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European Journal of Heart Failure 2007 9(4):343-351; doi:10.1016/j.ejheart.2006.10.007
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© 2007 European Society of Cardiology

Intramyocardial injection of vascular endothelial growth factor gene improves cardiac performance and inhibits cardiomyocyte apoptosis

Yin Ruixinga,*, Yang Dezhaib, Wu Haia, Huang Kaia, Wu Xianghonga and Chen Yuminga

a Department of Cardiology, Institute of Cardiovascular Diseases, The First Affiliated Hospital, Guangxi Medical University Nanning 530021, China
b Department of Molecular Biology, Medical Scientific Research Center, Guangxi Medical University Nanning 530021, China

* Corresponding author. Department of Cardiology, Institute of Cardiovascular Diseases, Guangxi Medical University, 22 Shuangyong Road, Nanning 530021, Guangxi, China. Tel.: +86 771 5358269; fax: +86 771 5353342. E-mail address: yinruixing{at}yahoo.com.cn


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background: Previous studies suggest that vascular endothelial growth factor (VEGF) is a regulator of naturally occurring angiogenesis. However, whether VEGF plays a role in cardiomyocyte apoptosis is not known.

Aim: To investigate the effects of intramyocardial injection of VEGF165 cDNA on cardiac performance and cardiomyocyte apoptosis in a rat model of acute myocardial infarction.

Methods: Forty male Sprague–Dawley rats underwent left coronary artery ligation and were randomised to receive VEGF165 cDNA (treated group) or pcDNA3.1 (control), injected directly into the border zone of the ischaemic myocardium. Twenty rats underwent thoracotomy and injection of pcDNA3.1, without coronary ligation (sham group). Haemodynamic and apoptotic parameters were measured two weeks after injection.

Results: Three sham, eight control, and five treated animals died. Haemodynamic parameters and microvessel counts in the treated group were significantly better than in the control (P<0.05 to 0.01). Apoptotic index in the treated group was less than in the control (P<0.01). Caspase-3 activation and mitochondrial cytochrome c release in the treated group were also lower than in the control (P<0.01). VEGF165 cDNA treatment significantly inhibited p53, Fas, Bax, and increased VEGF and Bcl-2 expression in the myocardium.

Conclusion: Intramyocardial injection of VEGF165 cDNA significantly improved cardiac performance, stimulated angiogenesis and reduced cardiomyocyte apoptosis, in a rat model of acute myocardial infarction.

Key Words: Myocardial infarction • Heart failure • Vascular endothelial growth factors • Gene therapy • Angiogenesis • Apoptosis

Received February 26, 2006; Revised July 13, 2006; Accepted October 4, 2006


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Despite continued advances in the diagnosis and management of ischaemic heart disease over the last three decades, especially early myocardial reperfusion by percutaneous transluminal coronary angioplasty, acute myocardial infarction continues to be the leading cause of morbidity and mortality in industrialized nations, and is of growing concern in developing countries. Myocardial infarction, which is usually caused by occlusion of coronary flow, induces the death of cardiomyocytes and replacement by fibrosis. Although this cardiomyocyte death is classically believed to be induced via necrosis, which is the mechanism of accidental cell death, the common view on how cardiomyocytes die during or after myocardial infarction has altered in recent years. A number of studies have indicated that apoptosis also plays an important role in the process of tissue damage subsequent to myocardial infarction [1-5]. Apoptosis can be influenced by a wide variety of regulatory stimuli. Many genes such as p53, Fas, and Bax have been shown to be directly involved in cell death after myocardial ischaemia [6-10]. Antiapoptotic interventions have been shown to delay ischaemic myocardial damage. A caspase inhibitor has been reported to be effective in reducing myocardial reperfusion injury, an action that was partially attributed to attenuation of cardiomyocyte apoptosis [10-15].

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a 34- to 46-kDa heparin-binding secreted growth factor that is angiogenic in vitro and in vivo, and has a unique target cell specificity for vascular endothelial cells [16-18]. At least four forms of human VEGF mRNA encoding VEGF proteins of 121, 165, 189, and 206 amino acids are produced as a result of alternative splicing from a single gene [19-21]. VEDF165 is the predominant isoform secreted by a variety of normal and transformed cells [19-21]. Consistent with the hypothesis that VEGF plays a major role in the biologic process of angiogenesis are the observations that VEGF [22-27] and VEGF receptor [28-31] expression are upregulated in areas of myocardial tissue ischaemia. Furthermore, local administration of VEGF protein [32-42] or gene [43-52] has been shown to augment the development of collateral blood vessels and to improve cardiac function in experimental animal models of myocardial ischaemia. However, whether VEGF plays an important role in postischaemic myocardial apoptosis in vivo has not been determined. Therefore, the present study was undertaken to determine the effects of VEGF165 cDNA on cardiac performance, cardiomyocyte apoptosis, as well as the expression of VEGF, p53, Fas, Bax and Bcl-2 proteins in a rat model of acute myocardial infarction.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Rat infarct model and injection of VEGF165 cDNA
Sixty adult male Sprague-Dawley rats weighing approximately 300 g were used for this study. Forty rats were subjected to left anterior descending coronary artery ligation which produced extensive acute myocardial infarction (about 35% of left ventricle) as previously reported [4,5,28,53]. The animals were anesthetized by intraperitoneal injection of ketamine hydrochloride (80 mg/kg), intubated via tracheotomy, and mechanically ventilated with a volume-cycled small-animal respirator. Anterolateral thoracotomy was performed, and the heart was rapidly exteriorized. One or two 6-0 silk sutures were snared in a blinded manner around the proximal left anterior descending coronary artery and then tightly ligated to occlude the vessel. Ten minutes after ligation, the rats were randomised to injection of either VEGF165 cDNA (n=20, treated group) or pcDNA3.1 (n=20, control group) (Academy of Military Medical Sciences, China), 10 µg/injection administered in 10 µl of phosphate-buffered saline solution, at pH 7.4. Each vector was injected at 6 sites, in the left anterior descending coronary artery distribution. The chest was closed in three layers with 4-0 silk sutures. Sham animals (n=20) underwent the same procedure and were injected with an equal volume of pcDNA3.1 except that the suture was passed under the coronary artery and then removed. The rats were kept under mechanical ventilation until they awoke from anesthesia. To prevent infection, benzylpenicillin sodium was also administered by intramuscular injection at a dose of 2x105 U/day for the first three days after surgery. The study protocol was approved by the Administrative Panel on Laboratory Animal Care of Guangxi Medical University, China.

2.2. Haemodynamic monitoring
Two weeks after surgery, the animals were again anesthetized. A longitudinal incision was then performed in the right cervical part. Through this incision, the right internal carotid artery was isolated and controlled using vessel loops. A polyethylene-50 catheter filled with heparin and saline (200 IU/ml) was then inserted through the right internal carotid artery into the left ventricular cavity and connected to a pressure transducer. Left ventricular pressure was continually recorded on a Power Macintosh computer via a data acquisition system. The left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), the maximum rate of left ventricular pressure rise or fall (±dP/dtmax), and heart rate (HR) were automatically analyzed. Mean arterial pressure (MAP) was calculated from the carotid arterial pressure recorded in the right internal carotid artery.

2.3. Measurement of infarct size
At the completion of physiologic measurements, the chest was opened and the heart excised. The atria were separated, the blood in the ventricular chambers removed, the left ventricle inclusive of the septum and right ventricle were dissected, and their weights measured. The left ventricle was cut into four transverse slices from apex to base. The slices were fixed at 24 h in 10% formalin and embedded in paraffin. Five-micron sections were cut and stained with Massons' trichrome stain and mounted. The endocardial and epicardial circumferences of the infarcted and noninfarcted circumference were determined with a planimeter Digital Image Analyzer. The infarcted circumference and the total left ventricular circumference of all four slices were summed separately for each of the epicardial and endocardial surfaces, and the sums were expressed as a ratio of the infarcted circumference to the left ventricular circumference for each surface. These two ratios were then averaged and expressed as a percentage for infarct size.

2.4. Intramyocardial microvessel counts
Myocardial samples were embedded in paraffin wax after fixing with 10% neutral formaldehyde solution using routine histological methods. Intramyocardial microvessels were identified by polyclonal antibodies against Factor VIII-related antigen to detect endothelial cells (Dako, Denmark) as previously described [26]. The sections were then counterstained with hematoxylin before coverslipping. Microvessels were defined as vessels with a total vessel diameter >10 µm. A total of 10 different fields from one myocardial section were counted under a 40x objective to determine the microvessel density (mean number of microvessels per high power field). The counting was performed in a blinded manner.

2.5. Terminal deoxynucleotidyl transferase-mediated dUTP-Biotin in situ nick-end labelling (TUNEL)
Four-micron sections were mounted on poly-L-lysine-coated slides. After deparaffinization and rehydration, tissue sections were incubated with proteinase K (20 µg/ml in 10 mM Tris-HCl, pH 7.6) for 30 min at 21 to 37 °C. Sections were rinsed twice with PBS and incubated with blocking solution (0.3% H2O2 in methanol) for 1 h at room temperature. DNA strand breaks were detected using the terminal deoxynucleotidyl transferase mediated dUTP nick end labelling method (TUNEL). The reagents were all from Roche Diagnostics (Hong Kong) Limited. Briefly, sections were covered with 50 µl of TUNEL reaction mixture, and incubated in this solution for 60 min at 37 °C in a humidified chamber. After rinsing in PBS, the sections were analyzed under a fluorescence microscope. Subsequently, the samples were covered with 50 µl of converter-POD (anti-fluorescein antibody, Fab fragment from sheep, conjugated with horse-radish peroxidase), and incubated in a humidified chamber for 30 min at 37 °C. Sections were rinsed three times with PBS. Finally, sections were covered with AEC-substrate solution (50 to 100 µl), and incubated for 10 min at room temperature. Sections were rinsed three times with PBS again. The sections were faintly counterstained with hematoxylin before coverslipping with AEC-mounting solution. The number of apoptotic cardiomyocytes and their percentage of total cardiomyocytes were counted with the use of a microscope. Cardiomyocytes from at least three sections per animal that were randomly selected were evaluated immunohistochemically to determine the number and percentage of cells exhibiting positive staining for apoptosis. For each slide, 5 fields were randomly chosen, and by using a defined rectangular field area (x40 objective), a total of 200 cells per field were counted. The apoptotic index was determined (i.e., number of apoptotic cardiomyocytes divided by the total number of cardiomyocytes counted x100) from a total of 15 fields per heart, and the assays were performed in a blinded manner.

2.6. Caspase-3 immunohistochemical staining
Immunohistochemical staining for Caspase-3 protein was also performed on three sections serially cut at 4-µm intervals from each animal. Briefly, this involved blocking endogenous peroxidase with 3% hydrogen peroxide, preincubation in blocking serum, and application of the primary antibody at 1:500 dilution overnight at 4 °C. A biotinylated secondary antibody was then applied for 30 min at room temperature followed by streptavidin-horseradish-peroxidase complex. After 30 min at room temperature, sections were rinsed with PBS and visualized by incubation with 0.05% (w/v) 3,3'-diaminobenzidine tetrahydrochloride dehydrate. Slides were counterstained with 0.5% methyl green before coverslipping. The value of optical density is monitored with computer-assisted image analysis (LEICA Q550IW). For each slide 10 fields were randomly chosen. The value of mean optical density was calculated. The assays were performed in a blinded manner.

2.7. Caspase-3 enzymatic assay
Heart tissues were homogenized with Telfon homogenizer in an extract buffer, which contained 25 mmol/l HEPES buffer (pH 7.4), 5 mmol/l ethylenediaminetetraacetate (EDTA), 2 mmol/l dithiothreitol (DTT), 0.1% CHAPS, 1.0 mmol/l phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml aprotinin. The homogenate was centrifuged at 20,000 g for 30 min. The supernatant was diluted with an assay buffer (50 mmol/l HEPES, 10 mmol/l DTT, 1 mmol/l EDTA, 100 mmol/l NaCl, 0.1% CHAPS, and 10% glyceral [pH 7.4]) and incubated at 37 °C with 200 µmol/l caspase-3 substrate I [N-Acetyl-Asp-Glu-Val-Asp-pNA (Ac-DEVD-pNA)]. Cleavage of the substrate was monitored at 405 nm using a microplate reader and recorded at 10-min intervals for 2 h. The specific activity was expressed in picomoles per minute per mg of protein.

2.8. Detection of mitochondrial cytochrome c release
Subcellular fractionation and Western blotting analysis were used to detect cytochrome c content in cytosol and mitochondria. The increase in the cytosol with a concomitant decrease in mitochondria is indicative of cytochrome c release from mitochondria. Briefly, heart tissues were excised and washed in cold PBS and then homogenized in cold lysis buffer supplemented with 250 mmol/l sucrose and incubated on ice for 30 min. The buffer contains 20 mmol/l HEPES (pH 7.5); 150 mmol/l NaCl; 1% NP-40; 0.1% SDS; 1 mmol/l EDTA; 1 mmol/l DTT; 2 µg each of the protease inhibitors aprotinin, leupeptin, and pepstatin A; and 0.5 µmol/l PMSF. The homogenate was centrifuged twice at 750 g at 4 °C for 10 min. Aliquots of the supernatant from the second 750 g were kept for assay of total protein, and the remaining was removed to fresh tubes and centrifuged at 10,000 g at 4 °C for 20 min. The supernatant of the 10,000 g spin was removed to clean tubes and centrifuged at 100,000 g for 1 h at 4 °C (the supernatant of this spin is the cytosolic light membrane fraction). The 10,000 g mitochondrial pellet was resuspended and lysed in lysis buffer containing 1% v/v Triton X-100 (this is the heavy membrane fraction). To probe possible cross-contamination, we measured a cytosolic marker enzyme, lactate dehydrogenase, and a mitochondrial marker, citrate synthase, in both fractions. Cross-contamination between the fractions was not detectable. The protein concentration was assayed with an aliquot of each, and the remaining was boiled in 2xSDS sample buffer. The protein samples from each fraction were separated via SDS/PAGE and subsequently transferred to nitrocellulose filters for Western blotting. Filters were probed using purified mouse anti-cytochrome c monoclonal antibodies and subsequently exposed to secondary HRP-conjugated IgG. Antigen-antibody complexes were then visualized by enhanced chemiluminescence.

2.9. Immunohistochemical staining of VEGF, p53, Fas, Bax, and Bcl-2 proteins
Immunohistochemical staining for VEGF, p53, Fas, Bax, and Bcl-2 proteins was performed on three cross-sections serially cut at 4-µm intervals from each animal. Briefly, this involved blocking endogenous peroxidase with 3% hydrogen peroxide, preincubation in blocking serum, and application of the primary antibody (Santa Cruz Biotechnology, Inc.) at the appropriate dilution (i.e., 1:80) overnight at 4 °C. A biotinylated secondary antibody was then applied for 30 min at room temperature followed by streptavidin-horseradish-peroxidase complex. After 30 min at room temperature, sections rinsed with PBS and visualized by incubation with 0.05% (w/v) 3,3'-diaminobenzidine tetrahydrochloride dehydrate. Slides were counterstained with hematoxylin before coverslipping. The value of optical density is monitored with computer-assisted image analysis (LEICA Q550IW). For each slide 5 fields were randomly chosen. The value of mean optical density was calculated. The assays were performed in a blinded manner.

2.10. Statistical analysis
All values in the text and tables are presented as mean±standard deviation (S.D.). All analyses were performed with SPSS 10.0 (SPSS Inc., Chicago, Illinois). Comparisons between multiple groups were carried out using analysis of variance (ANOVA) and then Newman-Keuls. Comparisons between two groups were carried out using Student's t-test. A value of P<0.05 was considered to have statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
Sixteen animals died during the experiment (three sham, eight control and five treated). The remaining 44 rats were included in the analysis.

3.1. Effect of VEGF165 cDNA on haemodynamics
Haemodynamic parameters changed markedly two weeks after left coronary artery ligation, but MAP, LVSP and ±dP/dtmax in the treated group were significantly higher than in the control group. In contrast, LVEDP in the treated group was significantly lower than that in the control group (Table 1).


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  		Table 1 Comparison of haemodynamic variables between the groups

 
3.2. Effect of VEGF165 cDNA on infarct size
Two weeks after coronary ligation, the infarct size in the treated group was significantly less than that in the control group (P<0.05). However, VEGF165 cDNA treatment did not affect HR, heart weight, body weight or the ratio of heart weight to body weight (Table 2).


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  		Table 2 Comparison of heart weight, infarct size and density of intramyocardial microvessels between the groups

 
3.3. Effect of VEGF165 cDNA on intramyocardial microvessels
The density of intramyocardial microvessels in the treated group was significantly higher than that in the control group (P<0.01, Table 2, Fig. 1).


Figure 01
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  		Fig. 1 Immunohistochemical (IHC) staining for Factor VIII-related antigen of myocardial sections from a treated animal (A) and control (B). The microvessels are more numerous and larger in the treated animal (A) than in the control (B). IHC-LSAB method x400.

 
3.4. Effect of VEGF165 cDNA on cardiomyocyte apoptosis
There was little evidence of apoptosis in the sham-operated rat hearts as evaluated by TUNEL staining, but many apoptotic cardiomyocytes were observed in the border zone of infarcted tissue in both control and treated animals. The apoptotic index in the treated group was significantly less than that in the control group (P<0.01, Table 3, Fig. 2).


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  		Table 3 Comparison of apoptotic index and mean optical density of myocardial VEGF, p53, Fas, Bax and Bcl-2 proteins between the groups

 


Figure 02
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  		Fig. 2 Determination of apoptotic cardiomyocytes with terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labelling (TUNEL) method. The number of TUNEL-positive cardiomyocytes (red fluorescence labelling of the nuclei) from treated rat (A) is less than that in control animal (B). TUNEL method x400.

 
Immunohistochemical staining for active caspase-3, another important indicator of apoptotic cell death, showed an increase in the immunoreactivity with the antibody against active caspase-3 in the heart of treated and control rats as compared with sham animals (Fig. 3A). The caspase-3 activation in the control group was higher than that in the treated group (P<0.01). The increased caspase-3 immunoreactivity was accompanied by increased caspase-3 activity [70±40 in sham, 105±45 in treated (P<0.05 in comparison with sham group), and 150±40 pmol/min/mg protein in the control group (P<0.01 in comparison with sham or treated group); respectively]. Increased cytosolic concentrations of cytochrome c were accompanied by decreased mitochondrial concentrations (Fig. 3B). The fraction of cytosolic and mitochondrial concentrations of cytochrome c was 0.13 vs. 0.87 in sham, 0.35 vs. 0.65 in treated (P<0.05 in comparison with sham group), and 0.55 vs. 0.45 in control group (P<0.01 in comparison with sham group; P<0.05 in comparison with treated group); respectively.


Figure 03
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  		Fig. 3 Detection of caspase-3 and mitochondrial cytochrome c release. Activation of caspase-3 was measured by immunohistochemical staining methods (A, Magnification x200) and mitochondrial cytochrome c release was measured by Western blotting (B) from the hearts of sham, control, and treated rats.

 
3.5. Effect of VEGF165 cDNA on the expression of VEGF, p53, Fas, Bax and Bcl-2
Immunohistochemical staining showed that there was little evidence of VEGF, p53, Fas, Bax and Bcl-2 protein expression in the sham-operated rat hearts. But VEGF immunoreactivity was found in the tissue adjacent to the infarcted zone in both control and treated groups. The mean optical density in the treated group was higher than that in the control group (Table 3, Fig. 4). The intensity of p53 protein expression correlated with the degree of cardiomyocyte apoptosis. Immunoreactive product of p53 was mainly located in the nuclei. The mean optical density in the treated group was lower than that in the control group; Fas immunoreactivity was yellow-brown product. The immunoreactive product was located in the membrane and cytoplasm of the cardiomyocytes. The mean optical density of Fas protein in treated group was lower than that in the control group; Bax immunoreactivity was yellow-brown reactive product located in the cytoplasm of the cardiomyocytes. The mean optical density of Bax protein in treated group was also lower than that in control group; Bcl-2 labelling in cardiomyocytes was distributed in the cytoplasm. The mean optical density of Bcl-2 protein in the treated group was significantly higher than that in the control group.


Figure 04
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  		Fig. 4 Immunohistochemical staining for VEGF of myocardial sections. VEGF immunoreactivity is stronger in treated rat (A) that in control animal (B). IHC-LSAB method x400.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The present study shows that intramyocardial injection of VEGF165 cDNA is capable of significantly increasing MAP, LVSP and ±dP/dtmax, and lowering LVEDP; stimulating angiogenesis; reducing infarct size; and inhibiting cardiomyocyte apoptosis in a rat model of acute myocardial infarction. VEGF165 cDNA treatment did not affect HR, heart weight, body weight or the ratio of heart weight to body weight. Immunohistochemical staining showed that the mean optical density of VEGF protein in the treated group was significantly higher than that in the control group, suggesting that VEGF165 cDNA can be transfected and continued to secrete bioactivity protein at least for 2 weeks.

The anti-apoptotic mechanism of VEGF remains uncertain. In a recent study, Hiasa et al. [54] reported that transplantation of bone marrow mononuclear cells did not affect capillary density after infarction, but reduced myocardial cell apoptosis and decreased myocardial infarction size through the release of VEGF. Yau et al. [55] have also revealed that transplantation of VEGF-expressing cells (bone marrow cell) into a myocardial scar reduced cardiomyocyte apoptosis and enhanced left ventricular function. Arsic et al. [56] showed that the delivery of the 165-aa isoform of VEGF-A cDNA using an adeno-associated virus vector markedly improved muscle fiber reconstitution with a dose-dependent effect, which was mediated by VEGFR-2. Yoon et al. [57] have indicated that replenished myocardial VEGF expression using naked DNA gene therapy via direct intramyocardial injection of plasmid DNA encoding VEGF (phVEGF165) increased capillary density, and decreased endothelial cell and cardiomyocyte apoptosis. Therefore, we surmise that the cardioprotection of VEGF might result from the comprehensive roles of improving haemodynamics [34,36,37], stimulating angiogenesis [26,31,34], reducing infarct size [39], and regulating the expression of apoptotic genes.

Apoptosis can be influenced by a wide variety of regulatory factors. Apoptotic genes may have an important function in regulating apoptosis. One hypothesised general molecular mediator of hypoxia-induced apoptosis is the tumour suppressor transcription factor p53. Exposure of cardiomyocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation [6]. Upregulation of p53 can induce cardiomyocyte apoptosis by stimulating the expression of Bax and/or repression of Bcl-2 expression [6,7]. The results in this investigation show that the expression of p53 in the myocardium increased significantly two weeks after coronary artery occlusion, and this phenomenon indicates that p53 may be involved in cardiomyocyte apoptosis after myocardial infarction.

Fas antigen belongs to the family of cell surface proteins that includes tumour necrosis factor receptor, nerve growth factor receptor, B-cell antigen CD40, and T-cell antigen OX40 [8]. Apoptosis can be induced by Fas antigen-antibody interaction in various cell lines. Tanaka et al. [9] showed that myocardial Fas messenger RNA levels in hypoxic conditions were upregulated twofold over controls, whereas those of nonmyocytes were downregulated, indicating that cardiomyocyte death by hypoxia can occur via apoptosis and that the Fas antigen may be associated with the mechanism of this apoptotic process. The results of the present study show that upregulation of the Fas protein was observed at two weeks after myocardial infarction. The extent of Fas protein expression correlated with the degree of cardiomyocyte apoptosis, indicating a role for Fas protein in the regulation of ischaemia-induced apoptosis in rats.

Bax is a member of the Bcl-2 family and, when overexpressed, accelerates cell apoptosis by competing with Bcl-2 [10,11]. As mentioned earlier, apoptosis is a highly regulated process in which several regulatory proteins play a part, and in which the balance between these regulatory proteins decides the fate of the cell. Recent studies have documented that the mechanism of apoptosis regulation by Bcl-2 and Bax depends not only on expression levels, but also the ratio of Bcl-2 to Bax. A high ratio of Bcl-2 to Bax can inhibit apoptosis, whereas the reverse can induce apoptosis [12]. While Bax-Bax homodimers act as apoptosis inducers, Bcl-2-Bax heterodimer formation evokes a survival signal for the cells. Both Bcl-2 and Bax are transcriptional targets for the tumour suppressor protein, p53, which induces cell cycle arrest or apoptosis in response to DNA damage. In all, the coordinated performance of these molecules is crucial for controlling the life and death of a cell [13,14]. The current investigation shows that the expression of Bax protein was significantly increased two weeks after myocardial infarction. Apoptosis was accompanied by an increase in the expression of Bax, indicating a role for this protein in the regulation of ischaemia-induced apoptosis in rats. VEGF165 cDNA treatment is capable of significantly inhibiting Bax expression as well as reducing cardiomyocyte apoptosis.

Bcl-2 is the most important gene that inhibits apoptosis. A recent report has indicated that ischaemic preconditioning resulted in upregulation of Bcl-2 gene. The upregulation of Bcl-2 significantly inhibited the extent of cardiomyocyte apoptosis induced by ischaemia/reperfusion [15], suggesting that Bcl-2 can protect cardiomyocytes. The present study shows that the expression of Bcl-2 protein was increased two weeks after myocardial infarction. VEGF165 cDNA injection is capable of significantly increasing Bcl-2 protein expression as well as reducing cardiomyocyte apoptosis, suggesting that Bcl-2 protein has an important role in the regulation of ischaemia-induced cardiomyocyte apoptosis. Induction of Bcl-2 expression from cardiomyocytes may become a new pathway of myocardial preservation.

In conclusion, the present study shows that intramyocardial injection of VEGF165 cDNA is capable of significantly improving cardiac performance, stimulating angiogenesis, reducing infarct size and cardiomyocyte apoptosis, inhibiting myocardial p53, Fas and Bax expression, and increasing VEGF and Bcl-2 expression in a rat model of acute myocardial infarction. At present, however, the exact mechanism by which VEGF165 cDNA causes these effects is still uncertain.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the special grants of "New Century Ten-Hundred-Thousand Talent Engineering" of Guangxi, China (No.: 2001212) and the research grants of Guangxi Natural Science Foundation, China (No.: 0007044 and 0448059).


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

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