Влияние активатора аденилатциклаз на изменение цитоскелета клеток в ходе барьерной дисфункции эндотелия, вызванной воздействием тромбина

This work executed on a model of endothelial monolayer in vitro, is devoted to the study of mechanisms leading to endothelial barrier dysfunction. The dysfunction inductor, thrombin, acts on endothelial cells through a specific receptor, triggering cascades of intracellular reactions (including cytoskeletal reorganization) resulting to increased of endothelial permeability. Thrombin-receptor interaction leads to dissociation of heterotrimeric G-protein subunits (Gi, Gq and G12/13), each of which is potentially capable to handle one or more possible ways of dysfunction development. In the present study we analyzed the role of Gi protein in the occurrence of cytoskeletal disturbances that lead to barrier dysfunction. According to our results, it can be assumed that the activity of Gi protein does not directly affect on the endothelial barrier properties.Работа, выполненная на модели эндотелиального пласта in vitro, посвящена исследованию механизмов, приводящих к барьерной дисфункции эндотелия. В качестве индуктора дисфункции использовался тромбин. Тромбин действует на эндотелиоциты через специфический рецептор, запуская каскады внутриклеточных реакций, приводящих к росту проницаемости эндотелиального барьера, сопровождаемому увеличением фосфорилирования легких цепей миозина и перестройкой цитоскелета эндотелиальной клетки (деполимеризацией периферических микротрубочек и формированием дополнительных стресс-фибрилл). Взаимодействие тромбина с рецептором приводит к диссоциации субъединиц гетеротримерных G-белков семейств Gi, Gq и G12/13, каждая из которых потенциально способна регулировать один или несколько возможных путей развития дисфункции. В настоящем исследовании анализировали роль Gi белка в возникновении цитоскелетных нарушений, приводящих к барьерной дисфункции. Активированный Gi белок уменьшает уровень внутриклеточного медиатора - циклического АМФ и регулирует проницаемость ионных каналов. Действие активатора аденилатциклаз форсколина показало, что уровень цАМФ в клетке не влияет на изменения актинового цитоскелета и микротрубочек, вызываемые тромбином. Таким образом, можно предположить, что низкий уровень цАМФ не является обязательным условием для изменения цитоскелета под действием тромбина и активность Gi белка напрямую не влияет на барьерные свойства эндотелия

The functional role of the microtubule/microfi lament cytoskeleton in the regulation of pulmonary vascular endothelial barrier

The endothelial cells (EC) lining the vessels are in close contact with each other, rendering the vascular wall into a tight barrier, which control such diverse processes as vascular tone, homeostasis, adhesion of platelets and leukocytes to the vascular wall and permeability of vascular wall for cells and fluids (Bazzoni and Dejana 2004, Dudek and Garcia 2001, Komarova and Malik 2010, Ware and Matthay 2000). Lung endothelium regulates movement of fluid, macromolecules, and leukocytes into the interstitium and subsequently into the alveolar air spaces. The integrity of the pulmonary EC monolayer, therefore, is a critical requirement for preservation of pulmonary function. This barrier is dynamic and highly susceptible to the regulation, by various stimuli, of physiological and pathological origin. Any breach in the EC barrier results in leakage of fluid from the lumen of the vessels into the interstitial tissue and/or alveolar lumen, severely impairing gas exchange. Disruption of endothelial barrier occurs during inflammatory disease states such as acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), which remains a major cause of morbidity and mortality with an overall mortality rate of 30-40% (Ware and Matthay 2000), results in the uncontrolled movement of fluid and macromolecules into the interstitium and pulmonary air spaces causing pulmonary edema (Ermert et al. 1995). Data of literature have proved that normal functioning of the endothelial barrier is provided by the balance between contracting and stretching forces generated by EC cytoskeleton (Bogatcheva and Verin 2008, Dudek and Garcia 2001, Komarova et al. 2007). In this review, we will analyze the cytoskeletal elements whose reorganization affects endothelial permeability, and emphasize the role of microtubules/microfilament crosstalk in lung EC barrier regulation. © 2014 by Taylor & Francis Group, LLC

The leading role of microtubules in endothelial barrier dysfunction: Disassembly of peripheral microtubules leaves behind the cytoskeletal reorganization

Disturbance of the endothelial barrier is characterized by dramatic cytoskeleton reorganization, activation of actomyosin contraction and, finally, leads to intercellular gap formation. Here we demonstrate that the edemagenic agent, thrombin, causes a rapid increase in the human pulmonary artery endothelial cell (EC) barrier permeability accompanied by fast decreasing in the peripheral microtubules quantity and reorganization of the microtubule system in the internal cytoplasm of the EC within 5 min of the treatment. The actin stress-fibers formation occurs gradually and the maximal effect is observed relatively later, 30 min of the thrombin treatment. Thus, microtubules reaction develops faster than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction development. Direct microtubules depolymerization by nocodazole initiates the cascade of barrier dysfunction reactions. Nocodazole-induced barrier disruption is connected directly with the degree of peripheral microtubules depolymerization. Short-term loss of endothelial barrier function occurs at the minimal destruction of peripheral microtubules, when actin filament system is still intact. Specifically, we demonstrate that the EC microtubule dynamics examined by time-lapse imaging of EB3-GFP comets movement has changed under these conditions: microtubule plus ends growth rate significantly decreased near the cell periphery. The microtubules, apparently, are the first target in the circuit of reactions leading to the pulmonary EC barrier compromise. Our results show that dynamic microtubules play an essential role in the barrier function in vitro; peripheral microtubules depolymerization is necessary and sufficient condition for initiation of endothelial barrier dysfunction. J. Cell. Biochem. 114: 2258-2272, 2013. © 2013 Wiley Periodicals, Inc. Copyright © 2013 Wiley Periodicals, Inc

The leading role of microtubules in endothelial barrier dysfunction: Disassembly of peripheral microtubules leaves behind the cytoskeletal reorganization

Disturbance of the endothelial barrier is characterized by dramatic cytoskeleton reorganization, activation of actomyosin contraction and, finally, leads to intercellular gap formation. Here we demonstrate that the edemagenic agent, thrombin, causes a rapid increase in the human pulmonary artery endothelial cell (EC) barrier permeability accompanied by fast decreasing in the peripheral microtubules quantity and reorganization of the microtubule system in the internal cytoplasm of the EC within 5 min of the treatment. The actin stress-fibers formation occurs gradually and the maximal effect is observed relatively later, 30 min of the thrombin treatment. Thus, microtubules reaction develops faster than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction development. Direct microtubules depolymerization by nocodazole initiates the cascade of barrier dysfunction reactions. Nocodazole-induced barrier disruption is connected directly with the degree of peripheral microtubules depolymerization. Short-term loss of endothelial barrier function occurs at the minimal destruction of peripheral microtubules, when actin filament system is still intact. Specifically, we demonstrate that the EC microtubule dynamics examined by time-lapse imaging of EB3-GFP comets movement has changed under these conditions: microtubule plus ends growth rate significantly decreased near the cell periphery. The microtubules, apparently, are the first target in the circuit of reactions leading to the pulmonary EC barrier compromise. Our results show that dynamic microtubules play an essential role in the barrier function in vitro; peripheral microtubules depolymerization is necessary and sufficient condition for initiation of endothelial barrier dysfunction. J. Cell. Biochem. 114: 2258-2272, 2013. © 2013 Wiley Periodicals, Inc. Copyright © 2013 Wiley Periodicals, Inc

New superparamagnetic fluorescent Fe@C-C5ON2H10-Alexa Fluor 647 nanoparticles for biological applications

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