[Transcription of DNA-Methyltransferases in Endothelial Cells Exposed to Mitomycin C].

DNA-methyltransferases catalyze DNA methylation in the CpG sites, which play an important role in the maintenance of genome stability. The association between DNA methylation and genotoxic stress resulting in the action of various clastogens has been shown. Genotoxic stress is one of the triggers of endothelial dysfunction. In this study, the transcription of DNMT1, DNMT3A and DNMT3B genes in coronary (HCAEC) and internal thoracic (HITAEC) artery endothelial cells exposed to alkylating mutagen mitomycin C was studied using quantitative polymerase chain reaction. In HCAEC exposed to mitomycin C, DNMT1 transcription is 1.7-fold higher compared to the unexposed control. After elimination of the mutagen from the cultures followed by 24-hours of cultivation, a 2-fold increase of transcription of DNMT3B in HCAEC exposed to mitomycin C compared to the control was observed. At the same time, no changes in transcription of the studied DNA-methyltransferases were found in HITAEC exposed to the mutagen. Thus, increased transcription of DNA-methyltransferase may be a possible molecular mechanism underlying endothelial dysfunction in response to mutagenic load in an in vitro experiment.

[Ultrastructure of neointima of native and artificial elements of the blood circulatory system]. / Ul'trastruktura neointimy nativnykh i iskusstvennykh elementov sistemy krovoobrashcheniya.

OBJECTIVE: To compare the neointima structure in conduits for coronary bypass grafting, bioprosthetic heart valves, tissue-engineered vascular grafts, and metal stents. MATERIAL AND METHODS: The objects of the study were the fragments of the human internal thoracic artery, experimental biodegradable vascular prostheses, leaflets of xenopericardial bioprostheses of heart valves, and fragments of stented vessels. Tissue samples were fixed in formalin and post-fixed in osmium tetroxide. After dehydration and epoxy resin embedding, the samples were ground and polished. Samples were counterstained with uranyl acetate and lead citrate and visualized by means of backscattered scanning electron microscopy. RESULTS: Neointimal pattern in all samples was similar. Neointima was comprised of endothelial cells, smooth muscle cells, fibroblasts, and the extracellular matrix. Endothelial cells showed significant diversity both between different elements of the circulatory system and within the same tissue, having either elongated or polygonal shape. Adhesion of leukocytes testified to the endothelial cell activation. In the absence of inflammation in the superficial layer of the neointima, the arrangement of smooth muscle cells and extracellular matrix fibers was parallel to the endothelium. Clusters of foam cells were frequently detected around the neointimal layers with solid inclusions (metal stents or calcium deposits). Thickening of the neointima was accompanied by the presence of capillaries and capillary-like structures. CONCLUSION: Neointima formation is a typical response to the damage inflicted to the elements of the circulatory system. Neointima underwent a constant remodeling characterized by an altered cellular composition, macrophage invasion, neovascularization, and calcification.

[Comparative study of vasa vasorum and neointima in conduits for coronary artery bypass grafting]. / Sravnitel'noe izuchenie vasa vasorum i neointimy v konduitakh dlia koronarnogo shuntirovaniia.

AIM: This study was undertaken to investigate the preoperative incidence and severity of intimal hypertrophy, as well as the level of blood supply of arterial and venous conduits for coronary artery bypass grafting. MATERIAL AND METHODS: Segments of the internal thoracic artery and great saphenous vein (n=13) were harvested pairwise during coronary artery bypass grafting and were then visualized by scanning electron microscopy in back-scattered electrons. The analysis of the incidence and thickness of intimal hypertrophy, as well as the calculation of the number and the area of the vasa vasorum were performed using the programme ImageJ. RESULTS: Intimal hypertrophy was more characteristic for the great saphenous vein as compared with the internal thoracic artery (9/13 (69.2%) and 7/13 (55.8%), respectively), although this difference did not reach statistical significance. The maximal-to-minimal neointimal thickness ratio correlated with the percentage of stenosis (r=0.875, p<0.0001), the area (r=0.45, p=0.023) and the number (r=0.47, p=0.015) of the vasa vasorum in the conduits, thus confirming the hypothesis on possible participation of these vessels in the development of intimal hypertrophy, with the area of the vasa vasorum being greater in the vessels with >10% stenosis (p=0.051). The number of the vasa vasorum in the great saphenous vein exceeded that in the internal thoracic artery (p=0.0005), with this difference remaining significant after adjustment for the area of the adventitia (p=0.027). The number of the vasa vasorum per the percentage of stenosis in the great saphenous vein also exceeded that in the internal thoracic artery (p=0.039) and more strongly correlated with intimal hypertrophy in the great saphenous vein as compared with that in the internal thoracic artery (r=0.53 and r=0.27, respectively). CONCLUSION: Intimal hypertrophy correlates with the area and number of the vasa vasorum in conduits. The great saphenous vein is characterised by a larger number and higher density of the vasa vasorum as compared with the internal thoracic artery. The number of the vasa vasorum is correlated with stenosis of the great saphenous vein more closely than with stenosis of the internal thoracic artery. This may be suggestive of significant predisposition of the great saphenous vein to the onset of adventitial inflammation followed by the development of intimal hypertrophy.

Backscattered Scanning Electron Microscopy Approach for Assessment of Microvessels under Conditions of Normal Microanatomy and Pathological Neovascularization.

We evaluated the efficiency of an original method for studying of the microvascular bed under conditions of normal microanatomy and pathological neovascularization. The blood vessels, tissues surrounding the stent in the pulmonary artery and subcutaneously implanted titanium nickelide plate, atherosclerotic plaque, and vascular stent with restenosis were examined. The specimens were fixed in formalin and stained in OsO4, embedded into fresh epoxy resin, grinded, polished, and counterstained with uranyl acetate and lead citrate. Numerous vasa vasorum were found in all native vessels. Around the pulmonary artery stent and metal plates, numerous newly formed vessels of small diameter were seen. The intensity of neovascularization in atherosclerosis and carotid stent restenosis differed significantly. Our technique can be successfully used for evaluation of the microvascular bed.

[Progression of precerebral atherosclerosis and predictors of ischemic complications in cardiac patients]. / Progressirovanie pretserebral'nogo ateroskleroza i prediktory ishemicheskikh oslozhnenii u patsientov kardiokhirurgicheskogo profilya.

OBJECTIVE: To identify predictors of progression of precerebral atherosclerosis in long-term period after coronary artery bypass surgery. MATERIAL AND METHODS: There were 97 procedures of carotid endarterectomy in patients after previous coronary artery bypass grafting for the period from 2006 to 2017. Inclusion criteria were previous CABG, no significant (over 60%) stenosis of internal carotid arteries at discharge after CABG. The control group included 447 patients without progression of precerebral atherosclerosis in long-term period after CABG. RESULTS: Careful monitoring of progression of precerebral atherosclerosis and therapeutic prevention of ischemic stroke are required in patients with mild-to-moderate ICA stenosis after CABG. The most significant predictors of progression of precerebral atherosclerosis after CABG were AF (OR=1.97, 95% CI 1.04-3.73), previous occlusion of stent (OR=7.89, 95% CI=2.3-27.0), chronic brain ischemia grade II or III (OR=22.45, 95% CI=11.9-42.3), chronic kidney disease (OR=15.8, 95% CI=5.04-49.5). CONCLUSION: It was revealed that the majority of predictors of adverse ischemic cerebral and myocardial events are indirectly associated with atrial fibrillation.

Effects of Deltorphin II and Its Retroenantio Analog on Cardiac Tolerance to Ischemia and Reperfusion.

Selective agonist of δ2-opioid receptors deltorphin II and its retroenantio analog (0.12 mg/kg intravenously) were preventively injected to male Wistar rats 15 min prior to 45-min coronary occlusion or 5 min before 120-min reperfusion. Administration of deltorphin II before artery occlusion and before reperfusion decreased the infarct size/area at risk ratio. Deltorphin II prevented the appearance of ischemia-provoked ventricular arrhythmias and exerted no effect on HR and BP (systolic and diastolic). The retroenantio analog of deltorphin II produced no antiarrhythmic or infarct-limiting effects, but reduced HR without affecting BP. Deltorphin II can be viewed as a promising prototype for a medicinal remedy to treat acute myocardial infarction.

[Prospects of Application of Remote Preconditioning at Heart Revascularization].

Remote ischemic preconditioning of the heart exerts anti-necrotic, antiarrhythmic, inotropic effects that have been demonstrated in clinical trials in cardiac surgery both in adults and children. However, so far there is no consensus between cardiologists regarding the impact of remote preconditioning on the incidence of intraoperative myocardial infarctions and mortality in the postoperative period. Until now there is no unanimity concerning choice of remote preconditioning protocol and timing of its application before cardiac surgery.

CB-Receptor Agonist HU-210 Mimics the Postconditioning Phenomenon of Isolated Heart.

CB receptor agonist HU-210 exhibits an infarction-limiting effect during in vitro reperfusion of the heart after focal ischemia. This effect is paralleled by a decrease in left-ventricular developed pressure and double product. In addition, HU-210 reduces end-diastolic pressure during the reperfusion period, which indirectly attests to reduced Ca2+ overload of cardiomyocytes.

[Neuroprotective and nephroprotective effects of remote postconditioning: Prospects for clinical use]. / Neiroprotektornyi i nefroprotektornyi éffekty distantnogo prekonditsionirovaniia. Perspektivy klinicheskogo primeneniia.

The results of experimental and clinical studies strongly suggest that remote ischemic preconditioning (RIP) has no neuroprotective effect during cardiac surgery performed under extracorporeal circulation. Remote preconditioning (RP) has no neuroprotective effect in hemorrhagic stroke. A randomized multicenter study is needed to evaluate the efficiency RIP in patients with ischemic stroke. RP reduces the severity of ischemia/reperfusion kidney injury during transplantation. RIP has been established to prevent contrast-induced nephropathy. There is a need for a multicenter trial to evaluate the efficiency of RIP in patients with abdominal aortic aneurysm repair. Analysis of the presented data indicates that RIP fails to prevent cardiorenal syndrome in infants and children during cardiac surgery. The data available in the literature on the capacity of RIP to provide nephroprotective effect in patients after coronary artery bypass surgery are discordant and indicative of the advisability of a multicenter study.

[CARDIOPROTRECTIVE EFFECT OF REMOTE POSTCONDITIONING: MECHANISMS AND POSSIBILITIES OF CLINICAL APPLICATION].

Experimental data indicate that postconditioning at a distance is an effective method for cardiac protection against reperfusion injury. Remote postconditioning prevents reperfusion necrosis and apoptosis of cardiomyocytes, decreases a probability of postinfarction remodeling of the heart. Cardioprotective effect of remote postconditioning depends on the release of tissue factor(s) increasing cardiac tolerance to long-term ischemia-reperfusion after transient ischemia. Clinical investigations show that postconditioning at a distance is an effective method for the prevention of reperfusion injury of the heart during coronary artery bypass surgery.

[Remote preconditioning phenomenon. prospects for use in pathology of lung and gastrointestinal tract].

The literature data on the effectiveness of remote ischemic preconditioning (RIP) in the prevention of lung injury are contradictory. Authors of some works argue that RIP prevents lung damage during surgical interventions, the authors of other publications claim that the RIP does not protect lung against pathological processes. It is obvious that there is an urgent need for multicenter, randomized trials aimed at studying RIP protective effects against pathological processes in lung. Also required is clinical evaluation of the effectiveness of RIP in the thromboembolism of pulmonary arteries, the transplantation of the lungs and intestinal infarction. Remote preconditioning prevents the intestine injury associated with abdominal aortic aneurysm repair. Experimental data indicate that RIP has the hepatoprotective effect during ischemia and reperfusion injury of liver, septic or haemorrhagic shock. The question of whether the DIP has a protective effect during ischemia-reperfusion of the pancreas remains open.

[Role of infectious agents in the emergence of malignant tumors].

According to the data of the International Agency for Research on Cancer (IARC), at least 6 virus species (HPV, EBV, HHV-8/KSHV, HTLV-1, HBV, HCV), 4 helminthes species (Schistosoma haematobium and japonicum, Opisthorchis viverrini, Clonorchis sinensis) and I bacterium species (Helicobacter pylori) have been proved to be capable of causing the development of cancer. The analysis of the data available shows that Merkel cell polyomavirus (MCV), herpes simplex virus (HSV), John Cunningham polyomavirus (JCV), monkey virus 40 (SV40), cytomegalovirus (CMV), xenotropic murine leukemia virus (XMRV), Helicobacter bilis and hepaticus, Campylobacter jejuni, Fusobacterium varium, enteropathogenic Escherichia coli, enterotoxigenic Bacteroides fragilis, Bacteroides vulgatus, Prevotella spp., Streptococcus bovis and anginosus, Treponema denticola, Salmonella typhi, paratyphi and typhimurium, Borrelia burgdorferi, Bartonella spp., Mycobacterium tuberculosis, Chlamydia pneumoniae, trachomatis and psittaci, Neisseria gonorrhoeae, Propionibacterium acnes, Tropheryma whippelii, Schistosoma mansoni, Opistorchis felineus, Strongyloides stercoralis, Taenia solium, Candida spp., Paracoccidioides brasiliensis, Histoplasma capsulatum and Trichomonas vaginalis can also be potential etiological agents of cancer. Apparently, detection of new associations between infectious agents and risk of the development of cancer will facilitate progress in elaboration of prophylaxis measures, early diagnostic methods and, probably, methods of treatment of malignant tumors.

Endothelial Dysfunction in the Context of Blood-Brain Barrier Modeling.

Here, we discuss pathophysiological approaches to the defining of endothelial dysfunction criteria (i.e., endothelial activation, impaired endothelial mechanotransduction, endothelial-to-mesenchymal transition, reduced nitric oxide release, compromised endothelial integrity, and loss of anti-thrombogenic properties) in different in vitro and in vivo models. The canonical definition of endothelial dysfunction includes insufficient production of vasodilators, pro-thrombotic and pro-inflammatory activation of endothelial cells, and pathologically increased endothelial permeability. Among the clinical consequences of endothelial dysfunction are arterial hypertension, macro- and microangiopathy, and microalbuminuria. We propose to extend the definition of endothelial dysfunction by adding altered endothelial mechanotransduction and endothelial-to-mesenchymal transition to its criteria. Albeit interleukin-6, interleukin-8, and MCP-1/CCL2 dictate the pathogenic paracrine effects of dysfunctional endothelial cells and are therefore reliable endothelial dysfunction biomarkers in vitro, they are non-specific for endothelial cells and cannot be used for the diagnostics of endothelial dysfunction in vivo. Conceptual improvements in the existing methods to model endothelial dysfunction, specifically, in relation to the blood-brain barrier, include endothelial cell culturing under pulsatile flow, collagen IV coating of flow chambers, and endothelial lysate collection from the blood vessels of laboratory animals in situ for the subsequent gene and protein expression profiling. Combined with the simulation of paracrine effects by using conditioned medium from dysfunctional endothelial cells, these flow-sensitive models have a high physiological relevance, bringing the experimental conditions to the physiological scenario.

[Cancer molecular epidemiology: new horizons of prophylaxis].

Perspectives of malignant neoplasm prophylaxis based on molecular biology achievements are discussed. Gene variants critical to development of hereditary cancer syndromes, genes modulating malignant neoplasm development risk without hereditary cancer syndrome development, and genes determining tendency of individuals for different malignant neoplasm progress risk increasing lifestyle factors are examined. Molecular epidemiology by using large scale population analysis of cancerogenesis linked genetic polymorphisms prevalence allows determination of risk groups at the most earlier stages of cell transformation or even before the onset of cell malignization and development of goal-based prophylaxis measures based on polymorphism and corresponding cancer type. Epidemiologic analysis of this type allows for earlier diagnostics in risk groups, therapy efficacy increase, disability decrease. Specific therapy on molecular level may be possible in the future.

[The gene expression signature in endothelial cells exposed to mitomycin C]. / Profil' gennoi ékspressii v éndotelial'nykh kletkakh, kul'tiviruemykh v prisutstvii mitomitsina S.

The expression of DNA repair (DDB1, ERCC4, ERCC5), leukocyte adhesion (VCAM1, ICAM1, SELE, SELP), endothelial mechanotransduction (KLF4), endothelial differentiation (PECAM1, CDH5, CD34, NOS3), endothelial-to-mesenchymal transition (SNAI1, SNAI2, TWIST1, GATA4, ZEB1, CDH2), scavenger receptors (LOX1, SCARF1, CD36, LDLR, VLDR), antioxidant system (PXDN, CAT, SOD1) and transcription factor (HEY2) genes in primary human coronary (HCAEC) and internal thoracic (HITAEC) arteries endothelial cells exposed to alkylating mutagen mitomycin C (MMC) was studied at two time points - after 6 h of incubation with MMC and after 6 h of the genotoxic load followed by 24 h of incubation in pure culture medium using the quantitative PCR. Immediately after MMC exposure, in the exposed HCAEC and HITAEC a decreased expression of almost all studied genes was noted excepted SNAI, which demonstrated a 4-told increase in its expression compared to the unexposed control. Elimination of MMC from the cultures, an increased expression of the VCAM1, ICAM1, SELE, SNAI2, KLF4 genes and a decreased the mRNA level of the PECAM1, CDH5, CD34, ZEB1, CAT, PXDN genes were observed in both cell lines. In addition, HITAEC cells were characterized by a decreased expression of the SOD1, SCARF1, CD36 genes and an increased expression of the SNAI1 and TWIST1 genes; in HCAEC, an increased mRNA level of the LDLR and VLDLR genes was noted. Thus, MMC-induced genotoxic stress is associated with the endothelial dysfunction.

A Novel Technique for Preparation, Staining, and Visualization of Tissue with Metal Implants and Extraskeletal Calcification Areas.

The aim of the study was to evaluate the efficacy of a novel technique for preparation, staining, and visualization of tissues containing extra-skeletal mineralization areas, all-metal implants or their prototypes for their subsequent examination using scanning electron microscopy in the backscattered electron mode. MATERIALS AND METHODS: After fixation in 10% formalin (24 h), the biomaterial (a titanium nickelide plate with the surrounding tissues after subcutaneous implantation, patented titanium alloy plates with the surrounding tissues after cranioplasty, primary and secondary calcified atherosclerotic plaques) were fixed with 1% osmium tetroxide (12 h) and then stained with 2% aqueous solution of osmium tetroxide (48 h). The samples were further stained with 2% alcoholic uranyl acetate (5 h), dehydrated with isopropanol (5 h) and acetone (1 h), impregnated with a mixture of acetone and epoxy resin Epon (1:1, 6 h) and then embedded into a fresh portion of epoxy resin (24 h), which was followed by polymerization at 60°C. After grinding and polishing, epoxy blocks were counterstained with lead citrate (7 min) and sputter-coated with carbon, then the samples were visualized by scanning electron microscopy in the backscattered electron mode. The elemental composition was studied using X-ray microanalysis. RESULTS: The developed technique allows obtaining high-quality images at five thousand-fold magnifications, provides the possibility to identify the shape and structure of intact metal and mineral inclusions, and to type the surrounding cells, distinguishing mesenchymal and immunocompetent cells by shape and cytoplasmic content. Apart from connective tissue capsule thickness and leukocyte infiltration, this technique makes it possible to estimate the number and area of newly formed small-caliber vessels representing a surrogate marker of inflammation. CONCLUSION: The proposed technique provides the possibility to investigate adequately the structure of samples when their sectioning is impossible or significantly complicated, with image quality remarkably higher than that obtained by light microscopy.

[PHENOMENON OF REMOTE PRECONDITIONING THE HEART AND ITS MAIN MANIFESTATIONS].

Remote ischemic preconditioning prevents reperfusion cardiomyocyte apoptosis and has the infarct-limiting effect which is maintained in the experiments on the isolated perfused heart. Remote preconditioning promotes to recovery the contractility of the heart during reperfusion, but did not affect the incidence of occlusion and reperfusion of ventricular arrhythmias. Remote preconditioning has a mild anti-inflammatory effect. Presented article is a review and formulated conclusions based on the published literature data.

[PROBLEM OF END EFFECTOR OF ISCHEMIC PRECONDITIONING OF THE HEART].

An analysis of the literature data performed by the authors shows that the main contenders for the role of the end effector of ischemic preconditioning of the heart are: (1) MPT pore (2) nexuses (3) cytoskeleton. Thus, almost all of the known intracellular molecular cascades eventually converge on MPT pore, on the components of the cytoskeleton and nexuses.

[SIGNIFICANCE OF OPIOID RECEPTORS IN THE CYTOPROTECTIVE ACTION OF CHRONIC HYPOXIA DURING ANOXIA-REOXYGENATION OF CARDIOMYOCYTES].

It was investigated the role of δ-, µ- и κ-opioid receptors (ORs) in the development of cytoprotective effect of chronic normobaric hypoxia (CNH) using anoxia/reoxygenation of isolated cardiomyocytes. Adaptation to CNH was achieved by the maintenance of rats for 21 days at atmosphere containing 12% O2. Anoxia/reoxygenation of isolated cardiomyocytes of intact rats evoked a death of 23% cells and enhancement of lactate dehydrogenase (LDH) release from cells. Anoxia/reoxygenation of isolated cardiomyocytes of adapting rats induced a death of only 2.5% cells and LDH release decreased by 25%. Preliminary incubation of cells with the OR blocker naloxone (300 nM) or the δ-OR antagonist TIPP(ψ) (30 nM) or the selective δ2-OR antagonist naltriben (1 nM) or the µ-OR antagonist CTAP (100 nM) 25 min prior to anoxia abolished adaptive enhancement of cell survival and a decrease in LDH release. The blocking of δ1-OR by BNTX (1 nM) or κ-OR by nor-binaltorphimine (3 nM) not affected on the cytoprotection at CNH. Consequently, cardiac cell δ2- and µ-opioid receptors are involved in the cytoprotective effect of chronic normobaric hypoxia.

[CHRONIC CONTINUOUS NOR-MOBARIC HYPOXIA AUGMENTS CELL TOLERANCE TO ANOXIA(REOXYGE-NATION: THE ROLE OF PROTEIN KINASES].

The study evaluated the role of protein kinase C, PI3-kinase and tyrosine kinases in the cardi-oprotective effect of the chronic continuous normobaric hypoxia (CCNH). Adaptation to CCNH was provided by placing the rats in an atmosphere with a low content of O2 (12 %) during 21 days. Anoxia-reoxygenation of isolated cardiomyocytes of intact rats caused the deaths of 16.5 % of the cells and the lactate dehydrogenase (LDH) release of them. A similar effect on isolated cardiomyocytes of adapted rats caused the death of only 6.8 % of the cells and less pronounced increase in LDH release. Preincubation of cells for 25 minutes with one of the protein kinases inhibitors: che-lerythrine (10 |mM/l); rottlerin (1 |j.M/l); genistein (50 |mM/l) eliminated the adaptive increase in cell survival and reduction of LDH release. Incubation of cells with PI3-kinase blocker wortman-nin (100 nM/l) had no effect on the percentage of cell death of adapted animals and LDH release from them after anoxia-reoxygenation. The authors believe that the cytoprotective effect of chronic normobaric hypoxia is realized through activation of protein kinase C-5 and tyrosine kinases. Kinase PI3 - is not involved in the implementation of protective actions CCNH.