in tissue ischemia, and that cell incorporation into vessels is not a prerequisite for these effects. Fujii et al. [26] transplanted smooth, ventricular or skeletal muscle cells into hearts infarcted by cryoinjury, and observed greater elastin content and better LV function compared to controls, twelve weeks after transplantation of the three cellular types. They concluded that cardiac function preservation and the high elastin content was due to paracrine factors released by transplanted cells.

Based on the results from cell therapy studies and considering future clinical applications, we speculated that the microenvironment for cell engraftment could be ameliorated with standard therapy using an angiotensin converting enzyme inhibitor (captopril). As expected, continuous captopril treatment decreased infarct size and improved diastolic filling evaluated by ECHO four weeks after infarction. The role of captopril treatment on large myocardial infarction has previously been demonstrated [22].

Since cardiac function only fully recovered in the rats treated with the combination of captopril plus MSC, we suggest that MSC and ACEi act synergistically to prevent or diminish myocardial remodelling, thus restoring and maintaining myocardial performance (ejection fraction ~60%). Another important observation is that the rats treated with captopril plus MSC normalized the ventricular depolarization vector (ÂQRS) 4 and 8 weeks after cell transplantation, in contrast to the group that did not receive cell transplantation which exhibited a sustained ÂQRS deviation to the right.

Fujii et al. [26] demonstrated that cell therapy preserved ventricular function in a similar way to an ACE inhibitor at 5 weeks. But in contrast, only cell therapy and not the ACE inhibitor, continued to be effective 12 weeks after infarction induced by cryoinjury. The authors suggested that the beneficial mechanism of cell transplantation was related to modifications in the remodelling process driven by the preservation of the elastic components (based on elastin measurement) in the myocardium. The transplanted cells modified the surrounding matrix and preserved or produced elastin, both in the centre of the infarct and in the border area surrounding the cryoinjury. Thus, cell transplantation prevented thinning and dilatation, which are hallmarks of cardiac remodelling after cryoinjury. Despite differences in the injury model and the cell type used, our study and that of Fujii can be considered equivalent in some respects. First, cardiac function was preserved by ACEi-treatment only at 4 weeks and not at 12 (in Fujii's study) or 8 (in our study) weeks after treatment. Second, cell transplantation was effective both early (4 weeks) and late (12 or 8 weeks) after treatment. Third, while cell transplantation prevented both thinning and dilatation in Fujii's study, in our study the cell therapies only prevented the thinning, without affecting ventricular dilatation. It is possible that the different models employed may account for this divergence. Finally, as suggested by the authors, normalization of the thickness and elasticity of the ventricular wall, preserved ventricular function and minimized cardiac dysfunction.

It is rational to assume that better results could be obtained if the scar tissue was also decreased after a large ischemic insult. This approach was pursued with the captopril treatment. In this new-scenario for cellular grafting, the beneficial effect of cell transplantation on ventricular function was enhanced. Boyle et al. [27], using the same model of coronary artery ligation in rats, demonstrated the efficacy of using a combination of standard medical therapy, i.e. angiotensin converting enzyme inhibitors and beta-blockers along with endothelial progenitor cells. The combined therapy resulted in a greater overall improvement in LV function on echocardiography than either therapy alone, over a short follow-up period (two weeks). Working with MSC in large myocardial infarction, we have extended the follow-up period to eight weeks after transplantation and conclude that cardiac function in the combined therapy (MSC+ACEi) group was not only higher than in the other experimental groups, but also closer to values obtained in sham-operated rats (EF% ~70-75). Whether other cell types have the same potential as MSC in combined therapy remains to be established.

Cell transplantation is currently undergoing evaluation in clinical trials. Although several questions remain unresolved, we consider that this study supports the hypothesis that cell transplantation with BMNC or MSC may stabilize heart function, and diminish dysfunction of the infarcted heart. In addition, there is a synergistic effect between MSC and captopril therapy, which restores ventricular geometry and improves cardiac performance in a rat model of heart failure induced by myocardial infarction. In further studies, it would be interesting to identify the paracrine factors released by bone marrow progenitor cells in this model. It would also help to elucidate and understand the mechanistic basis of the synergism between two completely different therapeutic approaches, i.e., ACEi-treatment and cellular cardiomyoplasty with bone marrow derived adult stem cells.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
Research supported by grants from FAPERJ, Instituto do Milênio de Bioengenharia Tecidual-MCT and NIH (RO1 HL73732-01). V.P.R. and K.C.R. are fellows from CAPES-MEC. E.L.O. and J.P.S.W.C. are fellows from CNPq.


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

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 Am. J. Physiol. Heart Circ. Physiol.Home page
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