Relationship between Cellular Oxygen Consumption and Atherosclerosis-Associated Mitochondrial Mutations (Variants of the Mitochondrial Genome).

BACKGROUND: Mitochondria are the main sites of cellular aerobic energy production through conjugation of respiration and oxidative phosphorylation. We have recently discovered mutations (genome variants) of mitochondrial DNA (mtDNA) associated with atherosclerosis. We have then investigated the possible mechanisms underlying such association and the role of mitochondrial mutations in atherogenesis. Mitochondrial dysfunction is a known component of the pathogenesis of chronic human diseases, including atherosclerosis. OBJECTIVE: The aim of the study was to explore whether there is a relationship between cellular oxygen consumption and atherosclerosis-associated mitochondrial mutations. The study of mitochondrial respiration abnormalities can help to understand the role of mtDNA mutations in pathology. METHOD: By using the polarographic method with Clark electrode, we tested the possibility of respiration impairment in permeabilized cells carrying the tested mtDNA variants using the cybrid (cytoplasmic hybrid) lines. Mitochondria introduced in the cybrid lines were obtained from atherosclerotic patients that differed in the profile of mtDNA mutations, which made it possible to compare the degree of mtDNA mutation load with the rate of oxygen consumption by cybrid cells. RESULTS: It was found that three of the studied mutations were individually associated with impaired respiration. Besides, some combinations of two specific mutations have a high probability of being associated with altered oxygen consumption. As a result, eight mutations were identified, individually or paired combinations of which were associated with high or low rates of cellular respiration, significantly different from control cells. CONCLUSION: The observed effect may be involved in the pathogenesis of atherosclerosis. The study of mtDNA mutations associated with atherosclerosis can help reveal pharmacological targets for the development of novel therapies.

Defective Mitophagy Impairs Response to Inflammatory Activation of Macrophage-Like Cells.

BACKGROUND AND AIMS: The role of mitophagy in atherosclerosis has been extensively studied during the last few years. It was shown that mitophagy is involved in the regulation of macrophages, which are important players as immune cells in atherosclerosis development. In this study, we investigated the relationship between mitophagy and response to inflammatory stimulation of macrophage-like cells. Six cybrid cell lines with normal mitophagy, that is, increasing in response to stimulation, and 7 lines with defective mitophagy not responding to stimulation were obtained. The objective of the study was to compare the nature of the inflammatory response in normal and defective mitophagy in order to elucidate the role of mitophagy defects in inflammation. METHODS: We used cytoplasmic hybrids (cybrids) as cellular models, created using mitochondrial DNA from different atherosclerosis patients. Mitophagy was stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and assessed as the degree of colocalization of mitochondria with lysosomes using confocal microscopy. Western blotting methods were used for the determination of proteins involved in the exact mechanism of mitophagy. Experiments with stimulation of mitophagy show a high correlation between these two approaches (microscopy and blotting). The pro-inflammatory response of cybrids was stimulated with bacterial lipopolysaccharide (LPS). The extent of the inflammatory response was assessed by the secretion of cytokines CCL2, IL8, IL6, IL1ß, and TNF measured by ELISA. RESULTS: Basal level of secretion of cytokines CCL2, IL8 and TNF was 1.5-2 times higher in cultures of cybrids with defective mitophagy compared to cells with normal mitophagy. This suggests a persistently elevated inflammatory response in cells with defective mitophagy, even in the absence of an inflammatory stimulus. Such cells in the tissue will constantly recruit other immune cells, which is characteristic of macrophages derived from monocytes circulating in the blood of patients with atherosclerosis. We observed significant differences in the degree and type of response to inflammatory activation in cybrids with defective mitophagy. These differences were not so much quantitative as they were dramatically qualitative. Compared with cells with normal mitophagy, in cells with defective mitophagy, the relative (to basal) secretion of IL8, IL6 and IL1b increased after the second LPS activation. This indicates a possible lack of tolerance to inflammatory activation in cells with defective mitophagy, since typically, re-activation reveals a smaller pro-inflammatory cytokine response, allowing the inflammatory process to resolve. In cells with normal mitophagy, exactly this normal (tolerant) inflammatory reaction was observed. CONCLUSION: Data on the involvement of mitophagy, including defective mitophagy, in disturbances of the inflammatory response in sepsis, viral infections, autoimmune diseases and other pathologies have previously been reported. In this work, we studied the role of defective mitophagy in non-infectious chronic inflammatory diseases using the example of atherosclerosis. We showed a dramatic disruption of the inflammatory response associated with defective mitophagy. Compared with cybrids with normal mitophagy, in cybrids with defective mitophagy, the secretion of all studied cytokines changed significantly both quantitatively and qualitatively. In particular, the secretion of 3 of 5 cytokines demonstrated an intolerant inflammatory response manifested by increased secretion after repeated inflammatory stimulation. Such an intolerant reaction likely indicates a significant disruption of the pro-inflammatory response of macrophages, which can contribute to the chronification of inflammation. Elucidating the mechanisms of chronification of inflammation is extremely important for the search for fundamentally new pharmacological targets and the development of drugs for the prevention and treatment of chronic inflammatory diseases, including atherosclerosis and diseases characteristic of inflammation. Such diseases account for up to 80% of morbidity and mortality.

Evidence for the Involvement of Gene Regulation of Inflammatory Molecules in the Accumulation of Intracellular Cholesterol: The Mechanism of Foam Cell Formation in Atherosclerosis.

BACKGROUND: The relationship between the cellular pro-inflammatory response and intracellular lipid accumulation in atherosclerosis is not sufficiently studied. Transcriptomic analysis is one way to establish such a relationship. Previously, we identified 10 potential key genes (IL-15, CXCL8, PERK, IL-7, IL-7R, DUSP1, TIGIT, F2RL1, TSPYL2, and ANXA1) involved in cholesterol accumulation in macrophages. It should be noted that all these genes do not directly participate in cholesterol metabolism, but encode molecules related to inflammation. METHODS: In this study, we conducted a knock-down of the 10 identified key genes using siRNA to determine their possible role in cholesterol accumulation in macrophages. To assess cholesterol accumulation, human monocyte-derived macrophages (MDM) were incubated with atherogenic LDL from patients with atherosclerosis. Cholesterol content was assessed by the enzymatic method. Differentially expressed genes were identified with DESeq2 analysis. Master genes were determined by the functional analysis. RESULTS: We found that only 5 out of 10 genes (IL-15, PERK, IL-7, IL-7R, ANXA1) can affect intracellular lipid accumulation. Knock-down of the IL-15, PERK, and ANXA1 genes prevented lipid accumulation, while knock-down of the IL-7 and IL-7R genes led to increased intracellular lipid accumulation during incubation of MDM with atherogenic LDL. Seventeen overexpressed genes and 189 underexpressed genes were obtained in the DGE analysis, which allowed us to discover 20 upregulated and 86 downregulated metabolic pathways, a number of which are associated with chronic inflammation and insulin signaling. We also elucidated 13 master regulators of cholesterol accumulation that are immune response-associated genes. CONCLUSION: Thus, it was discovered that 5 inflammation-related master regulators may be involved in lipid accumulation in macrophages. Therefore, the pro-inflammatory response of macrophages may trigger foam cell formation rather than the other way around, where intracellular lipid accumulation causes an inflammatory response, as previously assumed.

Condition "Vasa Vasorum" in Patients with Thoracic Aortic Aneurysm.

It is known that vasa vasorum contributes substantially to the blood supply and nutrition of one-third of the wall of the ascending thoracic aorta. Therefore, we focused on studying the relationship between inflammatory cells and vasa vasorum vessels in patients with aortic aneurysm. The material for the study was biopsies of thoracic aortic aneurysms taken from patients during an aneurysmectomy (34 men, 14 women, aged 33 to 79 years). The biopsies belonged to patients with non-hereditary thoracic aortic aneurysm. An immunohistochemical study was carried out using antibodies to antigens of T cells (CD3, CD4, CD8); macrophages (CD68); B cells (CD20); endothelium (CD31, CD34, von Willebrand factor (vWF)); and smooth muscle cells (alpha actin). Samples without inflammatory infiltrates contained less vasa vasorum in the tunica adventitia than samples with inflammatory infiltrates, and this difference was statistically significant p < 0.05. T cell infiltrates in the adventitia of aortic aneurysms were found in 28 of 48 patients. In the vessels of the vasa vasorum, surrounded by inflammatory infiltrates, T cells that adhered to the endothelium were found. The same cells were also localized in the subendothelial area. The number of adherent T cells in patients with inflammatory infiltrates in the aortic wall dominated the number of these cells in patients without inflammation of the aortic wall. This difference was statistically significant, p < 0.0006. Hypertrophy and sclerosis of the arteries of the vasa vasorum system, the narrowing of their lumen, and, as a result, impaired blood supply to the aortic wall, were found in 34 patients with hypertension. In 18 patients (both in patients with hypertension and in patients without hypertension), T cells that adhered to the vasa vasorum endothelium were found. In nine cases, massive infiltrates of T cells and macrophages were found, which surrounded and squeezed the vasa vasorum, preventing blood circulation. In six patients, parietal and obturating blood clots were found in the vasa vasorum vessels, which disrupted the normal blood supply to the aortic wall. We believe that this indicates the importance of the state of the vessels of the vasa vasorum in the development of an aortic aneurysm. In addition, pathological changes in these vessels may not always play a primary role, but always a very important role, in the pathogenesis of this disease.

The Role of Mitochondrial Mutations in Chronification of Inflammation: Hypothesis and Overview of Own Data.

Chronic human diseases, especially age-related disorders, are often associated with chronic inflammation. It is currently not entirely clear what factors are responsible for the sterile inflammatory process becoming chronic in affected tissues. This process implies impairment of the normal resolution of the inflammatory response, when pro-inflammatory cytokine production ceases and tissue repair process begins. The important role of the mitochondria in the correct functioning of innate immune cells is currently well recognized, with mitochondrial signals being an important component of the inflammatory response regulation. In this work, we propose a hypothesis according to which mitochondrial DNA (mtDNA) mutations may play a key role in rendering certain cells prone to prolonged pro-inflammatory activation, therefore contributing to chronification of inflammation. The affected cells become sites of constant pro-inflammatory stimulation. The study of the distribution of atherosclerotic lesions on the surface of the arterial wall samples obtained from deceased patients revealed a focal distribution of lesions corresponding to the distribution of cells with altered morphology that are affected by mtDNA mutations. These observations support the proposed hypothesis and encourage further studies.

Cholesterol Transport Dysfunction and Its Involvement in Atherogenesis.

Atherosclerosis is the cause of the development of serious cardiovascular disorders, leading to disability and death. Numerous processes are involved in the pathogenesis of atherosclerosis, including inflammation, endothelial dysfunction, oxidative stress, and lipid metabolism disorders. Reverse transport of cholesterol is a mechanism presumably underlying the atheroprotective effect of high-density lipoprotein. In this review, we examined disorders of cholesterol metabolism and their possible effect on atherogenesis. We paid special attention to the reverse transport of cholesterol. Transformed cholesterol metabolism results in dyslipidemia and early atherosclerosis. Reverse cholesterol transport is an endogenous mechanism by which cells export cholesterol and maintain homeostasis. It is known that one of the main factors leading to the formation of atherosclerotic plaques on the walls of blood vessels are multiple modifications of low-density lipoprotein, and the formation of foam cells following them.

The Role of the VEGF Family in Atherosclerosis Development and Its Potential as Treatment Targets.

The vascular endothelial growth factor (VEGF) family, the crucial regulator of angiogenesis, lymphangiogenesis, lipid metabolism and inflammation, is involved in the development of atherosclerosis and further CVDs (cardiovascular diseases). This review discusses the general regulation and functions of VEGFs, their role in lipid metabolism and atherosclerosis development and progression. These functions present the great potential of applying the VEGF family as a target in the treatment of atherosclerosis and related CVDs. In addition, we discuss several modern anti-atherosclerosis VEGFs-targeted experimental procedures, drugs and natural compounds, which could significantly improve the efficiency of atherosclerosis and related CVDs' treatment.

The origin of multiple clones in the parthenogenetic lizard species Darevskia rostombekowi.

The all-female Caucasian rock lizard Darevskia rostombekowi and other unisexual species of this genus reproduce normally via true parthenogenesis. Typically, diploid parthenogenetic reptiles exhibit some amount of clonal diversity. However, allozyme data from D. rostombekowi have suggested that this species consists of a single clone. Herein, we test this hypothesis by evaluating variation at three variable microsatellite loci for 42 specimens of D. rostombekowi from four populations in Armenia. Analyses based on single nucleotide polymorphisms of each locus reveal five genotypes or presumptive clones in this species. All individuals are heterozygous at the loci. The major clone occurs in 24 individuals and involves three populations. Four rare clones involve one or several individuals from one or two populations. Most variation owes to parent-specific single nucleotide polymorphisms, which occur as heterozygotes. This result fails to reject the hypothesis of a single hybridization founder event that resulted in the initial formation of one major clone. The other clones appear to have originated via post-formation microsatellite mutations of the major clone.

Clonal diversity and clone formation in the parthenogenetic Caucasian rock Lizard Darevskia dahli [corrected].

The all-female Caucasian rock lizard species Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa.

Clonal diversity and clone formation in the parthenogenetic Caucasian rock lizard Darevskia dahlia.

The all-female Caucasian rock lizard species Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa.