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European Journal of Heart Failure 2007 9(5):458-465; doi:10.1016/j.ejheart.2006.10.022
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© 2006 European Society of Cardiology

Angiopoietin-1 for myocardial angiogenesis: A comparison between delivery strategies{star}

Lei Yea, Husnain Kh Haiderb,*, Shujia Jiangb, Ru San Tanc, Wee Chi Toha, RuoWen Ged and Eugene K.W. Sime

a National University Medical Institutes, National University of Singapore Singapore
b Department of Pathology and Laboratory Medicine, Medical Science Building, University of Cincinnati Cincinnati, Ohio 45220, USA
c National Heart Centre, Singapore General Hospital Singapore
d Department of Biological Sciences, National University of Singapore
e Department of Surgery, National University of Singapore and Gleneagles JPMC Cardiac Center Brunei Darussalam

* Corresponding author. Tel.: +1 513 558 2029.


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
We compare the effectiveness of direct adenoviral angiopoietin-1 (Ad-Ang-1) injection with transplantation of skeletal myoblasts (SkMs) over-expressing angiopoietin-1 (Ang-1) for angiogenic response and improvement of heart function in an experimental porcine model of myocardial infarction (MI).

Methods: Ad-Ang-1 was used for intramyocardial injection or transduction of SkMs. Three weeks after coronary artery ligation in 32 female pigs, animals were grouped to receive multiple intramyocardial injections of DMEM without cells (group-1; n=7), or containing 3{sum}108 Lac-z labelled SkMs transduced with Ad-Null vector carrying no gene (group-2; n=7), or 1{sum}1010 PFU Ad-Ang-1 (group-3; n=9), or 3{sum}108 Lac-z labelled SkMs transduced with Ad-Ang-1 (group-4; n=9). The animals were immunosuppressed for 6-weeks. After euthanasia, their heart tissue was processed for histological studies.

Results: Extensive survival of Lac-z positive SkMs was observed in and around the infarct 6 and 12-weeks after transplantation. Fluorescent immunostaining for vWF-VIII at 6-weeks revealed increased blood vessel density ({sum}100) in group-4 (p<0.05) as compared with other groups. Regional blood flow (ml/g/min) in the peri-infarct area was improved in group-4 (2.7; p<0.05) as compared with group-1 (1.2±0.1), group-2 (1.1±0.4) and group-3 (1.7±0.1) at 6-weeks. Similarly, ejection fraction was significantly higher in group-4 (49.2±5.9%, p=0.03) as compared with group-1 (36.8±3%) at 6 weeks.

Conclusion: SkMs mediated Ang-1 delivery is associated with improved angiogenic response, regional myocardial perfusion and heart function as compared with direct Ad-Ang-1 administration.

Key Words: Angiopoietin-1 • Angiogenesis • Ischaemia • Myoblast

Received April 27, 2006; Revised July 26, 2006; Accepted October 30, 2006


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The angiopoietin family of growth factors is a critical regulator in the complex angiogenic cascade [1]. Recent studies have shown that vascular endothelial growth factor (VEGF) and Ang-1 are separately but sequentially produced during the development of mature blood vessels [2]. Indispensability of the role of Ang-1/Tie-2 receptor has also been reported in coronary artery development in the heart [3]. Ang-1 is a weak mitogenic factor for endothelial cells and may induce sprout formation synergistically with VEGF [4]. The multi-prong mechanism responsible for the effects of Ang-1 in new blood vessel development includes prevention of endothelial cell apoptosis via phosphatidylinositol 3-kinase activation, which in turn up regulates survivin, Akt activation and stabilization of nascent blood vessels to become leak resistant [5,6]. The role of endothelium derived nitric oxide has also been implied in Ang-1 derived angiogenic effects [7]. The interaction between Ang-1 and its Tie-2 receptor, activates intracellular kinases, a step which is critical for angiogenesis, vascular remodelling and maturation [8,9].

Plasma levels of VEGF, Ang-2 and Tie-2 receptors increase during the acute phase of myocardial infarction (MI) and lead to capillary growth in response to ischaemia [10,11]. Early studies defined Ang-1 as a maturation factor for the newly formed blood vessels. More recently, however, exogenously delivered Ang-1 has been shown to promote neovascularization in conjunction with endogenously expressed VEGF in the infarcted myocardium [12]. There is confirmatory data available which shows that delivery of Ang-1 along with VEGF promoted collateral formation in animal models [13]. Interestingly, the outcome of such an intervention is influenced by the sequence in which Ang-1 and VEGF are administered [14]. We have previously demonstrated the feasibility of bicistronic vector encoding for VEGF and Ang-1 for co-administration and co-expression of the two angiogenic growth factors [15].

Suri et al. achieved increased vascularization of skin with larger, more numerous and more branching blood vessels in a transgenic mouse model overexpressing Ang-1 [16]. Similarly, Takahashi et al. have shown that adenoviral transfer of the Ang-1 gene clearly promoted angiogenesis, reduced infarct size and alleviated progressive loss of cardiac performance in a rat model of acute MI [17]. Similar results have also been reported by other research groups using Ang-1 delivery [18]. Most of these studies have used direct viral vector injection encoding for Ang-1. Encouraged by our results of cellular delivery of Ang-1 using genetically modulated SkMs, we sought to compare this approach with direct viral vector administration of Ang-1 in terms of angiogenic effectiveness and effect on left ventricle (LV) performance in a porcine heart model of MI. We propose that cellular delivery of Ang-1 using genetically modulated SkMs overexpressing Ang-1 may be more effective in induction of angiogenesis and preservation of LV-heart function as compared with direct viral vector delivery.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Propagation and purification of Ad-Ang-1
Adenoviral vector carrying Ang-1 (Ad-Ang-1) and adenoviral null vector (Ad-Null) were kindly provided by A/Prof Rouwen Ge, Angiogenesis Laboratory, National University of Singapore. The vectors were propagated in HEK-293 cells. At 70% confluence, HEK-293 cells were infected with Ad-Ang-1 or Ad-Null. The cells were maintained for 72-96 h. The supernatant from HEK-293 cells was removed at the stipulated time, purified on cesium chloride gradient and kept at –80 °C until used for transduction. Ad-vector titration was carried out using an end-point assay described by Quantum Biotechnology, USA [19].

2.2. SkMs culture
SkMs from human male donor skeletal muscle biopsies were kindly provided by Cell Transplants Singapore Pte Ltd., Singapore. The cells were manufactured according to the in-house Standard Operating Protocol (Cell Transplants Singapore Pte. Ltd., Singapore) with a license of the USA Patent No. 5, 130, 141 [19]. The cells were regularly passaged (for up to 5 passages) every 48-72 h to prevent their pre-mature differentiation in vitro.

The purity of SkMs culture was assessed by flow cytofluorometry for desmin expression. After blocking with Ultra-V block from Ultravision detection system (Lab Vision, USA), SkMs or human fibroblasts (as a negative control) were incubated in phosphate buffered saline (PBS) with 1:50 rabbit anti-desmin primary antibody (Sigma, USA). After one and half hours incubation, cells were washed and incubated with 1:200 goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) for 1 h at room temperature. After thorough washing with PBS, cells were re-suspended in 0.5 ml PBS and analyzed using Coulter Flowcytometric Software (Scripps Res. Institute, USA) by technicians who were blinded to this experiment. The experiment was performed in triplicate and the data were analyzed using WinMDI version 2.8.

2.3. SkMs transduction with Ad-Ang-1
SkMs were cultured at 1x107 cell density in 225 mm2 tissue culture flask and transduction was performed as described earlier [19]. Briefly, SkMs were exposed to Ad-Ang-1 at a ratio of 1:1000 for 8 h. The viral infection medium was replaced with fresh Super-medium. The transduction procedure was repeated thrice to achieve optimum transduction efficiency.

2.4. Characterization of Ad-Ang-1 transduced SkMs in vitro
2.4.1. Fluorescent immunostaining
SkMs transduced with Ad-Ang-1 (Ang-1 SkMs) were grown on glass chamber slides for 24 h after final transduction and fixed with –20 °C cold methanol. Ad-Null transduced SkMs (Ad-Null SkMs) were used as a negative control. The cells were incubated at 37 °C with 1:100 rabbit anti-Ang-1 antibody (Chemicon Inc., USA) followed by incubation with goat anti-rabbit IgG conjugated with tetramethylrhodamine isothiocyanate (TRITC) (Sigma, USA). The expression of Ang-1 was visualized for stained and unstained cells using a fluorescent microscope (Olympus, Japan).

2.4.2. RT-PCR for Ang-1 gene expression from Ad-Ang-1 SkMs
Samples of Ang-1 SkMs were collected at days 1, 8, 18 and 30 after transduction for assessment of Ang-1 expression. Total RNA was isolated using Total RNA Isolation Kit (QIAGEN, Germany) and RT-PCR was performed by using QIAGEN One-step RT-PCR Kit (QIAGEN, Germany). The sequence of primers used for amplification of Ang-1 and human GAPDH is given in Table 1.


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  		Table 1 Primer sequences used in the study

 
2.5. SkMs labelling
For post transplant identification, SkMs were transduced with a retroviral vector carrying Lac-z gene (kindly provided by Dr. Colin Porter, UK) with nuclear localization signal. The supernatant containing retrovirus was collected, passed through 0.22 µm microfilter and used for transduction of SkMs [20].

2.6. Porcine heart model of MI
All animal procedures were carried out in accordance with Institutional Protocols and Guidelines approved by the National University of Singapore and the Institutional Animal Care and Use Committee (IACUC), Singapore General Hospital. A pig heart model of chronic MI was developed as described earlier [20]. Briefly, young female pigs (each weighing 30±5 kg) were anaesthetized and maintained using isoflurane (2%) and 100% oxygen (2 l/min). The heart was exposed by a limited left side thoracotomy between the 4th and 5th intercostal spaces. A branch of the left circumflex coronary artery was ligated using a #4.0 polypropylene suture. The success of the model was confirmed by coronary angiography. The catheter was inserted into the left femoral artery and passed into the ascending aorta. Angiography was performed before and after coronary ligation for confirmation of the complete occlusion of the ligated blood vessel.

2.7. Animal grouping and SkMs transplantation
Three weeks after coronary artery ligation, the animals were prepared for a second left side thoracotomy as described previously. Thirty two female pigs with experimental MI were randomised into 4 treatment groups as follows.

Basal DMEM (5 ml) without cells or viral particles (group-1, n=7) or containing 3x108 SkMs transduced with Ad-Null (group-2, n=7) or containing 2x1010 Ad-Ang-1 particles (group-3, n=9) or SkMs transduced with Ad-Ang-1 (group-4, n=9). Treatments were injected intramyocardially at 20 different sites in and around the infarct area. SkMs were labelled with Lac-z reporter gene before transplantation. Four pigs without MI served as normal control.

All animals were maintained on transient immunosuppression for 6-weeks after respective treatment using 5 mg/kg cyclosporine. The animals were euthanized at pre-determined time points as detailed in the Results section and the heart was explanted for molecular and histological studies.

2.8. Lac-z expression in pig heart
Cryo-sections of pig heart at 8~10 µm thickness were stained for Lac-z expression as described earlier [20].

2.8.1. PCR for human sry-gene in pig heart
DNA was isolated from the heart tissue according to the manufacturer's instructions using DNeasy-Tissue Kit (QIAGEN, Germany). PCR for human sry gene detection was performed using TaqPCR Master Mix Kit (QIAGEN, Germany). Briefly, the sequences of the primers for PCR amplification of human sry gene and pig {gamma}-actin are given in Table 1. The reaction conditions were maintained as: initial denaturation was at 94 °C for 3 min followed by 3-step cycling for a total of 30 cycles: denaturation for 1 min at 94 °C, annealing for 1 min at 63 °C (human sry gene) and 56 °C (pig {Upsilon}-actin) respectively and extension for 1 min at 72 °C. The final extension was performed for 10 min at 72 °C.

2.9. RT-PCR for Ang-1 expression in porcine heart
Total RNA was isolated from the heart tissue samples at the contra-lateral, center, and peri-infarct regions, using Total RNA Isolation Kit (QIAGEN, Germany) and RT-PCR was performed by using QIAGEN One-step RT-PCR Kit (QIAGEN, Germany). The sequences of the primers used for amplification of human Ang-1 and pig GAPDH are shown in Table 1.

2.10. Quantitative assessment of blood vessel density
Blood vessel density in the heart 6- and 12-weeks after respective treatment was quantified by dual fluorescent immunostaining for von Willebrand Factor-VIII (vWF-VIII) and smooth muscle actin (SMA) expression, using low power microscope field (x100). Blood vessels positive for vWF-VIII expression were counted in 3-4 randomly selected microscope fields in 10-12 microscope slides. For calculation of maturation index, the corresponding images for SMA (FITC) and vWF-VIII (TRITC) were superimposed for simultaneous expression of vWFactor-VIII and SMA. The maturation index was calculated as;


Formula

The measurement of blood vessel diameter was performed and categorized as: (a) diameter >10 µm (b) diameter between 6-10 µm and (c) diameter <6 µm. The percentage of blood vessels in each diameter category was calculated as: number of blood vessels (in each diameter category)/total blood vessels per microscope field.

2.11. Regional myocardial blood flow measurement (RMBF)
Fluorescent microspheres (Molecular Probes, USA) with three different spectra were injected (1.4x104 microspheres/kg body weight), at 0-week, 6-weeks and 12-weeks after SkMs transplantation and RMBF (ml/g/min) was calculated as described earlier [20].

2.12. Heart function studies
Echocardiography was performed using Vingmed Vivid-5 (GE, USA) by an investigator who was blinded to therapeutic intervention of each experimental group. The animals were anesthetized and kept in the right lateral position. From M-mode recordings, measurements of the LV end-diastolic diameter (LVEDD), end systolic diameter (LVESD), inter-ventricular septum (IVS), LV-cavity and posterior wall thickness (PW) were obtained. LV fractional shortening (LVFS) and LV ejection fraction (LVEF) were derived from:


Formula

2.13. Statistic analysis
Statistical analysis was performed using SPSS (version 11.0). All data were presented as mean±standard error of means (SEM) and analyzed by analysis of variance (ANOVA) between groups. Intra-group comparison at 0, 6 and 12 weeks after treatment was carried out using paired student t test. p<0.05 was considered as statistically significant.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. In vitro characterization
SkMs culture was >96% pure for desmin expression (Fig. A1a-c; Online Data Supplement). More than 95% Ad-Ang-1 SkMs were stained positive for Ang-1 expression (Fig. A2 a-b; Online Data Supplement). Ang-1 SkMs continuously expressed Ang-1 for 30-days of observation after transduction as revealed by RT-PCR (Fig. A2c; Online Data Supplement). Transduction efficiency for Lac-z reporter gene in human SkMs was >75~80%.

3.2. In vivo studies
There were no animal deaths related to viral vector injection or SkMs transplantation. All experimental animals completed the predefined study duration. We did not observe any gross or histological evidence of angioma formation until 12-weeks after respective treatment. The animals were euthanized at 6-weeks (n=4 per group) and 12 weeks (n=3 per group). Additionally, two animals from group-3 and group-4 were euthanized 2 weeks after cell transplantation or viral vector injection.

3.3. SkMs survival and Ang-1 expression in pig heart
Histochemical studies on the pig heart tissue sections revealed extensive survival of the Lac-z positive SkMs in group-2 and -4 until 12-weeks of observation (Fig. 1a). DMEM injected pig heart tissue was used as a negative control (Fig. 1b). The survival of donor SkMs was further confirmed by PCR for human sry-gene in pig heart (Fig. 1c). The donor SkMs were found to survive in both the infarct and peri-infarct regions at the site of the cell graft. Furthermore, RT-PCR showed continuous human Ang-1 expression in group-4 in the infarct and peri-infarct regions (Fig. 2 row-3). The expression of human Ang-1 was observed at 2 and 6 weeks in the peri-infarct region, but was discontinued by 12-weeks of observation. In contrast, human Ang-1 expression was observed for only 2-weeks in the group-3 animal hearts and not at 6 and 12-weeks after treatment (Fig. 2 row-2).


Figure 01
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  		Fig. 1 a-c: (a) Histochemical staining for Lac-z expression in pig heart for survival of human SkMs 12-weeks after cell transplantation using (b) DMEM injected pig heart tissue as a negative control (Magnification: 100x). (c) PCR for human sry-gene in pig heart tissue from group-1 (row 2 and 4), group-2 (row-2) at 6 and 12 weeks, and group-4 (row 4) at 2, 6 and 12-weeks after cell transplantation. Pig {gamma}-actin was used as house-keeping gene (rows 1 and 3). Lane 1=DNA marker; Lane 2=sry-gene from human male SkMs as a positive control; Lane 3, 6, 9, 12=Centre of the infarct; Lane 4, 7, 10, 13=Peri-infarct; and Lane 5, 8, 11, 14=Remote from infarct.

 


Figure 02
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  		Fig. 2 RT-PCR of pig heart tissue for Ang-1 expression at 2-weeks, 6-weeks and 12-weeks after treatment. Lane 1=DNA marker; Lane 2=Ang-1 expression from Ad-Ang-1 SkMs in vitro; Lane 3, 6, 9, 12=Centre of infarction; Lane 4, 7, 10, 13=Peri-infarction; and Lane 5, 8, 11, 14=Remote from infarction.

 
3.4. Blood vessel density
Our prime aim was to compare angiogenic response between direct injection of Ad-Ang-1 and transplantation of SkMs after transduction with Ad-Ang-1 or Ad-null. Blood vessel density quantification based on vWF-VIII expression in the peri-infarct area (Fig. A3a,d,g,j,m and Fig. A4a,d,g,j; Online Data Supplement) was highest in group-4 and increased from 39.9±3 at 6-weeks to 45.14±1.7 at 12-weeks (Fig. 3). This was followed by group-3 (30.6±2.1; 37.4±2.7; p<0.05 vs group-4), group-2 (26.5±2; 26.2±1.6) and group-1 (16±0.9; 13.4±0.9) respectively.


Figure 03
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  		Fig. 3 Blood vessel density in the peri-infarct area in pig heart after fluorescent immunostaining for vWF-VIII. *vs group-1, group-2 and group-3: p<0.05; $ vs group-1 and group-2: p<0.05).

 
The blood vessel density based on SMA expression (Fig. A3b,e,h,k,n and Fig. A4b,e,h,k; Online Data Supplement) demonstrated that SMA positive blood vessel in group-4 was 34.73±2.52 at 6-weeks and increased to 41.3±1.5 at 12-weeks. This was significantly higher (p<0.05) as compared to group-1 (7.8±0.5; 7.00±0.5), group-2 (20.1±1.6; 20.8±1.9) and group-3 (37.5±2 p=0.67; 35.8±2.7) respectively. An interesting feature of our observation was that group-3 had the highest maturation index at 6-weeks (98.5±4.2%) which was well-maintained at 12-weeks (96±2.2%). Similarly, group-4 showed progressive increase in maturation index from 85.2±2.8% at 6-weeks to 91.6±7.9% (p=0.14) at 12-weeks. This was significantly higher (p<0.05) as compared with group-2 (6-weeks=75.9±3.3%; 12-weeks=71.8±5.0%) and group-1 (6-weeks=49.2±4.7%; 12-weeks=52±5.9%).

The mean blood vessel diameter was 11.7±1.6 µm and 10.3±2 µm in group-1 and 13.7±1.4 µm and 10.6±1 µm in group-2 at 6-weeks and 12-weeks respectively. However, mean blood vessel diameters in group-3 and group-4 at 6-weeks were significantly smaller than those of group-1 and group-2 (p<0.05). Mean blood vessel diameter increased from 7.1±0.7 µm in group-3 and 7.9±0.2 µm in group-4 at 6-weeks to 10.9±1.2 µm (p<0.05) and 10.6±1.1 µm (p<0.05) at 12-weeks after treatment.

3.5. Measurement of RMBF
Mean RMBF (ml/g/min) in the centre of infarct in all four animal groups was 0.65±0.06 at 0-weeks (the time of therapeutic intervention which was 3-weeks after induction of the MI model). RMBF in group-4 increased to 1.5±0.08 and 2.1±0.07 at 6-weeks and 12-weeks respectively after cell transplantation. This was significantly higher than in group-3 (1.04±0.1; 1.5±0.2 p<0.05) at the same time points. In contrast, there were no significant changes in RMBF for both group-1 (0.6±0.06; 0.39±0.05) and group-2 (0.8±0.05; 1±0.07) at 6-weeks and 12-weeks. Mean RMBF in the peri-infarct region was 1.01±0.1 at 0-weeks which improved significantly in group-4 (2.7±0.03; 3.59±0.23 p<0.05) at 6-weeks and 12-weeks as compared with group-1 (1.2±0.17; 0.98±0.04) group-2 (1.1±0.04; 1.4±0.12) and group-3 (1.7±0.1; 2.1±0.2) at the same time-points (Fig. 4). RMBF in group-1 and group-2 was also significantly lower as compared with group-3 at 6-weeks and 12-weeks (p<0.05). Although RMBF in groups-1,2, and 3 showed insignificant change in the region away from the infarct, it changed significantly in group-4 at 6 and 12 weeks (p<0.05) after treatment.


Figure 04
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  		Fig. 4 Regional blood flow in peri-infarct region of the left ventricle. § vs group-1, group-2 at 6 and 12 weeks after treatment: p<0.05. *vs group-1, group-2, and group-3 at 6-weeks and 12-weeks after treatment: p<0.05.

 
3.6. LV-function assessment by echocardiogram
Pre- and post treatment echocardiography was carried out to assess heart function using four normal pigs as baseline control (LVEF=55.4±2.6% and LVFS=32±2.5%) (Fig. 5). The average LVEF (40.7±3.3%, p=0.005) and LVFS (23.9±2.1%, p=0.042) of the four groups was significantly reduced at 0-weeks. In group-1, LVEF and LVFS further declined to 36.8±3% and 20.4±1.8% at 6-weeks compared to (44.2±3.55%, p=0.2; 24.9±2.5%, p=0.176) in group-2 and (48.4±8.2%, p=0.1; 29.0±5.3%, p=0.076) in group-3, and (49.2±5.9%, p=0.037; 29.8±1.6%, p=0.04) in group-4.


Figure 05
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  		Fig. 5 Assessment of pig heart function by echocardiography showed markedly improved LVEF in group-4 at 6-weeks after treatment. Group-1 vs group-3 p=0.01; group-2 vs group-4 p=0.382; group-3 vs group-4 p=0.913).

 
The maximal enlargement of LV end diastolic dimension (LVEDD) (13.7±2.97%) was observed in group-1 at 6-weeks as compared with group-2 (7.5±1.6%, p=0.72), group-3 (6.6±3.01%, p=0.068), and group-4 (7.5±2%, p=0.069).


   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We have provided a comparison of Ang-1 delivery modalities; ex-vivo SkMs based delivery vs direct viral vector (Ad-Ang-1) injection. Our results support the feasibility and effectiveness of combining Ang-1 delivery together with SkMs transplantation in comparison with a monotherapy approach, to achieve myocardial angiogenesis and improvement of deteriorating LV function after chronic MI. SkMs based Ang-1 delivery gave superior overall prognosis in terms of angiogenic response, RMBF and LV function improvement.

Most previous studies aimed at angiogenic gene therapy have involved either the delivery of recombinant protein or the gene encoding for the respective growth factor using viral and non-vial vectors [21,22]. Despite ongoing phase-III clinical trials involving larger groups of patients, there are still multiple issues regarding the safety and effectiveness of the currently employed strategies, which require in depth elucidation.

With the encouraging results from heart cell therapy in experimental animal models as well as human studies, combining angiogenesis with cell transplantation has multiple advantages. It will have salutary effect on the survival of the donor cells in the early stages after transplantation and a subsequent alleviation of myocardial ischaemia due to improved regional blood flow. Indeed, the myogenic transdifferentiation of donor cells will generate a muscle fiber scaffold which will prevent the vicious cycle of LV remodelling and restore LV contractile function. Induction of angiogenesis may further improve efficacy of cellular cardiomyoplasty [21]. A more popular approach in this regard is the ex-vivo delivery of angiogenic genes [20,23], which provides an efficient way to achieve localized expression of the delivered gene with the added advantage of avoiding untoward events associated with the direct injection of viral vectors. In most cases, the cells used as carriers of transgene are inherently myogenic [20,24]. These cells have been purified from various species, including mice, rat, rabbit, sheep, pig and humans, and are characterized for their potential to improve deteriorating heart function and attenuation of LV remodelling after MI [25-27]. Moreover, they have been genetically manipulated for over-expression of reporter as well as therapeutic genes in pre-clinical studies [28,29].

Angiopoietins act as modulators of the angiogenic process by binding with endothelial cell-specific tyrosine kinase receptor Tie-2. Ang-1 efficiently increases angiogenesis in conjunction with VEGF released intrinsically in the infarcted myocardium. The presence of Ang-1 ensures leak resistant, mature, stable and functioning blood vessels with smooth muscle cell covering [8,17,30]. We have previously shown that VEGF delivery to the heart potently stimulated nascent vascular network formation, however, more than 30-35% of the newly formed blood vessels regressed by 12 weeks of observation [20]. Combining Ang-1 with VEGF using a novel bicistronic Ad-vector construct helped them to develop into functionally mature blood vessels [31]. These observations were in agreement with previous findings that increased perfusion was accompanied by the formation of stable and mature blood vessels as a result of combined VEGF and Ang-1 administration [29,30,32]. Although the role of Ang-1 as a modulator of the newly formed blood vessels has been well documented, its ability to initiate angiogenic response remains an area of intense debate. In an acute hind limb ischaemia model, blood vessel density in the Ang-1 treated group (108±12/mm2) was similar to that of VEGF treated group (117±10/mm2) [32]. The capillary to muscle fiber ratio and angiography score of the Ang-1 treated group (0.47±0.02) were the same as that of the VEGF-treated group (0.46±0.03). In most previous reports, co-administration of VEGF with Ang-1 gave the highest level of blood vessel formation. These observations also suggest that Ang-1 administration under normo-perfused condition will not induce neovascularization. It will however, potentiate the angiogenic response to exogenous (such as co-administration with VEGF) or endogenous angiogenic factors (VEGF, IGF) under hypoxia. Contrary to these findings, VEGF administered alone or in combination with Ang-1 induced significantly increased blood vessels, while over-expression of Ang-1 alone had no role in promoting neovascularization [29]. Our present study showed that Ang-1 administration, irrespective of the mode of delivery, had the potential to trigger angiogenic response, which resulted in the development of physiologically functioning blood vessels. We observed significantly improved RMBF in groups-3 and -4 using fluorescent microspheres. Although RMBF showed significant improvement in group-3 as compared with groups-1 and -2, it was still higher in group-4. These results signify the superiority of SkMs based delivery of Ang-1 as compared with direct Ad-Ang-1 injection. The difference may be attributed to SkMs which endogenously secrete platelet-derived growth factor (PDGF) [33], EGF-like growth factor [34], and VEGF [19]. Moreover, Ang-1 expression by SkMs based delivery continued for longer when compared with direct viral Ang-1 administration. In addition, the smaller mean diameter of the blood vessels observed in groups-3 and -4 as compared with groups-1 and -2 may be due to an increase in the number of new blood vessels. As the newly formed vessels matured the, mean diameter of the blood vessels continued to increase significantly thus leading to improved RMBF.

Adverse reactions from incomplete inactivation of the viral replication machinery may lead to inflammatory reactions [35-37] and haemangioma formation instead of normal blood vessels [38]. SkMs based angiogenic gene delivery is a safer alternative than direct injection of angiogenic gene carrying adenoviruses. We did not observe any untoward events after viral vector administration encoding for Ang-1. This might be because of the use of transient immunosuppression during the first 6-weeks after Ad-Ang-1 treatment. Our results also showed that only group-4 had significantly improved LVEF and LVFS as compared with group-1. However, no significant improvement in LVEDD was achieved by any group as compared with group-1; groups-2, -3 and -4 showed a slight increase in LVEDD.

In conclusion, we have demonstrated successful transduction of human SkMs to over express human Ang-1. A comparison of human SkMs based Ang-1 delivery with direct adenoviral vector has shown that both the delivery modalities induced mature, stable and functional blood vessel formation. However, the combined therapy approach of using SkMs over-expressing Ang-1 is better than either Ad-vector Ang-1 gene delivery or SkMs transplantation therapy alone.


   Acknowledgements
 
This work was sponsored by research grants from National Medical Research Council (NMRC): 0683/2002, 0748/2003 and Cardiovascular Program.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
{star} A part of the data in this paper was presented at the 54th International Congress for European Society of Cardiovascular Surgery, Athens Greece, May 19–22 2005 and awarded the Rabago Prize. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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