I. B. Alieva

Novel role of microtubules in thrombin-induced endothelial barrier dysfunction

Disturbances in endothelial cell (EC) barrier regulation are critically dependent upon rearrangements of EC actin cytoskeleton. However, the role of microtubule (MT) network in the regulation of EC permeability is not well understood. We examined involvement of MT remodeling in thrombin‐induced EC permeability and explored MT regulation by heterotrimeric G12/13 proteins and by small GTPase Rho. Thrombin induced phosphorylation of MT regulatory protein tau at Ser409 and Ser262 and peripheral MT disassembly, which was linked to increased EC permeability. MT stabilization by taxol attenuated thrombin‐ induced permeability, actin remodeling, and paracellular gap formation and diminished thrombin‐induced activation of Rho and Rho‐kinase. Expression of activated Gα12/13 subunits involved in thrombin‐mediated signaling or their effector p115RhoGEF involved in Rho activation caused MT disassembly, whereas p115RhoGEF‐specific negative regulator RGS preserved MT from thrombin‐induced disassembly. Consistent with these results, expression of activated RhoA and Rho‐kinase induced MT disassembly. Conversely, thrombin‐induced disassembly of peripheral MT network was attenuated by expression of dominant negative RhoA and Rho‐kinase mutants or by pharmacological inhibition of Rho‐kinase. Collectively, our data demonstrate for the first time a critical involvement of MT disassembly in thrombin‐induced EC barrier dysfunction and indicate G‐protein‐dependent mechanisms of thrombin‐induced MT alteration.—Birukova, A. A., Birukov, K G., Smurova, K., Adyshev, D., Kaibuchi, K., Alieva, I., Garcia, J. G. N., Verin, A. D. Novel role of microtubules in thrombin‐induced endothelial barrier dysfunction. FASEB J. 18, 1879‐1890 (2004)

Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: Role of Rho-dependent mechanisms

Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti‐cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND‐induced activation of Rho GTPase. Rho‐mediated activation of its downstream target, Rho‐kinase, induced phosphorylation of Rho‐kinase effector EC MLC phosphatase (MYPT1) at Thr696 and Thr850 resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho‐MLC, which induced acto‐myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho‐kinase by Y27632 abolished ND‐induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)‐Rho, or DN‐Rho‐kinase) attenuated ND‐induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho‐dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho‐kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND‐induced MT disassembly. J. Cell. Physiol. 201: 55–70, 2004.

Vertebrate primary cilia: a sensory part of centrosomal complex in tissue cells, but a “sleeping beauty” in cultured cells?

Primary cilium development along with other components of the centrosome in mammalian cells was analysed ultrastructurally and by immunofluorescent staining with anti‐acetylated tubulin antibodies. We categorized two types of primary cilia, nascent cilia that are about 1 μm long located inside the cytoplasm, and true primary cilia that are several μm long and protrude from the plasma membrane.

Protein Kinase C-α and Arginase I Mediate Pneumolysin-Induced Pulmonary Endothelial Hyperpermeability

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)(+/-)/arginase II (AII)(-/-) C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI(+/+)/AII(-/-) counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.

Molecular mechanisms involved in adenosine-induced endothelial cell barrier enhancement

Extracellular adenosine is a physiologically relevant agonist released by various sources, including endothelial cells (EC) and activated platelets, with complex effects mediated via activation of P1 purinergic receptors. Adenosine-induced EC production of glutathione peroxidase1 and nitric oxide is recognized, and an anti-inflammatory mechanism has been described. Effects of extracellular adenosine on the pulmonary EC barrier function and vascular permeability, however, remain poorly characterized. In this study, we demonstrated the adenosine-induced rapid dose-dependent barrier enhancement in human pulmonary artery EC (HPAEC) as measured by an increase in transendothelial electrical resistance (TER). We have shown that HPAEC express only A2A and A2B adenosine receptors. Pharmacological and siRNA depletion studies indicate that A2A, but not A2B receptor activation is required for the adenosine-induced TER increase. Depletion of Galphas with a specific siRNA significantly attenuated the adenosine-induced TER response in HPAEC. In contrast, depletion of either Galphaq or Galphai2 did not affect the adenosine-induced TER increase. This suggests that the adenosine-induced TER increase is cAMP-dependent. The adenosine-induced barrier enhancement effects were associated with a rearrangement of the EC F-actin component of the cytoskeleton, enhanced cell-surface presentation of cell-cell junctional protein VE-cadherin and an involvement of Myosin-light-chain phosphatase (MLCP). Our results suggest, for the first time, that adenosine regulates the EC barrier function via A2A receptors followed by Galphas engagement and is associated with cytoskeletal activation.

Effect of transitory glucose deprivation on mitochondrial structure and functions in cultured cerebellar granule neurons.

We found that 60-min glucose deprivation leads to progressive decrease in the mitochondrial membrane potential and increase in [Ca(2+)](i) in cultured cerebellar granule neurons. The latter effect was fully reversible, returning to the basal level 60 min after restoration of normal glucose level in the incubation medium, whereas mitochondrial membrane potential remained at 10.0+/-1.8% below the initial value. Electron microscopy indicated that glucose deprivation induced appearance of mitochondria with local lightening of the matrix and destruction of cristae. This mitochondrial conformation was preserved during the restoration phase after glucose level in the cultivation medium returned to the normal level. Neuronal death within a 24-h period after 60-min glucose deprivation was relatively small, being 14.0+/-4.4%.

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.

Microtubule system in endothelial barrier dysfunction: Disassembly of peripheral microtubules and microtubule reorganization in internal cytoplasm

Endothelial cell barrier dysfunction is associated with dramatic cytoskeletal reorganization, the activation of actomyosin contraction, and, finally, gap formation. Although the role of microtubules in the regulation of endothelial cell barrier function is not fully understood, a number of observations allow for the assumption that the reaction of the microtubule is an extremely important part in the development of endothelial dysfunction. These observations have forced us to examine the role of microtubule reorganization in the regulation of the endothelial cell barrier function. In quiescent endothelial cells, microtubule density is the highest in the centrosome region; however, microtubules are also present near the cell margin. The analysis of microtubule distribution after specific antibody staining using the method of measurement of their fluorescence intensity showed that, in control endothelial cells, the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of exponential regression. The edemagenic agent, thrombin (25 nM), caused the rapid increase of endothelial cell barrier permeability accompanied by a fast decrease in quantity of the peripheral microtubules and reorganization of the microtubule system in the internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress within as little as 5 min after the beginning of treatment). Both effects are reversible; within 60 min after the beginning of treatment, the microtubule network does not differ from the standard one. Thus, the microtubule system is capable of adapting to the influence of a natural regulator, thrombin. The reorganization of microtubules develops more quickly than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in the circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.

Solid state synthesis of carbon-encapsulated iron carbide nanoparticles and their interaction with living cells

Superparamagnetic carbon-encapsulated iron carbide nanoparticles (NPs), Fe7C3@C, with unique properties, were produced from pure ferrocene by high pressure-high temperature synthesis. These NPs combine the merits of nanodiamonds and SPIONs but lack their shortcomings which limit their use for biomedical applications. Investigation of these NPs by X-ray diffraction, electron microscopy techniques, X-ray spectroscopic and magnetic measurement methods has demonstrated that this method of synthesis yields NPs with perfectly controllable physical properties. Using magnetic and subsequent fractional separation of magnetic NPs from residual carbon, the aqueous suspensions of Fe7C3@C NPs with an average particle size of ∼25 nm were prepared. The suspensions were used for in vitro studies of the interaction of Fe7C3@C NPs with cultured mammalian cells. The dynamics of interaction of the living cells with Fe7C3@C was studied by optical microscopy using time-lapse video recording and also by transmission electron microscopy. Using novel highly sensitive cytotoxicity tests based on the cell proliferation assay and long-term live cell observations it was shown that the internalization of Fe7C3@C NPs has no cytotoxic effect on cultured cells and does not interfere with the process of their mitotic division, a fundamental property that ensures the existence of living organisms. The influence of NPs on the proliferative activity of cultured cells was not detected as well. These results indicate that the carbon capsules of Fe7C3@C NPs are air-tight which could offer great opportunities for future use of these superparamagnetic NPs in biology and medicine.

Microtubules growth rate alteration in human endothelial cells.

To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC) and “fast” (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.