[Direct human DNA damage by unfavorable environmental and climatic factors].

AIM: To study the impact of simulated climatic conditions of the 2010 summer in Moscow on the telomere repeats of chromosomes in human blood cells. MATERIALS AND METHODS: The climatic conditions of July-August 2010 in Moscow were simulated at the Medical Technical Complex, Institute of Biomedical Problems, Russian Academy of Sciences. The relative length of the telomeric repeats of blood cell chromosomes from 6 apparently healthy volunteers was measured by quantitative real-time polymerase chain reaction. RESULTS: These conditions were ascertained to lead to a statistically significant decline in the length of telomere repeats in the terminal portions of chromosomes by 15%. CONCLUSION: Environmental changes and abnormal temperature rises may result in oxidative stress accompanied by telomere shortening, which can be, in turn, a factor of premature aging.

[Prolongation of enoxaparin therapy to one month promotes recanalization of the occlusively thrombosed deep veins].

AIM: To compare effects of prolongation of the treatment with therapeutic doses of enoxaparin to 1 month on recanalization of occlusively thrombosed deep veins (OTDV) of the limbs with results of standard therapy with unfractionated heparin (UFH). Both treatments were followed by warfarin administration. MATERIAL AND METHODS: Thirty patients were selected from 111 patients with a history of deep vein thrombosis (DVT) and/or pulmonary artery embolism according to the following criteria: the presence of occlusive thrombosis of one deep vein minimum; the absence of DVT for 12 months of follow-up. Patients of group 1 (n = 15) received standard therapy (UFH for at least 5 days) with switch to warfarin. Patients of group 2 (n = 15) received therapeutic doses of enoxaparin (1 mg/kg each 12 hours) for 30 days minimum with switch to warfarin. Follow-up was 12 months. Ultrasonic duplex angioscanning of the limbs was made at baseline, 1, 3, 6 and 12 months after treatment start. RESULTS: After follow-up month 1, 3 and 6 number of patients with occlusive DVT was significantly less in group 2. All the patients given enoxaparin achieved recanalization of OTDV within 3 months of treatment. OTDV recanalization was not achieved in 20% patients of group 1 even 12 months after treatment start. CONCLUSION: Prolongation of enoxaparin treatment to 1 month followed by warfarin treatment is superior to standard UFH treatment followed by warfarin in providing recanalization of OTDV within 3 months of treatment. Moreover, this treatment predicts persistence of recanalization within 12 months of anticoagulant therapy.

[Polymorphisms of genes CYP2C9 and VKORC1 in patients with venous thromboembolic complications in Moscow population: effects on stability of anticoagulant therapy and frequency of hemorrhage].

AIM: To investigate frequency of carriage of genetic polymorphisms CYP2C9 and VKORC1 in patients with venous thromboembolic complications (VTEC) in Moscow population given warfarin treatment and effects of this carriage on stability of anticoagulation and frequency of hemorrhagic complications (HC) in warfarin treatment. MATERIAL AND METHODS: The study included 111 patients with the history of deep vein thrombosis and/ or pulmonary artery thromboembolism. All the patients received non-fractionated or low-molecular heparin for at least 5 days, then warfarin (target INR 2.0-3.0). Warfarin dose was selected empirically. Gene CYP2C9 and VKORC1 polymorphisms were studied. HC were endpoints. RESULTS: Genotype CYP2C9*1/*1 (a "wild" type) was detected in 94 (84.7%) patients. Of other genotypes - heterozygotes CYP2C9*1/*2 (4.5%) and CYP2C9*1/*3 (10.8%). Genotyping by VKORC1 detected genotype GG (a wild type) in 42.3%, genotype GA--in 48.6%, genotype AA--in 9.1% patients. A mean warfarin dose, supporting an adequaite INR, was asspciated with both genotype CYP2C9 and VKORC1. Warfarin doses were highest in carriers of wile genotypes CYP2C9 and VKORC1 (6,9 and 8,8 mg/day), the lowest--in patients with genotypes CYP2C9*1/*3 and VKORC1 (4,5 and 4,0 mg/day). The carriers of polymorphisms CYP2C9*1/*3 and VKORC1 showed less stable anticoagulation vs carriers of allele variants CYP2C9*1/*1, CYP2C9*1/*2 and genotypes GG, GA VKORC1. An HC rate depended, as a rule, on carriage of genotypes CYP2C9*1/*3 and AA VKORC1. The highest risk of HC was associated with genotype CYP2C9*1/*3. The results of multifactorial regression analysis also indicated that carriage of genotype CYP2C9*1/*3, a female gender and the range of INR in warfarin treatment > or = 2,66 are independent predictors of HC in VTEC patients on warfarin treatment. CONCLUSION: Carriage of gene CYP2C9 and VKORC1 polymorphisms affects suppoting dose of warfarin and rate of hemorrhage in patients with VTEC in Moscow population. Frequency of HC is the highest in carriers of genotypes CYP2C9*1/*3 and AA VKORC1, they need minimal supporting dose of warfarin. Carriage of genotype CYP2C9*1/*3 in line with a female gender and instability of INR is an independent predictor of HC in VTEC patients in Moscow population on warfarin treatment.

[Thrombin-activated inhibitor of fibrinolysis and efficacy and safety of long-term warfarin treatment in patients with venous thromboembolic complications].

AIM: To study effects of thrombin-activated fibrinolysis inhibitor (TAFI) on efficacy and safety of long-term anti-coagulant treatment in patients with venous thromboembolic complications (VTEC). MATERIAL AND METHODS: A total of 111 patients with a history of an episode of deep vein thrombosis (DVT) and/or pulmonary artery thromboembolism (PATE) entered the study. All the patients received unfractionated or low-molecular heparin for at least 5 days than switch on warfarin (target values of INR 2.0-3.0). Baseline blood levels of TAFI were measured. The patients were followed up for 18 months. Recurrent (DVT/TAFI and hemorrhagic complications (HC) were endpoints. Also, frequency of complete lysis of deep vein thrombi was assessed after 12 months of treatment. RESULTS: A TAFI level varied from 50 to 217% (median 106%, interquartile rage 90-133%). TAFI concentration positively correlated with fibrinogen and thromb size. The patients were divided into two groups depending on TAFI content: group 1 patients had low TAFI (under 25th percentile; < 90%); patients of group 2 had high TAFI (above 25th percentile; > 90%). Group 1 patients were characterized by less stable anticoagulation. This association did not depend on genetic characteristics which determine sensitivity to warfarin (CYP2C9 and VKORC1). Low TAFI was associated with reduced risk of DVT for 18 months and higher probability of complete lysis of the thrombi after 12 months of anticoagulant therapy compared to VTEC patients with high TAFI. No differences were found by TAFI level in patients with HC and without HC, but in HC patients low TAFI was associated with spontaneous hemorrhages and bleeding in therapeutic INR values. CONCLUSION: The results of this pilot study evidence that a TAFI level can be one of the factors influencing efficacy and safety of long-term anticoagulant therapy in patients with VTEC on warfarin treatment.

[A new method of quantitative estimation of mutant allele in mitochondrial genome].

A new original method of quantitative estimation of heteroplasmy levels in mitochondrial genome is necessary for determination cut-of values mutant allele in tissues, associated with the emergence and development of pathologies in human organism. This method is based on pyrosequencing of short DNA fragments. It is characterized by high accuracy and good reproducibility and helps to find out crucial differences in the level of heteroplasmy of mitochondrial genome in various organs and tissue fragments from the same individual.

[Influence of genetic factors on the development of target organ lesions in relation to age in diagnosis of arterial hypertension].

AIM: To analyze the impact of polymorphism of a group of genes encoding for endothelial function on the development of target organ lesions in arterial hypertension (AH) in relation to age. SUBJECTS AND METHODS: Six hundred and seventy-two AH patients (mean age 50.6 years; men 67%) were examined. Microalbuminuria (MAU) was estimated. Electrocardiography, echocardiography, and carotid ultrasonography were performed. A control group comprised 184 subjects. Single-nucleotide substitutions genotyping of the Glu298Asp endothelial NO synthase (eNOS), p22phox of NADPH oxidase subunit C242T, and angiotensin II type 1 receptor (ATR1) A1166C gene polymorphisms was conducted by a polymerase chain reaction (PCR) via restriction fragment length polymorphism analysis, and M235T substitution genotyping of the G-6A polymorphism of the angiotensinogen gene was performed by a real-time allele-specific PCR. The impact of the polymorphisms on the development of MAU, left ventricular hypertrophy (LVH), carotid lesion was analyzed in the groups: AH was diagnosed in subjects aged less than 35 years (n = 128) or older. The ultrasound signs of carotid lesion, LVH, and MAU were revealed in 65, 39, and 10.5% of the patients with AH, respectively. RESULTS: The subgroups showed differences in the distribution of polymorphisms of the study genes in relation to age at AH detection. CONCLUSION: In patients with AH diagnosed at less than 35 years of age, pathological changes in the carotid are associated with a G allele of the Glu298Asp eNOS polymorphism (odds ratio (OR) = 2.3; p = 0.016) and with an T allele of the p22phox of NADPH oxidase subunit C242T polymorphism (OR 1.7; p = 0.049). In this age subgroup, LVH was associated with an A allele of the Glu298Asp eNOS polymorphism (OR = 1.9; p = 0.037), MAU was with an A allele of the Glu298Asp eNOS polymorphism (OR = 3.6; p = 0.02) and a C allele of the ATR1 A1166C gene polymorphism (OR = 2.6; p = 0.034).

[ATR1 gene polymorphism in patients with hypertrophic cardiomyopathy and hypertensive heart].

Aim of the study was analysis of dependence of clinical picture and degree of severity of left ventricular hypertrophy (LVH) on polymorphism A/C of ATR1 gene in patients with hypertrophic cardiomyopathy (HCMP) and hypertensive disease (HD). With the method of polymerase chain reaction genotyping for polymorphic markers of A/C of ATR1 gene was carried out in 35 patients with HCMP and 33 patients with LVH developed at the background of long lasting HB. In the work we used clinico-instrumental methods of investigation (electrocardiography - ECG, echocardiography). It was revealed as result of the study that in HCMP type AA in comparison with type AC of ATR1 gene was associated with addition of arterial hypertension, presence of left ventricular outflow tract obstruction, greater severity of heart failure. In case of combination of HD with LVH type AA in comparison with types AC and CC of ATR1 gene is associated with more pronounced LVH.

[Delayed appearance of hypertension in spontaneously hypertensive rat (SHR) injected with CpG-rich DNA early in ontogenesis].

In this study we have investigated properties of blood serum extracellular DNA (cell-free DNA) from patients with essential arterial hypertension (AH). Cell-free DNA concentration was not changed in the control AH group compared to norma (healthy donors) but fragments of CpG-rich cell-free DNA marker content were increased at transcribed area of ribosomal repeat (TArDNA, CpG-DNA). To evaluate effect of CpG-DNA on AH development in 2-day SHR line and in control normotensive line (WKY), 700 ng of human TArDNA single subcutaneous injection were inoculated to obtain anti-CpG-DNA polyclonal antibodies. These antibodies could change CpG-DNA contents in total cell-free DNA. Blood pressure (BP) in 9-week SHR line rats immunized with CpG-DNA was equal to BP of WKY rats. Then BP of immunized SHR steadily increased with age and reached high value 8 weeks later compared to control SHR rats. Cell-free DNA analysis in 17-week SHR line rats showed significantly reduced concentrations of cell-free DNA and also showed decrease in small DNA fragments content, but increased content of CpG-DNA (rat TArDNA). These changes were accompanied with 3.5-fold blood endonuclease activity increase and decrease of free (unbound to cell-free DNA) anti-CpG-DNA antibodies quantity. Total anti-CpG-DNA antibodies quantity in immunized rats wasn't changed compared to control animals. Thus, observed effect of increase in stable BP elevation age in immunized SHR line rats doesn't relate to increase of anti-CpG-DNA antibody production. Possible reason of this effect is further discussed.

[Independent predictors of deep vein thrombosis (results of prospective 18 months study)].

We followed for 18 months 90 patients who had had deep vein thrombosis (DVE) and/or pulmonary embolism (PE) and received therapy with anticoagulants either for 3-12 months or for indefinitely long time. During follow-up rate of recurrent DVE was 16.7%, no recurrences of PE were registered. Predictors of recurrent PE were selected among 165 demographic, anthropometric, anamnestic, clinical, genetic, instrumental, and laboratory parameters, as well as risk factors of development of thromboembolic complications. According to results of multifactorial regression analysis we established the following independent predictors recurrent DVE during 18 months of follow-up: elevated level of DAdimer after 1 month of anticoagulant therapy (p=0.005; relative risk--relative risk [RR] 8.1, 95% confidence interval [CI] 1.9 to 34.8), homozygosity for C249T polymorphism in beta-fibrinogen gene (p=0.044; RR 8.4 95% CI 1.1 to 65.7), and percentage of all values of international normalized ratio within therapeutic interval 2.0-3.0 (p=0.009; RR 0.94, 95 CI 0.89 to 0.98).

[Study of the genetic markers of thrombosis in hypertensive patients of the native population in the Republic of Mordovia].

The genetic markers (encoding factor of blood coagulation, the PAI-1 gene, the prothrombin gene, the GP IIb/IIIa gene, and the MTHFR gene) of thrombosis were studied in the Mordovians and Russians with essential hypertension (EH), who were residents of the Republic of Mordovia. One hundred and forty patients with TH and 90 healthy individuals of three nationalities, such as Russian, Moksha-Mordovian, and Erzya-Mordovian, were examined. Along with the routine clinical and instrumental studies, alleles of polymorphic markers were identified by the polymerase chain reaction (PCR). The polymorphic markers of the Arg 506 Gln gene, encoding factor V of blood coagulation, 4G5G of the PAI-1 gene, the G 20210 A prothrombin gene, the GP IIb/IIIa gene, and the A 1298 C MTHFR gene were also studied. The data were statistically analyzed using a package of the programs: Statistica for Windows 6.0 (Stat Soft), SPSS (version 14.0), and MS Excel XP (Microsoft). The authors used a chi-2 (chi-square) test was used to compare the incidence of genotypes and alleles in the patient groups. Hypertensive and normotensive persons were found to have no significant differences in the distribution of genotypes (encoding factor of blood coagulation, the PAI-1 gene, the prothrombin gene, and the GP IIb/IIIa gene) with regard to ethnicity in the Russian, and Moksha-Mordovian, and Erzya-Mordovian groups). Meanwhile, there was a significant preponderance of MTHFR gene mutation with the unfavorable genotype for Moksha-Mordovian patients with EH. The findings suggest that drug therapy for preventing venous thrombosis in patients with EH should be adjusted in relation to ethnicity.

[Mitochondrial energy conversion disturbance with decrease in ATP production as a source of systemic arterial hypertension].

This review deals with the cellular mechanisms underlying decreased energy status documented in different tissues from experimental rat models of primary and secondary hypertension as well as the involvement of these abnormalities in the pathogenesis of the disease. Such analyses allow us to hypothesize that dysfunction of mitochondrial energy conversion, caused by distinct stimuli, including generalized disturbances of intracellular Ca2+ handling and mitochondria calcium overload found in primary hypertension, leads to uncoupling of oxidation and phosphorylation and attenuated ATP synthesis. Examples of arterial hypertension accompanied by mitochondrial uncoupling and cell ATP depletion (hyperthyroidism, cold hypertension, cyclosporine A intake, etc.) may be considered as an additional argument supporting this opinion. It means also that despite of differences in triggering mechanisms of mitochondrial dysfunction in all these models, the final outcome, i.e. decreased mitochondrial ATP production, is similar. Attenuated intracellular ATP content, in turn, results in the long-term maintenance of elevated BP by increased sympathetic outflow, whereas augmented ROS production following mitochondrial dysfunction lowers the capacity of the NO-dependent vascular relaxation. In the light of these data the cause of stationary elevated BP in chronic arterial hypertension should be regarded as a compensatory response to decreased mitochondrial ATP synthesis.

[The study of therapeutic effect of synthetic bradykinin "gene" contained in retrovirus vector on spontaneously hypertensive rats].

Administration of DNA plasmid pPS-3-neo (brd) with synthetic bradykinin " gene " to 2-days old male spontaneously hypertensive rats (SHR) leads to 2 weeks delay in development of arterial hypertension. Lowering of SBP and positive results of PCR DNA of various organs observed in synthetic bradykinin " gene " transgenic SHR but not in control SHR confirm therapeutic effect of synthetic bradykinin " gene " . This data indicate one of possible ways of gene therapy of arterial hypertension as well as other pathological states by introduction of transgene directly into genome of the organism.

[Calcium induced calcium release from liver mitochondria of spontaneously hypertensive rats].

BACKGROUND: Disturbances of ionic homeostasis of cells and recently discovered cellular energy deficiency due to reduced ATP-synthesizing ability of mitochondria are the most important components of pathogenesis of primary hypertension. Therefore it is essential to elucidate relationship between functioning of ionic transport systems especially that of calcium transport and ATP-synthesizing ability of mitochondria. AIM: To study calcium induced calcium release from liver mitochondria of spontaneously hypertensive rats at various initial calcium concentrations in medium. RESULTS: We observed 30% lowering of maximal level of calcium uptake from the medium with unchanged rate of calcium uptake. Under conditions of calcium overload the rate of calcium release into the medium was 30% higher than in control. INTERPRETATION: These results evidenced for changes of functioning of mitochondrial permeability transition pores (MPT-pores) during calcium overload appearing as acceleration of their switch to high conductance mode and equilibration of all ionic gradients on internal mitochondrial membrane. This can negatively affect ATP-synthesizing ability of mitochondria. Thus the presence of linkage between disturbance of ionic homeostasis and intracellular energy deficit was confirmed.

[Decreased ATP-synthesis ability of brain mitochondria in spontaneously hypertensive rats]. / Snizhennaia ATF-sinteziruiushchaia sposobnost' mitokhondrii kletok golovnogo mozga krys so spontannoi gipertenziei (SHR).

UNLABELLED: It has been shown previously that a decrease of ATP amount and changed balance of other macroergic phosphates occurs in different tissues of spontaneously hypertensive rats (SHR) compared with control normotensive rats (WKY). AIM: To assess the ability of SHR brain isolated mitochondria to synthesize ATP and to elucidate its relation to extramitochondrial calcium concentration. RESULTS: The present work shows for the first time that SHR brain mitochondria initially differ from WKY ones by decreased (by 30%) ATP synthesis rate. When calcium concentration is increased up to 20 or 40 micromole/l ATP synthesis rates in WKY and SHR mitochondria are about one half compared with the condition when the medium does not contain calcium, but the differences between SHR and WKY remain the same. CONCLUSION: Decreased ATP synthesis rate in SHR mitochondria may be considered as the main cause of cell energy deficiency observed in experimental variant of primary arterial hypertension.

[Decreased ATP-synthesis ability of liver mitochondria in spontaneously hypertensive rats (SHR): role of calcium overload of the mitochondria]. / Snizhennaia ATF-sinteziruiushchaia sposobnost' mitokhondrii pecheni spontanno gipertenzivnykh krys (SHR): rol' kal'tsievoi peregruzki mitokhondrii.

It has been shown previously, that a decrease of ATP amount and changed balance of the other macroergic phosphates are observed in different tissues of spontaneously hypertensive rats (SHR) compared with the normotensive controls (WKY). The aim of the present study was to assess the ability of SHR liver isolated mitochondria to synthesize ATP and to clarify its dependence on extramitochondrial calcium concentration. Macroergic phosphate concentrations were measured by high performance liquid chromatography (HPLC). The present work shows for the first time, that SHR liver mitochondria initially differ from WKY ones by decreased (by 30%) ATP synthesis rate. Respiratory control coefficient and phosphorylation rate in SHR and WKY mitochondria change in different manner when extramitochondrial calcium concentration increases from 0 to 50 microM. When calcium concentration is increased up to 50 microM both parameters in WKY mitochondria are about one half compared with the condition when the medium does not contain calcium. In the conditions, when the medium contains 50 microM of calcium, complete uncoupling of oxidation and phosphorylation in SHR mitochondria is observed. Thus the decrease of ATP synthesis rate in SHR mitochondria in conjunction with reduced tolerance to calcium overload (by respiratory control coefficient and phosphorylation rate) in comparison with WKY may be considered as the main cause of cell energy deficiency, observed in experimental variant of primary arterial hypertension.

[Changes in the energy state of tissues in spontaneously hypertensive rats]. / Izmeneniia énergeticheskogo sostoianiia tkanei pri spontannoi gipertenzii u krys.

The contents of adenine nucleotides (ATP, ADP, AMP), phosphocreatine (PCr) and creatine (Cr) in the heart, skeletal muscle, liver and spleen in spontaneously hypertensive (SHR) and normotensive (WKY) rats. The ATP/ADP ratio in cardiac tissue was lower in SHR compared with WKY, while myocardial contents of adenine nucleotides, PCr and Cr did not differ significantly between the groups. A lower ATP/ADP ratio in the skeletal muscle SHR of was accompanied by a reduction of PCr content comparing with these indices in WKY rats. The liver and spleen of SHR exhibited lower ATP contents and higher ADP and AMP levels compared with those ones in WKY rats, despite of the close values of adenine nucleotide pools (sigma AN = ATP + ADP + AMP). This redistribution of tissue adenine nucleotides was corresponded to lower energy charges (EC = (ATP + 0.5 ADP)/sigma AN) and ATP/ADP ratios in SHR group. The reduction of the energy state of tissues in SHR rats increased in the following rank: heart > skeletal muscle > liver > spleen, thus, reflecting progressive decrease of intensity of oxidative metabolism. The results suggest changes in the balance of rates of ATP formation and hydrolysis occur at the system level in primary hypertension. Probably, consequences of such rearrangement in energy metabolism are functional disturbances of plasma membrane and sacroplasmic reticulum well-documented in a number of experimental and clinical studies.

[The role of genome repetitive sequences in primary hypertension]. / Rol' povtoriaiushchikhsia posledovatel'nostei genoma v pervichnoi gipertenzii.

Genetic determining factors of essential hypertension seems to involve a dynamic mutation or a similar process. The mobile elements of the genome: moderately recurrent sequences, play a special role in initiation of such a process. A possible molecular mechanism of the initiation and development of the dynamic mutation, is described.