Nikolay V. Goncharov

Reactive oxygen species in pathogenesis of atherosclerosis.

The volume of publications on the role of reactive oxygen species (ROS) in biological processes has been increasing exponentially over the last decades. ROS in large amounts clearly have detrimental effects on cell physiology, whereas low concentrations of ROS are permanently produced in cells and play a role as signaling molecules. An imbalance in ROS production and defense mechanisms can lead to pathological vascular remodeling, atherosclerosis being among them. The aim of this review is to examine different sources of ROS from the point of view of their participation in pathogenesis of atherosclerosis and related cardiovascular risk. Among the possible sources of ROS discussed here are mitochondria, NADPH-oxidases, xanthine oxidase, peroxidases, NO-synthases, cytochrome P450, cyclooxygenases, lipoxygenases, and hemoglobin of red blood cells. A great challenge for future research is to establish interrelations, feedback and feed-forward regulation mechanisms of various sources of ROS in development of atherosclerosis and other vascular pathologies.

A new method for studying platelets, based upon the low-angle light scattering technique. 1. Theoretical and experimental foundations of the method

A new method is presented for simultaneous monitoring of changes in shape and aggregation of platelets. The signal of light scattering alterations at angles below six degrees was shown to be determined by platelet aggregation dynamics (aggregation, disaggregation, coagulation). Over a range of larger angles (6-15 degrees), cell shape changes also contributed to the signal: (i) spherization, and (ii) pseudopodia formation. The first stage was shown to be fast (t1/2 of few seconds) and correlated with (Ca 2+ ) increase. It was characterised by a narrow signal fluctuation and by a rapid increase (30-40%) in signal intensity. During the second stage, which was much slower, the signal decreased describing the aggregation process. The EC50 value for ADP-induced spherization was 40 nmol l −1 . Aggregation kinetics in saline solution under turbulent flow showed second order kinetics in relation to initial cell concentration. The rate constant depended on stirring conditions and on calcium concentration in the medium. Standardisation of the testing conditions made it possible to characterize the initial functional state of platelets by their sensitivity to agonists, with estimation of EC50 and maximum velocity of aggregation (Umax )v alues. The method has potential applications in pharmacology and toxicology research and in clinical practice, as a simple and highly sensitive functionality test for platelets.

On the enzymatic activity of albumin

Albumin molecules, unlike molecules of many other plasma proteins, are not covered with a carbohydrate shell. Albumin plays a crucial role in maintaining colloid osmotic blood pressure and can bind and transport various endogenous and exogenous molecules. The enzymatic activity of albumin, the existence and the role of which most researchers are still skeptical to accept, is our main interest. In this review, a history of the issue is traced, with particular attention paid to the esterase activity of albumin. The kinetic and thermodynamic characteristics of the interaction of albumin with some substrates are adduced, and the possibility of attributing albumin to certain groups of Enzyme Nomenclature is considered.

High-sensitivity determination of 2-chlorovinylarsonous acid in biomedical samples for retrospective detection of exposure to lewisite upon antidotal therapy

A procedure for determination of the lewisite metabolite 2-chlorovinylarsonous acid (CVAA) in biomedical samples, involving derivatization of the latter with propane-1,3-dithiol and head-space solid-phase microextraction of the derivative on a 100-I¼m PDMS fiber followed by GC-MS, was applied for the first time to analysis of in vivo samples. The detection limits of CVAA in urine, plasma and red blood cells were 0.1, 1.0 and 10 ng/ml, respectively. Upon exposure to lewisite at a dose of 1.6 mg/kg, CVAA could be detected in rat urine for about three months. Study of the effect of a single injection of the antidote unithiol on the CVAA excretion profile revealed more active CVAA excretion during the first two days after the injection, compared to that observed in the absence of antidotal therapy.

Microplate spectroscopic methods for determination of the organophosphate soman

Two microplate spectroscopic methods for determination of organophosphates, based on inhibition of acetylcholinesterase activity, have been elaborated and evaluated for determination of the chemical weapon agent soman. The principal difference between the methods is that one measures reaction substrate concentration (elaborated from Hestrin), while the other measures reaction product (elaborated from Ellman). The linear ranges of the two methods were found to be similar. Although the limit of quantification was lower for the Ellman method (110 pM), the sensitivity coefficient was in favor of the Hestrin method (1.55-fold higher). The effects of the main soman hydrolysis products were consistent for the two methods: both methylphosphonic acid and pinacolyl methylphosphonic acid did not inhibit acetylcholinesterase activity. The main components of decontaminating solutions showed differential effects: while monoethanolamine had no influence upon results obtained by either method, hydrogen peroxide interfered with the Ellman method at far lower concentrations than with the Hestrin method. In practical applications involving samples containing hydrogen peroxide, the method based on Hestrin should be regarded as much more specific for OP determination than the Ellman method.

A new method for studying platelets, based upon the low-angle light scattering technique. 2. Application of the method in experimental toxicology and clinical pathology.

A new method for studying platelets based on low-angle light scattering has been applied to studies in experimental toxicology and clinical pathology, using animal and human platelets. Index EC50 for ADP within control groups was estimated to be in the range of 100–180 nmol l–1 (healthy men, rabbits, rats). The refractory state of platelets was obtained by use ADP or ATP concentrations that just caused platelet activation. The time constant and the half-cycle of the refractory state of platelets were calculated. Kinetic parameters of platelet aggregation at interference of hemostasis (rabbit) and the influence of the age factors on the aggregation indices of platelets (rat) were studied. The method of low-angle light scattering was used as a diagnostic tool in experimental low-level intoxication by organophosphates and for interpretation of the pathogenesis of delayed effects. In human patients with ischemic heart disease or with prosthetic heart valves, significant changes in the functional state of platelets were observed. A model for cooperative binding of receptors with ligands on platelets from pregnant women with preeclampsia was developed.

Hydrogen peroxide modulates expression of surface antigens by human umbilical vein endothelial cells in vitro

The aim of this study was to determine the immunological phenotype of human umbilical vein endothelial cells in culture and dynamics of expression of inducible CD molecules of the endothelial cell surface exposed to hydrogen peroxide (H2O2, 100–300 μM) in vitro. PMA was applied as a positive control. There was an insignificant increase (9–21%) of CD31 expression 3 h after the addition of H2O2 to the culture medium, statistically significant decrease of CD309 expression upon exposure to 200 and 300 μM H2O2, whereas a day after the addition of H2O2 we found a strongly pronounced dose-dependent increase of CD54 expression, as well as a moderate enhancement of the expression of all CD molecules.

Necrotic and apoptotic volume changes of red blood cells investigated by low-angle light scattering technique

A low-angle light scattering technique, which has been applied previously to studies of blood platelets and Ehrlich ascite tumor cells, revealed differences in the dynamics of necrotic and apoptotic red blood cell death. Under hypotonic loading or in ammonia medium, red blood cells (RBC) swelled to a critical size (diameter approximately 13 µm) prior to hemolysis (necrosis). Under acidic loading, hemolysis occurred with less pronounced swelling of cells (diameter approximately 10 µm). Apoptosis induced by a calcium ionophore resulted in initial formation of echinocytes, followed by development of rounded red blood cells with uneven membrane, capable of agglomeration. In such a way, RBC aggregation can precede the final stages of the RBC apoptosis when small cellular fragments are generated. On the basis of erythrograms of the cells hemolysing in ammonia medium, the echinocytic (preapoptotic) and stomatocytic (prenecrotic) RBC were discerned due to the very high resistance of apoptotic RBC to osmotic (ammonia) loading.

A new method for studying platelets, based upon the low-angle light scattering technique. 3. Aggregation hypersensitivity of platelets (ADP agonist) and search for corrective agents.

A new method for studying platelets based on low-angle light scattering has previously revealed that platelets taken from pregnant women with preeclampsia are hypersensitive to ADP, with aggregation developing at concentrations of 7–15 nmol l−1. The method has been applied to further studies in experimental toxicology and clinical pathology. Toxicological experiments with fluoroacetate (FA), an inhibitor of TCA cycle, showed that the platelet hypersensitivity could also be caused by energy depletion. In modeling experiments, the low-angle light scattering method was applied to assessment of potential corrective agents of the pathological states related to hypersensitivity of platelets. Sodium glutamate (SG) was shown to be a potent antiaggregant in vitro, and subsequent in vivo studies demonstrated that SG can apparently serve as anaplerotic agent and normalize the platelet status of rats intoxicated with FA. Donators of nitric oxide (NO), such as isosorbide-5′-dinitrate, can also normalize in vitro the hypersensitive status of platelets taken from the patients with preeclampsia.

Dual proapoptotic and pronecrotic effect of hydrogen peroxide on human umbilical vein endothelial cells

Human umbilical vein endothelial cells were exposed in culture to hydrogen peroxide (H2O2), keeping them close to physiological conditions (high cell density, high serum content, H2O2 concentration not over 500 µM). Cell viability was assessed by flow cytometry using simultaneous staining with the fluorescent dye PO-PRO-1 to detect early apoptotic cells and DRAQ7 to detect late apoptotic and necrotic cells. The data obtained suggest that the primary mechanism of the cytotoxic response to H2O2 is apoptosis. The critical concentration of H2O2 causing death in a dense monolayer is 250 µM. Lower H2O2 concentrations (up to 200 µM) cause death of individual cells. The population of endothelial cell retains viability and response to calcium activating agonists does not change compared to control cells.