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Cardiovascular Research Advance Access originally published online on November 21, 2007
Cardiovascular Research 2008 77(3):525-533; doi:10.1093/cvr/cvm077
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007. For permissions please email: journals.permissions@oxfordjournals.org

Stable therapeutic effects of mesenchymal stem cell-based multiple gene delivery for cardiac repair

Jiang Shujia, Husnain Khawaja Haider, Niagara Muhammad Idris, Gang Lu and Muhammad Ashraf*

Department of Pathology and Laboratory Medicine, University of Cincinnati, 231-Albert Sabin Way, Cincinnati, OH 45267-0529, USA

* Corresponding author. Tel: +1 513 558 0145; fax: +1 513 558 0807. E-mail address: muhammad.ashraf{at}uc.edu

Received 26 January 2007; revised 23 October 2007; accepted 7 November 2007

Time for primary review: 25 days


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: We have previously shown that transplantation of mesenchymal stem cells (MSCs) co-overexpressing angiopoietin-1 (Ang-1) and Akt prevented cell apoptosis, enhanced angiogenesis, and improved left ventricular heart function. The present study was designed to determine the persistence of therapeutic benefits on longer term basis.

Methods and results: Acute myocardial infarction model was developed in 30 young female Fischer-344 rats by permanent ligation of the left anterior descending coronary artery. The animals were grouped (n = 10) to receive 70 µL Dulbecco’s modified Eagle’s medium (DMEM) without cells (DMEM group 1) or containing 3 x 106 non-transduced male MSCs (MSC group 2) or transduced MSCs co-overexpressing Ang-1 and Akt (MAA group 3). The injections were carried out intramyocardially in the free wall of left ventricle at multiple sites. Three months after cell transplantation, real-time polymerase chain reaction for the rat sry gene, confocal imaging, and immunohistochemical studies revealed the extensive survival and myogenic differentiation of the PKH67-labelled cell graft. Blood vessel density was significantly higher in the MAA group (P < 0.05) at 3 months compared with the other groups. Blood vessel maturation index as determined by double-fluorescent immunostaining for vWFactor VIII and smooth muscle actin showed that most of the newly formed vessels matured to develop a smooth muscle covering in MAA group. Sonographic assessment of heart function showed that heart function deteriorated in the DMEM group, whereas the functional benefits were stable over a period of 3 months following transplantation of transfected cells.

Conclusion: Engraftment of genetically modified MSCs co-overexpressing Ang-1 and Akt produced long-term histological and functional benefits in an infarcted heart.

KEYWORDS Apoptosis; Cell differentiation; Gene therapy; Stem cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Funding
 References
 
Marrow-derived mesenchymal stem cells (MSCs) are multipotent1 and their cytotherapeutic application for myocardial repair has shown promise in both experimental animal heart models and human studies.2–6 Despite encouraging results in short-term infarct repair, their longer term therapeutic effects remain less studied. A recent study has shown that the therapeutic benefits in terms of improved cardiac function after MSCs transplantation were transient and were related to their paracrine effects.7 These therapeutic benefits were lost over longer time period of observation. In other cases, the transplanted bone marrow stem cells were not even located at the site of the cell graft at 1 month after transplantation in rats, although improvement of heart function was observed.8 We posit that genetic modification of donor cells prior to transplantation may result in more stable and sustained prognosis.

MSCs are excellent carriers of therapeutic genes which in turn also support their survival besides angiomyogenic response post-transplantation in the animal heart.9–11 MSCs transduced to overexpress Akt transgene prevented apoptosis of the cell graft and enhanced their survival by many folds in an experimentally infarcted rat heart.12 Further validation of the effectiveness of Akt-transduced MSCs was performed in a porcine heart model of myocardial infarction with encouraging data.13 Contemplating that improved survival of the cell graft in the absence of restored regional blood flow to the ischaemic myocardium may be insufficient to achieve maximum functional benefits of cytotherapy, we carried out simultaneous expression of Akt and angiopoietin-1 (Ang-1) transgenes combined with MSCs for the treatment of experimentally infarcted rat heart.14 The study exploited the close interaction between Ang-1 and Akt during cell survival signalling and angiogenesis.15,16 We have successfully shown the beneficial effects of our approach for enhanced cell survival and improved cardiac function after 4 weeks of our observation. The present study has been designed to investigate the persistence of the beneficial effects of our combined therapy approach of using MSCs transplantation with Ang-1 and Akt overexpression on post-infarction failing hearts for three months. The results showed that multiple gene delivery approach combined with MSCs transplantation resulted in stable therapeutic effects in terms of survival of the cell graft, persistence of the myogenic effect, and maturation of angiogenic response for the treatment of ischaemic heart.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Preparation and characterization of mesenchymal stem cells for transplantation
MSCs purification and expansion were carried out from bone marrow obtained from young male Fischer rats (3–4 month old) as described earlier.14 Briefly, bone marrow cells were seeded into 150 mm dishes and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. After 6–8 days in culture, the non-adherent haematopoietic cells were discarded during the routine fresh medium replacement. The adherent, spindle-shaped cell population was expanded for further use during in vitro and in vivo studies. Not more than 3–4 passages were allowed for the purified MSCs propagation in vitro before transplantation.

The ex vivo expanded MSCs were analysed for their surface marker expression including CD29, CD90, CD117, CD31, and CD34 by fluorescence-activated cell sorter (FACSCalibur, Becton Dickinson) and immunostaining as described earlier.14 At least 10 000 cells for each sample were acquired and analysed using isotype-identical antibodies served as controls.

The adenoviral vector (Ad-null) without therapeutic gene (Ad-null) and the one encoding for Ang-1 (Ad-Ang-1) were provided by Dr Ge Ruowen, National University of Singapore, and Ad-vector for myristylated Akt (Ad-Akt) was provided by Dr Xu Meifeng, University of Cincinnati. These vectors were replication-deficient and were propagated in human embryonic kidney cells (HEK-293 cells), using DMEM cell culture medium supplemented with 15% FBS. The supernatant from 293 cells was collected, purified on caesium chloride gradient, and used for transduction of MSCs as described earlier.17 The Akt- and Ang-1-transduced MSCs (MAA) were characterized for co-overexpression of Ang-1 and Akt by immunostaining and reverse transcription polymerase chain reaction (RT–PCR).14

2.2 Rat model of acute myocardial infarction and cell transplantation
Young female Fischer-344 rats (n = 30) each weighing 180–200 g were used in this study. The present study conformed to 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 the protocol approved by the Institutional Animal Care and Use Committee, University of Cincinnati. All surgical manipulations were carried out under general anaesthesia.

Experimental model of acute myocardial infarction was developed by permanent occlusion of left anterior descending coronary artery, using prolene 6-0 suture.14 The animals were grouped (n = 10 per group) to receive injection of 70 µL basal DMEM without cells (DMEM group) or containing 3 x 106 non-transduced MSCs (MSC group) or 3 x 106 MSCs simultaneously over-expressing Akt and Ang-1 genes (MAA group) from male donor rats. For the identification and study of their fate after transplantation, male donor MSCs were labelled with PKH67 green fluorescent cell tracker dye, using Fluorescent Cell Linker Kit (Sigma Chemical Co., St Louis, MO, USA) as per the manufacturer’s instructions. Intramyocardial injections of donor cells or DMEM were carried out at multiple sites (an average of 4–5 sites per animal) in free wall of the left ventricle under direct vision. The chest of the animals was sutured and the animals were allowed to recover.

2.3 Physiological assessment of heart function
Transthoracic echocardiography was performed to study the change in the heart function indices for each animal. Twelve weeks after the respective treatment, at least six animals from each group were assessed for heart function. Echocardiography was performed as described previously.14 Briefly, each rat was anesthetized with ketamine/xylazine, placed in supine position, and their anterior chest wall was shaven. The heart was imaged in two-dimensional mode and recordings were obtained from the parasternal long-axis view at the level of the papillary muscles, using Compact Linear Array probe CL10-5 on HDII-5000 SONOS CT (HP). Anterior and posterior end-diastolic and end-systolic wall thicknesses and left ventricle internal dimensions [left ventricle end-systolic (LVESd) and end-diastolic LVEDd diameters] were measured according to the American Society of Echocardiology leading-edge method from at least three consecutive cardiac cycles. Left ventricle fractional shortening (LVFS) was calculated as (LVEDd–LVESd)/LVEDd x 100 and expressed as percentage. Left ventricle ejection fraction (LVEF) was calculated as following: LVEF (%) = [(LVEDd3–LVEDs3)/LVEDd3] x 100.

2.4 Real-time polymerase chain reaction for donor cell survival
Real-time PCR was performed for the quantification of sry gene on rat heart tissue samples at 3 months after cell transplantation.18 Briefly, rat heart samples from different groups were frozen in liquid nitrogen and powdered. The DNA purification was performed using Genomic DNA Isolation Kit (Qiagen), and concentration of the purified DNA was determined by spectrophotometry. Real-time PCR was performed using iQ SYBR-Green Supermix (Bio-Rad) in a Bio-Rad iQ5 optical module. The final concentration of MgCl2 used in the PCR mix was 3.5 mM. The cycling conditions were set at 3 min at 95°C for initial denaturation, 45 cycles of denaturation at 95°C for 30 s, annealing at 59.5°C for 40 s, and extension at 72°C for 50 s. The data were acquired during the extension step. Melting curves were obtained at the end of the reaction by gradually raising the temperature by 1°C/cycle (with dwell time of 15 s at each cycle) from 59.5–95°C over a period of 35 min. The primary curve method was used to calculate threshold cycle (Ct), which is defined as the cycle at which the fluorescence level reaches a predetermined threshold. Ct was measured for each reaction and used to calculate the fold change of each experimental sample compared with the control sample according to the equation: fold change=2–{Delta}Ct, where {Delta}Ct = CtMAA–CtMSC.

2.5 Histochemical and immunohistochemical studies
Using overdose of pentobarbital (100 mg/kg administered intra-peritoneally), the animals were euthanized to remove their hearts for morphological evaluation and to study the fate of the engrafted cells. After removal, the hearts were immediately excised and three transverse segments of equal thickness were cut from apex to base. Subsequently, the transverse segments were embedded in tissue-freezing medium and cut into 6 µm thick transverse sections for histochemical and immunohistochemical studies. Haematoxylin and eosin staining was carried out for structure elucidation. The images were used for infarct size measurements by dividing the sum of the planimetered endocardial and epicardial circumferences of the infarcted area by the sum of the total epicardial and endocardial circumferences of the left ventricle.18 Planimetry was performed with Image-J analysis software (version 1.366; NIH). The mean value calculated from the three cross-sections was used for statistical analysis.

The fate of the cell graft was identified by green fluorescence of PKH67 in the frozen histological sections from the hearts with myocardial infarction in MSC group 2 and MAA group 3. For elucidation of myogenic differentiation of the engrafted cells, immunostaining of tissue sections for myosin heavy chain (slow isoform), sarcomeric actin, and connexin-43 was carried out as previously described.19 The primary antibody reaction with respective antigen was detected by imaging using a Zeiss LSM 510 confocal microscope. Standard filters for FITC and rhodamine were used to detect green and red fluorescence (FITC excitation wavelength 488 nm, emission band pass filter 505–550 nm; rhodamine excitation 543 nm, emission long pass filter 560 nm). In some cases, fluorescence microscopy was performed with an Olympus BX41 (Olympus America Inc., Melville, NY, USA) equipped for epiflouresence microscopy, and images were recorded using a digital camera with MagnaFireTM 2.1 software.

Fluorescent in situ hybridization (FISH) on the histological sections was performed using rat 12/Y-chromosome paint labelled with Cy3 fluorescent label (cat CA1631) according to the instructions of the manufacturer.

The degree of therapeutically induced angiogenesis was assessed in rat hearts from different treatment groups by measuring the blood vessel density in tissue sections as described previously.17 Briefly, tissue sections from the respective hearts were embedded in Tissue-Tek OCT compound (Miles Inc., Elkhart, IN, USA) and snap-frozen in liquid nitrogen. Frozen histological sections (6 µm thick) were immunostained using specific antibodies for von Willebrand factor-VIII (vWFactor-VIII) and smooth muscle actin. Detection of the primary antibody–antigen reaction was carried out using fluorescently labelled specific secondary antibodies. The blood vessels positive for vWFactor-VIII and smooth muscle actin were counted in both infarct and peri-infarct regions. At least 40 microscopic fields (x200 magnification) in infarct and peri-infarct regions each were randomly selected and counted in each animal group. The number of blood vessels was averaged separately for infarct and peri-infarct regions, and blood vessel density was expressed as the number of vessels per surface area (0.74 mm2).

2.6 Statistical analysis
Data analysis was performed with SPSS for Windows (version 13.0). All data were described as mean ± SEM and analysed using Student’s t-test and one-way ANOVA with post hoc analysis, and a value of P< 0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Funding
 References
 
All animals survived full length of experiment. We did not observe any deaths related with MSCs or MAA transplantation.

3.1 In vitro studies
The purified MSCs were propagated for not more than 3–4 passages before engraftment into the experimentally infarcted heart. Flow cytometry revealed that the cultured adherent cells were rich in CD29+ and CD90+ and low in CD31, CD34, and CD117 positivity (Figure 1A–E). Our repeated transduction protocol achieved >95% cells co-overexpressing Ang-1 and Akt.


Figure 1
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  		Figure 1 Analysis of bone marrow-derived mesenchymal stem cells for CD29, CD90, CD117, CD31, and CD34 surface markers to assess the purity of the cell preparation by flow cytometry. The black line as an overlay in each histogram represents the unlabelled control cells for comparison with the cells labelled for each surface marker (solid black area).

 
3.2 In vivo studies
3.2.1 Donor cell survival and myogenesis
Extensive presence of the PKH67-labelled donor cells was observed in the infarct as well as peri-infarct regions in the MSC group 2 and MAA group 3 until 3 months after cell engraftment. None of the heart tissues from the DMEM group 1 showed PKH67 fluorescence. Real-time PCR for rat sry gene showed that survival of the donor cells in the MAA group 3 was significantly higher (more than two-folds) compared with the MSC group 2 (P=0.02) (Figure 2A). No sry gene was detected in the DMEM group 1. These results indicate that the co-overexpression of Ang-1 and Akt earlier on after transplantation during the acute phase of myocardial infarction contributes to graft survival or proliferation. FISH results further confirmed the long-term survival of male donor cells at the site of the cell graft in infarct as well as peri-infarct regions (Figure 2B–C). Fluorescent microscopy for PKH67 label (green fluorescence) of the rat heart tissue sections (6 µm thickness) from the MAA group 3 and MSC group 2 of animals showed that 3 months after cell transplantation, the transplanted cells were incorporated predominantly into the peri-infarct and infarct regions, whereas some of these cells were also detected in the non-infarcted myocardium. Confocal images after immunofluorescent staining for cardiac structural proteins demonstrated that the engrafted cells developed into mature muscle fibres. This was evident from co-localization of PKH67 label and {alpha}-actinin expression (Figure 2D–G). Fluorescent immunostaining of the heart tissue sections also revealed that the presence of connexin-43 was apparent transversely between the differentiating cells both in the peri-infarct and in the centre of the infarct (Figure 2H–J). These results clearly depict the potential of the myogenically differentiating transplanted cells to electromechanically couple with the host cardiomyocytes. In our 4 week study results, we failed to observe connexin-43 expression at the site of the differentiating MSCs, which implied that for the transplanted cells to fully differentiate, longer time duration may be required.


Figure 2
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  		Figure 2 (A) Real-time polymerase chain reaction for sry gene in the female heart tissue samples at 3 months after sex-mismatched cell transplantation. The fold change in sry gene expression in different animal groups was calculated as detailed in the Materials and methods section. The results showed significantly higher survival of the cell graft in the MAA group 3 (P = 0.02) compared with the MSC group 2. (B–C) Fluorescent in situ hybridization for rat Y-chromosome showed continued survival of the male donor cells in the infarct as well as peri-infarct regions (white arrowheads). (D–G) Confocal images of the rat heart tissue sections after fluorescent immunostaining for {alpha}-actinin expression [(D) red fluorescence] showed myogenic differentiation of PKH67-labelled MAA [(E) green fluorescence] at 3 months after engraftment. (F–G) represents merged image of (D) and (E) which reveals mingling of red and green fluorescence. (H–J) Fluorescent immunostaining for connexin-43 (red fluorescence) of the rat heart tissue after PKH67-labelled MAA (green fluorescence) transplantation in the (H) peri-infarct and (I) infarct region. (J) Newly developing fibres (green fluorescence) with intercellular connexin-43 (red fluorescence) in the centre of the infarct. Magnification: oil immersion.

 
3.3 Blood vessel density
Fluorescent immunostaining of the infarcted myocardium for vWFactor-VIII expression showed augmentation of neovascularization in the cell-transplanted groups of animals (Figure 3A–G). At 3 months after cell or DMEM injection, blood vessel density per surface area (0.74 mm2) in the infarct and peri-infarct zones was significantly greater in the MAA group 3 (76.57 ± 4.25; 182.19 ± 7.12, P < 0.05) compared with the MSC group 2 (63 ± 5.15; 101 ± 6.44) and DMEM group 1 (62.3 ± 4.51; 79.5 ± 5.47). A comparison of these results with our 4 week study convincingly showed the persistence of angiogenic response at 12 weeks after cell engraftment (Figure 3G). This observation was further confirmed after double-fluorescent immunostaining for vWFactor-VIII and smooth muscle actin expression which showed that most of the newly formed vascular structures matured to develop smooth muscle covering, both in the scar and in the peri-scar areas (Figure 4A–F). Although the maturation index (based on positivity of vessels for vWFactor-VIII and smooth muscle actin expression/total number of blood vessels) was higher in the DMEM group 1 and MSC group 2 in the peri-infarct region (76%) compared with the MAA group 3 (61%), the absolute number of double-positive vascular structures per surface area was highest in the MAA group 3 (116 ± 6.6) followed by the MSC group 2 (86.1 ± 6) and DMEM group 1 (60.4 ± 5) (Figure 4G).


Figure 3
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  		Figure 3 Fluorescent immunostaining for blood vessel density in the rat heart tissue at 3 months after mesenchymal stem cells and MAA transplantation. Numerous microvessels expressing vWF-VIII were observed both in the infarct and in the peri-infarct zones in the MAA group (A, D) and in the MSC group (B, E) relative to the control heart (C, F) at high-power microscopic field (x200). (G) The highest blood vessel density was observed mainly in the peri-infarct zone in the MAA group (* vs. $ and #: P = 0.001; != vs. § and ¥: P = 0.001).

 


Figure 4
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  		Figure 4 Double-fluorescent immunostaining of the rat heart from the MAA group 3. The tissue sections were immunostained using vWFactor-VIII and smooth muscle actin-specific antibodies and detected by TRITC (red) and Alexa Fluor-350 (blue) labelled secondary antibodies, respectively. (G) The total number of blood vessels was highest in the infarct and peri-infarct areas in the MAA group 3 compared with the MSC group 2 and DMEM group 1 (* vs. peri-infarct values of all other groups at 3 months: P < 0.05; {Phi} vs. infarct values of all other groups at 3 months: P <0.05).

 
3.4 Infarction size
Histological sectioning at the mid-papillary muscle level revealed transmural infarction in all the animals' hearts. Significant thinning of the left ventricle wall was observed in the DMEM group 1 (Figure 5A) compared with the MSC group 2 and MAA group 3 (Figure 5B–C). In the histological study, the size of the infarct was significantly reduced in the MAA group 3 (28.03 ± 2%) compared with the MSC group 2 (37.9 ± 2.5%, P < 0.043) and DMEM group 1 (47.5 ± 4.3%, P = 0.001). Severe fibrosis of the myocardium was observed in the DMEM group 1. The area of fibrosis in the MAA group 3 animals was 23.1 ± 4.6%, which was insignificantly higher compared with the MSC group 2 (28.65 ± 2.9%, P = 0.14) but significantly improved compared with the DMEM group 1 (33.5 ± 2.6%, P < 0.05).


Figure 5
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  		Figure 5 Infarct size measurement was carried out after Masson trichome staining of the rat heart tissue sections: (A) DMEM group 1, (B) MSC-transplanted group 2, (C) MAA group 3. (D) Infarct size reduced significantly in the MAA group 3 compared with group 2 (P = 0.043) and DMEM group 1 (P = 0.001).

 
3.5 Heart function studies
The indices of left ventricle function, LVEF, and LVFS were significantly improved in both the MAA group 3 (66.13 ± 2.23%; 30.39 ± 1.46%) and MSC group 2 (50.3 ± 2.38%, P < 0.001; 20.9 ± 0.86%, P < 0.001), respectively, compared with the DMEM group 1 (30.7 ± 2.84, P < 0.001; 11.5 ± 1.28, P < 0.001) (Figure 6A–B). Similarly, left ventricle end-diastolic (LVEDD) and end-systolic (LVESD) dimensions were lower in the MAA group 3 (0.69 ± 0.05 cm; 0.48 ± 0.057 cm) as well as in the MSC group 2 (0.71 ± 0.07 cm; 0.56 ± 0.06 cm) compared with the DMEM group 1 (0.8 ± 0.03 cm; 0.71 ± 0.05 cm) (Figure 6C–D). These results corresponded well with our previously reported results at 4 weeks post-transplantation.14


Figure 6
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  		Figure 6 Transthoracic echocardiography for heart function studies revealed indices of left ventricle functional improvement, and left ventricle dimensions remained significantly improved as compared with the DMEM group 1 [* vs. all other groups in (A) and (B): P <0.001].

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Funding
 References
 
The main finding of our study is the persistence of the therapeutic effects of cell-based delivery of Ang-1 and Akt until 3 months after engraftment of cells overexpressing these transgenes. We observed that (i) MSCs transduced to overexpress Ang-1 and Akt engrafted during the acute phase after myocardial infarction showed long-term survival; (ii) myogenic differentiation of the transplanted cells was prevalent; (iii) the increased neovascularization persisted with improved maturation index; (iv) left ventricle contractile function and remodelling showed sustained improvement.

The goal of cellular- and molecular-level therapeutic interventions is to compensate for the loss of functional cardiomyocytes and to induce myocardial angiogenesis for the regional perfusion of the ischaemic heart that cannot be adequately re-vascularized by conventional means.20 The complexity of the events in the infarcted myocardium necessitates a multi-faceted therapeutic strategy for the long-term functional benefits and viability of the rescued myocardium. A combined cell and gene therapy approach is hence the best strategy to restore the deteriorated heart function after an infarction episode.10,12 One of the important features of cell-based gene delivery is that it affords an extended and localized expression of the transgene besides the differentiation potential of bone marrow cells to adopt myogenic and endothelial phenotypes. The rationale of our study was to adopt multidisciplinary approach using cell-based delivery of transgenes encoding for angio-competent factors, Ang-1 and its downstream key player Akt to achieve cytoprotective, anti-apoptotic, and angiogenic effects.

Despite encouraging results of cytotherapeutic approach either alone or combined with therapeutic gene delivery, the stability of the beneficial effects in most of the studies to date has been assessed for a relatively short period of time. For adoption of a treatment modality in the clinical perspective, it is imperative that the beneficial effects should be stable for longer term. In the absence of stable and long-term cell engraftment, the infarcted heart may re-enter into the vicious cycle of post-infarction remodelling. Indeed, under such circumstances, the beneficial effects of cell therapy will remain limited to only delaying the inevitable onset of heart failure. The shortfall of beneficial effects over longer period has been attributed to the instability of the cell graft in the host tissue.21 The viability of the cell graft is affected by multiple adverse factors in the infarcted heart, including apoptosis, lack of nutrients, ischaemia, and host-related factors including inflammatory and immune responses. Bone marrow-derived MSCs have immunopriviledged status and have shown successful transplantation from non-autologous source into immunocompetent hosts.22–24 Similarly, their ability to engraft and undergo differentiation to adopt cardiac phenotype is compromised by the mechanical properties of the fibrotic microenvironment. Hence, there is a need for supportive manipulations of cells to promote their survival, engraftment, and differentiation.11,25 Most of these manipulations, however, are intended to promote cell survival during the earlier phase after cell engraftment, and the longer term survival of the cell graft remains an area less studied thus far.

Our approach of combined Ang-1 and Akt delivery is of significance, as it helped the donor cells to sustain in the hostile environment of the infarcted heart both on short and on longer term basis. Akt is a major angiogenic mediator downstream of Ang-1/Tie-2-signalling pathway.16 Hence, together they form an ideal combination of transgenes, simultaneous overexpression of which should significantly enhance the therapeutic efficacy of heart cell therapy via stimulation of multiple signalling pathways in which both Ang-1 and Akt are involved.26–28 We observed the presence of cell overexpressing Ang-1 and Akt in large number at 3 months after implantation into infarcted heart. However, it is important to note that we did not quantify the successful engraftment of donor cells in this study. Our results provide a qualitative correlation between the donor cell survival and angiomyogenic response and heart function improvement. Immunostaining of rat heart tissue for {alpha}-actinin expression revealed that some of these cells were developed into mature muscle fibres. Moreover, their alignment was observed to follow the host muscle architecture and not just random organization. The newly formed muscle fibres were positive for connexin-43, which implied their electrical coupling. These cells which expressed cardiac muscle proteins were located both in the infarct and in the peri-infarct regions; however, the ones in the infarct region were isolated from the viable myocardium, making it difficult to ascertain their role in the improvement of left ventricle contractile function.

Another important feature of our study was the persistence of improved blood vessel density in and around the area of infarct. Ang-1 is a modulator of the newly formed vascular structures and is crucial in their development as stable and leak-resistant blood vessels. Maturation index observed at 3 months was highest in the DMEM group compared with the cell-transplanted groups. This may be attributed to the regression of most of the vascular structures formed in response to the inflammatory reaction earlier on after infarction. On the contrary, extensive angiogenesis in the MAA group as a result of Ang-1 and Akt overexpression led to the formation of larger number of vascular structures, most of which matured under the influence of Ang-1. Despite lower maturation index, the total number of the mature blood vessels remained higher in the MAA group compared with the MSC and DMEM groups.

In conclusion, the results clearly depict that transplantation of MSCs genetically modulated to co-overexpress Ang-1 and Akt gave stable effects via angiomyogenesis. The transplanted cells after myogenic differentiation were integrated in the host myocardium. Presence of Ang-1 stabilized the angiogenic response for longer duration. These findings form the basis of future experimental studies using larger experimental animal models, and with an extended follow-up duration to assess the effectiveness and stability of the therapeutic outcome.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Funding
References
 
This work was supported by National Institutes of Health grants R37-HL074272; HL-23597 and HL-080686; HL087246 (to A.M.).


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

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  5. Gojo S, Gojo N, Takeda Y, Mori T, Abe H, Kyo S, et al. In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells. Exp Cell Res (2003) 288:51–59.[CrossRef][Web of Science][Medline]
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  7. Dai W, Hale SL, Martin BJ, Kuang JQ, Dow JS, Wold LE, et al. Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short- and long-term effects. Circulation (2005) 112:214–223.[Abstract/Free Full Text]
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