Effects of therapeutic angiogenesis with plasmid VEGF165 on ventricular function in a canine model of chronic myocardial infarction

Objective: Therapeutic angiogenesis is currently under investigation in ischemic heart disease. We examined the effect on left ventricular function induced by therapeutic angiogenesis by intramyocardial injection of plasmid VEGF165, in a canine model of chronic myocardial infarction. Methods: Left thoracotomy was performed in 10 mongrel dogs, and myocardial infarction induced by ligation of the major diagonal coronary artery. At 7 postoperative (p.o.) day (pre-treatment), left ventricular ejection fraction was assessed by echocardiogram, and a second procedure was done: saline or plasmid VEGF165 at 200 mg/mL was injected over 10 points of the ischemic areas of control or treated groups, respectively. Fourteen days later (post-treatment, day 21) a control echocardiogram was performed, the animals were sacrificed and histological examination was performed. Results: Ejection fraction was maintained in the treated group: 52.45 + 15.1% on day 7 and 48.53 + 11.74% on day 21 (P=0.59), and tended to decrease in the control group, from 59.3 + 4% to 39.37 + 19.43% (P=0.04), although absolute values did not differ significantly between groups. Histological examination revealed a non significant increase in capillary vessels number in all areas in treated group. Paradoxically, arterioles were significantly less in number in all areas of treated dogs. Conclusion: Intramyocardial injection of plasmid VEGF165, in this canine model of chronic myocardial infarction, resulted in preservation of left ventricular ejection fraction, contrary to the control group where left ventricular ejection fraction showed continuous decline during the experiment. Histological examination, however, was unable to explain completely these results. Descriptors: Angiogenesis inducing agents. Myocardial infarction. Animal experimentation. Myocardial ischemia. Gene therapy.

advantages, such as the low level of potential side effects and, depending on the type of transfection employed, the possibility to be used as adjuvant of well established therapeutic protocols. The effect intensity is influenced by route of administration and vector used. There are evidence, however, that naked DNA is sufficiently effective at the target organ and produces less global effects than when viral vectors are used [6].
Vascular endothelial growth factor (VEGF) is one of the best characterized angiogenic agents [7][8][9][10][11], employed alone or in association [12]. Expression of this factor is induced by hypoxia, and its mitogenic activity is specific for endothelial cells, suggesting that it is a natural mediator of angiogenesis in response to ischemia. VEGF is most commonly produced as a homodimer of polypeptides with 165 or 121 aminoacids (VEGF 165 e VEGF 121 , respectively), and both forms have been shown to improve colateral circulation in experimental models [4,8,10].
In this study, we evaluated the effects of myocardial administration of plasmidial VEGF 165 on maintenance of ventricular function, possibly due to generation of new INTRODUCTION Gene therapy represents an interesting therapeutic alternative for ischemic cardiomyopathy, using genes encoding angiogenic growth factors to promote the development of new blood vessels or the remodeling of existing vessels [1,2]. In spite of significant developments in the treatment of ischemic cardiomyopathy during the last two decades, some cases of advanced coronary arterial disease are not suitable for conventional therapy, so that alternative therapeutic strategies are necessary. In this regard, neoangiogenesis induced by alternative methods could provide some revascularization in ischemic areas for no-option patients.
Experimental studies of ischemic models have shown that angiogenic interventions represent a valuable approach to improve myocardial perfusion [3,4], as well as left ventricular function [5]. Genes encoding angiogenic agents may be successfully administered associated to liposomes, via viral vectors, or by direct intramyocardial injection of plasmid DNA. Myocardial injection presents some resultou em tendência para atenuar a perda de contratilidade consequente ao dano miocárdico, na fase crônica do IAM. O exame histológico da rede vascular, entretanto, não explica completamente os eventos funcionais.

Abstract
Objective: Therapeutic angiogenesis is currently under investigation in ischemic heart disease. We examined the effect on left ventricular function induced by therapeutic angiogenesis by intramyocardial injection of plasmid VEGF 165 , in a canine model of chronic myocardial infarction.
Methods: Left thoracotomy was performed in 10 mongrel dogs, and myocardial infarction induced by ligation of the major diagonal coronary artery. At 7 postoperative (p.o.) day (pre-treatment), left ventricular ejection fraction was assessed by echocardiogram, and a second procedure was done: saline or plasmid VEGF 165 at 200 mg/mL was injected over 10 points of the ischemic areas of control or treated groups, respectively.
Fourteen days later (post-treatment, day 21) a control echocardiogram was performed, the animals were sacrificed and histological examination was performed.
Results: Ejection fraction was maintained in the treated group: 52.45 + 15.1% on day 7 and 48.53 + 11.74% on day 21 (P=0.59), and tended to decrease in the control group, from 59.3 + 4% to 39.37 + 19.43% (P=0.04), although absolute values did not differ significantly between groups. Histological examination revealed a non significant increase in capillary vessels number in all areas in treated group. Paradoxically, arterioles were significantly less in number in all areas of treated dogs.
Conclusion: Intramyocardial injection of plasmid VEGF 165 , in this canine model of chronic myocardial infarction, resulted in preservation of left ventricular ejection fraction, contrary to the control group where left ventricular ejection fraction showed continuous decline during the experiment. Histological examination, however, was unable to explain completely these results.

Descriptors: Angiogenesis inducing agents. Myocardial infarction. Animal experimentation. Myocardial ischemia. Gene therapy.
veterinary clinic, when a second transthoracic echocardiogram and measurement of LVEF were performed (day 21). All echocardiograms were independently examined by two investigators, and in case of difference in measurements, mean values were considered.
On the final day the dogs were sacrificed and the hearts were excised and fixed in buffered formalin for histological analysis.

Histology and analysis of vascular density
Myocardial samples were collected from ischemic and transition areas, as well as from the posterior wall of the left ventricle, free from ischemia in both groups. Histological sections size of the ischemic area were 2.59 + 0.59 cm 2 and 2.38 + 0.76 cm 2 for treated and control groups, respectively; for the transition area, sections' size were 2.41 + 0.37 cm 2 and 2.83 + 0.94 cm 2 , and for the posterior wall, 1.33 + 0.22 cm 2 and 1.52 + 0.39 cm 2 . The sections were embedded in paraffin, cut in 5 micra sections and stained using the hematoxylin-eosin method. Vascular density and vessel size were electronically determined. The mean weight of the samples analyzed was 110.8 + 16.57 g and 132.4 + 26.89 g in treated and control groups, respectively. Vessels smaller than 25 mm in diameter were considered to be capillaries, vessels larger than 25 mm and smaller than 100 mm were considered arterioles, and those larger than 100 mm were considered to be arteries [13] (Figure 1). blood vessels (capillaries and arterioles) in a canine model of chronic ischemic cardiomyopathy.

Animals
Ten male dogs of unknown breed, weighing between 9 and 12 kg (9.2 + 2.14 kg), were used. The dogs received care in compliance with the Guide for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Academy of Sciences, Washington DC, and the ethical principles for animal experiments of the Brazilian Code for Animal Experimentation (COBEA).
This study was approved by the Internal Ethical Committee in 19/09/2001, under protocol number UP2891.

Production of pHuVEGF 165
The pHuVEGF 165 plasmid, a transient and non-viral vector that express the VEGF 165 gene under the control of the CMV promoter, was obtained from Genentech (San Francisco, CA, USA) and introduced in XL1-Blue Escherichia coli by standard heat-shock transformation. Three clones of transformed cells were analyzed with different restriction enzymes, and one of them was selected for further cloning. The resulting plasmid was extracted with the PureLink TM HiPure Plasmid Maxiprep Kit (Invitrogen, USA), which allows the isolation of a large amount of DNA (0.5-1 mg). Plamid integrity was analyzed by electrophoresis in agarose gel stained with ethidium bromide, and DNA was quantified by spectrophotometry at 260-280 nm.

Experimental methods
The animals were anesthetized with intravenous administration of thiopental (3 to 5 mg/Kg), propofol (3 mg/ Kg) and pancuronium (0.1 mg/Kg) and were mechanically ventilated with a volume respirator.
A lateral left thoracotomy through the fifth intercostal space was performed, and acute myocardial infarction was made by simple ligation of the main diagonal branch of the left anterior descending coronary artery with 5.0 monofilament propylene suture. The pericardium and chest were closed and the dogs were allowed to recover. The animals were maintained in a veterinary clinic, and seven days later (day 7) were submitted to transthoracic echocardiography for examination of the ischemic area and measurement of left ventricular ejection fraction (LVEF). On the same day, the dogs were submitted to a second surgical procedure, with a transmural injection of 1 mL saline (n=5, control group) or 200 µg of the plasmidial VEGF 165 in 1 mL saline solution (n=5, treated group), administered in 10 points in and around the ischemic area. The dogs were allowed to recover and remained for another 14 days at the

Transthoracic echocardiography
There were no significant differences in mean LVEF between treated and control groups during the study period. Mean LVEF on day 7, before treatment, was 59.3 + 4% in dogs of the control group and 52.45 + 15.1% in the treated group. On day 21, mean LVEF was 39.37 + 19.43% and 48.53 + 11.74% in the control and treated groups, respectively.
Comparison of the evolution within each group, however, showed that mean ejection fraction decreased significantly more in control dogs than in those receiving plasmidial VEGF 165 . When mean LVEF was compared in days 7 and 21, a reduction of 3.9 + 15% was observed in the treated group (P=0.13), whereas in the control group this reduction was 19.9 + 15% (P=0.04) ( Figure 2).
(P=0.029). This difference was not observed comparing the same areas of the control group (P=0.480).
An increased number of arterioles was observed in the ischemic and transition area of control when compared to the same areas of the treated group (P=0.016 and P=0.024, respectively). Control group showed higher arteriolar density in the ischemic area than in the posterior wall (P=0.018) .This difference was not observed comparing the same areas of the treated group (P=0.201).

DISCUSSION
The main target of VEGF is the endothelial cell. Recent studies in experimental models of myocardial ischemia involving mammals of medium size have shown that VEGF is arteriogenic, supporting their use to promote arteriole growth in patients with severe coronary disease [14]. Other roles have also been suggested for VEGF, such as the ability to induce cardiomyocyte cytokinesis, as revealed by cardiomyocyte hyperplasia in an experimental model for ischemia [15,16].
We have previously reported a canine model of ischemic cardiomyopathy with myocardial perfusion analysis through cintilography, in which the gene encoding the green fluorescent protein was successfully transfected [17]. With a similar model, we were able to induce myocardial angiogenesis by transmural injection of plasmidial VEGF 165 in areas of acute myocardial infarction (AMI) in dogs [18]. A similar method was used by Kawasuji et al. [13], but in that case intramyocardial basic fibroblast growth factor (bFGF) was administered.
The favorable results obtained in experimental studies, and the absence of adverse effects related to the use of VEGF 165 in initial clinical trials, indicate the potential clinical use of this therapeutic approach. Recent clinical studies showed that high doses of rhVEGF improve myocardial perfusion in patients with known severe coronary artery disease and provided evidence of a dose-dependent effect [19]. Clinical results obtained with the treatment of patients with stable exertional angina have shown significant

Vessel Density
As presented in Table 1, mean capillary density was higher in all areas of myocardium analyzed in the treated group when compared with the control group, this difference, however, was not significant. In the treated group, capillary density was higher in the transition than in the posterior wall FURLANI, AP ET AL -Efeito da angiogênese terapêutica com VEGF 165 sobre a função ventricular em infarto do miocárdio crônico improvement in angina class and a favorable trend in angina frequency and exercise treadmill test in patients receiving rhVEGF as compared with placebo [20].
The optimal method for administration of VEGF is unclear. Catheter-based local intracoronary transfer of adenoviral VEGF was shown to be safe and to improve myocardial perfusion during percutaneous transluminal balloon angioplasty and after 6-month follow up [21]. On the other hand, direct intramyocardial injection resulted in improved contractility of the ventricular wall as evaluated by NOGA and ventriculography [22]. In this study, the stress-induced myocardial perfusion was not affected, but the results suggest an anti-ischemic effect of the treatment.
The best approaches to evaluate the effects of tissue perfusion are not yet clear. Simple angiography is not able to detect changes at the microvascular level, but contrast myocardial echocardiography, along with the use of radiolabeled microspheres, has been successfully employed to analyze the beneficial effects of VEGF 121 therapy [11].
Although the initial expectations in the clinical testing of gene therapy with angiogenic factors in ischemic cardiomyopathy have not been fully met, there are evidences of consistent positive results for patients. These results include histological analyses, vascular neoformation and, more recently, myocardial hyperplasia. Improvement of functional parameters related to measurements of left ventricular function and diminished frequency of cardiovascular complications, as well as subjective improvements in symptoms of patients treated for advanced ischemic cardiomyopathy and with no other viable therapeutic options, have also been observed [10,21,22].
These results stress the importance of further studies about the effects of angiogenic factors in animal models and in controlled clinical trials.
This experimental model in dogs was established for this purpose and previously tested, as described above [17,18]. Canine models have limitations related to the establishment of abundant collateral circulation, that limits the extension and severity of the infarction. Our model however was adequate, since areas of infarction and transition were clearly identified under direct vision and microscopically. The above mentioned limitation of canine models, however, could be overcome by the control group, since all animals were submitted identical procedures for myocardial infarction production and left ventricular function assessment.
The functional evaluation of global left ventricular function, by echocardiographic measurement of left ventricular ejection fraction, showed stability in treated dogs (absolute reduction of 3.9%, NS) and a decrease in the control group of around 20% (19.9%, P = 0.04). So, the role of therapeutic angiogenesis on stabilization of LVEF has been demonstrated in this experiment. In control animals, on the other hand, significant decrease of LVEF was observed until the end of the experimental period. Regional contractility, not investigated in the present study, could give more detailed information about the specific effect of the treatment in the ischemic area.
Although the canine model of myocardial infarction have their recognized limitations, it has been used to evaluate gene therapy, as by Ferrarini et al. [23], who found improvement in cardiac tissue viability and functional Histological results showed no difference regarding capillary density in the ischemic and transition areas between treated and control groups. In treated group, capillary density was higher in the transition areas than in other regions. Arteriole density, on the other hand, showed results different from those expected, since a smaller number of arterioles was observed in samples from treated than control dogs, in all examined areas. We could not explain this phenomenon, that deserves further investigation. Some limitations occurred in histological analysis, including using a large area for counting vascular density, which contributed to higher variability between values and the method used for, based on staining with hematoxylin-eosin method and electronically counting, without imunnohistochemistry which is more specific. The small number of samples also limited the statistical analysis.
In a study of efficacy of therapeutic angiogenesis by intramyocardial injection of pCK-VEGF 165 in pigs, Choi et al. [24] found that at 30 days after, there were no significant differences in segmental perfusion, wall thickening, and wall motion between groups. In the VEGF group, all variables of perfusion, wall thickening, and wall motion were significantly improved at day 60 compared with those at day 30 (P<0.05), while there were no differences in the control group. At day 60, perfusion (P=0.018), wall motion (P=0.004), and wall thickening (P=0.068) of the VEGF group were improved compared with those of the control group. Histologic analysis showed that microcapillary density was significantly higher in the VEGF group than the control group (P < 0.001) and concluded that intramyocardial injection of pCK-VEGF 165 significantly augmented neoangiogenesis in the ischemic area and improved regional myocardial function as well as myocardial perfusion.

CONCLUSION
In conclusion, in this canine model of chronic myocardial ischemia, therapeutic angiogenesis induced by intramyocardial injection of plasmidial VEGF 165 resulted in stabilization and maintenance of left ventricular function. There was simultaneous increase in capillary density in all the myocardium, but particularly in the transition area between infarcted and normal myocardium. A smaller number of arterioles was, unexpectedly, observed in treated animals. These results stress the importance of continuing experimental studies and controlled clinical trials of gene therapy for ischemic cardiomyopathy.