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THE GENE THERAPEUTIC CORRECTION OF CORONARY INSUFFICIENCY
Shuman E.
Assistant at the Department of medical biology and genetics Ural State Medical University, Yekaterinburg, Russia
Korotkov A. Candidate of Medical Sciences, Associate Professor of the Department of Medical Biology and Genetics Ural State Medical University, Yekaterinburg, Russia
Sichkar D.
Assistant at Department of medical biology and genetics, Ural State Medical University, Yekaterinburg, Russia
Desyatova M.
Assistant at the Department of medical biology and genetics Ural State Medical University, Yekaterinburg, Russia
Kostyukova S. Candidate of Biological Sciences Associate Professor of the Department of Medical Biology and Genetics Ural State Medical University, Yekaterinburg, Russia
Makeev O.
Professor, Doctor of Medical Sciences Head of the Department of Medical Biology and Genetics of
Ural State Medical University, Yekaterinburg, Russia, Head of the Laboratory of Gene and Cellular Technologies of Institute of Medical Cell Technology, Yekaterinburg, Russia
ABSTRACT
The absence of neoangiogenesis in the ischemic myocardium is the reason for the widespread prevalence of cardiovascular diseases. The mechanism is the age-dependent epigenetic blockade of the corresponding genes. In model experiments, it was shown that transfection of ischemic myocardium with plasmid vectors carrying the HIFla, HIFlb, VEGF165, VEGF225 genes in a stoichiometric ratio of 1: 0.2: 0.5: 0.3, at a concentration of 400 |ig / ml of physiological solution at a rate of 200 ^g DNA per cm2 of ischemia zone and a step over an area of 210 mm, with the addition of an adjuvant 2-dimethylaminoethanol at a concentration of 2.5 mmol / L, restores full neoangiogenesis.
Keywords: coronary insufficiency, gene therapy.
Annotation.
Despite the successes of pharmacology and surgery, coronary artery disease remains the leading cause of death. This is due to a genetically determined absence of vascular neoplasm in response to hypoxia in the tissues of the heart. The introduction of gene therapeutic agents into the myocardium that carry the genes responsible for the processes of neoangiogenesis allows the formation of a complete vasculature in the ischemic myocardium and normalize disturbed metabolic processes.
Introduction
Vascular diseases, and primarily coronary heart disease and stroke, are one of the main causes of mortality in the adult population of industrialized countries [2].
The aforementioned is associated with genome-dependent shutdown of long-term adaptation to local myocardial ischemia in most of the human population, since only one in four patients with vascular insufficiency develop collateral vessels [7]. A promising direction for the treatment of coronary insufficiency is the development of gene therapy technology. However, the introduction of individual genes, as was shown in a number of placebo-controlled studies, [3, 4, 5, 6], was not accompanied by a significant clinical effect. The inefficiency of monogenic therapy is due to the fact that products encoded by more than thirty genes participate in the formation of a complete vasculature.
The aim of the study was to find ways to achieve complete neoangiogenesis by activating a complex of growth factors involved in angiogenesis.
Materials and methods
The experiments were carried out on male Chinchilla rabbits weighing 2.8-3.2 kg and 1-1.2 years old. The animals, in order to ensure incomplete occlusion of the anterior descending artery of the heart, performed its ligation on the mandra, which narrowed the lumen of the vessel by 80%. Immediately after ligating, the two experimental animal groups No. 1 (n = 10) and No. 2 (n = 10) were injected intramiocardially once with the vectors of growth factors VEGF165 (group No. 1) at a concentration of 100 ^g / ml physiological saline at a rate of 50 ^g / cm2 of the ischemic zone with a step over the area of the zone of 2-10 mm uniformly throughout the ischemic zone or in the second group at the same time a single complex of four genes HIF1a, HIF1b, VEGF165, VEGF225 in a stoichiometric ratio of 1: 0.2: 0.5: 0, 3, group No. 2 at a concentration of 400 ^g / ml of physiological saline based on 200 ^g of DNA per cm2 of ischemic zone and a step over an area of 2-10 mm, with the addition of adjuvant. As an adjuvant, 2-dimethylaminoethanol at a concentration of 2.5 mmol / L was used. The control group of animals (n = 10) was injected with only the adjuvant solution in the corresponding volume of saline.
The level of angiogenesis was evaluated on the 30th day after surgery. Blood vessels and their relationships with cardiac muscle fibers were detected using the method of intravascular injection with contrasting suspensions followed by histological processing [1]. The study of pO2 in the open heart injury zone was carried out polarographic. The radioactivity of the tissue after a tight filling of the vascular bed with a solution of uranyl acetate-238 was determined on a GSU-1M scintillation counter and expressed in kBq per gram of tissue (dry weight). Additionally, after 30 days, the animals underwent a pharmacological stress test with dipyridamole in a total dose of 0.75 mg per 1 kg of body weight of a 0.5% solution and the electrocardiogram was recorded. he experiments were carried out on male chinchilla rabbits weighing 2.8-3.2 kg and 1-1.2 years old. The animals, in order to ensure incomplete occlusion of the anterior descending artery of the heart, performed its ligation on the mandra, which narrowed the lumen of the vessel by 80%. Immediately after ligating, the two experimental animal groups No. 1 (n = 10) and No. 2 (n = 10) were injected intramiocardially once with the vectors of growth factors VEGF165 (group No. 1) at a concentration of 100 ^g / ml physiological saline at a rate of 50 ^g / cm2 of the ischemic zone with a step over the area of the zone of 2-10 mm uniformly throughout the ischemic zone or in the second group at the same time a single complex of four genes HIF1a, HIF1b, VEGF165, VEGF225 in a stoichiometric ratio of 1: 0.2: 0.5: 0, 3, group No. 2 at a concentration of 400 ^g / ml of physiological saline based on 200 ^g of DNA per cm2 of ischemic zone and a step over an area of 2-10 mm, with the addition of adjuvant. As an adjuvant, 2-dimethylaminoethanol at a concentration of 2.5 mmol / L was used. The control group of animals (n = 10) was injected with only the adjuvant solution in the corresponding volume of saline.
The level of angiogenesis was evaluated on the 30th day after surgery. Blood vessels and their relationships with cardiac muscle fibers were detected using the method of intravascular injection with contrasting suspensions followed by histological processing [1]. The study of pO2 in the open heart injury zone was carried out polarographic. The radioactivity of the tissue after a tight filling of the vascular bed with a solution of uranyl acetate-238 was determined on a GSU-1M scintillation counter and expressed in kBq per gram of tissue (dry weight). Additionally, after 30 days, the animals underwent a pharmacological stress test with dipyridamole in a total dose of 0.75 mg per 1 kg of body weight of a 0.5% solution and the electrocardiogram was recorded.
Results
The results obtained (Table 1 and Fig. 1) indicate that transfection with the VEGF-165 gen alone is accompanied by the development of the microvasculature
and an increase in pO2 in the ischemic zone compared (Fig. 1 B), pronounced perivascular edema is observed, with the control group of animals. At the same time accompanied by "collapse" of the vessel.
Table 1
Parameters of the microvasculature of the ischemic myocardium
Control Introduction of VEGF-165 Introduction of HIF1a, HIF1b, VEGF165 and VEGF225 Significance of differences at p <0.05
intact group group of animals No. 1 group of animals No. 2
The number of capillaries n (1 mm2 per cut) 3661,0+-92,0 4180.0+-60.0 5154.0+-282.0 *, +
Diameter of open capillaries d (microns) 6,5+-0,3 6.80+-1.10 6.90+-0.80
Capillary length l (mm / mm3) 2123,0+-80,0 2600.0+-76.0 4018.0+-201.0 *, +
Capillary exchange surface area S (mm2/mm3) 43,30+-0,94 55.50+-3.11 87.10+-4.20 *, +
pO2 mmHg 18,0+-4,8 32.3+-4.9 45.4+-5.8 *, +
Radioactivity of uranyl ace-tate-238 per gram of tissue (dry weight), kBq 0,79+-0,06 1.04+-0.10 1.35+-0.05 *, +
* - significance of differences from the control group, + - significance of differences from the group of animals No. 1
"¡8. • '•
► 3 .
«■ W Sr.
A
*
»
B
Figure 1. A - control group of animals. In the upper part, the ischemic zone is clearly visible, which developed as a result of occlusion of the anterior descending branch of the coronary artery (Van Giesz stain, x100). B - formation in the zone of vascular ischemia (indicated by arrows) under the influence of hyperexpression of the VEGF165 gene. Pronounced perivascular edema (hemotoxylin / eosin, x200).
Transfection of cells with a combination of four genes of vascular growth factors has a more pronounced effect than transfection only with the VEGF-165 gene (Table 1 and Figure 2). In addition, along with the formation of the capillary network, transfection
with a combination of vascular growth factors is accompanied by the formation of arterioles (Fig. 3). as well as the formation of anastomoses between the newly formed vessels and vessels of intact ischemia of the heart tissue.
B
Fig. 2. A - formation of vascular ischemia in the zone (indicated by arrows) under the influence of VEGF 165 overexpression. Severe perivascular edema (hemotoxylin / eosin, x200). B - vascular formation under the influence of overexpression of the HIF1a, HIF1a, VEGF 165, VEGF 225 genes. There is no edema, the capillaries are filled with red blood cells (hemotoxylin / eosin, x100)
In addition, along with the formation of the capil- arterioles (Fig. 3). as well as the formation of anasto-lary network, transfection with a combination of vascu- moses between the newly formed vessels and vessels of lar growth factors is accompanied by the formation of intact ischemia of the heart tissue.
Fig. 3. A - Formation offull (arteries and venules) vessels - the formation of layers of the vascular wall is traced as a result of overexpression of the genes HIF1a, HIF1a, VEGF165, VEGF 225 (hemotoxylin / eosin, x100). B - Formation of anastamoses (indicated by a triangle, hemotoxylin / eosin, x100).
During the formation of a full-fledged vascular bed, normalization of metabolic processes in the area of the damaged myocardium is observed, as evidenced by the dynamics of changes in the electrocardiogram (ECG) during the performance of the load test. An ECG analysis after the introduction of a vector with the VEGF165 gene after 5 minutes of loading (test with di-pyridamyl) indicates the appearance of a horizontal depression of the ST (V4) segment by 0.3 mV (3 mm, a sign of myocardial ischemia). With the introduction of a complex of vectors with the genes HIFla, HIFlb, VEGF165, VEGF225, a negative ECG test is observed - the absence of ST segment depression.
Conclusion
Thus, the use of four genes integrated into the vectors of the complex allows more efficiently stimulating angiogenesis and remodeling the vasculature in an is-chemic myocardium than the technical solution of the prototype. In turn, the epigenomic localization of gene constructs and the limited duration of stay in the cell are sufficient for the expression of the introduced genes and the formation of a complete vasculature.
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