Properties and Activity of Peptide Derivatives of ACE2 Cellular Receptor and Their Interaction with SARS-CoV-2 S Protein Receptor-Binding Domain.

The aim of this work was to design and characterize peptides based on the α-helices h1 and h2 of the ACE2 receptor, forming the interaction interface between the receptor-binding domain (RBD) of the SARS-CoV-2 S protein and the cellular ACE2 receptor. Monomeric and heterodimeric peptides connected by disulfide bonds at different positions were synthesized. Solubility, RBD-binding affinity, and peptide helicity were experimentally measured, and molecular dynamics simulation was performed in various solvents. It was established that the preservation of the helical conformation is a necessary condition for the binding of peptides to RBD. The peptides have a low degree of helicity and low affinity for RBD in water. Dimeric peptides have a higher degree of helicity than monomeric ones, probably due to the mutual influence of helices. The degree of helicity of the peptides in trifluoroethanol is the highest; however, for in vitro studies, the most suitable solvent is a water-ethanol mixture.

Features of the Population of Mouse Peritoneal Macrophages Isolated after Stimulation with Concanavalin A and Thioglycolate.

Murine peritoneal macrophages isolated from the lavage fluid after administration of thioglycolate and concanavalin A are presented by two populations of cells of different diameters. Polarization of macrophages into a proinflammatory (M1) phenotype is accompanied by an increase in number of small cells. Macrophages obtained after administration of thioglycolate demonstrate higher tendency to anti-inflammatory (M2) phenotype, while macrophages isolated after administration of concanavalin A are committed in the proinflammatory direction. Lactate level is increased in M1 macrophages in comparison with M2 cells, which indicates predominance of glycolytic metabolism. Macrophages obtained after administration of concanavalin A have reduced mitochondrial potential, which reflects a tendency to apoptosis. Autophagy activation and inhibition neutralize the differences in pro- and anti-inflammatory properties of polarized macrophages obtained after thioglycolate administration, but have less pronounced effect on macrophages obtained after administration concanavalin A. Autophagy inhibitor increases mitochondrial potential in non-polarized macrophages obtained after administration of concanavalin A. These results demonstrate divergent properties of macrophages obtained after administration of glycolate and concanavalin A due to the difference in the mechanisms of experimental peritonitis.

Hypoxia - as a Possible Regulator of the Activity of Epicardial Mesothelial Cells After Myocardial Infarction.

Aim      To study the effect of hypoxia on the activity of epithelial-mesenchymal transition (EMT) in epicardial cells, which provides formation of a specialized microenvironment.Material and methods   This study used a model of experimental myocardial infarction created by ligation of the anterior descendent coronary artery. The activity of epicardial cells after a hypoxic exposure was studied with the hypoxia marker, pimonidazole, bromodeoxyuridine, immunofluorescent staining of heart cryosections, and in vitro mesothelial cell culture.Results The undamaged heart maintained the quiescent condition of mesothelial cells and low levels of their proliferation, extracellular matrix protein production, and of the EMT activity. Acute ischemic injury induced moderate hypoxia in the epicardial/subepicardial region. This caused a global rearrangement of this region due to the initiation of EMT in cells, changes in the cell composition, and accumulation of extracellular matrix proteins. We found that the initiation of EMT in mesothelial cells may result in the formation of smooth muscle cell precursors, fibroblasts, and a population of Sca-1+ cardiac progenitor cells, which may both participate in construction of new blood vessels and serve as a mesenchymal link for the paracrine support of microenvironmental cells. In in vitro experiments, we showed that 72­h hypoxia facilitated activation of EMT regulatory genes, induced dissembling of intercellular contacts, cell uncoupling, and increased cell plasticity.Conclusion      The epicardium of an adult heart serves as a "reparative reserve" that can be reactivated by a hypoxic exposure. This creates a basis for an approach to influence the epicardium to modulate its activity for regulating reparative processes.

Latent Inflammation and Defect in Adipocyte Renewal as a Mechanism of Obesity-Associated Insulin Resistance.

Obesity is a major risk factor for type 2 diabetes and metabolic syndrome and an essential medical and social problem. In the first part of the review, we briefly highlight the biochemical basis of metabolic disbalance in obesity and evolution of our views on the mechanisms of insulin resistance development in insulin-sensitive tissues. Because obesity relates to the disturbance in the normal physiology of fat tissue, the second part of the review focuses on latent inflammation that develops in obesity and is supported by immune cells. Finally, the problem of adipocyte hypertrophy, reduced regenerative potential of fat progenitor cells, and impaired renewal of fat depots is discussed in the context of type 2 diabetes pathogenesis.

Chemical Inducers of Obesity-Associated Metabolic Stress Activate Inflammation and Reduce Insulin Sensitivity in 3T3-L1 Adipocytes.

Obesity is accompanied by dyslipidemia, hypoxia, endoplasmic reticulum (ER) stress, and inflammation, representing the major risk factor for the development of insulin resistance (IR) and type 2 diabetes. We modeled these conditions in cultured 3T3-L1 adipocytes and studied their effect on insulin signaling, glucose uptake, and inflammatory response via activation of stress-dependent JNK1/2 kinases. Decreased insulin-induced phosphorylation of the insulin cascade components IRS, Akt, and AS160 was observed under all tested conditions (lipid overloading of cells by palmitate, acute inflammation induced by bacterial lipopolysaccharide, hypoxia induced by Co2+, and ER stress induced by brefeldin A). In all the cases, except the acute inflammation, glucose uptake by adipocytes was reduced, and the kinetics of JNK1/2 activation was bi-phasic exhibiting sustained activation for 24 h. By contrast, in acute inflammation, JNK1/2 phosphorylation increased transiently and returned to the basal level within 2-3 h of stimulation. These results suggest a critical role of sustained (latent) vs. transient (acute) inflammation in the induction of IR and impairment of glucose utilization by adipose tissue. The components of the inflammatory signaling can be promising targets in the development of new therapeutic approaches for preventing IR and type 2 diabetes.

Gene therapy of type 2 diabetes mellitus: state of art.

Type 2 diabetes mellitus (T2DM) and other metabolic diseases are essential links in the structure of morbidity and mortality in the modern world. The accepted strategy for the correction of T2DM and insulin resistance is drug therapy aimed at delivering insulin from the outside, stimulating the secretion of own insulin and reducing the concentration of blood glucose. However, modern studies demonstrate a great potential for the use of gene therapy approaches for the correction of T2DM and insulin resistance. In the present review, the main variants of plasmid gene therapy of T2DM using the genes of adiponectin and type 1 glucagon-like peptide, as well as the main variants of viral gene therapy of T2DM using the genes of type 1 and leptin are considered. T2DM gene therapy is currently not ready to enter into routine clinical practice, but, subject to improvements in delivery systems, it can be a powerful link in combination therapy for diabetes.

Interleukin-4 Restores Insulin Sensitivity in Lipid-Induced Insulin-Resistant Adipocytes.

Obesity and latent inflammation in adipose tissue significantly contribute to the development of insulin resistance (IR) and type 2 diabetes. Here we studied whether the antiinflammatory interleukin-4 (IL-4) can restore insulin sensitivity in cultured 3T3-L1 adipocytes. The activity of key components of the insulin signaling cascade was assessed by immunoblotting using phospho-specific antibodies to insulin receptor substrate IRS1 (Tyr612), Akt (Thr308 and Ser473), and AS160 (Ser318) protein that regulates translocation of the GLUT4 glucose transporter to the plasma membrane. IR was induced in mature adipocytes with albumin-conjugated palmitate. IR significantly reduced phosphorylation levels of all the above-mentioned proteins. Addition of IL-4 to the culturing medium during IR induction led to a dose-dependent stimulation of the insulin-promoted phosphorylation of IRS1, Akt, and AS160. At the optimal concentration of 50 ng/ml, IL-4 fully restored activation of the insulin cascade in IR cells, but it did not affect insulin signaling activation in the control cells. IL-4 neither upregulated expression of key adipogenesis markers GLUT4 and PPARγ nor caused lipid accumulation in the adipocytes. These results demonstrate that IL-4 can restore insulin sensitivity in adipocytes via mechanisms not associated with induced adipogenesis or de novo formation of lipid depots.

ISOLATION AND CHARACTERIZATION OF CARDIAC PROGENITOR CELLS OBTAINING FROM MYOCARDIAL RIGHT ATRIAL APPENDAGE TISSUE.

Resident stem cells of the heart are denoted as heterogeneous population of immature cells, which reside in the myocardium and characterized by their ability to self-renewal and are multipotent differentiation capacity into cardiomyocyte-like and vascular like cells. CSCs were originally isolated directly by long enzymatic digestion of heart tissue and selection using stem cell markers. However, long exposure to enzymatic digestion and small myocardial sample size can affect the possibility of obtaining a significant amount of viable cells. To avoid these problems, we developed a method consisting of growing of the CPC in explant culture and subsequent immunomagnetic selection.

Molecular Mechanisms of Latent Inflammation in Metabolic Syndrome. Possible Role of Sirtuins and Peroxisome Proliferator-Activated Receptor Type γ.

The problem of metabolic syndrome is one of the most important in medicine today. The main hazard of metabolic syndrome is development of latent inflammation in adipose tissue, which promotes atherosclerosis, non-alcoholic fatty liver disease, myocarditis, and a number of other illnesses. Therefore, understanding of molecular mechanisms of latent inflammation in adipose tissue is very important for treatment of metabolic syndrome. Three main components that arise during hypertrophy and hyperplasia of adipocytes underlie such inflammation: endoplasmic reticulum stress, oxidative stress, and hypoxia. Each of these components mediates activation in different ways of the key factor of inflammation - NF-κB. For metabolic syndrome therapy, it is suggested to influence a number of inflammatory signaling components by activating other cell factors to suppress development of inflammation. Such potential factors are peroxisome proliferator-activated receptors type γ that suppress transcription factor NF-κB through direct contact or via kinase of a NF-κB inhibitor (IKK), and also the antiinflammatory transcription factor AP-1. Other possible targets are type 3 NAD+-dependent histone deacetylases (sirtuins). There are mutually antagonistic relationships between NF-κB and sirtuin type 1 that prevent development of inflammation in metabolic syndrome. Moreover, sirtuin type 1 inhibits the antiinflammatory transcription factor AP-1. Study of the influence of these factors on the relationship between macrophages and adipocytes, macrophages, and adipose tissue-derived stromal cells can help to understand mechanisms of signaling and development of latent inflammation in metabolic syndrome.

[UROKINASE SYSTEM INVOLVES IN VASCULAR CELLS MIGRATION AND REGULATES THE GROWTH AND BRANCHING OF CAPILLARIES].

Urokinase system representing urokinase-type plasminogen activator (urokinase, uPA) and urokinase re- ceptor (uPAR) plays an important regulatory role in the vascular wall and has the ability to run a proteolytic cascade, degradation of extracellular matrix and activate intracellular signaling in vascular cells. In this work, we have firstly shown a fundamental mechanism of urokinase system-dependent regulation of the trajectory of growth and branching of blood vessels what may be of particular importance in the growth of blood vessels in early embryogenesis and in adults during the repair/regeneration of tissues.

[Therapeutic Angiogenesis Using Growth Factors: Current State and Prospects for Development].

Therapeutic angiogenesis has been in use for treatment of ischemic diseases for about 15 years. During this period of successes and failures this field has accumulated a significant amount of published and ongoing surveys giving insights and raising new questions and problems. One of the most utilized methods for therapeutic angiogenesis suggests introduction of angiogenic growth factors (VEGF, bFGF, angiopoietin-1 etc.) into ischemic tissues. Still, there is a whole range of problems regarding the efficacy of therapeutic angiogenesis. These can be potentially circumvented by use of new delivery methods, development of combined approaches and use of more relevant pre-clinical animal models. Present review gives a brief analysis of crucial achievements and issues that has been recently raised in experimental and clinical studies focusing on therapeutic angiogenesis. Final part brings some possible directions for development that can give an opportunity to circumvent current obstacles and provide further development.

Mitochondrial dynamics keep balance of nutrient combustion in thermogenic adipocytes.

Non-shivering thermogenesis takes place in brown and beige adipocytes and facilitates cold tolerance and acclimation. However, thermogenesis in adipose tissue also was found to be activated in metabolic overload states for fast utilization of nutrients excess. This observation spurred research interest in mechanisms of thermogenesis regulation for metabolic overload and obesity prevention. One of proposed regulators of thermogenic efficiency in adipocytes is the dynamics of mitochondria, where thermogenesis takes place. Indeed, brown and beige adipocytes exhibit fragmented round-shaped mitochondria, while white adipocytes have elongated organelles with high ATP synthesis. Mitochondrial morphology can determine uncoupling protein 1 (UCP1) content, efficiency of catabolic pathways and electron transport chain, supplying thermogenesis. This review will highlight the co-regulation of mitochondrial dynamics and thermogenesis and formulate hypothetical ways for excessive nutrients burning in response to mitochondrial morphology manipulation.

[Peptide inhibitors of the interaction of the SARS-CoV-2 receptor-binding domain with the ACE2 cell receptor]. / Peptidnye ingibitory vzaimodeistviia retseptor-sviazyvaiushchego domena virusa SARS-CoV-2 s kletochnym retseptorom ASE2.

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22-23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which edge cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of the molecule of each of the precursor peptides. The precursors h1 and h2 modelled amino acid sequences of α1- and α2-helices of the extracellular peptidase domain of ACE2, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their RBD-peptides (particularly in dependence of the middle and edge methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified (to 95-97% purity), and characterized by HPLC and MALDI-TOF mass spectrometry. The binding of the peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. Binding to the original RBD of the Chinese variant was detected in three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant with micromolar constants. The antiviral activity of the proposed peptides in Vero cell culture was also evaluated.

Plasmid-based gene therapy with hepatocyte growth factor stimulates peripheral nerve regeneration after traumatic injury.

Peripheral nerve injury remains a common clinical problem with no satisfactory treatment options. Numerous studies have shown that hepatocyte growth factor (HGF) exerts neurotrophic effect in motor, sensory, and parasympathetic neurons in addition to mitogenic, morphogenic, angiogenic, antiapoptotic, antifibrotic, and anti-inflammatory effect on various tissues and cells. In our study we examined efficacy of gene therapy with HGF-bearing plasmid (pC4W-hHGF) to improve consequences of traumatic nerve injury in mice. Treatment by pC4W-hHGF led to restoration of nerve structure and functional recovery compared to similar parameters in control animals. Compound action potentials (CAP) in experimental groups treated with 100 or 200 µg of pC4W-hHGF demonstrated increased amplitude and latency decrease compared to spontaneous recovery control group. In HGF-treated mice histological analysis showed a three-fold increase in axon number in nerve portion located distal to the lesion site compared to control. Moreover, significant functional recovery of n. peroneus communis triggered by pC4W-hHGF gene therapy was observed using the footprints analysis. Obtained results provide evidence for plasmid-based HGF gene therapy as a potential treatment for traumatic injury of peripheral nerve.

Latent Inflammation and Insulin Resistance in Adipose Tissue.

Obesity is a growing problem in modern society and medicine. It closely associates with metabolic disorders such as type 2 diabetes mellitus (T2DM) and hepatic and cardiovascular diseases such as nonalcoholic fatty liver disease, atherosclerosis, myocarditis, and hypertension. Obesity is often associated with latent inflammation; however, the link between inflammation, obesity, T2DM, and cardiovascular diseases is still poorly understood. Insulin resistance is the earliest feature of metabolic disorders. It mostly develops as a result of dysregulated insulin signaling in insulin-sensitive cells, as compared to inactivating mutations in insulin receptor or signaling proteins that occur relatively rare. Here, we argue that inflammatory signaling provides a link between latent inflammation, obesity, insulin resistance, and metabolic disorders. We further hypothesize that insulin-activated PI3-kinase pathway and inflammatory signaling mediated by several IκB kinases may constitute negative feedback leading to insulin resistance at least in the fat tissue. Finally, we discuss perspectives for anti-inflammatory therapies in treating the metabolic diseases.

Increased expression of uPA, uPAR, and PAI-1 in psoriatic skin and in basal cell carcinomas.

There is substantial evidence implicating the urokinase system in tissue remodeling during neo-vascularization, inflammation, tumor invasion, and metastasis. Regulated degradation of the extracellular matrix at the leading edge of migrating cells, mediated by uPA and uPAR, is required for tissue remodeling, invasiveness, and angiogenesis. Psoriasis and basal cell carcinoma (BCC) are the most common skin diseases. Pathogenesis of both of them is associated with keratinocyte hyperproliferation, inflammatory cell migration, and angiogenesis-processes in which the plasminogen system (uPA, uPAR, tPA, and PAI-1) plays a crucial role. In the present study, the comparative analysis of uPA, uPAR, tPA, and PAI-1 expression in the normal skin, in the biopsies of patients with psoriasis vulgaris, and BCC was carried out. uPA, uPAR, and PAI-1 expression was up-regulated in the epidermis of psoriatic skin and in tumor cells in BCC. Increased uPAR expression was detected in the derma of psoriatic lesions and in the stroma surrounding tumor cells in BCC. Increased expression of uPA in epidermal cells in psoriasis and in tumor cells in BCC suggests an important role of the uPA system for aggressively proliferating and invading cells of epidermal origin. A possible activation of the stroma, as a result of uPA-uPAR interaction between tumor cells and the surrounding stroma, is suggested.

Plasminogen activators in vascular remodeling and angiogenesis.

This review considers cellular and molecular mechanisms of the involvement of plasminogen activators in extracellular proteolysis and cell migration and proliferation. The role of plasminogen activators in vascular remodeling in atherosclerosis, restenosis, and angiogenesis is discussed.