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

Abstract

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.

Characterization of vascular permeability using a biomimetic microfluidic blood vessel model

“…51 Further treatment is known to decrease the F-actin quantity and return to the control case (no thrombin) level. 54 Quantifying F-actin content in thrombin activated ECs has shown an increase in actin polymerization during the initial 30 min of thrombin treatment followed by a reduction in total F-actin content by 60 min. Figures 5 and 6 provided a quantitative understanding of the relative increase in intercellular gap area for BBV and static transwell cases.…”

Cytoskeletal mechanisms regulating vascular endothelial barrier function in response to acute lung injury

“…45,93,95 In the thrombin model of EC permeability microtubule disassembly precedes actin stress fiber formation. 96 Thrombin may induce microtubule dissolution via stimulation of G 12/13 / p115RhoGEF cascade, followed by Rho/Rho kinase activation, resulting in phosphorylation of the microtubule-associated protein, tau. 93 In its unphosphorylated form, tau promotes assembly of microtubules and inhibits the rate of depolymerization.…”

Staphylococcus aureus–induced endothelial permeability and inflammation are mediated by microtubule destabilization

“…The MT cytoskeleton plays a critical role in the control of cell division and intracellular trafficking of organelles and proteins. Increasing evidence suggests that MT also play an active role in the regulation of endothelial permeability via cross-talk with the actin cytoskeleton (15,16). MT cycle between polymerized and depolymerized states, which is determined by post-translational modifications such as the acetylation/deacetylation of tubulin and by numerous MT-associated proteins (17).…”