Skip Navigation

European Journal of Heart Failure 2005 7(6):945-952; doi:10.1016/j.ejheart.2005.03.012
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ye, L.
Right arrow Articles by Sim, E. K.W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ye, L.
Right arrow Articles by Sim, E. K.W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2005 European Society of Cardiology

Reversal of myocardial injury using genetically modulated human skeletal myoblasts in a rodent cryoinjured heart model{star}

Lei Yea, Husnain Kh. Haiderb, Shujia Jiangc, Lieng H. Lingd, Ruowen Gee, Peter K. Lawf and Eugene K.W. Sima,*

a Department of Cardiothoracic and Vascular Surgery National University of Singapore-117597, Singapore
b National University Medical Institutes, Clinical Research Centre 10-Medical Drive, National University of Singapore-117597, Singapore
c National Heart Centre Singapore
d Division of Cardiology, Department of Medicine National University of Singapore-119074, Singapore
e Departement of Biological Sciences, National University of Singapore Singapore
f Cell Therapy Research Foundation Memphis TN 38117-7126, USA

* Corresponding author. Department of Cardiothoracic and Vascular Surgery, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074, Singapore. Tel.: +65 772 5214; fax: +65 776 6475. E-mail address: sursimkw{at}nus.edu.sg


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A.
 References
 
Background: We hypothesized that combination therapy using human myoblasts and VEGF165 will lead to better prognosis in a failing heart.

Methods: Forty-eight female Wistar rats with cryoinjured hearts were randomized into non-treated normal (group-1, n=12), DMEM injected (group-2, n=10), myoblast-transplanted (group-3, n=12) and myoblast–hVEGF165 (group-4, n=14). Ten days after cryoinjury, 200 µl DMEM containing 3 x 106 cells or without cells was injected into the injured myocardium. Animals were maintained on cyclosporine for 6 weeks post cell transplantation. Heart function was assessed by echocardiography. Animals were sacrificed and hearts were processed for histochemical and immunohistochemical studies.

Results: Histological examination showed survival of the donor myoblasts expressing lac-z and hVEGF165 in rat cardiac tissue. Fluorescent immunostaining for vWillebrand Factor-VIII and smooth muscle actin expression at low power microscope (x 100) showed significantly higher blood vessel density in group-4 (31.25±1.82; 24.63±0.92) as compared to group-2 (13.29±1.0; p<0.001; 9.71±0.81, p<0.001) and group-3 (16.50±1.43, p<0.001; 14.5±1.34, p<0.001). Echocardiography showed that ejection fraction and fractional shortening of group-3 (93.36±1.52%, p=0.005; 75±3.75%, p=0.024) and group-4 (94.8±1.62%, p=0.003; 76.13±2.15%, p=0.011) significantly improved as compared to group-2 (81.8±3.3%, 55.1±7.18%).

Conclusion: Myoblasts carrying of hVEGF165 are potential therapeutic transgene carriers for cardiac repair.

Key Words: Angiogenesis • Myogenesis • Myoblast • Gene therapy • Cryoinjury

Received July 1, 2004; Revised December 29, 2004; Accepted March 2, 2005


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A.
 References
 
Cell transplantation for cellular myocardial reconstruction has been extensively studied, both in animals and humans, as a novel treatment modality [1–3]. Most studies have invariably shown improved myocardial performance after transplantation [4–6]. Reports have also been published showing the angiogenic effect of cell transplantation [7,8]. Cells from various sources have shown differential ability to enhance vascular density related to their intrinsic ability to express growth factors and angiogenic cytokines. Endothelial progenitor cells have attracted attention for therapeutic angiogenesis in treatment of myocardial infarction [9]. These cells have been shown to induce new blood vessel formation in the infarct-bed (vasculogenesis) and proliferation of pre-existing vasculature (angiogenesis) after experimental myocardial infarction [10]. Similarly, human embryonic stem cells differentiate into endothelial cells and promote cellular angiogenesis [11].

More recently, multipotential bone marrow derived stem cell implantation has been shown to impart improved contractility as well as perfusion to the myocardium in ischemic and infarction animal models [3,8,12]. Fuchs and co-workers demonstrated that bone marrow cells secrete angiogenic factors that induce endothelial cell proliferation via vascular endothelial growth factor (VEGF) and macrophage chemo-attractant protein-1 [13]. The ability of autologous bone marrow cell transplantation to improve injured heart function is by restoration of regional blood flow through angiogenesis and improved regional wall thickening by myogenesis [14]. Therapeutic angiogenesis using genetically engineered cells has more advantages, including localized, regulatable expression, reduced inflammation, and achieving concurrent myogenesis and angiogenesis. For this purpose, purified myoblasts may be genetically modulated to express recombinant genes of interest. Various studies have assessed the effectiveness of transfected rat primary myoblasts carrying human VEGF165 (hVEGF165) for the improvement of injured heart [15,16]. Our present study has been carried out to assess the potential of human skeletal myoblasts (HM) as carriers of hVEGF165 to repair cryoinjured rodent heart.


   2. Materials and methods
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A.
 References
 
2.1. Adenovirus vector propagation and purification
Replication deficient adenoviral vectors carrying hVEGF165 gene (Ad–hVEGF165) or without hVEGF165 (Ad-Null) were provided by Dr Ge Rouwen, Angiogenesis Laboratory, National University of Singapore. The vectors were propagated in 293 cells. At 70% confluence, 293 cells were infected with Ad–hVEGF165 or Ad-Null. The cells were maintained for 72–96 h. At stipulated time the supernatant from 293 cells was removed, purified on CsCl2 gradient and kept at –80 °C until used for transduction.

2.2. HM plating and passaging in vitro
HM from human male donor skeletal muscle biopsies were prepared and expanded in vitro according to the in-house Standard Operating Protocol (SOP) (Cell Transplant Pvt Ltd. Singapore) and trade secrets with a license of the U.S. Patent No. 5,130,141 and with a license of the Singapore Patent No. 34490 (WO 96/18303). The cells were propagated and maintained using patented Super Medium containing 10% FCS at 37 °C in 5% CO2 and frequently passaged until the required number of the cells was obtained [17]. The purity of the HM culture was assessed by immunostaining for desmin expression.

2.3. Lac-z and hVEGF165 genes transduction of HM
HM were cultured at 1x106 cell density in 75-mm2 tissue culture flasks and grown to achieve 80% confluence. The cells were exposed to 15 ml transduction medium containing 1x106 particles/ml retroviral vector carrying lac-z reporter gene or 1x109 particles/ml adenoviral vector carrying hVEGF165. At 8 h post-infection, the viral infection medium was replaced with DMEM basal medium for 24 h. The transduction procedure was repeated three times. Transduction efficiency was assessed by histochemical staining for lac-z expression and for hVEGF165 by ELISA using hVEGF sandwich ELISA kit (Chemicon Int., USA). ELISA was carried out as described earlier [18].

2.4. Histochemistry for β-galactosidase expression
Lac-z transduced HM were fixed with 0.5% glutaraldehyde for 15 min at room temperature. After thorough rinsing with PBS containing 1 mM MgCl2, the cells were incubated at 37 °C overnight with 5-bromo-4-chloro-3indoyl-β-D galactosidase (X-gal, Sigma) (50 mg/ml in dimethylformamide) in X-gal buffer containing 35 mM each of potassium ferricyanide and potassium ferrocyanide, 2 mM MgCl2 and 0.1% sodium dodecyl sulfate in PBS. The cells were rinsed in PBS and observed under microscope for bluish green stained nuclei.

2.5. BrdU labeling
Half of the HM for transplantation were labeled with the thymidine analogue 5-bromo-2deoxyuridine (BrdU) (Zymed). The cells were incubated with Super Medium containing of 1% BrdU solution overnight. Biotinylated mouse anti-BrdU was used to investigate the cell survival.

2.6. Experimental groups
Forty-eight adult female Wistar rats, each weighing ~250 g, were included in the study and were randomized into four groups; normal animals (with no cryoinjury) (group-1, n=12); DMEM injected without cells (group-2, n=10); HM (transfected with lac-z reporter gene only) injected (group-3 (as a control), n=12) and HM (transfected with lac-z reporter gene and hVEGF165) injected (group-4, n=14). All the animals underwent thoracotomy twice, first for the development of cryoinjury model and secondly for the injection of DMEM or the HM treatment 10 days after cryoinjury. All animal procedures were carried out in accordance with the Institutional Protocols and Guidelines approved by National University of Singapore and the Institutional Animal Care and Use Committee (IACUC) Singapore General Hospital, and assisted by a veterinary doctor.

2.7. Cryoinjury model in rats
The animals were anesthetized using sodium pentobarbital (50 mg/kg, i.p.), intubated and mechanically ventilated using a rodent ventilator. The chest of the animal was opened through left side thoracotomy under anesthesia. The heart was exposed and epicardium was removed. An 8 mm steel rod was pre-cooled in liquid nitrogen and placed in contact with the anterior wall of the left ventricle for 10 s. After the tissue was thawed, the probe was applied again. The procedure was repeated three times at an interval of 2 min to reliably produce transmural injury of the left ventricular wall. The chest was closed and the animal was allowed to recover. The animal was kept on cephalosporin for 5 days for prophylactic treatment of infection.

2.8. HM preparation and implantation
HM were harvested by trypsinization and re-suspended in serum free DMEM at a cell concentration of 3x106 cells/200 µl and kept on ice until transplantation. The animal was prepared for the second thoracotomy. The heart was exposed and the cells were injected at five different sites in and around the injured region, intramyocardially under direct vision. The chest was closed and the animal was allowed to recover. The animals were maintained on a daily dose of cyclosporine (5 mg/kg body weight) starting 5 days prior to until 6 weeks after myoblast treatment.

2.9. Histochemical and immunohistochemical studies
At the end of the study, the animals were euthanized using an overdose of sodium pentobarbitol and the heart was explanted, and conditioned at 4 °C for 24 h in 30% sucrose–phosphate buffer saline solution. The atria and right ventricle were removed and each heart was sectioned into 5 mm blocks along the longitudinal axis and frozen in liquid nitrogen cooled isopentane. Thin sections of 6–8 µm were cut from the frozen slices and stained for β-gal expression by standard protocol. Hematoxylin and eosin staining was carried out for visualization of muscle. Masson trichome staining was carried out to delineate fibrous tissue from the normal tissue. Immunostaining for BrdU was carried out to ascertain the survival of the cells.

2.10. PCR for human Y-chromosome in rat heart
Rat heart tissue samples were collected at 6 weeks after HM transplantation and used for human Y-chromosome detection by PCR using HM as positive control. Total DNA was isolated according to manufacturer's instruction using DNeasy Tissue Kit (QIAGEN, Germany). The PCR for human Y chromosome was carried out using TaqPCR Master Mix Kit (QIAGEN, Germany). Template of total DNA of each sample (1 µg) was used. Primer sequences for PCR amplification of human Y-chromosome was; (forward) 5'-CATGAACGCATTCAT CGTGTGGTC-3', (reverse) 5'-CTGCGGGAAGC AAACTGCAATTCTT-3'; rat GAPDH (forward) 5'-ATGGGAGCTGGTCATCAAC, (reverse) 5'-CCACAGTCTTCTGAGTG GCA-3'.

2.11. RT–PCR for VEGF165 expression in rodent heart
Total RNA of rat heart tissue (n=4 from each group) was isolated according to the manufacturer's instructions using Total RNA Isolation Kit (QIAGEN). The RT–PCR analysis of VEGF165 expression in rodent heart tissue was carried out by using One-step RT–PCR Kit (QIAGEN) with ad-VEGF165 transduced HM as positive control. Template total RNA of each sample (1 µg) was added into master mix and 30 cycles were performed. Primers for RT–PCR amplification of hVEGF165 were (forward) 5'-ATGAAC TTTCTGCTGTCTTGGGTG-3', (reverse) 5'-TCACCGCCTCGGCTTGTCACA-3'; rat GAPDH (forward) 5'-TTCTTGTGCAGTGCCAGCCTCGTC-3', (reverse) 5'-TAGGAAC ACGGAAGGCCATGCCAG-3'.

2.12. Quantitative histological assessment for vascular density
In each group, half of the animals were assessed for quantification of blood vessel density after 6 weeks at low power microscope (x100). Dual fluorescent immunostaining for vWillebrand Factor-VIII (vWF-VIII) and smooth muscle actin (SMA) expression was carried out in the centre and border zone area of the infarct to assess the neovascularization of the three groups with normal rat heart as baseline. After fixing the tissue sections, 1:500 rabbit anti-human vWF-VIII (Dako, Denmark) and 1:400 mouse anti-SMA (Sigma, USA) were applied to slides overnight. Then 1:500 goat anti-rabbit IgG conjugated with TRITC (Sigma, USA) and 1:400 goat anti mouse IgG conjugated with FITC (Sigma, USA) were applied for 1.5 h and observed under fluorescent microscopy. The blood vessel density (number of the blood vessels per low power microscopic field) was calculated at low power magnification (x100). The assessment was based on counting the TRITC and FITC positive blood vessels in 2–3 random microscopic fields on 10–12 microscope slides. For maturation index, the corresponding images from FITC and TRITC were superimposed to see the simultaneous fluorescence for vWFactor-VIII and SMA expression.

2.13. Functional assessment studies
Left ventricle functional assessment and dimension measurements were carried out by two dimensional echocardiography (n=8 in each group). The animals were anesthetized using sodium pentobarbital (50 mg/kg, i.p.), the chest of the animals was shaven and a layer of acoustic coupling gel was applied to the thorax. The animals were placed in a supine position, taking care to avoid any unnecessary stretch or stress on the thorax. The heart was imaged in two-dimensional mode at minimum depth settings. Parasternal long axis views were captured making sure that mitral and aortic valves and the apex were visualized. From the correct image obtained with well-defined continuous interfaces of septum and posterior wall at higher frame rate, numeric acquisition was performed at the hard disc of the echocardiographic machine. The following measurements were performed on-line at end diastole (at the time of maximum cavity dimension) and end systole (at the time of maximum motion of the posterior wall) by use of cine-loop feature to obtain adequate visualization of the following, maximum left ventricular length on long axis and endocardial area tracings (by using the leading edge method). This data was used to calculate left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) by using the single phase area length method: EF=(LVEDV2–LVESV2)/LVEDV2, where EF is the ejection fraction. All assessments were made by one investigator who was blinded to the treatment groups.

2.14. Statistical analysis
All values are given as mean±standard error (mean±S.E.M.) and processed using SPSS 11.0. One-way ANOVA was performed to assess the significant difference among groups. A p-value less than 0.05, was considered statistically significant.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A.
 References
 
All the animals survived the full length of the experiment. Macroscopic examination of the hearts at the time of euthanasia revealed no signs of angioma formation. Thirteen animals died during the development of the model. No deaths occurred due to HM transplantation. In some animals, we observed adhesion of the heart to the chest wall, however without any angiomatous structures. The repeated freeze and thaw of the left ventricle wall yielded a uniformly injured area with diameter of 8 mm.

3.1. In vitro characterization
HM culture was 97.79% pure for desmin expression when gated for control as 0.97%, using cytofluorimetry for desmin expression (Fig. 1A–B, electronic version). Immunostaining for hVEGF165 expression in the HM–hVEGF165 showed that >98% of HM were transduced with Ad–hVEGF165. The Ad-Null transduced myoblasts did not stain positive for VEGF expression. The HM–hVEGF165 continuously secreted hVEGF165 up to 18 days after transduction as revealed by ELISA.

3.2. Survival of HM in rat heart
Histochemical staining for lac-z expression and BrdU showed extensive survival of HM in rat heart at 2 days, and 2 and 6 weeks after cell transplantation in and around the infarct region (Fig. 2A–C, electronic version). PCR for human Y-chromosome in rat heart revealed survival of donor HM at 6 weeks after transplantation (Fig. 3A, electronic version). RT–PCR analysis of rat heart tissue revealed that HM–hVEGF165 transplanted in rat heart secreted hVEGF165 for up to 4 weeks after HM transplantation (Fig. 3B, electronic version). It was strongest at 1 week, declined at 4 weeks and stopped secreting at 6 weeks after cell transplantation. This is consistent with RT–PCR analysis of HM–VEGF165 in vitro.

3.3. Quantitative blood vessel density
The capillary density at low power microscopic field (100x) in group-4 was 31.25±1.82 and 24.63±0.92, respectively, for vWFactor-VIII and SMA immunostaining which were significantly higher than group-2 (13.29±1.0, p=<0.001; 9.71±0.81 p<0.001), group-3 (16.50±1.43, p<0.001; 14.5±1.34, p<0.001) and normal rat heart (20.17±1.61, p<0.001; 17.25±1.17 p<0.001) (Fig. 4A, electronic version). Superimposition of the respective images of SMA and vWF-VIII of the same sections revealed that some of the vessels were simple tubular structures of the endothelial cells whereas the others were surrounded by SMA suggesting the formation of mature vessels (Fig. 1A–L). However, the percentage of mature blood vessels was highest in group-3 (87.62±1.64%), followed by normal animals in group-1 (85.59±3.27%) and group-4 (79.61±2.28%). The percentage of mature blood vessels in the DMEM injected group, group-2 (73.93±4.80%, p=0.029) was significantly lower than in the normal rat group (group-1) and in group-3 (p=0.018 and p=0.017, respectively) (Fig. 4B, electronic version).


Figure 1
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 (A–L) The vascular density analysis using double fluorescent immunostaining for vWF-VIII (red fluorescent) and SMA (green fluorescence) at low power microscopic field (100x) showed significantly higher blood vessel density in group-4 as compared to the other groups. The merged images (I, J, K, L) showed that maturity index was highest in group-3 animals but the total number of mature blood vessels was highest in group-4 animals.

 
3.4. Improvement in cardiac function
ANOVA revealed significantly improved LVEF and LVFS (between groups: p=0.001 and p=0.009, respectively). Post hoc test showed that LVEF in group-2 decreased to 81.8±3.3% as compared to the normal animals in group-1 (96.75±2.36%, p<0.001) (Fig. 2A). Compared to group-2, animals in group-3 and group-4 showed improved cardiac function (93.63±1.52%, p=0.005; 94.8±1.62%, p=0.003, respectively). Similar observations were made for LVFS in group-3 and group-4 animals (75.0±3.75%, p=0.024; 76.13±2.15%, p=0.011, respectively) as compared to the group-2 (55.1±7.18%) which showed significant reduction as compared to group-1 (81±7.69%, p<0.001). The global heart function of rats receiving myoblast and HM–VEGF165 showed significant recovery.


Figure 2
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 (A and B) Echocardiographic assessment of heart function. Heart function deteriorated in the animals injected with DMEM without cells (group-2) as compared to (group-1) whereas HM and HM–VEGF165 transplanted rats (Groups-3 and -4) showed significant improvement in ejection fraction and fractional shortening as compared to group-2.

 
Similar results were also achieved for LVESD (p=0.015 between groups by ANOVA) and LVEDD (p=0.081, between groups by ANOVA). Post hoc test showed that LVESD in group-2 increased to 0.27±0.039 cm as compared to the normal animals (0.10±0.04 cm, p=0.005) (Fig. 2B). Compared to group-2, animals in group-3 and group-4 showed a smaller increase in LVESD (0.175±0.028 cm, p=0.045; 0.135±0.028 cm, p=0.006, respectively). Moreover, a smaller increase in LVEDD was observed in group-3 and group-4 animals (0.626±0.045 cm, p=0.45; 0.60±0.03 cm, p=0.22, respectively) as compared to group-2 (0.667±0.04 cm). However, LVEDD in group-2 significantly increased as compared to group-1 (0.493±0.03, p=0.013).


   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A.
 References
 
Therapeutic approaches aimed at promoting angiogenesis and myogenesis are currently being explored [19–21]. Cell therapy to enhance viable and functioning myocyte count in a failing heart may be more effective if the grafted cells are supported by constitutive improvement in muscle perfusion through angiogenesis. The principal objective of combining angiogenesis and myoblast transplantation is primarily to evaluate safety, feasibility and efficacy of this novel approach to achieve better prognosis. This strategy may provide cardioprotective effects for ischemic myocardium through VEGF induced vasodilatation in the early phase of infarction followed by an angiogenic effect in salvaging the host myocardium, in conjunction with the functional benefits of cellular cardiomyoplasty from the newly formed myoblast derived muscle fibers. The concept of combining the two separate therapies is attractive, and exciting data has been generated to support this as an effective mode of cardiac repair. The present study demonstrated that transplantation of HM carrying hVEGF165 gene efficiently improved heart function in a xeno-transplantation rat heart model of cryoinjury. The genetically modulated HM concurrently achieved myogenesis and angiogenesis. The results imply that localized and transient delivery of hVEGF165 via HM is sufficient to initiate and maintain therapeutic angiogenesis in cardiac repair. The expected synergistic effect of the two approaches may also obviate the undesirable remodeling process that is a common feature associated with heart failure. Most research on angiogenesis is focused on the use of VEGF, which is a potent stimulator of angiogenesis [20,22].

Most previous studies have used autologous or allogenic or stable cell lines of HM for transplantation. This has left the in vivo behavior of donor HM very poorly defined and uncharacterized. Keeping this fact in mind, we opted to use HM in the present study. The cryoinjury model was used in the present study to overcome the difficulty in achieving a standardized and uniform ligation of rat coronary arteries which otherwise may result in uncontrolled and highly variable infarct sizes. Moreover, the cryoinjury model is easy to create and more uniform and reproducible size of the necroses can be produced. Cryoinjury differs from the ischemic insult inflicted through coronary artery ligation in that cryoinjury arises from the epicardium whereas ischemic injury originates endocardially and traverses across towards the epicardium. The early stages of cryoinjury, however, closely resemble myocardial necrosis due to ischemia, with characteristics similar to those seen after acute myocardial infarction, including reduced blood flow, inflammation and myocyte death [23]. These characteristics make cryoinjury a viable and reliable experimental tool in terms of microvascular injury, healing and remodeling and to evaluate cardiomyoplasty.

One of the hypotheses explaining the causal relationship between cell transplantation and cardiac function improvement is related to the intrinsic ability of the donor cells to release growth factors and the angiogenic factors. Our in vitro data has shown that untransduced HM also express VEGF. However, this angiogenic trigger is insufficient to enhance neovascularization in the host myocardium anyway beyond what has been observed in the control animals.

In the present study, HM transduced with Ad–hVEGF165 efficiently secreted hVEGF165 for up to 18 days. The hVEGF165 level was 19 ng/ml 30 days after transduction, which suggests a sustained level of VEGF165 expression [18]. These transduced myoblasts were able to secrete hVEGF165 in rat heart up to 4 weeks after cell transplantation (Fig. 3B, electronic version). Thus, the surviving HM works as a reservoir efficiently expressing VEGF165 which in turn induces neovascularization. Thus, it is anticipated that enhanced neovascularization can be achieved by HM–VEGF165 transplantation. Macroscopic examination revealed no angioma or tumor formation which contrary to previously published data [24,25]. Schwartz and colleagues reported angiomatous growth at the site of injection and related this to high transfection and expression of VEGF, accentuated by higher vector dose delivery [24]. In another study, transplantation of murine HM transduced with VEGF in an immunodeficient murine model, led to the development of intramural vascular tumors at the site of transplantation [25]. However, unlike us, the authors used retroviral VEGF transduction which resulted in longer term up-regulation of VEGF at the site of the graft. Similar results were reported by Springer et al. [26] from constitutive expression of VEGF after transplantation of genetically modulated HM into non-ischemic skeletal muscle [26]. No such side effects were reported by Suzuki et al. [15] or Yau et al. [16]. As stated before, the adenovirus transduced HM expressed hVEGF165 in vivo for up to 4 weeks post-transplantation. We hypothesized that this expression time was sufficient to induce neovascularization. We observed significantly higher blood vessel density in animals receiving hVEGF165 carrying HM transplantation as compared to the group-3 animals which received myoblasts transduced with lac-z reporter gene only to serve as a control (Fig. 4A–B, electronic version). This suggests that HM carrying hVEGF165 system efficiently induced neovascularization and may be an ideal strategy for VEGF therapy. Although the percentage of mature blood vessel of group-3 was highest among all groups (Fig. 4B, electronic version), the total number of the mature blood vessels was highest in group-4 (Fig. 4A, electronic version). This observation raises the probability that HM mediated VEGF delivery stimulated mature blood vessel formation (Fig. 1L). On the other hand, the percentage of mature blood vessels in DMEM group-2 was significantly lower than the normal group-1.

Using the approach of ex vivo delivery of VEGF via HM in a porcine model of myocardial ischemia, we have already shown that delivery of the VEGF not only resulted in increased coronary vascular density but also led to an enhanced regional coronary blood flow and more pronounced improvement of cardiac function in the VEGF165-HM transplanted group [17]. Putting together the results of these two studies with similar fundamental hypothesis and experimental approach but in different species, our data substantiate the feasibility of HM based VEGF delivery for cardiac repair. The subtle differences between groups-3 and -4 in heart function in the present study may be model/species related.

The improved cardiac performance in group-3 was attributed to the therapeutic effect of donor HM combined with the paracrine effect that the donor HM exert on the extra cellular matrix through intrinsic low level VEGF expression. This resulted in better preservation of the myocardial architecture in the damaged area. The delivery of exogenous angiogenic factor further potentiated these effects through improved neovascularization in and around the area of infarct. HM expressing hVEGF165 may provide cardio-protective effects for ischemic myocardium through VEGF induced vasodilatation in the early phase after transplantation. This is followed by an angiogenic effect in salvaging the host myocardium, in conjunction with the functional benefits of cellular cardiomyoplasty from the newly formed HM derived muscle fibers. Germani et al. showed that VEGF enhanced C2C12 myoblast migration [27]. They also found that VEGF administration to differentiating C2C12 myoblasts prevented apoptosis in vitro. We observed significantly improved global heart function as indicated by left ventricular ejection fraction and fractional shortening. The improvement of left ventricular function resulted from the beneficial effects on the remodeling process.

Although there was no significant difference between group-3 and group-4, global heart function recovered slightly better in group-4 than in group-3 (Fig. 2A and B). In summary, we demonstrated that transplantation of HM carrying hVEGF165 resulted in a localized, sustained but transient expression of hVEGF165 for myocardial repair. This strategy led to increased vascular density and improved heart function. This strategy may be promising for myocardial infarction patients.


   Appendix A.
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A.
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejheart.2005.03.012.


   Acknowledgements
 
The work was carried out at the Animal Unit Facilities of National University of Singapore and Department of Experimental Surgery, Singapore General Hospital, Singapore and approved by the Institutional Animal Care and Use Committee (IACUC).


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A.
 References
 
{star} Source of funding: Partially funded by NMRC (Singapore) Grant # R-176-000-042-213 and NUMI Cardiovascular Research Program Grant # R-364-000-021-213. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A.
 References
 

  1. Al-Radi O.O., Rao V., Li R.K., Yau T., Weisel R.D. Cardiac cell transplantation: closer to bedside. Ann Thorac Surg (2003) 75:S674–S677.[Abstract/Free Full Text]
  2. Hagege A.A., Carrion C., Menasche P., Vilquin J.T., Duboc D., Marolleau J.P., et al. Viability and differentiation of autologous skeletal myoblast grafts in ischaemic cardiomyopathy. Lancet (2003) 361:491–492.[CrossRef][Web of Science][Medline]
  3. Stamm C., Westphal B., Kleine H.D., Petzsch M., Kittner C., Klinge H., et al. Autologous bone marrow stem-cell transplantation for myocardial regeneration. Lancet (2003) 361:45–46.[CrossRef][Web of Science][Medline]
  4. Al Attar N., Carrion C., Ghostine S., Garcin I., Vilquin J.T., Hagege A.A., et al. Long-term (1-year) functional and histological results of autologous skeletal muscle cells transplantation in rat. Cardiovasc Res (2003) 58:142–148.[Abstract/Free Full Text]
  5. Taylor D.A., Atkins B.Z., Hungspreugs P., Jones T.R., Reedy M.C., Hutcheson K.A., et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med (1998) 4:929–933.[CrossRef][Web of Science][Medline]
  6. Tomita S.J., Li R.K., Weisel R.D., Mickle D.A.G., Kim E.J., Sakai Tl. Autologous transplantation of BMC improves damaged heart function. Circulation (1999) 100:II247–II256.[Medline]
  7. Kobayashi T., Hamano K., Li T.S., Katoh T., Kobayashi S., Matsuzaki M., et al. Enhancement of angiogenesis by the implantation of self bone marrow cells in a rat ischemic heart model. J Surg Res (2000) 89:189–195.[CrossRef][Web of Science][Medline]
  8. Tse H.F., Kwong Y.L., Chan K.F., Lo G., Ho C.L., Lau C.P. Angiogenesis in ischemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet (2003) 361:47–49.[CrossRef][Web of Science][Medline]
  9. Condorelli G., Borello U., De Angelis L., Latronico M., Sirabella D., Coletta M., et al. Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration. Proc Natl Acad Sci U S A (2001) 98:10733–10738.[Abstract/Free Full Text]
  10. Kocher A.A., Schuster M.D., Szabolcs M.J., Takuma S., Burkhoff D., Wang J., et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med (2001) 7:430–436.[CrossRef][Web of Science][Medline]
  11. Levenberg S., Golub J.S., Amit M., Itskovitz-Eldor J., Langer R. Endothelial cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A (2002) 99:4391–4396.[Abstract/Free Full Text]
  12. Strauer B.E., Brehm M., Zeus T., Kostering M., Hernandez A., Sorg R.V., et al. Repair of infarcted myocardium by autologous intra coronary mononuclear bone marrow cell transplantation in humans. Circulation (2002) 106:1913–1918.[Abstract/Free Full Text]
  13. Fuchs S., Baffour R., Zhou Y.F., Shou M., Pierre A., Tio F.O., et al. Trans-endocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J Am Coll Cardiol (2001) 37:1726–1732.[Abstract/Free Full Text]
  14. Tomita S., Mickle D.A., Weisel R.D., Jia Z.Q., Tumiati L.C., Allidina Y., et al. Improved heart function with myogenesis and angiogenesis after autologous porcine bone marrow stromal cell transplantation. J Thorac Cardiovasc Surg (2002) 123:1132–1140.[Abstract/Free Full Text]
  15. Suzuki K., Murtaza B., Smolenski R.T., Sammut I.A., Suzuki N., Kaneda Y., et al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation (2001) 104:I207–I212.[Web of Science][Medline]
  16. Yau T.M., Fung K., Weisel R.D., Fujii T., Mickle D.A., Li R.K., et al. Enhanced myocardial angiogenesis by gene transfer with transplanted cells. Circulation (2001) 104:I218–I222.[Web of Science][Medline]
  17. Haider H. Kh., Lei Y., Shujia J., Ge R., Law P.K., Chua T., et al. Angiomyogenesis for cardiac repair using human myoblasts as carriers of human vascular endothelial growth factor. J Mol Med (2004) 82:539–549.[Web of Science][Medline]
  18. Lei Y., Haider Kh.H., Shujia J., Ge R., Law P.K., Sim E.K.W. High efficiency transduction of humanVEGF165 into human skeletal myoblast cells: in vitro studies. Exp Mol Med (2003) 35:412–420.[Web of Science][Medline]
  19. Hassink R.J., Brutel dela Riviere A., Mummery C.L., Pan D. Transplantation of cells for cardiac repair. J Am Coll Cardiol (2003) 41:711–717.[Abstract/Free Full Text]
  20. Koransky M.L., Robins R.C., Blau H.M. VEGF gene delivery for treatment of ischemic cardiovascular disease. Trends Cardiovasc Med (2002) 12:108–114.[CrossRef][Web of Science][Medline]
  21. Reffel mann T., Kloner R.A. Cellular cardiomyoplasty. Cardiovasc Res (2003) 58:358–368.[Free Full Text]
  22. Hedman M., Hartikainen J., Syvanne M., Stjervall J., Hedman A., Kivela A., et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of post angioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT). Circulation (2003) 107:2677–2683.[Abstract/Free Full Text]
  23. Huwer H., Nikoloudakis N., Rissland J., Vollmar B., Menger M.D., Schaffer H.J. In vivo analysis of microvascular injury after myocardial cryothermia. J Surg Res (1998) 79:1–7.[CrossRef][Web of Science][Medline]
  24. Schwarz E.R., Speakman M.T., Patterson M., Hale S.S., Isner J.M., Kedes L.H., et al. Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat angiogenesis or angioma formation. J Am Coll Cardiol (2000) 35:1323–1330.[Abstract/Free Full Text]
  25. Lee R.J., Springer M.L., Blanco-Bose W.E., Shaw R., Ursell P.C., Blau H.M. VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation (2000) 102:898–901.[Abstract/Free Full Text]
  26. Springer M.L., Chen A.S., Kraft P.E., Bednarski M., Blau H.M. VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Mol Cell (1998) 2:549–558.[CrossRef][Web of Science][Medline]
  27. Germani A., Di Carlo A., Mangoni A., Straino S., Giacinti C., Turrini P., et al. Vascular endothelial growth factor modulates skeletal myoblast function. Am J Pathol (2003) 163:1417–1428.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Exp Biol MedHome page
L. Ye, H. K. Haider, W. B. Esa, P. K. Law, W. Zhang, L. Su, Y. Zhang, and E. K. W. Sim
Nonviral Vector-Based Gene Transfection of Primary Human Skeletal Myoblasts
Exp Biol Med, December 1, 2007; 232(11): 1477 - 1487.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
L. Ye, H. Kh Haider, R. Tan, W. Toh, P. K. Law, W. Tan, L. Su, W. Zhang, R. Ge, Y. Zhang, et al.
Transplantation of Nanoparticle Transfected Skeletal Myoblasts Overexpressing Vascular Endothelial Growth Factor-165 for Cardiac Repair
Circulation, September 11, 2007; 116(11_suppl): I-113 - I-120.
[Abstract] [Full Text] [PDF]

Home page
 Asian Cardiovasc. Thorac. Ann.Home page
E. K. Sim, L. Ye, and H. K. Haider
New Strategy for Cardiac Repair: Genetically Modified Skeletal Myoblasts
Asian Cardiovasc Thorac Ann, June 1, 2007; 15(3): 183 - 184.
[Full Text] [PDF]

Home page
 Eur J Heart FailHome page
L. Ye, H. K. Haider, S. Jiang, R. S. Tan, W. C. Toh, R. Ge, and E. K.W. Sim
Angiopoietin-1 for myocardial angiogenesis: A comparison between delivery strategies
Eur J Heart Fail, May 1, 2007; 9(5): 458 - 465.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Thorac. Surg.Home page
L. Ye, H. Kh. Haider, C. Guo, and E. K.W. Sim
Cell-Based VEGF Delivery Prevents Donor Cell Apoptosis After Transplantation
Ann. Thorac. Surg., March 1, 2007; 83(3): 1233 - 1234.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ye, L.
Right arrow Articles by Sim, E. K.W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ye, L.
Right arrow Articles by Sim, E. K.W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?