[Transcription Factor NRF2 in Endothelial Functions].

The transcription factor NRF2 is a major regulator of cell antioxidant defense. NRF2 is activated by various stimuli, such as oxidants and electrophiles, to induce transcription of a number of genes whose products are involved in xenobiotic metabolism and contribute to the reduction of oxidative stress. NRF2 is one of the key transcription factors that ensure the endothelial cell function. The endothelium is a cell layer that lines the lumens of blood vessels and performs various homeostatic functions, controlling migration of leukocytes, regulating thrombosis and vascular tone, and playing a role in angiogenesis. Endothelial dysfunction is often accompanied by inflammation and oxidative stress, which may lead to cell aging and cell death by apoptosis, necrosis, or ferroptosis. Endothelial dysfunction contributes to the development of diabetes and common cardiovascular disorders, such as hypertension and atherosclerosis. Many pathophysiological processes in the endothelium, including senile changes, are associated with decreased NRF2 activity, leading to inflammatory activation and decreasing activity of the cell antioxidant defense systems. Activation of the NRF2 signaling pathway generally contributes to the resolution of inflammation and oxidative stress. The review focuses on the role that NRF2 plays in basic functions of the endothelium in normal and pathological conditions. Advantages and disadvantages of NRF2 activation as a way to prevent and treat cardiovascular diseases are discussed additionally.

Mitochondrion-targeted antioxidant SkQ1 prevents rapid animal death caused by highly diverse shocks.

The response to stress involves the activation of pathways leading either to protection from the stress origin, eventually resulting in development of stress resistance, or activation of the rapid death of the organism. Here we hypothesize that mitochondrial reactive oxygen species (mtROS) play a key role in stress-induced programmed death of the organism, which we called "phenoptosis" in 1997. We demonstrate that the synthetic mitochondria-targeted antioxidant SkQ1 (which specifically abolishes mtROS) prevents rapid death of mice caused by four mechanistically very different shocks: (a) bacterial lipopolysaccharide (LPS) shock, (b) shock in response to intravenous mitochondrial injection, (c) cold shock, and (d) toxic shock caused by the penetrating cation C12TPP. Importantly, under all these stresses mortality was associated with a strong elevation of the levels of pro-inflammatory cytokines and administration of SkQ1 was able to switch off the cytokine storms. Since the main effect of SkQ1 is the neutralization of mtROS, this study provides evidence for the role of mtROS in the activation of innate immune responses mediating stress-induced death of the organism. We propose that SkQ1 may be used clinically to support patients in critical conditions, such as septic shock, extensive trauma, cooling, and severe infection by bacteria or viruses.

Effect of Antioxidants on the Production of MCP-1 Chemokine by EA.hy926 Cells in Response to IL-6.

An elevated level of circulatory interleukin 6 (IL-6) is a biomarker for cytokine storm of various etiologies, including COVID-19, and contributes to poor prognosis. Vascular endothelial cells are one of the main targets of pathological action of IL-6. IL-6 activates the trans-signaling pathway via the formation of the IL-6/sIL-6Ra/gp130 receptor complex and subsequent activation of the JAK/STAT3 signaling pathway and PI3K/AKT and MEK/ERK kinases in some cases. Previously, it was shown by the authors' group and other researchers that reactive oxygen species (ROS), including mitochondrial ROS (mito-ROS), contribute to the induction of IL-6 expression in the endothelium, mainly due to increased activation of the transcription factor NF-kB. We have also shown that the mitochondria-targeted antioxidant SkQ1 (Plastoquinolyl-10(6'-decyltriphenyl)phosphonium) prevented tumor necrosis factor (TNF)-induced cytokine storm and death in mice. In the aortas of these animals, SkQ1 also prevented the increase in the expression of NF-kB-dependent genes, including the cytokine IL-6 and the chemokine MCP-1. In the current work, the hypothesis of mito-ROS involvement in the IL-6-signaling-mediated proinflammatory gene expression in endothelial cells is tested. SkQ1 suppressed the expression and secretion of the MCP-1 chemokine, induced by IL-6 in combination with sIL-6-Ra, but not the expression of ICAM-1 adhesion molecules in EA.hy926 human endothelial cells. Using specific inhibitors, the authors have shown that, in EA.hy926 cells, IL-6-induced expression of MCP-1 and ICAM-1 depends on the signaling protein and transcription activator STAT3 and, in some cases, on JNK, PI3K, and MEK1/2 kinases and is independent of p38 kinase. In this model, IL-6 induced rapid STAT3 activation, while ERK1/2 activation was less pronounced, and there was no IL-6 effect on Akt and JNK activation. SkQ1 partially suppressed STAT3 and ERK1/2 activation. Thus, we have shown that SkQ1 suppresses not only NF-kB-dependent expression of IL-6 and other proinflammatory genes but also IL-6-induced activation of JAK/STAT3 and STAT3-dependent expression of MCP-1, which probably contributes to the overall therapeutic effect of SkQ1.

Transcription Factor Nrf2 as a Potential Therapeutic Target for Prevention of Cytokine Storm in COVID-19 Patients.

Nrf2 is a key transcription factor responsible for antioxidant defense in many tissues and cells, including alveolar epithelium, endothelium, and macrophages. Furthermore, Nrf2 functions as a transcriptional repressor that inhibits expression of the inflammatory cytokines in macrophages. Critically ill patients with COVID-19 infection often present signs of high oxidative stress and systemic inflammation - the leading causes of mortality. This article suggests rationale for the use of Nrf2 inducers to prevent development of an excessive inflammatory response in COVID-19 patients.

A Combination of Kidney Ischemia and Injection of Isolated Mitochondria Leads to Activation of Inflammation and Increase in Mortality Rate in Rats.

We studied the development of acute kidney injury and animal death in the model of combined injury caused by kidney ischemia/reperfusion with simultaneous systemic administration of mitochondria. It was found that intraperitoneal injection of isolated mitochondria led to the appearance of mitochondrial DNA in the peripheral blood that could activate innate immunity. After administration of mitochondria, as well as after renal ischemia/reperfusion, proinflammatory changes were observed, primarily leukocytosis and granulocytosis. The combination of ischemia/reperfusion with injection of mitochondria caused a sharp increase in animal death, which may indicate a direct link between activation of TLR-signaling and high mortality of patients with combined injuries and multiple-organ failure in intensive care units. Treatment with mitochondria-targeted antioxidant increased animal survival, which indicated the participation of mitochondrial ROS in the development of systemic inflammatory response and death caused by acute renal failure.

COVID-19 and Oxidative Stress.

Pathogenesis of the novel coronavirus infection COVID-19 is the subject of active research around the world. COVID-19 caused by the SARS-CoV-2 is a complex disease in which interaction of the virus with target cells, action of the immune system and the body's systemic response to these events are closely intertwined. Many respiratory viral infections, including COVID-19, cause death of the infected cells, activation of innate immune response, and secretion of inflammatory cytokines. All these processes are associated with the development of oxidative stress, which makes an important contribution to pathogenesis of the viral infections. This review analyzes information on the oxidative stress associated with the infections caused by SARS-CoV-2 and other respiratory viruses. The review also focuses on involvement of the vascular endothelium in the COVID-19 pathogenesis.

Mitochondria-Targeted Antioxidant SkQR1 Reduces TNF-Induced Endothelial Permeability in vitro.

Prolonged or excessive increase in the circulatory level of proinflammatory tumor necrosis factor (TNF) leads to abnormal activation and subsequent damage to endothelium. TNF at high concentrations causes apoptosis of endothelial cells. Previously, using mitochondria-targeted antioxidants of SkQ family, we have shown that apoptosis of endothelial cells is dependent on the production of reactive oxygen species (ROS) in mitochondria (mito-ROS). Now we have found that TNF at low concentrations does not cause cell death but activates caspase-3 and caspase-dependent increase in endothelial permeability in vitro. This effect is probably due to the cleavage of ß-catenin - an adherent junction protein localized in the cytoplasm. We have also shown that extracellular matrix metalloprotease 9 (MMP9) VE-cadherin shedding plays a major role in the TNF-induced endothelial permeability. The mechanisms of the caspase-3 and MMP9 activation are probably not related to each other since caspase inhibition did not affect VE-cadherin cleavage and MMP9 inhibition had no effect on the caspase-3 activation. Mitochondria-targeted antioxidant SkQR1 inhibited TNF-induced increase in endothelial permeability. SkQR1 also inhibited caspase-3 activation, ß-catenin cleavage, and MMP9-dependent VE-cadherin shedding. The data suggest that mito-ROS are involved in the increase in endothelial permeability due to the activation of both caspase-dependent cleavage of intracellular proteins and of MMP9-dependent cleavage of the transmembrane cell-to-cell contact proteins.

Priming of Human Neutrophils Is Necessary for Their Activation by Extracellular DNA.

Extracellular plasma DNA is thought to act as a damage-associated molecular pattern causing activation of immune cells. However, purified preparations of mitochondrial and nuclear DNA were unable to induce neutrophil activation in vitro. Thus, we examined whether granulocyte-macrophage colony-stimulating factor (GM-CSF) acting as a neutrophil priming agent can promote the activation of neutrophils by different types of extracellular DNA. GM-CSF pretreatment greatly increased p38 MAPK phosphorylation and promoted CD11b/CD66b expression in human neutrophils treated with mitochondrial and, to a lesser extent, with nuclear DNA. Our experiments clearly indicate that GM-CSF-induced priming of human neutrophils is necessary for their subsequent activation by extracellular DNA.

Mitochondrial Dysfunction in Neocortex and Hippocampus of Olfactory Bulbectomized Mice, a Model of Alzheimer's Disease.

Structural and functional impairments of mitochondria in brain tissues in the pathogenesis of Alzheimer's disease (AD) cause energy deficiency, increased generation of reactive oxygen species (ROS), and premature neuronal death. However, the causal relations between accumulation of beta-amyloid (Aß) peptide in mitochondria and mitochondrial dysfunction, as well as molecular mechanisms underlying deleterious effects of both these factors in sporadic AD, the most common form in humans, remain unknown. Here we used olfactory bulbectomized (OBX) mice of NMRI strain as a model for sporadic AD. Five weeks after surgery, the OBX mice developed major behavioral and biochemical features of AD neurodegeneration, including spatial memory loss, increased brain levels of Aß, and energy deficiency. Mitochondria isolated from the neocortex and hippocampus of OBX mice displayed severe functional impairments, such as low NADH oxidation rate, reduced transmembrane potential, and decreased cytochrome c oxidase (complex IV) activity that correlated with high levels of soluble Aß1-40. Mitochondria from OBX mice showed increased contents of lipid peroxidation products, indicative of the development of oxidative stress. We found that neurodegeneration caused by olfactory bulbectomy is accompanied by energy metabolism disturbances and oxidative stress in brain mitochondria similar to those occurring in transgenic animals - familial AD models and patients with sporadic AD. Therefore, OBX mice can serve as a valid AD model for investigating the mechanisms of AD neurodegeneration, drug testing, and development of therapeutic strategies for AD treatment.

Mitochondrial Genome and Longevity.

The mitochondrial genome provides not only respiratory chain function, but it also ensures the impact of mitochondria on nearly all crucial metabolic processes. It is well known that mitochondria regulate aging and lifespan. However, until now there were no direct experimental data concerning the influence of various mitochondrial DNA variants on lifespan of animals with identical nuclear genome. In a recent paper of J. A. Enríquez and coworkers (Latorre-Pellicer, A., et al. (2016) Nature, 535, 561-565), it was shown that mice carrying nuclear DNA from one strain and mitochondrial DNA from another had longer median lifespan and retarded development of various aging traits. This review critically analyzes that paper and considers some aspects of the crosstalk between the nuclear and mitochondrial genomes. We also discuss new perspectives of gerontology in the light of the discovery made by Enríquez's group.

Role of Reactive Oxygen Species in Mast Cell Degranulation.

Mast cells are a heterogeneous multifunctional cellular population that promotes connective tissue homeostasis by slow release of biologically active substances, affecting primarily the permeability of vessels and vascular tone, maintenance of electrolyte and water balance, and composition of the extracellular matrix. Along with this, they can rapidly release inflammatory mediators and chemotactic factors that ensure the mobilization of effector innate immune cells to fight against a variety of pathogens. Furthermore, they play a key role in initiation of allergic reactions. Aggregation of high affinity receptors to IgE (FcεRI) results in rapid degranulation and release of inflammatory mediators. It is known that reactive oxygen species (ROS) participate in intracellular signaling and, in particular, stimulate production of several proinflammatory cytokines that regulate the innate immune response. In this review, we focus on known molecular mechanisms of FcεRI-dependent activation of mast cells and discuss the role of ROS in the regulation of this pathway.

DNA Methylation, Mitochondria, and Programmed Aging.

DNA methylation is a key epigenetic process involved in the regulation of nuclear gene expression. Progress in the study of genomic DNA methylation led to the precise identification of methylation sites reflecting biological age of cells and tissues. However, the functional significance of mitochondrial DNA (mtDNA) methylation remains unknown. Growing evidence suggests that mtDNA methylation is linked to aging and oxidative stress. This mini-review summarizes information about the methylation of nuclear and mtDNA in mammals, indicating the connection of these processes to programmed aging.

Effect of SkQ1 on Activity of the Glutathione System and NADPH-Generating Enzymes in an Experimental Model of Hyperglycemia.

We studied the effect of mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on the antioxidant activity of the glutathione system and NADPH-generating enzymes in liver and blood serum of rats with hyperglycemia induced by protamine sulfate. It was found that intraperitoneal injection of SkQ1 prevented both decrease in reduced glutathione level and increase in activity of glutathione system enzymes--glutathione peroxidase, glutathione reductase, and glutathione transferase. Activity of NADPH-generating enzymes--glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase--was also attenuated by SkQ1. Probably, in this model of hyperglycemia, decreased level of reactive oxygen species in mitochondria led to the decreased burden on the glutathione antioxidant system and NADPH-generating enzymes. Thus, SkQ1 appears to be a promising compound for the treatment and/or prevention of the adverse effects of hyperglycemia.

Low Concentrations of Uncouplers of Oxidative Phosphorylation Prevent Inflammatory Activation of Endothelial Cells by Tumor Necrosis Factor.

In endothelial cells, mitochondria play an important regulatory role in physiology as well as in pathophysiology related to excessive inflammation. We have studied the effect of low doses of mitochondrial uncouplers on inflammatory activation of endothelial cells using the classic uncouplers 2,4-dinitrophenol and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, as well as the mitochondria-targeted cationic uncoupler dodecyltriphenylphosphonium (C12TPP). All of these uncouplers suppressed the expression of E-selectin, adhesion molecules ICAM1 and VCAM1, as well as the adhesion of neutrophils to endothelium induced by tumor necrosis factor (TNF). The antiinflammatory action of the uncouplers was at least partially mediated by the inhibition of NFκB activation due to a decrease in phosphorylation of the inhibitory subunit IκBα. The dynamic concentration range for the inhibition of ICAM1 expression by C12TPP was three orders of magnitude higher compared to the classic uncouplers. Probably, the decrease in membrane potential inhibited the accumulation of penetrating cations into mitochondria, thus lowering the uncoupling activity and preventing further loss of mitochondrial potential. Membrane potential recovery after the removal of the uncouplers did not abolish its antiinflammatory action. Thus, mild uncoupling could induce TNF resistance in endothelial cells. We found no significant stimulation of mitochondrial biogenesis or autophagy by the uncouplers. However, we observed a decrease in the relative amount of fragmented mitochondria. The latter may significantly change the signaling properties of mitochondria. Earlier we showed that both classic and mitochondria-targeted antioxidants inhibited the TNF-induced NFκB-dependent activation of endothelium. The present data suggest that the antiinflammatory effect of mild uncoupling is related to its antioxidant action.

Pure Mitochondrial DNA Does Not Activate Human Neutrophils in vitro.

Excessive activation of the innate immune system often leads to fatal consequences and can be considered as one of the phenoptotic events. After traumatic injury, various components of mitochondria are released into the circulation and stimulate myeloid cells of the innate immunity. Presumably, mitochondrial DNA (mtDNA) might activate immune cells (Zhang, Q., et al. (2010) Nature, 464, 104-107). In the present study, we investigated the role of mtDNA as a direct activator of human neutrophils, as well as a prognostic marker in patients with severe trauma. Quantitative determination of mtDNA in the plasma of these patients revealed its significant increase (p < 0.02) in the group of survivors compared to non-survivors. Highly purified mtDNA was not able to induce activation of human neutrophils, thus possibly indicating the existence of additional factor(s) ensuring the recognition of mtDNA as a damage-associated molecular pattern.

Age-associated murine cardiac lesions are attenuated by the mitochondria-targeted antioxidant SkQ1.

Age-related changes in mammalian hearts often result in cardiac hypertrophy and fibrosis that are preceded by inflammatory infiltration. In this paper, we show that lifelong treatment of BALB/c and C57BL/6 mice with the mitochondria-targeted antioxidant SkQ1 retards senescence-associated myocardial disease (cardiomyopathy), cardiac hypertrophy, and diffuse myocardial fibrosis. To investigate the molecular basis of the action of SkQ1, we have applied DNA microarray analysis. The global gene expression profile in heart tissues was not significantly affected by administration of SkQ1. However, we found some small but statistically significant modifications of the pathways related to cell-to-cell contact, adhesion, and leukocyte infiltration. Probably, SkQ1-induced decrease in leukocyte and mesenchymal cell adhesion and/or infiltration lead to a reduction in age-related inflammation and subsequent fibrosis. The data indicate a causative role of mitochondrial reactive oxygen species in cardiovascular aging and imply that SkQ1 has potential as a drug against age-related cardiac dysfunction.

Mitochondria-targeted antioxidants prevent TNFα-induced endothelial cell damage.

Increased serum level of tumor necrosis factor α (TNFα) causes endothelial dysfunction and leads to serious vascular pathologies. TNFα signaling is known to involve reactive oxygen species (ROS). Using mitochondria-targeted antioxidant SkQR1, we studied the role of mitochondrial ROS in TNFα-induced apoptosis of human endothelial cell line EAhy926. We found that 0.2 nM SkQR1 prevents TNFα-induced apoptosis. SkQR1 has no influence on TNFα-dependent proteolytic activation of caspase-8 and Bid, but it inhibits cytochrome c release from mitochondria and cleavage of caspase-3 and its substrate PARP. SkQ analogs lacking the antioxidant moieties do not prevent TNFα-induced apoptosis. The antiapoptotic action of SkQR1 may be related to other observations made in these experiments, namely SkQR1-induced increase in Bcl-2 and corresponding decrease in Bax as well as p53. These results indicate that mitochondrial ROS production is involved in TNFα-initiated endothelial cell death, and they suggest the potential of mitochondria-targeted antioxidants as vasoprotectors.

[The effects of aerobic exercise upon genes expression in skeletal muscle of trained and untrained men].

It is well recognized the PGC-1 alpha is a key regulator of mitochondrial biogenesis. Mechanical and metabolic perturbations in a skeletal muscle during and after aerobic exercise lead to increase PGC-1alpha expression. In addition to that an increase of PGC-1alpha expression after exercise depends on relative workload intensity and does not depend on fitness level. The aim of the study was to compare the expression of regulators of mitochondrial biogenesis PGC-1alpha, TFAM and TFB2M and of proteolysis-related genes FOXO1 and Atrogin-1 in skeletal muscle untrained and trained men after aerobic exercise with the same relative workload. The study showed that after exercise the PGC-1alpha expression did not differ between groups but TFAM and TFB2M expression was higher in untrained muscle than trained. On the contrary proteolysis-related genes FOXO1 and Atrogin-1 expression increased only in the muscle of trained men.