The defects in the mitochondrial genome are associated with different pathologies, including atherosclerosis. It is generally believed that atherosclerosis leads to premature cell senescence, but there is increasing evidence that cell senescence, the biomarker of which is the mutational load of mitochondrial genome, is itself a mechanistic factor of atherogenesis. The basic scientific problem addressed is an examination of functional consequences of mitochondrial DNA (mtDNA) damage based on the analysis of variability of mitochondrial genome. The paper is devoted to identification of genetic markers of mtDNA mutation burden, molecular and cellular markers of disorders in functional properties of cells arising from mutations in the mitochondrial genome, and identification of combinations of genetic markers that lead to violation of functional properties of human monocyte-macrophages. New data are presented, which resulted from whole genome sequencing of mtDNA from atherosclerotic lesions of arterial intima, the analysis of the relationship of mtDNA damage with the changes in cellular composition of arterial intima and expression of apoptosis- and inflammation-related genes, retrieving the data on mitochondrial genome variability in patients with atherosclerosis, and the study of functional activity of human blood monocytes differing substantially by the degree of mtDNA mutation burden.
- mitochondrial DNA
- mitochondrial dysfunction
The defects in the mitochondrial genome are often associated with various human pathologies. There is the evidence that such a relationship can be in the case of atherosclerosis. The nature and the mechanisms of this association remain unclear. The basic scientific problem, to which the given chapter is aimed, is the study of functional consequences of mtDNA damage based on the analysis of variability of the mitochondrial genome. The disclosure of those associations will provide new knowledge on atherogenesis as an age-related degenerative pathological process and will create a basis for development of new approaches to the diagnosis, prevention, and treatment of atherosclerosis.
Chronic age-related degenerative diseases account for the major share of mortality in society. The most frequent pathology is atherosclerosis, which is classified as a typical disease of aging, and its prevalence in the elderly reaches 100%. It is generally believed that atherosclerosis leads to premature biological aging of cells, but there exists also the increasing evidence that cell aging, one of the indicators of which is the mutational load of mitochondrial genome, is a mechanistic factor of atherogenesis. In such a case, the association of mitochondrial DNA (mtDNA) variability with the disturbances in the functional properties of the artery wall cells participating in atherogenesis remains poorly understood. The results of the study of the association of mtDNA damage and functional properties of cells based on the analysis of the mitochondrial genome variability will make it possible to establish the features and patterns of the pathology of vascular aging and to justify the assumption that the prevention of accelerated cell aging is a therapeutic target in atherosclerosis.
Mammalian cells contain dozens, hundreds, and even thousands of mtDNA copies. It was previously assumed that all mtDNA molecules are identical at birth [1, 2]. However, later studies have shown that about 25% of healthy people inherit a mixture of wild-type and variant mtDNA (the phenomenon of heteroplasmy) and the latter may imply potentially pathogenic variants in the coding region of mtDNA [3, 4]. The mitochondrial genome is characterized by exaggerated instability, with an estimated mutation rate of at least 5–15 times higher than that of the nuclear genome. This is partly a result of proximity to the electron transport chain, which is the main intracellular source of free oxygen radicals, and partly due to relatively limited protection and reparation of mtDNA mechanisms . The high frequency of mutations determines the high level of mtDNA variability, as well as the appearance of somatic mutations of mtDNA with the aging of human organism . The proportion of mutant copies of mtDNA carrying both inherited and somatic mutations may be changed during lifetime due to unequal separation of mitochondrial genotypes during cytokinesis in dividing cells (vegetative segregation) or in nondividing cells during mtDNA replication. An increase in the degree of heteroplasmy represents a kind of “clonal expansion” of low-level inherited variants, which occurs due to preferential replication of mtDNA carrying certain types of mutations . Exceeding the critical threshold level of the degree of heteroplasmy of pathogenic mutations in the cell is manifested by biochemical defects in the mitochondrial respiratory chain . The high level of mtDNA mutations manifests itself in the form of multi-system mitochondrial diseases  and degenerative age-related diseases and undoubtedly contributes to biological aging processes .
The basic scientific problem addressed by this chapter is an examination of functional consequences of mitochondrial DNA (mtDNA) damage based on the analysis of variability of mitochondrial genome. The described results are devoted to identification of genetic markers of mutational load of mitochondrial genome, molecular and cellular mechanisms of disorders in functional properties of cells arising from mutations in the mitochondrial genome, and identification of combinations of genetic markers that lead to violation of functional properties of human monocyte-macrophages.
2. The markers of mitochondrial DNA mutation load in atherosclerotic lesions
It has been shown earlier that several mutations of mitochondrial genome have significantly higher prevalence and mean value in lipofibrous plaques as compared to non-atherosclerotic intima and therefore are associated with atherosclerosis [11–13].
In continuation of the above studies, the samples of human thoracic aorta (32 samples, 19 men, 13 women aged 53–78 years old) were taken at autopsy performed within 6–16 h after sudden death. After macroscopic identification of unaffected and atherosclerotic areas of the aorta (initial lesions, fatty streaks, lipofibrous and fibrous plaques), autopsy aortic samples (2 × 2 cm, 4–9 sites per autopsy sample) were excised for further studies. In addition, five aortas were subjected to complete morphological mapping, that is, from whole samples approximately 8 × 12 cm in size, 25–56 sites differing by the degree of atherosclerotic lesion were obtained. The total of 297 samples was obtained. Histological and immunocytochemical analysis of the samples was carried out; DNA and RNA were isolated from aortic intima. For the analysis of mtDNA mutations, the pyrosequencing technology was used.
The analysis of heteroplasmy of mitochondrial genome of cells from unaffected and atherosclerotic regions of aortic intima was performed for 42 mutations previously known from the MITOMAP database (m.652delG, m.652insG, m. 716T>G, m. 750A>G, m.961insC, m.961delC, M.1555A>G, m.3256C>T, m.3258T>C, m.3271T>C, m.3280A>G, m.3285C>T, m.3316G>A, m.3336T>C, m. 5132insAA, m.5178C>A, m.5540G>A, m.5692T>C, m.5814T>C, m.6489C>A, m.8362T>G, m.8363G>A, m.8993T>G, M.8993T>C, m.9379G>A, m.9480del15, m.9537insC, m.12315G>A, m.13513G>A, m.14459G>A, m.14482C>G, m.14482C>A, M.14484T>C, m.14487T>C, m.14709T>C, m.14846G>A, m.15059G>A, m.15084G>A, m.15452C>A, m.15498del24, m.15723G>A, and m.15762G>A). Thus, most of the mitochondrial DNA genes encoding enzymes (9 of 13) were covered, except for the
The analysis showed that mtDNA variants m.652delG, m.3256C>T, m.12315G>A, m.14459G>A, and m.15059G>A were significantly associated with the lipofibrous plaque (
It was found that the same mtDNA variants associated with the advanced atherosclerotic lesions are also associated with the initial atherosclerotic lesions (fatty dots and fatty streaks). The mutation m.13513G>A was antiatherosclerotic, as it correlated negatively with the degree of atherosclerotic lesion. Mutation m.1555A>G negatively correlated with initial atherosclerotic lesions and lipofibrous plaques (
To determine the relationship between the mutational load and the degree of atherosclerotic lesion, a linear regression analysis was performed. For each mutation in each aorta, the distribution of the heteroplasmy level was assessed, and interquartile boundaries were determined. When estimating the integral mutation burden for 11 mutations, the linear regression model reached statistical significance at
Further, whole genome sequencing of mtDNA from unaffected and atherosclerotic intima of the thoracic aorta, obtained in autopsy (11 cases) within 6–16 h after sudden death, was carried out using next-generation sequencing (NGS) technology on the Roche 454 GS Junior Titanium platform (Roche Applied Science, USA). The following samples were analyzed: unaffected areas of intima (
It has been established that the mitochondrial genome has a significant individual variability and has a much larger number of heteroplasmic mutations than was previously assumed in the literature. Most mitochondrial DNA mutations found by whole genome sequencing had a low degree of heteroplasmy, but the results of the analysis suggest that there are both general and individual variants associated with atherosclerotic lesions .
Thus, heteroplasmic mtDNA mutations associated with atherosclerotic lesions were identified by NGS, and the integral mutation burden of the mitochondrial genome was assessed. Ten heteroplasmic mutations of mtDNA were detected that occurred simultaneously in different parts of the intima of the aorta (m.368A>G, m.751delA, m.8404T>C, m.8485G>C, m.9720delT, m.10663delT, m.14160G>A, m.14207delG, m.16086T>C, and m.16389G>A). The level of heteroplasmy of these mutations ranged from 2 to 26%, with more than half of the values not exceeding 6%. Among the heteroplasmic mutations found, the majority occurred in the coding region, namely, in the mitochondrial 12S-RNA genes, the ATP synthase subunit 8, the cytochrome oxidase subunits 3 and 4L, and the NADH dehydrogenase subunit 6. One mutation was synonymous, and the rest were either deletions or missense mutations. For mutations occurring in genes encoding mitochondrial respiratory chain proteins, the levels of heteroplasmy did not exceed 15%, while mutations in the hypervariable segment 1 (HVS1) and in adjacent noncoding region were characterized by heteroplasmy values of 20–26% .
The distribution of mtDNA mutations in various parts of the aortic intima was of a mosaic nature, and in some cases, even neighboring parts of the intima were characterized by different compositions of heteroplasmic mutations. Unaffected areas of the intima and areas with initial lesions (fatty dots) were characterized by an almost complete absence of heteroplasmic mutations of mtDNA. In areas with fatty streaks and fibrous and lipofibrous plaques, heteroplasmic variants were more common, and the levels of heteroplasmy increased. The highest frequency of heteroplasmic mutations was found in lipofibrous plaques, while in areas with fatty streaks in close proximity to lipofibrous plaques, an increased frequency of mutations was also observed. The fact of the presence of heteroplasmic deletions, previously not described in the literature (m.9720delT, m.10663delT, and m.14207delG in the coding region of mtDNA with a level of heteroplasmy up to 15%), was revealed. Because of the reading frame shift, the presence of deletions in mtDNA should lead to completely defective synthesis of the mtDNA-encoded proteins, and the level of heteroplasmy at a 15% for deletion means that the synthesis of respiratory chain proteins should be reduced by 15%. It should be noted that heteroplasmic deletions in the coding region of mtDNA were detected only in fatty streaks and lipofibrous plaques. As an example, the distribution of m.9720delT mutation, the deletion in the gene encoding cytochrome oxidase subunit 3, may be described: it was found with a similar level of heteroplasmy (12–15%) only in areas with fatty streaks and lipofibrous plaques, located relatively close to each other .
Taking into account the results of pyrosequencing and whole genome sequencing of mtDNA, the genes were identified, the damage of which due to point mutations is observed in atherosclerotic lesions, and heteroplasmic mtDNA mutations were revealed being associated with atherosclerotic lesions: m.368A>G (
3. Cellular composition of atherosclerotic lesions
The use of a set of antibodies covering most of the cells found in the intima made it possible to characterize the qualitative and quantitative changes that occur with resident intimal cells and cells of hematogenous origin in the same site of the vessel. By the results of immunocytochemical typing, the proportion of smooth muscle cells containing α-actin was determined, which was about half of the total cell population in both normal and atherosclerotic intima. In the row “unaffected intima—initial lesions—fatty streak—lipofibrous plaque—fibrous plaque,” the proportion of cells expressing smooth muscle α-actin was 46.7% (SD = 9.8), 47.2% (SD = 7.9), 42.2% (SD = 9.2), 47.0% (SD = 9.9), and 42.4% (SD = 9.0), respectively. Accordingly, the rest of the cells (about 50% of the total population) could not be identified with this smooth muscle cell marker. About two-thirds of the cells of the muscular-elastic intimal sublayer were positively stained with anti-smooth muscle α-actin antibodies, whereas in the proteoglycan (subendothelial) intimal sublayer, the percentage of cells containing α-actin was significantly lower. Cells of hematogenous origin (lymphocytes and macrophages) were localized only in the subendothelial part of the intima in the proteoglycan sublayer. Their proportion increased in atherosclerotic lesions and accounted for about 20% of all cells. The percentage of cells expressing CD68, the antigen of macrophages, increased with the progression of atherosclerotic lesion: 3.9% (SD = 1.7), 6.1% (SD = 6.3), 13.2% (SD = 3.2), 13.1% (SD = 6.3), and 18.2% (SD = 8.8), respectively .
Resident intimal cells also expressed antigens uncommon for the typical smooth muscle cells of intima media. In areas with advanced atherosclerotic lesions (fatty streaks and lipofibrous and fibrous plaques), the cells expressing 2A7 antigen characteristic for pericytes from active regions of angiogenesis were present. In unaffected intima, such cells could not be identified. The proportion of cells expressing the antigen of activated pericytes (2A7) increased with the progression of atherosclerotic lesion in the row “unaffected intima—initial lesions—fatty streak—lipofibrous plaque—fibrous plaque”: 0.0% (SD = 0.2), 1.2% (SD = 1.3), 3.0% (SD = 2.8), 27.0% (SD = 9.9), and 24.0% (SD = 8.6). The maximum proportion of 2A7-positive cells was reached in the lipofibrous plaques. In addition to antibodies against 2A7 antigen, antipericytic antibodies 3G5 characteristic for resting pericytes were used. In atherosclerotic lesions, the number of cells expressing 3G5 antigen was significantly less than in intact intima. The proportion of cells expressing 3G5 antigen decreased with progression of atherosclerotic lesion in the row “unaffected intima—initial lesions—fatty streak—lipofibrous plaque—fibrous plaque”: 31.3% (SD = 7.0), 16.3% (SD = 4.5), 11.7% (SD = 8.0), 5.0% (SD = 2.8), and 3.9% (SD = 3.6). The total number of cells expressing antigens 2A7 and 3G5 remained relatively constant, but their ratio varied. The total number of 3G5-positive cells and 2A7-positive cells was about 30% of the entire cell population in the areas of unaffected intima and atherosclerotic plaques. In the initial lesions and fatty streaks, the number of resting and activated pericytes was a smaller part. This pattern of expression of two pericyte antigens allows us to assume that when atherosclerotic lesions are formed, the functional state of the pericyte-like cells of the proteoglycan intima sublayer changes, accompanied by a change in the antigens that they express. With double immunocytochemical staining, the CD68 antigen of macrophages was expressed not only in cells of hematogenic origin but also in some resident subendothelial cells. This suggests that the presence of CD68 antigen is not only a sign of monocytic origin of cells but also a marker of their phagocytic activity, which increases when atherosclerotic lesions are formed .
Thus, smooth muscle cells, macrophages, and lymphocytes are present both in unaffected arterial intima and in the areas of atherosclerotic lesions. In contrast to popular beliefs, no predominance of monocyte-macrophages among cells inhabiting atherosclerotic lesion has been detected. There is a special type of cells expressing pericytic antigens, the number of which constitutes a significant proportion of intimal cells. Pericyte-like cells, together with smooth muscle cells, represent the majority of cellular population of subendothelial intima. In the areas of atherosclerotic lesion, the behavior of pericyte-like cells changes—they capture lipids and begin to proliferate. This is accompanied by a change in expression of antigens specific for pericytes.
4. Expression of apoptosis-related genes in atherosclerotic lesions
In the aortic intima samples differing by the degree of atherosclerotic lesion, the mRNA expression level of several genes associated with apoptosis (
A monotonous decrease in the expression level of the mRNA of the
It was found that the level of expression of the
There were no significant changes in the level of expression of the mRNA of the
The presented data of immunocytochemical analysis of changes in cellular composition in atherosclerotic lesions can partly explain the patterns of changes in the expression of apoptosis-related genes. It can be assumed that if the most significant changes in the cellular composition, namely, an increase in the proportion of cells of hematogenic origin, are observed in atherosclerotic plaques, the activation of pericyte-like cells is accompanied by corresponding changes in the expression of
5. Expression of inflammation-related genes in atherosclerosis
The expression of genes associated with inflammation (
Rather poorly expressed bell-shaped dependence was observed for the expression of the mRNA of the
6. Interaction of mitochondrial DNA mutation burden, cellular composition, and expression of apoptosis- and inflammation-related genes in atherosclerotic lesions
An analysis was made to examine the relationship between the mtDNA variability, the qualitative and quantitative changes in intimal cell composition that occur in atherosclerosis, and the expression of apoptosis- and inflammation-related genes. The analysis used an adaptive neural network model based on artificial neural networks and the theory of neuro-fuzzy interactions . It was found that there are rather similar but interdependent patterns of association of variables with the type of atherosclerotic lesion. The pattern of the first type (the bell-shaped relationship with the maximum for fatty lesions) combines and similarly characterizes the mRNA expression of
Thus, at least three groups of nonequilibrium interactions were identified: between the mtDNA mutation burden and mRNA expression of
7. Mitochondrial genome variability and subclinical atherosclerosis
The study of the degree of mtDNA heteroplasmy by detected atherosclerosis-related mutations was performed in nonrelative women. At the first stage of the study, 183 apparently healthy women were screened. Study participants had no clinical manifestations of cardiovascular diseases, and an ultrasonographic examination of carotid arteries was performed, followed by a quantitative measurement of carotid intima-media thickness (cIMT). The participants of the study were genotyped for 37 heteroplasmic mtDNA variants. In this study, five mutations of the mitochondrial genome m.3256C>T, m.14709G>A, m.12315G>A, m.13513G>A, and m.14846G>A were in focus, and the level of heteroplasmy was associated with the degree of preclinical atherosclerosis. The total mutation burden of the mitochondrial genome assessed by these mutations accounted for 68% of the cIMT variability, while the combination of conventional risk factors for cardiovascular disease accounted for only 8% of the variability of cIMT. Data were obtained on the correlation of the degree of heteroplasmy in these mutations with each other, which indicated the predominantly hereditary nature of these mutations, as well as the existence of proatherosclerotic haplotypes of the mitochondrial genome .
Further, those women with a high mtDNA mutation burden, who were asked to participate in genetic screening of the maternal relatives, were selected. In total, 37 families (1 family of 4 generations, 27 families of 3 generations, 7 families of 2 generations) were examined. Monocytes were isolated from the mononuclear fraction by affinity separation using magnetic CD14 affinity microparticles on LS Separation Columns (Miltenyi Biotec, Germany). Genotyping was carried out on heteroplasmic variants m.1555A>G, m.5178C>A, m.3256C>T, m.13513G>A, m.12315G>A, m.14846G>A, and m.15059G>A. The choice of mtDNA variants was due to their high occurrence and the level of heteroplasmy sufficient for evaluation in the mathematical model of inheritance of mutations. Heteroplasmy of mtDNA by selected variants was determined by qPCR. The probability of the hereditary nature of these variants was calculated, as well as the probability that the above variants are mutations that arise in any generation and may be transmitted by the maternal line with accumulation from generation to generation.
When analyzing the results of genotyping mtDNA samples from maternal relatives, it was established that the probability of hereditary nature of variant m.1555A>G approximates to 100%; m.5178C>A, 100%; m.3256C>T, 92%; m.13513G>A, 99%; m.12315G>A, 99%; m.14846G>A, 100%; and m.15059G>A, 96%. The probability that the above variants may be somatic arising in any generation, for variant m.1555A>G accounted for 8%; m.5178C>A, for 5%; m.3256C>T, for 19%; m.13513G>A, for 10%; m.12315G>A, for 12%; m.14846G>A, for 4%; and m.15059G>A, for 11%. Thus, the prevalent variants of the mitochondrial genome are thought to be heritable with a high degree of probability.
The differences in the degree of mtDNA heteroplasmy in various types of blood cells (monocytes, neutrophils, lymphocytes, platelets, and the total fraction of leukocytes) obtained from non-related individuals were studied. Seventy-one study participants (32 men, 39 women) were examined. The leukocyte fraction of cells was obtained from the whole citrated blood by gradient centrifugation using Ficoll-Paque (GE Healthcare Life Sciences, UK). Blood cells of various types were isolated from the leukocyte fraction by affinity separation using magnetic affinity microparticles on LS Separation Columns (Miltenyi Biotec, Germany). Heteroplasmy of mitochondrial genome was determined by pyrosequencing of short-chain mtDNA fragments by mtDNA variants m.13513G>A, m.3256C>T, m.3336T>C, m.12315G>A, and m.1555A>G. There were no significant differences in the level of heteroplasmy of these mutations studied between different types of blood cells from the same participant within the study. The only statistically significant difference was observed between neutrophils and lymphocytes by mutation m.1555A>G. The obtained data indicate that these mutations are not accumulated during differentiation of blood cells, but more probably are inherent in the progenitor cells in the bone marrow. Thus, for the studies of single nucleotide substitutions of mtDNA in various pathologies, it is possible to use whole fraction of white blood cells rather than certain types of them .
Further, the variability of mtDNA from blood leukocytes was evaluated by NGS (Roche 454 GS Junior Titanium platform) in a sample of 80 non-related study participants (37 healthy individuals and 43 patients with subclinical carotid atherosclerosis, the diagnosis being established by ultrasound scanning of the common carotid arteries and measurement of cIMT). Arterial hypertension, diabetes mellitus, and myocardial infarction were criteria of exclusion in sample formation. Despite of the relatively low percentage of mapped readings during sequencing, the mean 70-fold mitochondrial genome coverage was provided sufficiently to detect single nucleotide substitutions. The possibility of sequencing using Roche 454 technology made it possible to determine as accurately as possible the haplogroup of the mitochondrial genome, considering all single nucleotide substitutions throughout the mtDNA. As a result, mitochondrial haplogroups H, U, T, and J were the most common (85.7% of cases), which corresponds to general data from the Russian population. Haplogroups I, W, R, and D were represented only among persons with preclinical atherosclerosis. In turn, haplogroups N, V, and M were found only among apparently healthy individuals. When calculating the odds ratio for preclinical atherosclerosis, it was established that belonging to haplogroup H is associated with an increased risk of atherosclerosis (
According to the data of whole mtDNA sequencing, the list of variants of the mitochondrial genome presumably determining the individual genetic predisposition to atherosclerosis was compiled. The methodology used to assess the variability of the mitochondrial genome as a marker of predisposition to atherosclerosis involved the use of data on the presence of 12 mtDNA variants and considered the calculation of the cumulative mutation burden for those mtDNA variants.
The ROC analysis was performed to assess the diagnostic effectiveness of this method for estimating the total mutation burden of mtDNA to determine the predisposition to atherosclerosis. The following results were obtained: the area under the curve was 0.791, sensitivity of the test was 73.3%, and specificity of the test was 75.0%. The accuracy of the method was 74.0%, the proportion of true positive results was 42.9%, and the proportion of true negative results was 31.1%. The odds ratio for the risk of atherosclerosis was 8.25 (95% confidence interval 2.9–23.29), and the relative risk was 2.41. The relationship between mtDNA variants and their combinations with atherosclerosis, conventional risk factors for cardiovascular diseases, and age-gender variation was characterized. There were no significant differences between the mean values of blood lipids, both between persons belonging to different haplogroups and between groups with the presence and absence of preclinical atherosclerosis. Nevertheless, the mean value of low-density lipoprotein (LDL) to high-density lipoprotein (HDL) ratio in the group of patients with preclinical atherosclerosis was slightly increased in comparison with healthy subjects (3.0 ± 1.7 versus 2.7 ± 1.1,
8. Mitochondrial DNA mutation burden and functional activity of cells
Based on the results of whole genome sequencing and pyrosequencing of mtDNA from normal and atherosclerotic sites of intima of the aorta, as well as of genotyping of mtDNA from the blood cells of the above study participants, valuable information was obtained on proatherosclerotic mtDNA variants (m.652delG, m.3256C>T, m12315G>A, m.14459G>A, m.15059G>A, m.368A>G, m.751delA, m.8404T>C, m.8485G>C, m.9720delT, m.14160G>A, m.14207delG, M.16086T>C, and m.16389G>A) and antiatherosclerotic mtDNA variants (m.1555A>G, m.5178C>A, m.13513G>A, and m.14846G>A). These data were used to determine whether the study participants belonged to persons with a low, neutral, or high mtDNA mutation burden, as judged by combinations of the proatherosclerotic or antiatherosclerotic alleles of mtDNA variants, or the highest possible matching to Cambridge reference sequence of the mitochondrial human genome, version NC_012920.1. Of the examined and genotyped persons (a total of 525 subjects), 139 (26.5%) met the above criteria. Of these, 97 subjects signed informed consent and donated venous blood for the study of functional activity of blood monocytes. Among them, 32 were included in the group with a low mtDNA mutation burden; 29, in the group with a neutral mtDNA genotype; and 36, in the group with a high mtDNA mutation burden (Sobenin, personal communication).
It is known that atherosclerotic phenotype at the cellular level for cells actively involved in the formation of atherosclerotic lesions is characterized by increased proliferative activity, increased protein and connective tissue matrix synthesis, and accumulation of esterified cholesterol. Recently, the markers of cell activation by pro-inflammatory (M1) and anti-inflammatory (M2) types, reflecting the processes of chronic inflammation in atherogenesis, have been added to phenotype description.
Mononuclear cells were isolated from the leukocyte fraction by affinity separation using magnetic CD14 affinity microparticles on LS Separation Columns (Miltenyi Biotec, Germany) and cultured in serum-free X-Vivo 10 medium (Lonza). To stimulate cell activation into pro-inflammatory phenotype, interferon-gamma was added to the medium at a final concentration of 100 ng/ml (1-day incubation). To stimulate cell activation into anti-inflammatory phenotype, interleukin-4 was added to the medium at a final concentration of 10 ng/ml (6-days incubation). After incubation, concentrations of TNF-α (the marker of activation into pro-inflammatory phenotype) and chemokine CCL18 (the marker of activation into anti-inflammatory phenotype) were measured in culture medium by enzyme-linked immunosorbent assay (ELISA) technique. To measure the expression of
Significant differences were found between the cells for such parameters as proliferative activity, synthetic activity, synthesis of matrix components (collagen), the ability of cells to accumulate cholesterol from modified low-density lipoproteins, stimulated secretion of anti-inflammatory chemokine CCL18,
The cells with a low and neutral mtDNA mutation burden did not differ significantly by proliferative and synthetic activity. The cells with a high mtDNA mutation burden were characterized by 1.8-fold increased proliferative activity (
The ability of cells to accumulate cholesterol from modified low-density lipoprotein was the highest in cells with a high mtDNA mutation burden; the intracellular cholesterol content increased by 1.7-fold in comparison with control cells,
Thus, by traditional cellular markers of atherosclerosis (fibrosis, proliferation, lipidosis), the cells with a high mtDNA mutation burden typically demonstrated proatherosclerotic phenotype, except for the dissonance with respect to collagen synthesis (Sobenin, not published). On the one hand, monocytes-macrophages are not professional producers of matrix in tissues, but on the other hand, this dissonance may reflect the dysregulation of cell adaptation, possibly related specifically to the presence of a high mtDNA mutation burden. Indirect confirmation of the latter assumption may be the characteristics of cell activation by an anti-inflammatory or pro-inflammatory phenotype.
The cells did not differ significantly by basal secretion of TNF-α (the marker of pro-inflammatory type of activation); there was a tendency to decrease of basal TNF-α secretion in the row “low-neutral-high mtDNA mutation burden,” which did not reach statistical significance (
Basal secretion of CCL18 (the marker of anti-inflammatory type of activation) was practically absent, which is normal, as the cells usually do not secrete CCL18 in the absence of stimulation. The cells with a neutral mtDNA mutation burden were the best responders to stimulation with interleukin-4 (
Ambiguous dissonance was observed not only at the level of expression of the product but also at the level of expression of the coding genes. The expression of the
Among the remaining characteristics, the expression of the
Obviously, such combinations of phenotypic cellular markers of atherosclerosis and mtDNA mutation burden should be associated with mitochondrial dysfunction. Indeed, such a dysfunction was detected, and it was increasing in the row “low-neutral-high mtDNA mutation burden,” by manifesting the decrease of the coefficient of respiratory control, the phosphorylation coefficient, and the rate of oxygen consumption. Correlation coefficients between these indices were
In the analysis of the relationship between mtDNA damage and functional impairment of cells with the use of an adaptive neural network model, the similar and interdependent patterns of relationships were identified (Sobenin et al., not published). The pattern of type 1 is compliant to the typical atherosclerotic phenotype (increased proliferation, increased synthetic activity, the ability of cells to accumulate cholesterol, the unchanged expression of
The data obtained from the described studies allow to analyze the relationship of mtDNA damage and its variability; qualitative and quantitative changes in the cellular composition of arterial intima, occurring in atherosclerosis; as well as expression of apoptosis- and inflammation-related genes. In maternal relatives in 2–4 generations, the differences in the degree of mtDNA heteroplasmy in different types of blood cells obtained from the same individuals helped to identify heritable mutations accumulated in the cells, and somatic mutations arising during the life of the individual. The relationship of mtDNA variants with atherosclerosis, traditional risk factors for cardiovascular disease, and age-gender variation was identified and characterized. In cell culture studies, the relationship of individual mtDNA mutation burden with functional activity of cells was studied. Taken together, the results of these studies strongly support the hypothesis on proatherosclerotic role of mtDNA mutations in atherogenesis.
This study was supported by the Russian Science Foundation, Grant #14-14-01038.
The author also acknowledges the efforts of the research group members (Margarita A. Sazonova, PhD; Andrey V. Zhelankin, PhD; Tatiana V. Kirichenko, MD, PhD; Zukhra B. Khasanova, BS; Konstantin Y. Mitrofanov, BS; Vasily V. Sinyov, BS; Nikita G. Nikiforov, BS; and Valeria A. Barinova, BS) in described in studies.
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Sazonova MA, Sinyov VV, Barinova VA, Ryzhkova AI, Zhelankin AV, Postnov AY, Sobenin IA, Bobryshev YV, Orekhov AN. Mosaicism of mitochondrial genetic variation in atherosclerotic lesions of the human aorta. BioMed Research International. 2015; 2015:825468. DOI: 10.1155/2015/825468
Sazonova MA, Zhelankin AV, Barinova VA, Sinyov VV, Khasanova ZB, Postnov AY, Sobenin IA, Bobryshev YV, Orekhov AN. Dataset of mitochondrial genome variants associated with asymptomatic atherosclerosis. Data in Brief. 2016; 7:1570-1575. DOI: 10.1016/j.dib.2016.04.055
Sobenin IA, Bobryshev YV, Korobov GA, Borodachev EN, Postnov AY, Orekhov AN. Quantitative analysis of the expression of caspase 3 and caspase 9 in different types of atherosclerotic lesions in the human aorta. Experimental and Molecular Pathology. 2015; 99:1-6. DOI: 10.1016/j.yexmp.2015.05.004
Karpenko AP, Moor DA, Mukhlisulina DT. Multicriteria optimization based on neural network, fuzzy and neuro-fuzzy approximation of decision maker’s utility function. Optical Memory and Neural Networks. 2012; 21:1-10. DOI: 10.3103/S1060992X12010031
Sazonova MA, Chicheva MM, Zhelankin AV, Sobenin IA, Bobryshev YV, Orekhov AN. Association of mutations in the mitochondrial genome with the subclinical carotid atherosclerosis in women. Experimental and Molecular Pathology. 2015; 99:25-32. DOI: 10.1016/j.yexmp.2015.04.003
Sinyov VV, Malyar NL, Kosogorova SA, Postnov AY, Orekhov AN, Sobenin IA, Sazonova MA. Mitochondrial genome variability in different types of human blood cells. International Research Journal. 2013; 19:58-61. DOI: 10.18454/IRJ.2227-6017
Zhelankin A, Khasanova Z, Barinova L, Sazonova M, Postnov A, Sobenin I. Mitochondrial DNA haplogroup H is associated with subclinical carotid atherosclerosis in Russian population. Journal of Hypertension. 2015; 33(Suppl 1):e2. DOI: 10.1097/01.hjh.0000467356.04711.50
Zhelankin AV, Sazonova MA, Khasanova ZB, Sinyov VV, Mitrofanov KY, Sobenin IA, Orekhov AN, Postnov AY. Analysis of mitochondrial haplogroups in persons with subclinical atherosclerosis based on high-throughput mtDNA sequencing. Patologicheskaia Fiziologiia i Eksperimentalnaia Terapiia. 2015; 59:12-16