[Genotoxic stress leads to the proinflammatory response of endothelial cells: an in vitro study]. / Genotoksicheskii stress vyzyvaet formirovanie éndotelial'nymi kletkami provospalitel'nogo fenotipa: issledovanie in vitro.

It was shown, that genotoxic stress can trigger endothelial disfunction and atherosclerosis, but the molecular genetic mechanisms of this process are poorly investigated. At the same time, inflammation also plays the important role in atherogenesis. This study aimed access of inflammatory marker expression in the endothelial cells exposed to alkylating mutagen mitomycin C (MMC). Primary human coronary (HCAEC) and internal thoracic artery endothelial cells (HITAEC) exposed to 500 ng/ml MMC (experimental group) and 0.9% NaCl (control) were used in this research. A gene expression profile was evaluated by quantitative reverse transcription PCR after 6 h exposure of endothelial cells to MMC (or 0.9% NaCl) followed by subsequent 24 h incubation in the mutagen-free cell growth media. The cytokine profile of endotheliocytes was studied by dot blotting. We found that MIF, IL-8, MCP-1, IP-10 and PDGFB were upregulated both in HCAEC and HITAEC, while MIP-1ß release remained unchanged. TIMP-2 was upregulated in HCAEC but not in HITAEC. sTNF RI was expressed only in HCAEC. According to gene expression analysis, HCAEC exposed to MMC are characterized by the increased mRNA level of IL-8, MCP-1 and IP-10; decreased expression of TIMP-2 and no differences in the expression of MIF, MIP-1ß and PDGFB compared to the control. In HITAEC, increased mRNA level of IL-8 and IP-10; decreased expression of MIF and TIMP-2, no differences in the expression of MCP-1, MIP-1ß and PDGFB was shown. TNF-RI expression was not detected in both cell lines. Thus, genotoxic stress in endothelial cells induced by MMC leads to differential inflammatory response that can trigger endothelial dysfunction.

[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.

[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.

[The expression level of cytokine genes in the cases of native heart valves in infectious endocarditis]. / Otsenka urovnia ékspressii genov tsitokinov v stvorkakh nativnykh klapanov serdtsa pri infektsionnom éndokardite.

The expression level of IL1B, IL6, IL8, IL10, IL12A, IL12B, IL18, IL23, IL33, CCL2, and IL1RL1 has been investigated using biopsies of native mitral, aortic, and tricuspid valves obtained during surgical correction of acquired defect from 25 patients with infectious endocarditis. Biopsies of native mitral and aortic valve cusps from 12 patients who underwent surgical correction of acquired heart disease of non-infectious etiology were used as control. We used quantitative PCR with fluorescent dye SYBR Green for determination of the cytokine gene expression level. This study revealed that genes could be subdivided into three groups: (i) genes with increased expression (IL1B, IL6, and IL8); (ii) genes with reduced expression (IL33 and IL1RL1); (iii) genes with unchanged expression (IL12A, IL18, IL23, and CCL2). The IL8 gene expression was characterized by the most pronounced increase (9.83 times versus control), while the IL1RL1 gene demonstrated the most pronounced decrease in its expression (4.17 times). Expression of IL10 and IL12B genes was negligible in all samples.

[Predicting the development of adverse events in patients with acute coronary syndrome including genetics in the long-term follow-up].

Aim To study a relationship of several factors (clinical and genetical markers) with unfavorable outcomes in patients with non-ST-segment elevation acute coronary syndrome (NSTE-ACS) in long-term follow-up.Material and methods This full-design, prospective study included 415 patients with NSTE-ACS. 266 patients were evaluated for the presence of multifocal atherosclerosis (MFA). Typing of polymorphic variants rs1041981 LTA, rs1800629 TNF, rs4986790, and rs498679 TLR4, and also rs3024491 and rs1800872 IL10 was performed. Follow-up period lasted for 67±4 months. By the end of this period, information about clinical outcomes for 396 patients became available.Results During the entire follow-up period, unfavorable outcomes were observed in 239 (57.5 %) patients with NSTE-ACS. The following clinical signs were associated with unfavorable outcomes: history of myocardial infarction, age >56 years, left ventricular ejection fraction (LV EF) ≤50 % and GRACE score ≥100, significant stenosis of brachiocephalic arteries, MFA, carriage of genotype А / А rs1041981 LTA (OR, 6.1; р=0.02) and allele А (OR, 1.9; р=0.01). According to results of a multifactorial analysis, the most significant predictors included LV EF <50 %, MFA, and carriage of genotype А / А rs1041981 LTA.Conclusion Stratification of patients with NSTE-ACS into groups of high or low risk for having an unfavorable outcome within the next 6 years is possible using the prognostic model developed and presented in this study. The model includes the following signs: LV EF <50 %, MFA, and carriage of genotype А / А rs1041981 LTA.

[Genetic Predisposition to Early Myocardial Infarction].

The aim of the study was to identify the features of the genetic structure of myocardial infarction (MI) susceptibility depending on age ("early MI" denoting individuals who had the first MI before the age of 60 years, and "late MI" the group of patients with the first "MI after 60 years"). A total of 355 patients were examined (n = 121 early MI and n = 234 late MI) and 285 residents of the Siberian region (as a control group). Genotyping of 58 single nucleotide variants (SNPs) was performed using mass spectrometry using the Agena (ex Sequenom) MassARRAY® System. Statistical analysis was performed using Statistica 8.0 ("StatSoft Inc.", USA), as well as the "stats" and "genetics" packages in the R environment. The regulatory potential of SNPs was evaluated using the rSNPBase online service (http://rsnp.psych.ac.cn/). eQTL loci were identified using data from the Genotype-Tissue Expression (GTEx) project (http://www.gtexportal.org/) and the Blood eQTL online service (https://genenetwork.nl/bloodeqtlbrowser/). The GG genotype of ITGA4 rs1143674, the CC genotype of CDKN2B-AS1 rs1333049, and the CC genotype of KIAA1462 rs3739998, are generally associated with MI. The AA genotype of ADAMDEC1 rs3765124 (OR = 2.03; 95% CI 1.23-3.33; p = 0.004) and the GG genotype of AQP2 rs2878771 (OR = 2.24; 95% CI 1.23-4.09; p = 0.006) are associated with the development of MI at an early age, and the TT genotype of TAS2R38 rs1726866 (OR = 1.82; 95% CI 1.11-2.89; p = 0.009) was the high-risk genotype for the late MI. Genetic variants associated with MI are regulatory SNP (rSNP) and affect the affinity of DNA binding to transcription factors, carry out post-transcriptional control of gene activity and change the level of gene expression in various tissues. Thus, early and late MI are based on both common genetic variants of ITGA4, CDKN2B-AS1, KIAA1462 genes and specific ones (ADAMDEC1 and AQP2 for early MI and TAS2R38 for late MI).

[Efficiency of pharmacogenetic approach to anticoagulant therapy in patients with prosthetic heart valves].

BACKGROUND: This study examined clinical, demographic, anthropometric, and inheritance factors that influence individual sensitivity to warfarin therapy after heart valve surgery. The clinical significance of the pharmacogenetic approach was assessed using the individual time frame and time spent in the INR therapeutic range. AIMS: We determined the clinical outcome of the pharmacogenetic approach at the start of warfarin therapy in patients with prosthetic heart valves. MATERIALS AND METHODS: The study included 915 patients, of which 512 women and 403 men (mean age 56±10 years), living in Western Siberia. Rheumatic heart disease was the main diagnosis that caused the acquired defect. Mechanical prostheses were used in 70% of cases of cardiac surgery. Real-time polymerase chain reaction used for molecular genetic testing. RESULTS: The frequencies of the alleles and genotypes of CYP2C9 and VKORC1 in the study population of patients with heart valves prosthetic correspond to the distribution in Caucasoid populations. The use of pharmacogenetic testing results at the beginning of warfarin therapy reduced the time required for selecting a therapeutic dose of anticoagulant by 2 times and increased the duration of stay in the INR therapeutic range by 20.2%. CONCLUSION: The use of the pharmacogenetic approach at the begin­ ning of warfarin therapy contributes to the effectiveness and safety of anticoagulant therapy in this category of patients.

[Infective Endocarditis Causing Native and Prosthetic Heart Valve Dysfunction].

Infective endocarditis (IE) is the disease that has high inhospital mortality. Heart valves dysfunction - both native and prosthetic - is the primary IE complication requiring a surgical intervention. The IE causes and its course have been discussed in this review. In particular, the role of concomitant infectious foci in the formation and development of IE have been considered, the mechanisms of mutual transition of subacute and acute clinical forms have been described. Modern diagnostic principles and methods based on the Duke criteria system have been mentioned, as well as the difficulties that follow the patient's clinical status evaluation. The normobiotic microbiota participation, as well as the possibilities for their identification using blood culture and PCR technique, have been closely reviewed. According to modern researches and publications, there have been made the conclusion about the contribution of obligate anaerobic bacteria, fungi and viruses to the development of endocarditis. There have been described the hypothesis about the presumptive strategy for the cardiac dysfunction formation as a result of the IE causative agents cells metabolic activity based on a literature data analysis in the article: vegetation formed by Staphylococcus aureus can lead to the heart valve stenosis, and the influence of hyaluronidases, collagenases on a heart valve structure can lead to regurgitation. The pathogens cells ability to avoid the human immune system response is caused by the biofilms, fibrin vegetations formation and the enzymes production - cytotoxins (streptolysins, leukocidin, etc.). It has been suggested that the mediators of inflammation and leukocyte cells participate in the destruction of native and prosthetic tissues due to an IE pathogens inaccessibility for immunocompetent cells.

[Microflora of peripheral blood obtained from patients with infective endocarditis.]

Infective endocarditis is a serious inflammatory disease associated with damage of heart valves and other parts of endocardium. This study included 91 patients with a confirmed diagnosis of «infectious endocarditis¼ and hospitalized at the Research Institute of Complex Problems of Cardiovascular Disease (Kemerovo, Russia) from 2010 to 2015. The determination of the spectrum of microorganisms in the samples of patients' peripheral blood was carried out using the PCR method using reagents produced by Lyteh Ltd. (Moscow, Russia) allowing detect Staphylococcus spp., Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalacticae, Enterobacter spp., Klebsiella spp., Proteus spp., Haemophilus influenza, Enterococcus faecalis, Enterococcus faecium in the samples of biological material. Statistical analysis was performed in the StatSoft STATISTICA 10 software. 83.5% of patients were characterized by presence Staphylococcus spp. in the peripheral blood; the other pathogens were detected much less often (from six cases of enterococci detection to one case of Streptococcus agalacticae and Proteus spp.). In nine patients, none of the analyzed pathogens was detected, and a number of patients were characterized by the simultaneous presence of several pathogens. There was no reliable data on the difference in microflora structure depending on sex, drug addiction, the type of infective endocarditis and the damaged valve. It was established that the results of PCR of peripheral blood samples and microbiological examination of the tissues of damaged valves that were removed during cardiac surgery, differ significantly. The obtained results testify to the need for more in-depth studies including PCR analysis of peripheral blood samples, flushing from damaged valves, and also homogenized valve tissue samples, which will allow us to obtain more detailed data and conclude that PCR can be used as a diagnostic test for early detection microbiological agent as causative.

[Proliferative and secretory activity of human umbilical vein endothelial cells cultured under varying degrees of hypoxia].

In this study we examined the impact of 3-day hypoxia of varying degrees on the viability, proliferative and secretory activity of endothelial cells in human umbilical vein (HUVEC). The gas mixture of the three components (%) was used: 1) 10 O2, 5 CO2, 85 Ar; 2) 5 O2, 5 CO2, 90 Ar and 3) 1 O2, 5 CO2, 94 Ar. The HUVEC, cultivated in CO2-incubator under conditions of atmospheric oxygen (21% O2) were the controls. Comprehensive assessment of the results after has shown that 3-day HUVEC cultivating in the presence of 1% O2 led to pathological activation of endotheliocytes: increased NO synthesis combined with the marked secretion of endothelin-1, IL-6, IL-8 and TNF-alpha, sVCAM-1, sE-cadherin and of sE-selectin, VEGF-A and bFGF, and slow proliferation. When HUVEC were cultivated at 10% O2 and 5% O2, the level of basal secretion of the substances listed above was the least against the background of increased proliferative activity. The results showing the changes in the secretory activity of endothelial cells when cultivated under the conditions of atmospheric oxygen levels have demonstrate HUVEC activation, because the secretion of NO, IL-6, IL-8 and von Willebrand factor after 3 days of cultivation in 21% 02 exceeded that in the case of 10 and 5% O2. Thus, a gaseous medium with reduced oxygen content of up to 5% provides more physiological conditions for HUVEC cultivation. The maximum proliferative activity of HUVEC with minimal basal secretion proved such a composition to be comfortable. Increasing the oxygen content to the atmospheric level leads to the activation of endotheliocytes with signs of endothelial dysfunction, and the critical reduction in oxygen to 1% causes the development of endothelial dysfunction and reduces the proliferative potential.