Mitochondrial dna as a prognostic sign of atherosclerotic coronary heart disease

 

The invention relates to medicine and relates to damage mitochondrial DNA as a prognostic sign of atherosclerotic coronary heart disease. The invention shows that damage to mitochondrial DNA occur prior to the development of atherosclerotic lesions or simultaneously with them, that damage mitochondrial DNA in the aorta increases with age and that of genotype and diet affect the level of damage of mitochondrial DNA. Therefore, the present invention shows that damage to mitochondrial DNA occur at an early stage of atherogenesis and may be the initiating event in atherogenesis, and presents methods for predicting atherosclerotic coronary heart disease, based on a quantitative level of damage to mitochondrial DNA, the advantage of the invention is to increase the sensitivity of the method for predicting atherosclerotic coronary heart disease. 3 S. and 6 C.p. f-crystals, 3 tab., 15 Il.

Background of the invention

Description Federal funding

The present invention was made in part with the use of the certain rights in the present invention.

The scope of the invention

The present invention generally relates to the field of plant physiology and molecular biology. More specifically the present invention relates to DNA damage and the effect of DNA damage on the development of atherosclerosis.

Description of this field of technology

It was found that reactive oxygen species play a key role in the pathogenesis of atherosclerotic lesions [1-6], but the underlying mechanisms have not been clarified. For example, mechanisms, mediated by reactive oxygen species, apparently, are an important factor in the oxidation of LDL (ox-LDL), which is a key event in atherogenesis [3, 7, 8]. Studies have shown that superoxide (O-2), and peroxynitrite (peroxynitrite: formed from-2+ nitric oxide) are able to oxidize LDL [9-11]. Therefore, reactions involving nitric oxide and/or-2considered to play a key role in the pathogenesis of atherosclerotic lesions and dysfunction of blood vessels (e.g., dysfunction of endothelial cells), and the mechanisms of action of the products of their oxidation (H2O2, peroxynitrite) is not precisely defined.

The mitochondria is the main source of reactive oxygen species (O-2), cared to damage the membranes of mitochondria and proteins [17-19], suppressing key cell functions, including mitochondrial respiration, which, as modified, leads to increased formation of reactive oxygen species [20-22], Poreba thus lipid peroxidation [23, 24] and DNA damage [25, 26]. Due to the fact that the ability of mitochondria to oxidative phosphorylation (C-FOS) is reduced due to damage mitochondrial DNA (mtDNA) and its accumulation of mutations with age [6, 27-29], damage to the mitochondria and the formation of active forms of oxygen can act as catalysts associated with the age of degenerative diseases such as coronary heart disease (CHD). Was offered the hypothesis that free radicals formed in the environment of endothelial cells and smooth muscles, mediate damage to the mitochondria in these cells, triggering a dangerous cycle of further education of reactive oxygen species and damage to the mitochondria, leading to dysfunction of vascular cells.

Atherosclerotic coronary heart disease is the leading cause of death in Western countries. Although there is a significant contradiction in the definition of the exact sequence of events leading to atherosclerosis of the coronary suck the mi, mediated reactive oxygen species. Macrophages recognize and internalized ox-LDL involving receptor-scavengers”, becoming kantamneni cells. The accumulation of such xantana cells is associated with lasting changes in the physiology of the blood vessels, including the migration and proliferation of smooth muscle cells, synthesis of extracellular matrix proteins and further dysfunction of endothelial cells, and this is the Central components of atherosclerotic plaques. Similarly, many of the risk factors for coronary heart disease are associated with increased production of reactive oxygen species (for example, Smoking, and hypercholesterolemia). In artery development of active forms of oxygen may be the induction of metabolic processes (oxidative phosphorylation in mitochondria, activation of cytokines or growth factors, stimulation of macrophages or neutrophils (inflammatory response) and the interaction of nitric oxide with superoxide to form peroxynitrite, which, in turn, produces singlet oxygen and hydroxyl radical. Therefore, although there are a number of processes that are important from the point of view of atherogenesis, mechanisms, mediated by reactive oxygen species, and their manifest vulnerable target for reactive oxygen species. The relationship of mitochondrial DNA matrix area of the inner membrane makes it susceptible to damage membranes and potential targets for electrophilic factors formed on the membrane. In addition to the close relationships of mitochondrial DNA with the inner membrane and OK-FOS, additional factors that make it susceptible to damage, are the lack of protective histones and nonhistone proteins and its limited repair ability. Previous research has shown that mitochondria are susceptible to damage mediated by reactive oxygen species, resulting in the intensification of lipid peroxidation and damage of mitochondrial DNA. In particular, it was found that the effect of reactive oxygen species in endothelial cells leads to the preferred damage to mitochondrial DNA, reducing the concentration of transcripts with mtDNA and disruption of mitochondrial OK-FOS.

In the prior art, there is a lack of ways to measure oxidative stress, which affects atherogenesis. The present invention fills this long-existing gap and the need of this technology.

Summary of invention

This is OK umbilical vein endothelium human (HUVEC) cells and smooth muscle aorta person (HASMC) in vitro. DNA damage, gene expression and the synthesis of mitochondrial proteins was assessed in cells treated with N2O2, peroxynitrite and-2. Mitochondrial DNA in the cells of both types were more sensitive to acute doses of reactive oxygen species compared to nuclear DNA, which was associated with lower (40-60%) levels of transcripts encoded by mtDNA proteins involved in OK-FOS (ND2 and cytochrome-b). The synthesis of mitochondrial proteins was suppressed by exposure to peroxynitrite, and cells treated with active forms of oxygen, were also characterized by significantly reduced levels of ATP and intensity of mitochondrial respiration (complex II): this is consistent with the fact that reactive oxygen species impair the function of mitochondria. The present invention shows the relationship between oxidative damage to mitochondrial DNA, altered gene expression and mitochondrial dysfunction in vitro, indicating that oxidative violation of the cells and damage to the mitochondria involved in vascular dysfunction and atherogenesis.

Taking into account the fact that mitochondrial DNA is more susceptible to damage mediated by reactive oxygen species, and given that reinforced oxidate, is the mitochondrial DNA in the tissues of the aorta, ready to be atherosclerotic, characterized by increased damage. For this purpose, the levels of damage of mitochondrial DNA present in the tissue of the aorta in hypercholesterinemia mouse model of atherosclerosis (zero apolipoprotein-E mouse), compared with values in healthy control mice of similar age. Assessment of DNA damage showed that in the tissues of the aorta in mice apoE has been a significant increase in the level of damage to mitochondrial DNA before and after the development of pathologically detectable damage (in comparison with healthy control).

In addition, the level of damage to mitochondrial DNA is increased with age in all mice, but only in mice apoE has a significant (P<0.05) relationship increase damage with age. On the contrary, the diet correlated with the level of damage to mitochondrial DNA only in mice C57BL at the age of 10 weeks, and the "Western" diet has led to an increase in damage. Finally, it was found that reducing dietary protein significantly (P<0,05) correlated with reduced damage to mitochondrial DNA in the aorta and in apoE-and control mice. Was not identified occelli the Neu DNA. Histochemical analysis of art in each group showed the presence of atherosclerotic lesions only in aged mice apoE, bicarbonates mixed (4% fat) or "Western" (21% fat) diets. As expected, the levels of lipid peroxides and cholesterol was significantly increased in mice apoE compared with control C57BL at the same age (p<0,05). However, the levels of lipid peroxides were not significantly increased in the group of apoE with high fat "Western" diet compared to a mixed diet. Thus, the data obtained confirm that: (1) in murine models of atherosclerosis in vivo mitochondrial DNA occur prior to the development of atherosclerotic lesions or simultaneously with it; (2) damage to mitochondrial DNA in the aorta increase with age in vivo; (3) apoE genotype has a greater impact on the level of damage of mitochondrial DNA compared with the composition of the diet; and (4) the effect of diet on mitochondrial DNA appears to be more pronounced in young C57BL mice. Therefore, damage to mitochondrial DNA occur in the early stages of atherosclerosis and may be the initiating event in atherogenesis.

One object of the present invention tion of the disease, caused by oxidative stress, based on the degree of damage to mitochondrial DNA, or similar measurements dysfunction of the mitochondria, which are caused by damage to mitochondrial DNA, including changes in the development of mitochondrial proteins, changes in mitochondrial oxidative phosphorylation or changes in produce ATP in the mitochondria.

The implementation of the present invention is a method for predicting atherosclerosis in a subject having an increased risk, including the following stages: (a) selection of subject samples tissue of interest; (b) quantifying the level of damage of mitochondrial DNA (mtDNA) in the tissue of interest; and (C) comparing the quantitative level of damage to mitochondrial DNA in the tissue of interest of a subject with an increased risk with quantitative level of damage to mitochondrial DNA in the tissue of interest of the controlling entity, which atherosclerosis is missing; where a higher quantitative level of damage of mitochondrial DNA of a subject with an increased risk, compared to a control subject, is a prognostic sign of atherosclerosis subject to risk.

Another implementation of the present educause the following stages: (a) the selection of the sample of interest to tissue from a subject; (b) determination of the level of damage of mitochondrial DNA (mtDNA) in the tissue of interest; (C) determining a quantitative level of DNA damage in nuclear gene in the tissue of interest; and (d) comparison of quantitative, based on the length of the DNA, the level of DNA damage between mitochondrial DNA and nuclear genome, where a higher quantitative level of damage to mitochondrial DNA in the calculation of the length of the DNA, rather than the level of damage of nuclear DNA per length of DNA is an indicator of elevated levels of oxidative stress in the subject.

Another implementation of the present invention is a method of determining the effectiveness of treatment, designed to reduce the risk of developing coronary heart disease, in a subject, comprising the following stages: (a) selection of peripheral blood leukocytes from the subject before treatment; (b) the selection of the sample of interest tissue from the subject after the treatment; and (C) determining a quantitative level of damage to mitochondrial DNA (mtDNA) in any tissue taken before treatment and after treatment, where the reduction of the level of damage of mitochondrial DNA as a result of treatment is an indication that the treatment has reduced the risk of developing coronary heart disease.

Brief description of figures

In order for the material in which the above features, advantages and objects of the present invention, as well as others that will become apparent, was understood in detail, more particular description of the invention briefly described above may be performed by reference to some realizations, which are illustrated in the attached figures. These figures form part of this application. It should be noted, however, that supplied firery only illustrative of the preferred embodiment of the invention and therefore should not be construed as limiting its scope.

Figure 1A shows an example of DNA damage associated with effects on HUVEC hydrogen peroxide. Cells were treated for 60 minutes with 0.2 mm H2O2collected and spent Capcr. Control cultures were incubated only in serum-free medium. Fewer product shows an increase in the distortion matrix. The figure 1B shows the analysis of the processed H2O2of HASMC cells and HUVEC depending on time. Cells were treated with 0.2 mm H2O2within 0-60 minutes, were extracted genomic DNA, and the surface is left class). In each experiment was performed at least two PCR per sample. Asterisks (*) indicate statistically significant (P<0.05) increase in damage compared to the untreated control. Values are average values (± s.e.).

Figure 2A shows a sample of DNA damage due to exposure to peroxynitrite in cells HUVEC and HASMC. Cells were treated for 60 minutes with the indicated doses of peroxynitrite, collected and spent Capcr. Control cultures were incubated only in serum-free medium. Fewer product indicates high damage matrix. Figure 2B shows the effects of 1 mm SIN-1 on HUVEC and HASMC. SIN-1 forms an equimolar amount of nitrous oxide and O-2. Samples SD/SIN-1 was pre-treated with 3 units/ml SOD to handle SIN-1. Fewer product indicates high damage matrix.

The figure 3 shows the levels of transcripts of mitochondrial DNA in cells treated with peroxynitrite. The chart represents the relative levels of transcripts (mean ± s.e.; normalized to-actin) 16S-pPHK, ND2 and Cyt-b after a 60-minute treatment (0.1 mm and 0.5 mm; cultural environment without si is Odom Northern blotting and hybridization with the corresponding radioactively labeled probe. Asterisks (*) indicate the levels of transcripts that were statistically significantly (P<0,05) different from untreated control.

The figure 4 shows the inclusion of35S-methionine into newly synthesized mitochondrial proteins. Figure 4A shows an example of a labeling protein after exposure to peroxynitrite in HASMC cells (left part). Cells were treated with 0.1 mm or 0/5 mm peroxynitrite within 60 minutes and then mark35S-methionine for about 2 hours, followed by electrophoresis in LTO-PAG. In the right part shows the same gel, colored Kumasi blue. Figure 4B shows a table summarizing the percent of including35S-methionine in cells and HUVEC and HASMC, treated with active forms of oxygen, compared with the control (empty processing in serum-free medium). Abbreviations: n/d - no data.

Figure 5 shows a diagram showing mitochondrial respiration (estimated recovery MTT) and total cellular ATP content. The HASMC cells and HUVEC were treated with appropriate concentrations of peroxynitrite for 1 hour at 37°C. figure 5A shows a diagram showing the relative levels of recovery MTT. After treatment, cells were washed in FSB and incubated in the course is the absorption at 570 nm. Restoration of MTT (mean ± s.e.) given in percentage of recovery in the untreated control. Figure 5B shows the determination of the total ATP content in HASMC and HUVEC treated with peroxynitrite. After this treatment, the cells were treated with ATP-releasing reagent (Labsystems), and the ATP content was determined using luminescence (Molecular Probes). Values are shown relative to untreated cells (100%).

In figure 6 it is shown that mouse apoE are characterized by much greater damage of mitochondrial DNA in the aorta and tissues of the heart compared to controls of the same age. Figure 6A shows the damage to mitochondrial DNA, estimated by the method of Capcr, in the aorta from apoE - and control mice, on the basis of which was determined by the level of amplification relative to the control group, 10 weeks of age. A smaller amount of the amplified product corresponds to the increased damage of mitochondrial DNA. Asterisks (*) indicate a statistically significant difference between aortae in the control group and apoE. Figure 6B shows the damage to mitochondrial DNA, estimated by the method of Capcr, in the left ventricle of mice apoE and control, on the basis of which was determined by the level amplificate increased damage of mitochondrial DNA. Asterisks (*) indicate a statistically significant difference between aortae apoE and control.

The figure 7 shows that reducing the absorption of food protein correlates with a lower level of damage to mitochondrial DNA in control and apoE-mice. Control and apoE-mice fed a diet with 16% or 24% protein for 4 weeks starting at 6 weeks of age, and mitochondrial DNA was evaluated by the method Capcr. Figure 7A shows the level of amplification of mitochondrial DNA in aorto control and apoE-mice compared to the control group, vykarmlival diet with 24% protein. A smaller amount of the amplified product indicates the increasing level of damage to mitochondrial DNA. Asterisks (*) indicate a statistically significant difference between mice vyernikovskaya at 16% and 24% protein. P-values determined using student's criterion, are given in parentheses. Figure 7B shows the relative level of amplification of the mitochondrial DNA control and apoE in the left ventricle compared with the control group, bicarbonates at 24% protein. A smaller amount of the amplified product increases damage of mitochondrial DNA. Asterisks (*) indicate omashu student criterion, are given in parentheses.

The figure 8 shows the increase in mtDNA damage in aorta in 3-week-old mice apoE-/- compared with control. Products 16,0, p. N. and 0.08, p. N. represent long and short Capcr-mtDNA fragments, respectively. The table below shows the relative frequency of damage (including damage by 10, etc., ad) in aorta 3-week control (zero class: A0) and apoE-/- mice. Values are average ± s.E.

In figure 9 it is shown that lipid peroxidation significantly increased in mice apoE compared with the control mice, although lipid peroxidation significantly increased in control mice who were given the "Western" diet (21% fat). Chart illustrates the level of lipid peroxidation in apoE - and control mice when feeding on a mixed (4% fat) and "Western" (21%) diets. Values are expressed relative to control mice, Wycherley on a mixed diet. Although mouse apoE was characterized by significantly higher levels of lipid peroxidation compared to control mice, regardless of diet, significant differences in the level of lipid peroxidation between apoE mice who were given smiling is Alice significantly higher levels of lipid peroxidation (marked “*”) compared to control mice, receiving a mixed diet.

In figures 10A, 10B and 10C shown that hybrid mice apoE-/-, SOD2-/+ were more intense atherosclerotic damage and damage to mtDNA. In figure 10A the whole aorta of the related hybrids apoE-/-, SOD2-/+ and apoE-/- were stained with oil red O. In the upper left part shows painted with oil red O aorta 17-week-old male apoE-/-, SOD2-/+ (4% fat in the diet), and in the upper right part shows the data related to male apoE-/- (4% fat in the diet). On the lower left and right portions shown painted aorta 34-week related individuals, respectively apoE-/-, SOD2-/+ and apoE-/-. Black arrows indicate the presence of large atherosclerotic lesions in apoE-/-, SOD2-/+ (while missing the 17-week relative apoE-/- the same litter), and the red arrow in each part points to the example of small lesions formed on arterial branches, which are more frequent and characteristic mice apoE-/-, SOD2-/+. Figure 10B is a diagram showing the frequency of atherosclerotic plaques in the whole aorta mice apoE-/-, SOD2-/+ and apoE-/-, visualizating staining with oil red O (n=4). Figure 10C shows the relative Powergen who was in control, therefore, the frequency of injuries denoted as “zero class” (Aabout). Damage frequencies are given as mean ± s.E.

The figure 11 shows the increase in the occurrence of damage to mitochondrial DNA in patients characterized by the presence of risk factors of myocardial infarction. Blood samples were selected during coronary angiography to determine the level of damage to mitochondrial DNA method Capcr. In this figure, the levels of damage to mitochondrial DNA significantly increased relative to the average for “normal” population. The graph depicts the proportion of patients in each category is characterized by damage of mitochondrial DNA. The increased damage to mitochondrial DNA is more frequent in subjects with arterial hypertension, Smoking, diabetes and a combination of these factors that determine the risk of myocardial infarction.

The figure 12 shows the damage of mitochondrial DNA before and after the 20-mile training run“. Samples were taken blood sample leukocyte film, the material of which the level of damage to mitochondrial DNA was determined with the aid of the data, their level quickly returns to normal.

The figure 13 shows that athletes spermatophores has a significantly lower level of damage of mitochondrial DNA after inclusion in the diet of high fat food compared to control subjects. In all cases, damage to mitochondrial DNA were assessed before, immediately after eating fatty foods and after 4-6 hours. Identified levels of damage to mitochondrial DNA in the control reflect the immediate effect of food assimilation of ROS in the form of fat and cholesterol. The relative “security” found at spermatophores, there may be positive regulation of antioxidant protective mechanisms in these people.

The figure 14 shows that spermatophores has a significantly lower level of damage to proteins after consuming a high fat meal compared to the control subjects.

In figures 15A and 15C shows the results Capcr mtDNA from normal and atherosclerotically aortic person. Figure 15A shows the results Capcr-compare “normal” and atherosclerotically aorta at the same age. Products 16,2, etc., N. (top row) and 0.22, etc., N. (bottom row) are long and co, 1999; Yakes, 1997). The figure 15 shows estimates of mtDNA damage per 10, etc., N. from healthy and atherosclerotically aortic person. The average frequency of injuries (± s.e.) for atherosclerotically of art expressed relative to the normal group (designated as “class zero” - 0,0±0.06 damage by 10, etc., ad). Statistical analysis (independent student test) showed significant differences between normal and ateroskleroticalkie aortae.

Detailed description of the invention

It was found that reactive oxygen species play a key role in the pathogenesis of atherosclerotic lesions, but the underlying mechanisms have not been clarified. In the environment of the blood vessels superoxide ion (O2-) interacts with nitric oxide at a rate limited by the level of diffusion, forms peroxynitrite (peroxynitrite), or, alternatively, it can be reduced to hydrogen peroxide (H2O2under the control of the enzyme superoxide dismutase. Hence, H2O2and peroxynitrite are active forms of oxygen that are relevant to studies of the vascular system. The present invention shows that hydrogen peroxide and the cells and smooth muscle aorta person (HASMC) and therefore, participate in running events of atherogenesis. Cells were treated with H2O2and peroxynitrite with the assessment of DNA damage, gene expression and protein synthesis. Mitochondrial DNA (mtDNA) is the predominant way damaged compared with-globinom gene cluster, which is transcriptionally active nuclear genome, and such damage was associated with dependent on dose reduction (40-60%) levels of transcripts encoded by mtDNA genes OK-FOS. The synthesis of mitochondrial proteins was reduced slightly (10%) effect of 0.1 mm peroxynitrite in a more significant reduction (55-70%) with 0.5 mm peroxynitrite. The impact of reactive oxygen species also resulted in lower levels of cellular ATP and mitochondrial respiratory complex II, succinate dehydrogenase): this is consistent with the fact that reactive oxygen species cause mitochondrial dysfunction. The obtained results confirm the link between damage to mitochondrial DNA, gene expression and dysfunction of mitochondria and thereby give a starting point for research dysfunction of vascular cells and atherogenesis.

DNA damage can be assessed in the tissue of the aorta 10 and the SJ high-affinity ligand for lipoprotein receptors, which is important for the uptake of LDL from the blood stream. Therefore, these mice are characterized by significantly increased serum levels of cholesterol and triglycerides, and they atherosclerotic plaques begin to develop at the age of 20 weeks. To determine the level of damage to nuclear and mitochondrial DNA in the aorta in each of the groups genomic DNA was extracted from the proximal and distal regions of the aorta and subjected to quantitative PCR (Capcr). Mouse apoE were characterized by the increased damage of mitochondrial DNA in aorta compared with control C57BL mice in all groups except the age of 10 weeks, vykarmlival to the "Western" diet. Despite the fact that the level of damage to mitochondrial DNA is increased with age in all mice, only mice apoE were statistically significant (P <0.05) level of damage of mitochondrial DNA associated with age, "Western" diet correlated with increased damage of mitochondrial DNA (compared with mixed ration) only in 10-week-old C57BL. On the contrary, there were no significant differences in nuclear DNA (-globin). In addition, the levels of lipid peroxides I), and histochemical analysis of art showed the presence of atherosclerotic plaques in the age groups of apoE in the "Western" and mixed rations. The data obtained confirm that: (1) mitochondrial DNA are associated with atherogenesis in a mouse model of atherosclerosis in vivo; (2) mitochondrial DNA of the aorta increases with age in vivo; (3) apoE genotype has a more significant impact on the level of damage of mitochondrial DNA compared with the factor of the diet; and (4) the effect of diet on mitochondrial DNA, apparently, has a stronger impact in young C57BL mice. Therefore, damage to mitochondrial DNA are considered as initiating atherogenesis event.

The present invention relates to methods for predicting the development of atherosclerotic coronary heart disease on the basis of the levels of damage to mitochondrial DNA. More specifically, the present invention relates to a method for assessing “atherosclerotic status of a subject, comprising the following stages: (a) the selection of the sample of interest specified tissue of the subject; (b) quantifying the level of damage to mitochondrial DNA in the specified tissue of interest; and (C) drawnincest level of damage to mitochondrial DNA in the tissue of interest of the controlling entity, has no atherosclerosis, where higher quantitative level of damage to mitochondrial DNA in the specified subject with an increased risk than the specified control subject is an indicator of atherosclerosis in the specified subject. Although damage to mitochondrial DNA may be determined using any method known to specialists in this field of technology, one of their examples is quantitative PCR. This method can be used to identify and quantify the level of damage to mitochondrial DNA in any subject. Preferably the target entity is associated with at least one risk factor associated with atherosclerosis. These risk factors are well known in the art and include, for example, Smoking or chewing tobacco, hypertension, diabetes, obesity, hypercholesterolemia and hyperlipidemia.

Also the present invention relates to a method for determining the quantitative level of oxidative stress in a subject, comprising the stage of: (a) the selection of the sample of interest specified tissue of the subject; (b) quantifying the level of damage to mitochondrial DNA in the specified yuusha tissue; and (d) comparing the quantitative level of damage per length of DNA in the indicated mitochondrial DNA and the nuclear gene, where a higher quantitative level of damage to mitochondrial DNA in the calculation of the length of the DNA compared to that found in nuclear DNA per length of DNA is an indicator of elevated levels of oxidative stress in the specified subject. Nuclear gene selected from the group which includes-glubinoy locus, transcriptionally active or inactive genes that depends on whether you want to determine the level of damage to nuclear DNA in actively transcribers genes or group of nuclear genes. Damage to mitochondrial DNA and DNA damage in these nuclear genes can be determined using quantitative PCR. In General, high quantitative levels of oxidative stress are predictive signs of atherogenesis, hypertension, diabetes, hypercholesterolemia, Smoking, degenerative diseases associated with aging, and cancer.

Also the present invention is directed to a method of determining the efficacy of a drug for reducing the risk of ater the introduction of specified drugs specified subject; (b) determining a quantitative level of damage to mitochondrial DNA in a specified interest tissue taken as a sample, where the reduction of damage of mitochondrial DNA after the specified treatment is an indication that the treatment reduces the risk of atherosclerosis.

In this text the term “atherogenesis” or “atherosclerosis” refers to a biological process, which leads to the formation of plaques, stenosis and occlusion of peripheral, coronary or cerebral arteries, which leads to ischemia, myocardial infarction, stroke and subsequent painful condition and mortality.

In this text the term “interested in fabric” refers to any hematopoietic cell or tissue sample.

In this text the term “oxidative stress” refers to the pathophysiological effects of reactive oxygen species, such as H2O2, superoxide, peroxynitrite, their derivatives and other active forms of oxygen, the normal function of cells. Targets of oxidative stress can be proteins, antigens, lipids, RNA, DNA, or any other cellular component.

In this text the term “introduction of an antioxidant” specifies Glee its pathophysiological manifestations.

In this text the term “low-protein diet” generally refers to the ration containing at least 16% protein, but which can be adjusted in accordance with the amount of protein contained in the analyzed diet.

In this text the term “mtDNA damage” generally denotes any type of damage (e.g., nucleotide change, apurinovaya sites, strand breaks, the formation of adducts, etc.,) or the mutation of mtDNA size (deletions, insertions, and duplications), which potentially can be detected either directly by using Capcr (by blocking polymerase or education Capcr-product, the amount of which differs from that expected due to mutations of mtDNA size) or in combination with enzyme treatment (e.g., DNA before Capcr you can handle FAPY-glycosylase to identify 8-oxoguanine).

To a person skilled in the art it will be clear that the determination of the level of damage to mitochondrial DNA is only one possible way to identify oxidative stress. Any “next set” or the net effect of damage to mitochondrial DNA will reflect the same process development Zab is on phosphorylation or changes in ATP production in the mitochondria will face the same problem.

The following examples are meant to illustrate different variants of the present invention and are not intended anyway to limit the present invention.

Example 1

Cells in vitro and mouse

The cells of the umbilical vein endothelium human (HUVEC) and smooth muscle cells of the aorta person (HASMC) were maintained at 37°C and 5% CO2+ 95% air in a modified on Dulbecco culture medium Needle (HASMC: Cell Gro) or medium M199 (HUVEC: Cell Gro), supplemented with 10% (HASMC) or 20% (HUVEC) V / V heat inactivated fetal calf serum (Gibco BRL), HEPES buffer (10 mm), glutamine, penicillin and streptomycin. Bulb standard image divided every 3-4 days and was disassociable for experiments using trypsin-EDTA (Gibco BRL). Cells were treated with various reactive oxygen species (“empty” control treatment - serum-free medium) when the degree of confluence of 70-80% between 5-7-mi subcultures.

“Off” apolipoprotein-E mouse (apoE) (-/-), has been shown to be an effective model of atherosclerosis. Mouse apoE(-/-) lack of apolipoprotein-E, which is the high-affinity ligand for lipoprotein receptors, which play an important role in the binding of LDL from the blood stream. SL is the potassium, and atherosclerotic plaque begins to develop at the age of 20 weeks when feeding on Western high fat diet. Among murine models of atherosclerosis line mouse apoE(-/-) most accurately the parameters for human atherosclerosis.

Control S and mouse apoE (on the basis of C57BL) were purchased (Jackson Laboratories, Bar Harbor) at 5 weeks of age, after which their acclimatized in vivarium UTMB Animal Research Facilities during the week and then fed or mixed (4% fat: diet Harlan Teklad 7001) or "Western" (21% fat: Harlan Teklad # 88137) rations, starting from 6 weeks of age for 4 young, a 10-week group) or 28 (old, 34-week group) weeks before sampling tissue. Experiment with protein rations 6-week-old mice fed diets with either 16% (16% protein, 4% fat: NIH31 # 101034) or 24% (24% protein, 4% fat: diet Harlan Teklad # 7001) protein for 4 weeks (when the killing of age was 10 weeks). Each group consisted of 4 mouse apoE and 4 mouse control C57BL. Tissue samples were taken after intraperitoneal injection ketaset-xylazine (1 mg, and 20 mg). On one side of the aorta from the group used for histochemical analysis, while samples of the remaining proximal and distal parts and -80°C to use. Plasma samples were taken and used to determine total cholesterol (Boehringer Mannheim, IN) and the content of lipid peroxides (Calbiochem-Novabiochem, La Jolla, CA).

Example 2

Treatment of the active forms of oxygen

The concentrated mother liquor H2O2(30%, Fisher) was diluted in phosphate-saline buffer (FSB), and the concentration was determined by absorption at 230 nm [30]. The peroxynitrite was synthesized from sodium nitrite and acidified with H2O2and analyze it quantitatively [31]. As donors low dose Of-2and nitrous oxide were used, respectively, the xanthine oxidase/lunasin and spermin-NONO-at. SIN-1 (3-morpholinosydnonimine hydrochloride: Molecular Probes) was used to generate higher equimolar levels of nitric oxide and O-2. Monolayer cultures on 60-mm plates (level merge 70-80%) was subjected to exposure to specific concentrations of reactive oxygen species to determine, depending on the dose and changes over time, using serum-free, free of phenol red minimal medium (MEM) at 37°C. Control monolayers were subjected to “empty” processing only the MEM medium containing no serum and phenol red. After treatment, cells etc is Capcr measure the average level of DNA damage per chain for two matrix circuits of interest genomic segment. Detection of DNA damage using Capcr is based on the following. (1) On the premise that any DNA matrix, including damage, will stop thermostable polymerase or directly [25,32], or in combination with the influence of the enzyme before Capcr (i.e., processing of samples FAPY-glycosylate will identify using Capcr damage type 8-oxoguanine), and (2) that the dimensional mutations (deletions and insertions) will change the size of the expected Capcr product, which will lead to reduced output expected Capcr-product (because of deletions in mtDNA will lead to the formation of Capcr products sizes that are smaller than the size of the expected product). Therefore DNA damage (i.e., breaks chains, modification of nucleotides, DNA adducts and apurinovaya sites) and dimensional mutations (i.e., deletions of mtDNA) will either block the polymerase (e.g., damage), or to give PCR products resized (for example, deletions of mtDNA), which will reduce the amplification of a target sequence (expected size). Therefore, only those DNA matrix, which does not include dimensional DNA mutations and/or detectable DNA damage, will give the expected amplificatoare mitochondrial DNA, and in the nuclear genome is the product of the length of 17.7 etc. N. of a gene cluster-Globino. Increased DNA damage is associated with a decrease in output amplication products. Because of differences in Capcr amplification can sometimes be associated with differences in the number of copies of the matrix, or simply the quality of the DNA with no regard to the damage caused in vivo and in vitro, with the purpose of quality control perform quantitative amplification of a small segment [32]. It is unlikely that small segments of the target in the composition of the DNA susceptible to some anomalies, and therefore, they can serve as indicators of the relative number of copies and the quality of the PCR genomic extract.

You can also use alternative means for the quantitative analysis of products Capcr: they may include fluorescence, luminescence, radioisotopic and immunological tools (antibodies). Examples of this are the use of fluorogenic probes, labeled with the quencher and/or reporter dye labeled antibodies or oligonucleotide probes, methods of binding (for example, biotinylated probes), etc., This type of quantitative PCR reduces the need for electrophoresis and obtained the government of analysis can also be used as a method for quantifying damage in individual DNA strands.

Example 4

DNA isolation and Capcr

Total cellular DNA was isolated using a kit Qiagen genomic tip 20G kit as described by the manufacturer. In the DNA extraction this method are the genomic preparations suitable for long Capcr. The concentration of total cellular DNA was determined by fluorescence bromide in Atidim using fluorimetry with A4 filter, whose filter band pass excitation is configured to 360 nm, and the cut-off emission filter at 600 nm (Optical Technology Devices, Inc., Elmsford, NY), using as a standard-HindIII DNA. At the beginning, before Capcr, the amount of DNA was assessed by electrophoresis in a pulsating field. The sample was also analyzed using Capcr 222-BP fragment of mitochondrial DNA and 84-nucleotide fragment genes-Globino (primers for mitochondrial DNA 14619FOR, 1484REV; primers for-Globino - 48440FOR, 48634REV), expecting equal concentration matrices will give similar concentrations (short) Capcr products. In experiments on mice, the quality of the samples was analyzed using Capcr 80-nucleotide fragment of the mitochondrial DNA and 143-n 13281-13306, reverse 13335-13361; primers for-Globino - direct 21582-21605, reverse 21704-21725).

Capcr spent in the system GeneAmp PCR 2400 with a set of reagents GeneAmp XLPCR (Perkin Elmer). The reaction mixture contained 15 ng of genomic DNA as template. The parameters of reagents for Capcr described [25,32] for the product of mitochondrial DNA length of 16.2, etc., N. (coordinates direct primer - 15149-15174; coordinates reverse primer - 14841-14816) and product-globin length of 17.7, etc., N. (coordinates direct primer - 44330-44351; coordinates reverse primer - 61989-61968). Each reaction contained on 1x XL buffer II (Perkin Elmer Cetus), 1.1 mm MD(SLA)2, 0.1 mg/ml BSA, 0, 6 mm primers, 2 MCI-32R-dATP (Dupon-NEN) and 1 unit of rTth polymerase (Perkin Elmer Cetus). Each Capcr initiated with a hot start at 75°with the addition of DNA polymerase, rTth. In experiments on mice quantitative monitoring and a half (7.5 ng) genomic control matrix was included in each PCR series to provide quantitative terms. Upon completion Capcr 15 µl of each Capcr product was dispersed (using vertical electrophoresis in 1% agarose gel (TBE) at a voltage of 80 to 90 volts for 4 hours. Dried gels PhosphoImager 425). Although described using for Capcr primers specific localization and relative positions, a specialist in the art will be able to construct many other primernih sequences and conditions that will be dictated by the basic approach described in this application.

Calculate the frequency of DNA damage [33]. Briefly, amplification of damaged samples (Ad) normalized to amplification of intact controls (Aabout), resulting in the received relative degree of amplification (Aaboutfor groups ages 10 and 34 weeks was the control group at the age of 10 weeks; Andaboutfor 3-week-old mice were mouse S at the age of 3 weeks; Andaboutfor mice apoE-/-, SOD2-/+ and apoE-/- were mouse apoE-/-). Having a random distribution of damage and using the Poisson equation [f(x)=ex/x!, whereequal to the average frequency of injuries], for the undamaged matrix (i.e., for “zero class”: x=0) was determined average frequency of damage per DNA chain:=-lnAd/Ao. Statistical analysis was performed with use the differences in DNA damage in the proximal and distal aorta in the composition of each group of apoE mice or C57BL, when carrying out inter-group comparisons eort results for each part (proximal and distal) were combined for each group.

Example 5

Analysis of the transcripts using Northern blot

Control and treated with peroxynitrite culture was collected using a 4 M guanidinosuccinic, and total cellular RNA was isolated by centrifugation through 5.7 M cesium chloride [34]. To analyze the stability of transcripts of 2.5 mg/ml of actinomycin-D was added before the addition of peroxynitrite. The heart tissue was solubilisate using tissue solubilizer transmitter station 4 M guanidinosuccinic. Then the homogenates were centrifuged for 15 minutes (3000 g), and supernatant fractions were collected to isolate total cellular RNA by centrifugation through 5.7 M cesium chloride [34]. Total RNA was dispersed using agarose gel electrophoresis, transferred to nylon membrane and was prehybridization and hybridized [35] with the corresponding probe. Probes for transcripts of mitochondrial DNA was obtained on the material purified mitochondrial DNA [36] using PCR (16S-rRNA: forward primer - 2005-2022, reverse - 2982-3001; ND2: direct primer - 4831-4847, reverse - 5464-5481; Cyt-b: direct primer - 14730-14749, reverse - 15845-15863) and cleaned what was strayaway fabric mouse purified in the gel of PCR products covering the sites of genes 16S-rRNA (forward primer - nucleotide 1330-1354, reverse primer - nucleotide 2072-2097), ND2 (direct primer - nucleotide 4234-4259, reverse primer - nucleotide 4916-4941) and Cyt-B (direct primer - nucleotide 14196-14220, reverse primer - nucleotide 14967-14992), were used as matrices for randomly marked32R-dctv probes (Stratagene). Filters were exposed to Khodakovsky XAR film at -80°C. the RNA Levels in each sample were normalized by hybridization with probe-actin, commercially available (Clontech). Autoradiographically prints scanned on a densitometer (densitometer SI from Molecular Dynamics) and was evaluated quantitatively using the program IMAGEQUANT (Molecular Dynamics). In connection with the quantitative assessment of the levels of mitochondrial transcripts can be used any method of quantitative analysis of the levels of mRNA transcripts. Statistical analysis was performed using independent student's criterion.

Example 6

The synthesis of mitochondrial proteins

Analysis methods of synthesis of mitochondrial proteins described [37]. Briefly, control and treated cells were washed does not contain methionine culture medium and incubated for the ith 20-30 minutes casinha 0.1 mm cold L-methionine.

Cells were treated with trypsin and washed in the FSB. Collected cell centrifugate, resuspendable them in solubilizing buffer (4% LTOs), were treated with ultrasound (6 pulses at 30% load, exit 5) and total protein was determined. Equal amounts (total protein) tagged fusion products were dispersed electrophoretic 10-20% gradient LTO-PAG. The gels were dried using Whatman filter paper and exposed on film Kodak XAR at -80°C. the Percentage of labeling products broadcast was determined by densitometric (Molecular Dynamics Densitometer SI) for all bands for treated and untreated samples. The amount of labeling bands for each sample were used to calculate relative levels of inclusion. From the point of view of quantitative determination of the levels of mitochondrial proteins is any suitable method used for the quantitative analysis of the synthesis of mitochondrial proteins. Statistical analysis was performed using independent student's criterion.

Example 7

Tests on MTT and ATP

Recovery MTT complex II was used to assess mitochondrial respiration [38-42]. Cells were planted in 96-well tablets at a density of 8000 cells per well and incubated at 37&#b>O2, 0.1 mm, 0.5 mm or 1.0 mm peroxynitrite, 1 hour, and the reaction of the restoration carried out in conditioned medium for 1 h with MTT at a final concentration of 2.0 mg/ml, was literally and measured the absorbance at 570 nm [25]. Indicators absorption was transformed into recovery MTT using a calibration curve constructed for known amounts of living cells. Recovery MTT for treated samples then were normalized with untreated control samples and were expressed as the percentage of control. The total ATP content was determined using a kit for determining the content of ATP (Molecular Probes, A-6608) and microinjecting luminometer MicroLumat Plus LB (EG&G Berthold). Briefly, this test is based on the luciferin-luciferase bioluminescence (560 nm) in the presence of ATP. This test is extremely sensitive: most luminometers can identify at least 0.1 picomole existing ATP or ATP after generation in kinetic systems. Statistical analysis was performed using independent student's criterion.

Example 8

Histological analysis, lipid peroxidation and cholesterol levels

One aorta from each g is Aravali, was registrirovali and stained with hematoxylin and eosin, and then analyzed the development of atherosclerotic lesions. The levels of lipid peroxidation were determined in plasma samples using a colorimetric test (586 nm: Calbiochem-Novabiochem, La Jolla, CA) specific for malonic aldehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE), which are the end products resulting from the peroxidation of polyunsaturated fatty acids and related esters. The measurement of such products provides an index of lipid peroxidation. The samples were compared with the calibration curves for 4-HNE and MDA. The levels of total cholesterol were determined in plasma samples of mice by means of a calibration curve using a set of reagents for determination of cholesterol (Boehringer Mannheim, Indianapolis, IN) based on the instructions provided by the manufacturer.

Example 9

The results of experiments in vitro

In the environment of vascular superoxide (O-2) interacts with nitric oxide and depending on the rate of diffusion of forms peroxynitrite, or, alternatively, it can be restored under the control of the enzyme superoxide dismutase to form hydrogen peroxide (H2O2). In connection with EB/sup>2in order to show that hydrogen peroxide and peroxynitrite mediates damage to mitochondria dysfunction in vitro, and therefore, they may participate in the initiation events of atherogenesis.

Effect of hydrogen peroxide led to increased damage of mitochondrial DNA in both cell lines, whereas damage to nuclear DNA also occurred in endothelial cells. And HUVEC and HASMC were treated with 0.2 mm hydrogen peroxide for 1 hour and evaluated on mitochondrial DNA and nuclear DNA (-globin) compared with the untreated control (table. 1, figs.1). The level of damage to mitochondrial DNA and agnc (gene cluster-globin) was statistically significant (compared to untreated control, P<0,005) in HUVEC cells, while in HASMC significant damage were only in mitochondrial DNA (P<0,005). On the contrary, in HASMC no statistically significant increase damage agnc relative to the untreated control (P=0,695).

Analysis of the temporal dynamics of the effects of hydrogen peroxide showed that damage to mitochondrial DNA occur rapidly in both cell period of time (Fig.1). Reliable damage mitochondrial DNA occurred within 10 minutes in HUVEC cells (P=0,037) and 15 minutes in HASMC (P=0,047) compared with untreated control. In contrast, locus-globin showed rapid accumulation of damage agnc (Fig.1) because you want a 60-minute treatment in order to achieve manifestations reliable damage level in HUVEC cells (P=0.005). Therefore, damage to mitochondrial DNA occur quickly and in HUVEC and HASMC, subjected to the action of reactive oxygen species in vitro.

Treatment with peroxynitrite resulted in preferential damage to mitochondrial DNA in HUVEC and HASMC. To analyze the effect of peroxynitrite cells were treated with 0.1 mm and 0.5 mm peroxynitrite in for 1 hour (table.1, figs.2). Treatment of HUVEC 0.1 mm and 0.5 mm peroxynitrite caused a significant increase in the damage of mitochondrial DNA (P<0.005 percent), while exposure to 0.5 mm peroxynitrite in all cases led to significant damage in HASMC (P<0.005 percent) compared with untreated samples (table.1).

In an attempt to evaluate the effect of permanent low doses of peroxynitrite in vitro cells HUVEC and HASMC were treated with donors About-2and nitric oxide (reaction Of--2(2 mm/ml/min) caused a significant damage of mitochondrial DNA (P<0.05) in HUVEC cells (PL. 1) that was prevented by pretreatment with catalase or SOD: it shows that About-2and the formation of hydrogen peroxide in the recovery Of-2partly involved in mediating damage to mitochondrial DNA. Moreover, cells HUVEC subjected to continuous formation of nitric oxide and O-2(0.5 mm/ml/min 2 mm/ml/min, respectively) showed significant levels of damage to mitochondrial DNA (P<0.05) as compared with untreated HUVEC (table.1). Similarly, the development of higher acute doses of sodium oxide and About-2in equimolar amounts (1 mm SIN-1) resulted in a significant distortion (P<0.001) in HUVEC, but not in HASMC (PL.1, figs.2). Pretreatment of cells SOD prevented due to SIN-1 mitochondrial DNA in cells HUVEC (table.1, figs.2). Therefore, constant low dose and the development of high acute doses Of-2and OK is lowlevel significant reduction in levels of transcripts encoded by mitochondrial DNA genes NADP-dehydrogenase-2 (ND2) and cytochrome-b (Cyt-b), but not 16S-pPHK. Treatment of HASMC cells 0.5 mm peroxynitrite resulted in a reduction of 55% of the levels of transcripts ND2 and Cyt-b, while the lower levels of 16S-pPHK amounted to 14% (Fig.3). Similarly treated HUVEC samples were characterized by reduced levels of transcripts ND2 and Cyt-b 45-50% when processing 0.5 mm peroxynitrite, whereas the levels of 16S-pPHK declined by 26% relative to untreated controls (Fig.3). When using smaller doses of peroxynitrite (0.1 mm) levels of transcripts 16S-pPHK, ND2 and Cyt-b was reduced by 25-30%, and a decrease in HASMC was 5-15% (Fig.3).

Pre-treatment of cells with actinomycin-D, which is a transcriptional inhibitor, showed that exposure to peroxynitrite causes and inhibition of transcription (ND2 and Cyt-b), and the destruction of individual transcripts (Cyt-b). In contrast, the levels of transcripts of nuclear gene-actin from treatment with peroxynitrite was independent. Therefore, peroxynitrite is capable of differentially suppress and transcription, and the stability of the transcripts in relation to mitochondrial genes.

Treatment of the active forms of oxygen also led to the weakening of the synthesis of mitochondrial proteins in both centralnyj proteins on 23-33%, while processing 0.5 mm peroxynitrite was manifested in 55-70% loss of synthesis of mitochondrial proteins compared with untreated cells (Fig.4). Smaller doses of peroxynitrite (0.1 mm) in HASMC showed a slight decrease by 12% - on35S-methionine. Therefore, the effect of reactive oxygen species may also be due to reduced synthesis of mitochondrial proteins.

Exposure to peroxynitrite resulted in lower overall levels of ATP and reduce mitochondrial respiration (complex II) in HUVEC and HASMC (Fig.5). The estimation of total ATP levels showed that, while the dose of 0.1 mm peroxynitrite does not lead to a significant decrease in total ATP in any cell line, dose 0.5 mm caused a significant reduction in the amount of ATP (HUVEC-P=0.02; HASMC-P=0.04). Similarly, exposure to 0.1 mm peroxynitrite did not lead to a significant recovery in MTT complex II (mitochondrial respiration), while the dose of 0.5 mm caused a significant decrease (HUVEC-P=0.02; HASMC-P=0.008).

Restoration of MTT by succinate dehydrogenase component of complex II of the OK-FOS) is the indicator function of the mitochondria and is often used as a means to assess respiratory and okislityelnoye ability of mitochondria. In parallel experiments, cells were stained Trifanova blue to determine the degree of cell death associated with each impact of active forms of oxygen. Both cell lines virtual was not characterized by cell death (<5%) in those times, when evaluated by MTT and ATP. Therefore, it is assumed that the processing of reactive oxygen species has an impact on the total ATP content and respiration in HUVEC and HASMC.

Table I shows the frequency of injuries (10, etc., ad), estimated in mitochondrial DNA and agnc (cluster genes-globin), compared to the “zero class” untreated controls in the case of impacts peroksida hydrogen (H2O2and peroxynitrite. Destroyed peroxynitrite indicates peroxynitrite, proskurovsky in culture medium for 1 hour at room temperature before adding (0.5 mm) to cultured cells. Values are given as median (± s.e.).

Table II shows the estimated frequency of damage (10, etc., ad) in mitochondrial DNA and agnc (cluster genes-globin), related swania About-2(2 mm/ml/min), while spermine-POPO-at is used for the formation of nitric oxide (0.5 mm/ml/min). SIN-1 (1 mm) used for the formation of equimolar amounts of nitric oxide and O-2. In a variant of SOD+SIN-1 cells pre-treated with 3 units/ml SOD with the subsequent addition of SIN-1. Abbreviations: * - “statistically significantly different from control (P<0,05); ** - “statistically significantly different from control (P<0,005); n/d - not determined; a negative value (-) indicates that, compared with control was identified fewer injuries.

Example 10

DNA damage in mice

Mice with hypercholesterolemia apoE and healthy control mice were divided into two age groups (age at the time of killing 10 weeks or 34 weeks) and fed or mixed (4% fat), or "Western" (21% fat) diet since the age of 6 weeks. Tissue of the aorta and the heart (the left ventricle) were evaluated for DNA damage using Capcr, while blood plasma was used to determine the levels of cholesterol and lipid peroxidation (4-HNE and MDA). One aorta from each group were fixed for histological examination.

Tissue of the aorta of apoE mice with hypercho the calling of mitochondrial DNA compared with controls (P<0,05; Fig.6A). These differences were found in mice at the age of 10 and 34 weeks: it clearly showed that the aorta from apoE retain a higher level of DNA damage in vivo compared with healthy mice. In the aorta 10-week-old mice apoE revealed reduction of amplification by 61% (reduced amplification correlates with increased DNA damage) in relation to the zero class control, or the estimated level of mitochondrial damage

DNA 10 thousand base pairs (etc., ad) was 0,582±0,123 compared to 0,0±0,198 damage by 10, etc., N. 10-week-old control mice (P=0,018). Similarly, 34-week specimens group apoE was characterized as 3-fold increase in estimated damage of mitochondrial DNA compared with the control group of the same age (1/325±0,257 damage by 10, etc., ad against 0,453±rate £ 0.162 damage by 10, etc., ad: P=0.007). The same parameters were also detected in the tissue of the heart (left ventricle; Fig.6B). For example, a 10-week mouse apoE was characterized by a 67% reduction in amplification compared with zero class control (0,685±0,093 damage by 10, etc., against N. 0,0±0,048 damage by 10, etc., H.: P<0,001), while the 34-week-old mice apoE remained approximately 4-fold increase oceny 10, etc., N. against 0,213±0,295 damage by 10, etc., ad: P=0,056).

The influence of "Western" diet was also assessed in apoE and control mice (Fig.6B). In control mice, the "Western" diet was associated with higher levels of damage to mitochondrial DNA in aorta 10-week-old control mice compared to control mice, brought up on a mixed diet (0,31±0.17 damage by 10, etc., against N. 0,0±0.20 damage by 10, etc., N. accordingly, when the "Western" and mixed ration), but this difference was less significant (P=0,25). These differences were less apparent in the control mice at the age of 34 weeks (namely 0,45±0.16 damage by 10, etc., N. and 0.49±0.25 damage by 10, etc., N. when mixed and "Western" diet, respectively: R=0,90). Similarly, 10-week-old control mice were characterized by increased identified the level of damage to the mitochondrial DNA of the heart in the "Western" diet (Fig.6B; reducing the amplification 58%: 0,54±0.23 damage by 10, etc., N. and 0,0±0,05 damage by 10, etc., N. in "Western" and a mixed diet, respectively), and similar to the data obtained for the aorta, no difference was found in control mice at the age of 34 weeks (0,21±0.29 damage by 10, etc., N. and 0.25±0.21 damage by 10, etc., N. when the mixture is through DNA in connection with the "Western" diet or in the aorta, neither in the heart tissue in mice apoE. Therefore, increasing the fat content of food, apparently due to the increased damage of mitochondrial DNA only from control mice at age 10 weeks.

Age was also associated with increased damage to mitochondrial DNA in aorta and apoE, and the control mice (table. 2). Estimated 2,3-5,8-fold increase in the number of lesions was observed in aorta of apoE mice aged 10 weeks and 34 weeks (mixed diet, P=0.013; "Western" diet - P=0.011), whereas control mice did not show significant increasing levels of damage, although the increase was significant (table. 2). Older age, apparently, affects the control mice, Wycherley on a mixed diet (0,0±0.20 damage by 10, etc., ad against 0,45±0.17 damage by 10, etc., B.C. at the age of 10 and 34 weeks, respectively, P=0,09), while smaller differences were identified with respect to the age when feeding mice on Western diet (0,31±0.17 damage by 10, etc., ad against 0,49±0.25 damage by 10, etc., B.C. at the age of 10 and 34 weeks, respectively, P=0,556). On the contrary, in the tissues of the heart statistically significant relationships have been identified. Therefore, age correlated significantly with increased poei feeding mice a mixed diet.

As demonstrated no significant differences in damage of mitochondrial DNA between mixed and "Western" diets with the same age and genotype, we examined the effect of protein on mice aged 10 weeks. And apoE mice, and control mice C57BL fed on diets containing 16% or 24% protein for 4 weeks, starting from the age of 6 weeks. Although both diets were associated with a higher damage of mitochondrial DNA in mice apoE compared with control (aorta - P=0.015; heart - P=0.005), a diet with lower protein led to a significant reduction of damage in the aorta and in the control (P=0.007), and apoE (P<0,001) (Fig.7). A similar trend was also found in the heart of apoE (P=0.002). Therefore, a diet with less protein content is associated with reduced damage of mitochondrial DNA in both types of mice.

Table III shows estimated [mean (s.e.)] the magnitude of mtDNA damage per 10, etc., ad compared to control group (S) when the diet with 4% fat. All rations were given to mice beginning at age 6 weeks, and always up to the age of 10 or 34 weeks. For statistical analysis used independent the 10-week-old control mice C57BL, brought up on a mixed diet (damage " zero grade). Data are given as mean values (±s.e.). By P-values of student's criterion are given for comparisons between groups at the age of 10, as opposed to 34 weeks for each genotype (apoE or S) and diet. Asterisks (*) indicate statistically significant differences from the 10-week reference option.

Example 11

Damage to mtDNA in the aorta in 3-week-old mice apoE-/- and control mice

Damage to mtDNA aortic also was evaluated in 3-week-old mice apoE-/- and control mice. Aorta 3-week-old mice apoE-/- were characterized by significantly higher levels of mtDNA damage compared to control animals of the same age (Fig.8; P=0.001): this confirms that the damage to mtDNA occur at an early age in aorta predisposed to the development of atherosclerotic lesions.

Example 12

Cholesterol in mice

As expected, cholesterol levels were significantly increased in all groups of mice apoE compared with control mice (above about 4-5 times: P<0,001). Although "Western" diet was associated with higher cholesterol levels in all mice, this increase (in relation to mixed ration) was a statistician is P=0,0019). In contrast, control mice C57BL showed not much more than high cholesterol levels (above about 1.6 times; mixed at 53.4±15.1 mg/DL; the "Western" - to 85.2±10,8 mg/DL) when using "Western" diet, although the differences were not statistically significant (P=0,17). Therefore, mouse apoE was characterized by significantly higher levels of cholesterol compared to control C57BL the same age, and higher levels of cholesterol have been associated with the Western diet.

Example 13

Lipid peroxidation in mice

The levels of lipid peroxidation was measured by determining the levels of MDA and 4-HNE. Mouse apoE with hypercholesterolemia were characterized by significantly elevated levels of lipid peroxides compared to control mice (P<0,05). However, in contrast to the results obtained for total cholesterol levels, the degree of lipid peroxidation was not increased significantly in mice apoE in comparison with the "Western" mixed ration (Fig.9). In contrast, the control mice, Wycherley to the "Western" diet, remained elevated lipid peroxidation compared with mixed ration (P<0,05). Therefore, although the mouse apoE often have higher levels of peroxides Lucae use "Western" diet compared to a mixed diet.

Example 14

Histology mice

The aorta from each group were embedded in paraffin and prepared cross-sections with a thickness of 5 μm, which were stained with hematoxylin and eosin. Although atherosclerotic damage was absent in all groups of 10-week-old mice (control and apoE), regardless of diet (mixed or "Western"), atherosclerotic damage was present in both groups 34-week-old mice apoE, but was absent in 34-week-old control mice. On a quantitative level and frequency, and the size of the lesions was obviously increased in mice apoE, Wycherley to the "Western" diet compared to mice apoE, vcarmlivaniu on a mixed diet.

Example 15

Analysis of atherogenesis and mtDNA damage in mice apoE-/-, SOD2-/+

To determine whether the dysfunction of mitochondria and antioxidant activity (MnSOD, SOD2) on atherogenesis, formed a line of mice apoE-/-, SOD2-/+. Earlier studies have shown that heterozygous for SOD2 mice (-/+) are characterized by reduced function of mitochondria and the activity of SOD2 in comparison with wild-type mice. Hybrid mouse apoE-/-, SOD2-/+ had 40% of the activity of SOD2 from that identified in mice apoE-/-. The development of atherosclerotic what is that in mice apoE-/-, SOD2-/+ observed a 2.5-fold increase (P=0.02) in the number of atherosclerotic plaques compared with relatives of apoE-/- (Fig.10V). Therefore, the damage of mtDNA aorta was increased in mice apoE-/-, SOD2-/+ compared to similar mice genotype apoE-/- (Fig.10C; P=0.006).

Example 16

Discussion in vitro experiments

The present study was conducted to demonstrate that hydrogen peroxide and peroxynitrite mediates damage and mitochondrial dysfunction in cells HUVEC and HASMC in vitro. Mitochondrial DNA was reliable in both types of cells exposed to hydrogen peroxide, and HUVEC were significant and damage to nuclear DNA. Similarly, mitochondrial DNA is significantly damaged by the effect of acute doses of peroxynitrite on HUVEC (0.1 mm and 0.5 mm) and HASMC (0.5 mm). Moreover, under the action of permanent low concentrations of peroxynitrite significant damage to mitochondrial DNA compared to controls were detected in HUVEC cells, while the effect on agnc was absent. Handling SIN-1 resulted in a significant increase in the damage of mitochondrial DNA in HUVEC. In contrast, handling SIN-1 cells HASMC not determined what ricinol such differences can be relative ability HASMC to restore About-2and hydrogen peroxide compared to HUVEC. This may be consistent with the results of the actions XO/LZ on HASMC, which did not lead to significant levels of damage, while the same treatment of HUVEC caused significant damage mitochondrial DNA (PL.1). These results, along with the fact that, in General, HUVEC cells presumably more sensitive to the effects of reactive oxygen species, may be in accord with this view.

The transcript level ND2 and Cyt-b was decreased after exposure to peroxynitrite in both types of cells. Although the levels and ND2 and Cyt-b was decreased significantly, only the lower levels of transcript Cyt-b was statistically significant (HASMC-P=0.033; HUVEC-P=0.001). The decrease in the content of the transcripts ND2 and Cyt-b, apparently, is temporary, as most cultures restored the content characteristic of the untreated control, after removal of the medium, treated with active forms of oxygen, and 2-hour stay in a conditioned environment. Therefore, the reduction of the transcript level is directly related to the acute treatment of active forms of oxygen. Because the transcript Cyt-b farthest from the site of transcription initiation polycistronically level) suppressed damage to mitochondrial DNA. On the other hand, the stability of the transcripts in the presence of specific active forms of oxygen can affect the levels of RNA. The results of the experiments with actinomycin-D confirm that inhibition of transcription, and the instability of transcripts occur in response to exposure to peroxynitrite in the case of Cyt-b, while the transcript ND2, apparently, less susceptible to instability induced by peroxynitrite. Finally, mitochondrial rRNA are subject to the preferred expression [47, 48], potentially making them less sensitive to the effects of treatment with peroxynitrite conducted in this study.

Consistent with reduced levels of transcription, a similar weakening of the synthesis of mitochondrial proteins was detected in HUVEC and HASMC after treatment of acute doses of active forms of oxygen. Protein synthesis was decreased by 23-33% in both cell lines under the influence of 0.2 mm H2O2, while treatment with 0.5 mm peroxynitrite caused a more significant loss in protein tagging (HASMC - 30% of control; HUVEC - 45% of control). Also consistent with these data, the decreased production of cellular ATP and intensity of breath that occurs after exposure to the active form of CI is to a number of related events, related to the functioning of the mitochondria in the cells of HUVEC and HASMC, which ultimately leads to dysfunction of cells.

Although mitochondria are the main producers of cellular ATP, they are also permanent generators of reactive oxygen species [12-16], forming About-2during transport of electrons (metabolic processes). Then-2removed with the participation of mitochondrial Mn-dependent superoxide dismutase (MnSOD, SOD2), although this reaction produces hydrogen peroxide, which accumulates in the mitochondria. As an alternative,-2interacts with nitric oxide near the speed of diffusion (6,7×109M-1with-1) with the formation of peroxynitrite [49].

Although it is known that nitric oxide primarily performs antiatherogenic role [50-52], these effects are attenuated in atherosclerotically vessels due to the interaction of nitric oxide with O-2with the formation of peroxynitrite. The peroxynitrite easily oxidizes LDL and depletes some antioxidants [10, 11]. Therefore, peroxynitrite, formed in the wall of the artery may contribute directly to the formation of ox-LDL, which, in turn, may lead to the dysfunction Mitko dothelial cells clinical levels of ox-LDL increase the formation of hydrogen peroxide 4-12 times [54]. In vivo studies confirmed that oxidants derived from nitric oxide (e.g., peroxynitrite), are formed at the person in the coronary arteries and, moreover, are concentrated in xantana cells and around them within atomnogo deposits, as well as in early fat stripes under the intima [55, 56]. In addition, peroxynitrite mediates narisovanie MnSOD (mitochondrial form of SOD) on tyrosine residues, which leads to inactivation of this enzyme [57, 58]. The effects of peroxynitrite on recombinant human MnSOD resulted in complete suppression of catalytic activity and education and nitrotyrosine, and dityrosine [57, 58]. Therefore, since atherosclerotically vessels scarce generation of nitric oxide by the endothelium [59] and because the ratio of nitric oxide to About-2it is important for the activity of nitric oxide as an anti - or prooxidant [5, 60], the biological activity of nitric oxide in these cells may be more Pro-oxidant, rather than an antioxidant, thereby triggering the cycle of continuous damage of endothelial cells. Additional studies have compared levels of damage to mitochondrial DNA and agnc on a limited number of atherosclerotically and zdorovyakam damage levels of mitochondrial DNA (P=0,0023), consistent with these messages. Thus, peroxynitrite, apparently, mediates a number of harmful actions on the cell, including mitochondrial DNA, varying levels of transcripts and proteins and reduced mitochondrial anti-oxidant activity.

Role of nitric oxide was also investigated by removing oxidants originating from mitochondria of cells trophoblast human placenta (NRST) [61]. When the production of nitric oxide is reduced in placental cells trophoblast human effects of L-NAME, which synthase inhibitor of nitric oxide, as expected, the formation of cellular oxidants increases. Treatment of cells trophoblast human placenta CuZnSOD and catalase does not weaken induced oxidative response due to suppression of NOS. Additional inhibitors of xanthine oxidase, cyclooxygenase, and mitochondrial OK-FOS shows that only inhibitors of mitochondrial OK-FOS increase oxidative response induced suppression synthase nitric oxide, while the HO inhibitors and cyclooxygenase this effect is not apparent. This means that reactive oxygen species generated in the mitochondria are the main damaging agents is aroda, formed in the mitochondria.

In oxidative environment artery endothelial cells and vascular smooth muscle exposed to chronic stress caused by reactive oxygen species. Therefore, cells that are no longer able to adequately respond to oxidative stress have a higher risk of adverse manifestations, caused by damage mediated by reactive oxygen species. Here it was shown that HASMC and HUVEC (especially HUVEC) are sensitive to such damage in vitro and, therefore, that chronic production in vitro reactive oxygen species with the greatest likelihood of causing cell dysfunction of blood vessels due to the initiation of the weakening of the mitochondria, resulting in the loss of many important cellular functions.

Example 17

Discussion of experiments on mice

The original aim of this study was to investigate the extent of DNA damage in tissues of the aorta and heart in mice with hypercholesterolemia apoE and control mice of the same age. The apoE mice developed atherosclerotic damage similar to the human type. In General, the accelerated atherogenesis when mice apoE feed on "Western" diet with high fat content on crawte not more than 6 ("Western" diet) or 8 (mixed diet) weeks, then Santanyi cells formed no later than age 8 ("Western" diet) or 10 (mixed diet) weeks and injuries develop no later than age 15 ("Western" diet) or 20 (mixed diet) weeks. The degree and progression of atherogenesis in mice apoE used in this series of experiments, consistent with earlier observations obtained.

Damaged mitochondrial DNA were statistically significantly increased in aorta and 10 - and 34-week-old mice apoE with hypercholesterolemia, Wycherley on a mixed diet compared with control C57BL the same age. Age was also associated with an immediate increase in the detected frequency of damage to mitochondrial DNA in mice apoE (P<0,05), regardless of diet. As additional studies that compared the relative levels of damage to mitochondrial DNA between the proximal and distal ends of the aorta from apoE mice, showed no significant differences in the damage of mitochondrial DNA within each group, it is unlikely that damage to mitochondrial DNA identified in aorta apoE, are an artifact of atherogenic factors specific to the Proxima what s in apoE: therefore, it is not a product of the development of atherosclerotic lesions, but is a potential factor involved in atherogenesis.

In mice apoE, brought up on the "Western" diet, there was a significant increase damage of mitochondrial DNA compared with the variant of apoE, brought up on a mixed diet. In this regard, in each group evaluated the levels of total cholesterol and lipid peroxidation. Although "Western" diet in mice apoE was also associated with significantly higher levels of cholesterol compared to a mixed diet (P<0.05), and this did not lead to significant increase of lipid peroxidation in mice apoE compared to a mixed diet. Therefore, the data obtained confirm that the levels of lipid peroxidation already peaked in apoE mice on a mixed diet (perhaps due to their genotypic predisposition to hypercholesterolemia). Accordingly, the "Western" diet with higher fat content are not able to increase lipid peroxidation in mice apoE compared with mixed ration, which is consistent with the data on damage to mitochondrial DNA, which did not allow to determine razevat increase in total cholesterol compared with mixed ration, however, the "Western" diet substantially does not increase the total levels of peroxide okislenii lipids in mice apoE, which, therefore, may reflect the fact that the damage of mitochondrial DNA in mice apoE more closely correlated with genotype and age than with diet. As expected, mouse apoE with hypercholesterolemia characterized by significantly elevated levels of cholesterol (4.2-5.7 times higher: P<0.001) and lipid peroxidation (1.9-2.7 times higher: P<0.05) as compared with healthy controls of the same age. Although it has been shown that, compared with control mice of the same age is associated with age, the increased lipid peroxidation in mice apoE, failed to find messages that compared the levels of lipid peroxidation when mixed and "Western" diets. Therefore, the results presented here indicate that, although the "Western" diet results in mice apoE to significantly elevated levels of cholesterol compared to a mixed diet, it does not change the overall level of products of lipid peroxidation.

Although age and high fat diet were not significantly associated with a significant increase damage m the control on a mixed diet in relation to age, but the trend toward increased damage when using "Western" diet was observed in mice at age 10 weeks. Apparently, the higher the age, the influence of the control mice on a mixed diet, while in mice, Wycherley "Western" diet, were found smaller differences. Control mice 10 weeks of age, vcarmlivaniu to the "Western" diet, were also characterized by more highly appreciated by the frequency of damage to mitochondrial DNA in comparison with feeding on a mixed diet, while unable to detect the connection between diet and damaged mitochondrial DNA in a 34-week-old control mice: this confirms that the effect of diet on mitochondrial DNA has the greatest effect in young control mice. In contrast to data obtained for mice apoE, the use of "Western" diet was associated with significant (P<0,05) increased levels of lipid peroxidation in control mice compared with mixed ration: this suggests that lipid peroxidation is not “out” at the maximum level in healthy mice, brought up on a mixed diet, and the increase in the content of substra, that may be consistent with the increased damage of mitochondrial DNA, were detected in control mice on Western diet.

Caloric restriction and reduced content of dietary protein, as is known, has a positive value for different organisms, increasing the life span. Therefore, the fact that both lines of mice were characterized by a lower level of damage to mitochondrial DNA when feeding on diets with lower protein compared to a high-protein diet, confirms this view. For a more complete understanding of this fact it is necessary to conduct long-term studies in mice apoE using low-calorie diets. Because low calorie diets are associated with reduced levels of reactive oxygen species, these observations are consistent with the view, according to which the reduction of damage from reactive oxygen species, inhibits atherogenesis.

Over the last decade have been accumulating evidence of the role of mitochondria (their damage and dysfunction) in various chronic age-related diseases. The main idea is that damage to the mitochondria accumulate totime as production of reactive oxygen species, mediated OK-FOS increases, leading to dysfunction of cells. Although it has been shown that the mitochondria of endothelial cells sensitive to damage in vitro, mediated by active oxygen forms, there is information about the increase in the number of pathogenic mutations of mitochondrial DNA in cardiovascular tissues in people with atherosclerosis, as well as information about pathogenic mutations of mitochondrial DNA associated with increased risk factors of heart disease (age, Smoking, diabetes, and others). Interestingly, risk factors such as Smoking, hypertension, hypercholesterolemia, and so forth, mediate increased formation of reactive oxygen species, and in recent studies it has been shown that mitochondria are susceptible target for damage. Although atherogenesis is really a complex process involving various mandatory stage, in this application it is assumed that the main mechanism for the development of atherosclerotic lesions begins with the accumulation of damaged mitochondria in tissues of blood vessels, which ultimately leads to dysfunction of OK-FOS and reduction of energy performance. Together, these processes lead to an increase of the vascular cells, thereby creating atherogenic environment in the artery.

Presents the first report of studies level of damage to mitochondrial DNA in the tissues of the aorta in a murine model of atherosclerosis when compared with controls of similar age. The results show that a distinct increase damage of mitochondrial DNA associated with atherogenesis and age. On the basis of the presence of a substantial level of damage to mitochondrial DNA in mice apoE at the age of 10 weeks, you can assume that in this model of atherosclerosis in vivo damage occurs before the development of atherosclerotic damage to or simultaneously with it. In addition, the damage of mitochondrial DNA in the aorta increases with age in vivo. Moreover, apoE genotype, apparently, has a stronger influence on the level of damage of mitochondrial DNA compared with the diet, although the influence of diet on mitochondrial DNA can occur in young healthy mice C57BL. Therefore, damage to mitochondrial DNA detected in mice apoE may ultimately lead to the violation of the processes of OK-FOS, thereby initiating a dysfunction of endothelial cells, which is key sobytiemi coronary angiography, were selected for in vivo studies of damage to mitochondrial DNA. All selected patients gave informed consent for the taking of additional samples of blood during coronary angiography. The entire group consisted of 75 patients. Leukocytes were isolated using standard methods (drug, “leukocyte film”) with the release of their DNA (nuclear and mitochondrial). Mitochondrial DNA was determined by real-time quantitative PCR using and short fragments of mitochondrial DNA, and damage within gene-globin as controls. In Fig.11 illustrates an increased incidence of damage to mitochondrial DNA in patients grouped according to risk factors associated with myocardial infarction.

Based on initial results, some level of damage to mitochondrial DNA was determined as the norm. Exceeding this norm was seen as a high level of damage. It was then determined the proportion of patients who have damage to mitochondrial DNA is a function of the risk factors of heart disease. For example, among smokers, about half had increased damage meterialnoy DNA. In some cases the influence was additive in nature: for example, 100% of smokers diabetics reported a higher damage of mitochondrial DNA. In the analysis revealed no significant difference between those who have the risk factors, and those who do not have them (for example, Smoking - P<0.05; Smoking plus diabetes P<0,01; Smoking plus hypertension - P<0,05). Also found a trend aimed at the existence of coronary heart disease based on the degree of damage of mitochondrial DNA. However, to determine the degree of significance of this trend needs to be examined a larger group of patients.

To further examine the degree of damage of mitochondrial DNA under various conditions in vivo were investigated 5 “spermatophores” and a group of 6 subjects of similar age, healthy and leading an active lifestyle, but not running spermataphore. First, mitochondrial DNA was determined from spermatophores before and after the 20-mile training run“. Samples were taken of the blood of obtaining leukocyte film and damage assessment of mitochondrial DNA.

In Fig.12 shows that there has been a slight increase povrezhdennosti spermataphore and control subjects received high fat food. High fat food was presented dish "El Grande Platter" from a local restaurant with Mexican food, the caloric value of which amounted to 3000 calories, 60-70% of which is accounted for calorie fats. Blood samples were taken just before eating and after 2 and 6 hours after it. Received leukocyte film and was isolated DNA. Also quantitatively evaluated mitochondrial proteins using as a source leukocyte mitochondrial protein. In Fig.13 and 14 shows that there is a gradual small increase in damage of mitochondrial DNA control, as well as increasing damage of mitochondrial proteins after 2 hours, with a decrease after 4 hours. Spermatophores damage to mitochondrial DNA and protein damage is actually reduced below background levels at 2 o'clock and starting to return to background levels after 6 hours. Presumably, because spermataphore exposed to increased oxidative stress during running, their antioxidant protective system more efficient. Thus, the effects of the incentive to DNA damage in spermatophores is even less damage than normal. Therefore, they are activated antioxidant is from damage to mtDNA in atherosclerotically and healthy aorta

To determine whether increased mtDNA damage in atherosclerotically human tissues, analysis of DNA damage was performed on tissues eort in groups of healthy and atherosclerosis of people similar in age composition. Aorta from each group were fixed in 4% paraformaldehyde, embedded in paraffin, cooked slices with a thickness of 5 μm, DEPA-definitional, rehydratable, were stained with hematoxylin and eosin and evaluated the development of atherosclerotic lesions.

The analysis method Capcr showed a significant increase in mtDNA damage in atherosclerotically tissues compared with artami healthy control group (Fig.15A and 15C). On the contrary, a marker of damage to nuclear DNA gene cluster-globin - did not show significant damage in atherosclerotically aorta compared with control (P=0.15). Therefore, mtDNA showed a significant and preferred damaging in atherosclerotically aorta person.

Example 20

The influence of mtDNA damage in atherogenesis

Comparison of the levels of mtDNA damage in tissues atherosclerotically aorta with controls of similar age shows that increased the damage of the MTD is ASTA 3 and 10 weeks is considered, what damage occurs in vivo to the development of atherosclerotic damage to or simultaneously with it. Moreover, since the frequency of atherosclerotic lesions, and damage to mtDNA in the aorta increased hybrids apoE-/-, SOD2-/+ compared with related species apoE-/-, it is clear that the function of mitochondria is important from the point of view of atherogenesis. Therefore, the data obtained confirm that: (1) damage to mtDNA is significantly increased in atherosclerotically aorta and in mice, and humans; (2) the damage of mtDNA in the aorta increases with age in vivo; (3) damage to mtDNA occur in vivo to the development of atherosclerotic plaques or in concert with them; (4) damage to the mitochondria is an important factor in atherogenesis.

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Any patents or publications mentioned in this application, is defined in accordance with the level of vision experts in the field of technology, which converts the invention. In addition, these patents and publications are included here for information in the form of bibliographic references to the same extent that each publication separately and specifically identified as included to information reference.

To a person skilled in the art will understand that the present invention fully adapted to obtain its objects and achieve the ultimate goals and advantages mentioned above as well as those objects, the ultimate goals and benefits arising from it. Examples presented here, along with the methods, procedures, obrabotkami, are examples and are not intended to limit the scope of the present invention. Changes in these and other ways the application will be clear to experts in the field of technology, and they correspond to the essence of the present invention, which is defined by the scope of the claims.

Claims

1. Method of assessment of atherosclerotic condition in the subject, which includes the following stages: (a) taking a blood sample from a specified entity; (b) quantifying the level of damage to mitochondrial DNA in a specified sample of blood by the method of quantitative PCR, where the specified DNA is treated with glycosylate FAPY to the specified PCR amplification for determination of 8-oxo-G lesions or the quantification of the level of damage of nuclear DNA by the method of quantitative PCR for the amplification product of the size of 16.2, etc., ad; (C) comparison of the quantitative level of damage to mitochondrial DNA in the blood sample of the specified entity with the quantitative level of damage to mitochondrial DNA in the blood sample of a control subject not suffering from atherosclerosis, where higher quantitative level of damage to mitochondrial DNA in the specified subject with elevated riskpool under item 1, moreover, the specified subject has at least one risk factor associated with atherosclerosis.

3. The method according to p. 2, and the specified risk factor selected from a group that includes Smoking, hypertension, diabetes, obesity, hypercholesterolemia and hyperlipidemia.

4. The method according to p. 1, and these mitochondrial DNA is assessed by measuring selected from the group which includes the measurement of the synthesis of mitochondrial mRNA, the measurement of the synthesis of mitochondrial proteins, measurement of changes in mitochondrial oxidative phosphorylation and dimension changes of ATP synthesis in mitochondria.

5. The method of quantitative measurement of the level of oxidative stress in the subject, which includes the following stages: (a) taking a blood sample from a specified entity; (b) quantifying the level of damage to mitochondrial DNA in a specified sample of blood by the method of quantitative PCR, where the specified DNA is treated with glycosylate FAPY to the specified PCR amplification for determination of 8-oxo-G lesions or the quantification of the level of damage to mitochondrial DNA by the method of quantitative PCR for the amplification product of the size of 16.2, etc., ad with Mr. the blood sample according to the method of quantitative PCR, where specified DNA is treated with glycosylate FAPY to the specified PCR amplification for determination of 8-oxo-G lesions or the quantification of the level of damage of nuclear DNA by the method of quantitative PCR for the amplification product with a size of 17.7 etc. N. of a gene cluster-globin; (d) comparing the quantitative level of DNA damage per length of DNA between the specified mitochondrial DNA and the specified nuclear genome, where a higher quantitative level of damage to mitochondrial DNA in the calculation of the length of the DNA compared to the level of damage to nuclear DNA in the calculation of the length of the DNA is indicative of the increased level of oxidative stress in the specified entity.

6. The method according to p. 5, with high quantitative levels of oxidative stress are predictive signs of atherogenesis, hypertension, diabetes, hypercholesterolemia, Smoking, degenerative, age-related diseases and malignant tumors.

7. The method according to p. 5, and these mitochondrial DNA is assessed by measuring selected from the group which includes the measurement of the production of mitochondrial mRNA measurement generation Micah the changes of ATP synthesis in mitochondria.

8. The method of determining the efficacy of a drug in reducing the risk of development of atherosclerosis in a subject, which includes the following stages: (a) taking a blood sample from the specified subject before and after administration of specified drugs specified subject; (b) quantifying the level of damage to mitochondrial DNA in the specified taken a blood sample according to the method of quantitative PCR, where the specified DNA is treated with glycosylate FAPY to the specified PCR amplification for determination of 8-oxo-G lesions or the quantification of the level of damage to mitochondrial DNA by the method of quantitative PCR for the amplification product of the size of 16.2, etc., N. with matrix mitochondrial DNA, where the reduction of the quantitative level of damage of mitochondrial DNA after the specified treatment is an indication that the treatment leads to decrease the risk of atherosclerosis.

9. The method according to p. 8, and specified damage to mitochondrial DNA is assessed by measuring selected from the group that includes measurement of the synthesis of mitochondrial mRNA, the measurement of the synthesis of mitochondrial proteins, measurement of changes in mitochondrial oxidative phosphorylation and of the

 

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