Publications by authors named "Pavel Avdonin"

13 Publications

  • Page 1 of 1

The Use of Fluorescently Labeled ARC1779 Aptamer for Assessing the Effect of H2O2 on von Willebrand Factor Exocytosis.

Biochemistry (Mosc) 2021 Feb;86(2):123-131

Koltsov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, 119334, Russia.

Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel-Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca2+. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H2O2 formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H2O2 on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H2O2 for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H2O2 acts as a mediator that activates exocytosis of Weibel-Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.
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http://dx.doi.org/10.1134/S0006297921020012DOI Listing
February 2021

The Role of Two-Pore Channels in Norepinephrine-Induced [Ca] Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction.

Cells 2019 09 25;8(10). Epub 2019 Sep 25.

Koltsov Institute of Developmental Biology RAS, Moscow 119334, Russia.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca]) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of -NED 19 and endolysosmal probe LysoTracker in SMCs. Both - or -NED 19 inhibited the rise of [Ca] in SMCs induced by 100 μM NE by 50-60%. IC for - and -NED 19 were 2.7 and 8.9 μM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of -NED 19 several times higher than that of -NED 19. Inhibition by -NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. - and -NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca] in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca] rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.
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http://dx.doi.org/10.3390/cells8101144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829401PMC
September 2019

Rational in silico design of aptamers for organophosphates based on the example of paraoxon.

Comput Biol Chem 2019 Jun 13;80:452-462. Epub 2019 May 13.

Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, pr. Torez 44, St. Petersburg 194223, Russia; Research Institute of Hygiene, Occupational Pathology and Human Ecology, bld.93 p.o.Kuz'molovsky, Leningrad Region 188663, Russia.

Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of the poison entering the body could be achieved with the help of DNA- or RNA-aptamers, which are able to bind poisons in the bloodstream. The aim of the research was to develop an approach to rational in silico design of aptamers for OPs based on the example of paraoxon. From the published sequence of an aptamer binding organophosphorus pesticides, its three-dimensional model has been constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy have been calculated using MD trajectories and MM-PBSA approach. On the basis of the energy values, two sequences that bind paraoxon most efficiently have been selected. The value of free binding energy of paraoxon with peripheral anionic site of acetylcholinesterase (AChE) has been calculated as well. It has been revealed that the aptamers found bind paraoxon more effectively than AChE. The peculiarities of paraoxon interaction with the aptamers nucleotides have been analyzed. The possibility of improving in silico approach for aptamer selection is discussed.
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http://dx.doi.org/10.1016/j.compbiolchem.2019.05.004DOI Listing
June 2019

Enhancement by Hydrogen Peroxide of Calcium Signals in Endothelial Cells Induced by 5-HT1B and 5-HT2B Receptor Agonists.

Oxid Med Cell Longev 2019 11;2019:1701478. Epub 2019 Feb 11.

Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, Saint Petersburg, Russia.

Hydrogen peroxide, formed in the endothelium, acts as a factor contributing to the relaxation of blood vessels. The reason for this vasodilatory effect could be modulation by HO of calcium metabolism, since mobilization of calcium ions in endothelial cells is a trigger of endothelium-dependent relaxation. The aim of this work was to investigate the influence of HO on the effects of Ca-mobilizing agonists in human umbilical vein endothelial cells (HUVEC). We have found that HO in concentration range 10-100 M increases the rise of [Ca] induced by 5-hydroxytryptamine (5-HT) and carbachol and does not affect the calcium signals of ATP, agonist of type 1 protease-activated receptor SFLLRN, histamine and bradykinin. Using specific agonists of 5-HT1B and 5-HT2B receptors CGS12066B and BW723C86, we have demonstrated that HO potentiates the effects mediated by these types of 5-HT receptors. Potentiation of the effect of BW723C86 can be produced by the induction of endogenous oxidative stress in HUVEC. We have shown that the activation of 5-HT2B receptor by BW723C86 causes production of reactive oxygen species (ROS). Inhibitor of NADPH oxidases VAS2870 suppressed formation of ROS and partially inhibited [Ca] rise induced by BW723C86. Thus, it can be assumed that vasorelaxation induced by endogenous HO in endothelial cells partially occurs due to the potentiation of the agonist-induced calcium signaling.
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http://dx.doi.org/10.1155/2019/1701478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388333PMC
May 2019

VAS2870 Inhibits Histamine-Induced Calcium Signaling and vWF Secretion in Human Umbilical Vein Endothelial Cells.

Cells 2019 02 23;8(2). Epub 2019 Feb 23.

Sechenov Institute of Evolutional Institute of Evolutionary Physiology and Biochemistry, Saint Petersburg 194223, Russia.

In this study, we investigated the effects of NAD(P)H oxidase (NOX) inhibitor VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine) on the histamine-induced elevation of free cytoplasmic calcium concentration ([Ca]) and the secretion of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVECs) and on relaxation of rat aorta in response to histamine. At 10 μM concentration, VAS2870 suppressed the [Ca] rise induced by histamine. Inhibition was not competitive, with IC50 3.64 and 3.22 μM at 1 and 100 μM concentrations of histamine, respectively. There was no inhibition of [Ca] elevation by VAS2870 in HUVECs in response to the agonist of type 1 protease-activated receptor SFLLRN. VAS2870 attenuated histamine-induced secretion of vWF and did not inhibit basal secretion. VAS2870 did not change the degree of histamine-induced relaxation of rat aortic rings constricted by norepinephrine. We suggest that NOX inhibitors might be used as a tool for preventing thrombosis induced by histamine release from mast cells without affecting vasorelaxation.
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http://dx.doi.org/10.3390/cells8020196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406370PMC
February 2019

Markers and Biomarkers of Endothelium: When Something Is Rotten in the State.

Oxid Med Cell Longev 2017 23;2017:9759735. Epub 2017 Nov 23.

Koltsov Institute of Developmental Biology RAS, Moscow, Russia.

Endothelium is a community of endothelial cells (ECs), which line the blood and lymphatic vessels, thus forming an interface between the tissues and the blood or lympha. This strategic position of endothelium infers its indispensable functional role in controlling vasoregulation, haemostasis, and inflammation. The state of endothelium is simultaneously the cause and effect of many diseases, and this is coupled with modifications of endothelial phenotype represented by markers and with biochemical profile of blood represented by biomarkers. In this paper, we briefly review data on the functional role of endothelium, give definitions of endothelial markers and biomarkers, touch on the methodological approaches for revealing biomarkers, present an implicit role of endothelium in some toxicological mechanistic studies, and survey the role of reactive oxygen species (ROS) in modulation of endothelial status.
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http://dx.doi.org/10.1155/2017/9759735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733214PMC
July 2018

Serum Albumin Binding and Esterase Activity: Mechanistic Interactions with Organophosphates.

Molecules 2017 Jul 18;22(7). Epub 2017 Jul 18.

Koltsov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilova str., Moscow 119334, Russia.

The albumin molecule, in contrast to many other plasma proteins, is not covered with a carbohydrate moiety and can bind and transport various molecules of endogenous and exogenous origin. The enzymatic activity of albumin, the existence of which many scientists perceive skeptically, is much less studied. In toxicology, understanding the mechanistic interactions of organophosphates with albumin is a special problem, and its solution could help in the development of new types of antidotes. In the present work, the history of the issue is briefly examined, then our in silico data on the interaction of human serum albumin with soman, as well as comparative in silico data of human and bovine serum albumin activities in relation to paraoxon, are presented. Information is given on the substrate specificity of albumin and we consider the possibility of its affiliation to certain classes in the nomenclature of enzymes.
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http://dx.doi.org/10.3390/molecules22071201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6151986PMC
July 2017

Inhibition of protein tyrosine phosphatases unmasks vasoconstriction and potentiates calcium signaling in rat aorta smooth muscle cells in response to an agonist of 5-HT2B receptors BW723C86.

Biochem Biophys Res Commun 2017 01 13;483(1):700-705. Epub 2016 Dec 13.

Koltsov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia. Electronic address:

In blood vessels, serotonin 5-HT2B receptors mainly mediate relaxation, although their activation by the selective agonist BW723C86 is known to exert contraction of aorta in deoxycorticosterone acetate (DOCA)-salt and N(omega)-nitro-l-arginine (l-NAME) hypertensive rats [Russel et al., 2002; Banes et al., 2003] and in mice with type 2 diabetes [Nelson et al., 2012]. The unmasking effect on vasoconstriction can be caused by a shift in the balance of tyrosine phosphorylation in smooth muscle cells (SMC) due to oxidative stress induced inhibition of protein tyrosine phosphatases (PTP). We have demonstrated that BW723C86 which does not cause contraction of rat aorta and mesenteric artery rings, evoked a vasoconstrictor effect in the presence of PTP inhibitors sodium orthovanadate (NaVO) or BVT948. BW723C86 induced a weak rise of [Ca] in the SMC isolated from rat aorta; however, after pre-incubation with NaVO the response to BW723C86 increased more than 5-fold. This effect was diminished by protein tyrosine kinase (PTK) inhibitor genistein, inhibitor of Src-family kinases PP2, inhibitor of NADPH-oxidase VAS2870 and completely suppressed by N-acetylcysteine and 5-HT2B receptor antagonist RS127445. Using fluorescent probe DCFH-DA we have shown that NaVO induces oxidative stress in SMC. In the presence of NaVO BW723C86 considerably increased formation of reactive oxygen species while alone had no appreciable effect on DCFH oxidation. We suggest that oxidative stress causes inhibition of PTP and unmasking of 5-HT2B receptors functional activity.
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http://dx.doi.org/10.1016/j.bbrc.2016.12.079DOI Listing
January 2017

Reactive oxygen species in pathogenesis of atherosclerosis.

Curr Pharm Des 2015 ;21(9):1134-46

School of Allied Health Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, UK.

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.
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http://dx.doi.org/10.2174/1381612820666141014142557DOI Listing
September 2015

Control of thrombotic thrombocytopenic purpura by sirolimus in a child with juvenile myelomonocytic leukemia and somatic N-RAS mutation.

Pediatr Blood Cancer 2014 Oct 3;61(10):1871-3. Epub 2014 Mar 3.

Federal Research Center for Pediatric Hematology, Oncology and Immunology, Moscow, Russia.

We describe an infant who developed juvenile myelomonocytic leukemia (JMML) at the age of 6 months. Myeloproliferation was effectively controlled by low-dose cytosine arabinoside and 13-cis retinoic acid therapy. Two years after therapy for JMML was stopped, at the age of 5 years, the patient developed autoimmune thrombotic thrombocytopenic purpura (TTP). TTP was transiently controlled by plasma exchange, prednisolone, rituximab, and cyclophosphamide, but relapsed within a short time. Long-term control of TTP was established by sirolimus. Somatic N-RAS G38A→Gly13Asp substitution was restricted to hematopoietic cells. The somatic N-RAS mutation may link myeloproliferation and autoimmunity.
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http://dx.doi.org/10.1002/pbc.25013DOI Listing
October 2014

Calcium ions as a factor of cell-cell cooperation in hepatocyte cultures.

Cell Biol Int 2003 ;27(12):965-76

Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, 117808, Moscow, Russia.

Ultradian protein synthesis rhythm was used as a marker of cell cooperation in synchronous dense and non-synchronous sparse hepatocyte cultures. Phenylephrine (2 microM, 2 min), an alpha (1)-adrenoreceptor agonist, which exerts [Ca(2+)](cyt)elevation from intracellular stores, affected protein synthesis rhythm in sparse cultures, i.e. initiated cooperative activity of the cells. The same effect was produced by 2,5-di(tertiary-butyl)-1,4-benzohydroquinone (10 microM, 2 min), which increases [Ca(2+)](cyt)by a non-receptor pathway. Pretreatment of dense cultures with the intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'- tetraacetic acid (acetoxymethyl) ester (BAPTA-AM) at 10-20 microM for, 30-60 min resulted in loss of the rhythm of protein synthesis, i.e. loss of cooperative activity between the cells. The medium conditioned by control dense cultures initiated rhythm in sparse cultures, whereas the conditioned medium of cultures pretreated with BAPTA-AM did not. [Ca(2+)](cyt)increase is known to occur with monosialoganglioside GM1 treatment. By ELISA estimation, the GM1 content in 3 h conditioned medium was similar in control dense cultures to that in cultures pretreated with BAPTA-AM. Bearing in mind data on the Ca(2+)-dependence of vesicle formation and shedding, the conditioned medium was separated by 150000 g centrifugation to supernatant containing monomers and micelles, and a pellet containing vesicular form of gangliosides. Only the latter initiated cooperative activity of the cells of sparse cultures. These cultures were also synchronized by GM1-containing liposomes at lower concentrations than added free GM1, 0.0003 and 0.06 microM respectively. Thus, GM1 and calcium are both involved in cell-cell synchronization. Activation of gangliosides, including GM1 and elevation of [Ca(2+)](cyt,)is known to lead to changes of protein kinase activity and protein phosphorylation resulting in modulation of oscillations in protein metabolism.
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http://dx.doi.org/10.1016/j.cellbi.2003.07.006DOI Listing
July 2004

Enhanced G(i) signaling selectively negates beta2-adrenergic receptor (AR)--but not beta1-AR-mediated positive inotropic effect in myocytes from failing rat hearts.

Circulation 2003 Sep 15;108(13):1633-9. Epub 2003 Sep 15.

the Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Md 21224, USA.

Background: Myocardial contractile response to beta1- and beta2-adrenergic receptor (AR) stimulation is severely impaired in chronic heart failure, in which G(i) signaling and the ratio of beta2/beta1 are often increased. Because beta2-AR but not beta1-AR couples to G(s) and G(i) with the G(i) coupling negating the G(s)-mediated contractile response, we determined whether the heart failure-associated augmentation of G(i) signaling contributes differentially to the defects of these beta-AR subtypes and, if so, whether inhibition of G(i) or selective activation of beta2-AR/G(s) by ligands restores beta2-AR contractile response in the failing heart.

Methods And Results: Cardiomyocytes were isolated from 18- to 24-month-old failing spontaneously hypertensive (SHR) or age-matched Wistar-Kyoto (WKY) rat hearts. In SHR cardiomyocytes, either beta-AR subtype-mediated inotropic effect was markedly diminished, whereas G(i) proteins and the beta2/beta1 ratio were increased. Disruption of G(i) signaling by pertussis toxin (PTX) enabled beta2- but not beta1-AR to induce a full positive inotropic response in SHR myocytes. Furthermore, screening of a panel of beta2-AR ligands revealed that the contractile response mediated by most beta2-AR agonists, including zinterol, salbutamol, and procaterol, was potentiated by PTX, indicating concurrent G(s) and G(i) activation. In contrast, fenoterol, another beta2-AR agonist, induced a full positive inotropic effect in SHR myocytes even in the absence of PTX.

Conclusions: We conclude that enhanced G(i) signaling is selectively involved in the dysfunction of beta2- but not beta1-AR in failing SHR hearts and that disruption of G(i) signaling by PTX or selective activation of beta2-AR/G(s) signaling by fenoterol restores the blunted beta2-AR contractile response in the failing heart.
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http://dx.doi.org/10.1161/01.CIR.0000087595.17277.73DOI Listing
September 2003
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