Fluorescence Methods for the Analysis of Microtubule/Microfilament Involvement in the Regulation of Endothelial Barrier Function

The endothelial monolayer is located on the inner surface of blood vessels and provides an important barrier function controlling the transport of metabolites and nutrients through the vessel wall, both toward the circulating blood and in the direction of the underlying tissues. This function is provided by cytoskeletal structures that generate contractile and tensile forces, existing in equilibrium in the intact endothelium. In the case of cytoskeleton rearrangements, there are changes in the shape of cells and the formation of intercellular spaces, which lead to endothelial dysfunction. Deep understanding of endothelial barrier function maintaining is a crucial problem because this phenomenon is common for a number of pathological states and diseases (inflammation, asthma, sepsis, acute lung injury, diabetes, etc.) and can lead to severe organ dysfunction, as well as a complication upon the treatment by a number of anticancer pharmacological drugs. Microtubules and the actin cytoskeleton function in cooperation in normal endothelium and under conditions of the barrier loss. In this review, we describe the application of modern fluorescence methods for investigation and analysis of the individual characteristics of cytoskeletal elements whose reorganization affects endothelial permeability, to emphasize the role of microtubules/microfilament crosstalk in EC barrier regulation

The “Third Violin” in the Cytoskeleton Orchestra—The Role of Intermediate Filaments in the Endothelial Cell’s Life

The endothelium plays an important role in the transcytosis of lipoproteins. According to one of the theories, endothelial injury is a triggering factor for the development of atherosclerosis, and intracellular structures, including components of the endotheliocyte cytoskeleton (microtubules, actin, and intermediate filaments), are involved in its development. In contrast to the proteins of tubulin-based microtubules and actin microfilaments, intermediate filaments are comprised of various tissue-specific protein members. Vimentin, the main protein of endothelial intermediate filaments, is one of the most well-studied of these and belongs to type-III intermediate filaments, commonly found in cells of mesenchymal origin. Vimentin filaments are linked mechanically or by signaling molecules to microfilaments and microtubules by which coordinated cell polarisation and migration are carried out, as well as control over several endotheliocyte functions. Moreover, the soluble vimentin acts as an indicator of the state of the cardiovascular system, and the involvement of vimentin in the development and course of atherosclerosis has been demonstrated. Here we discuss current concepts of the participation of vimentin filaments in the vital activity and functioning of endothelial cells, as well as the role of vimentin in the development of inflammatory processes and atherosclerosis

Huntingtin and Other Neurodegeneration-Associated Proteins in the Development of Intracellular Pathologies: Potential Target Search for Therapeutic Intervention

Neurodegenerative diseases are currently incurable. Numerous experimental data accumulated over the past fifty years have brought us closer to understanding the molecular and cell mechanisms responsible for their development. However, these data are not enough for a complete understanding of the genesis of these diseases, nor to suggest treatment methods. It turns out that many cellular pathologies developing during neurodegeneration coincide from disease to disease. These observations give hope to finding a common intracellular target(s) and to offering a universal method of treatment. In this review, we attempt to analyze data on similar cellular disorders among neurodegenerative diseases in general, and polyglutamine neurodegenerative diseases in particular, focusing on the interaction of various proteins involved in the development of neurodegenerative diseases with various cellular organelles. The main purposes of this review are: (1) to outline the spectrum of common intracellular pathologies and to answer the question of whether it is possible to find potential universal target(s) for therapeutic intervention; (2) to identify specific intracellular pathologies and to speculate about a possible general approach for their treatment

Colocalization Analysis of Cytoplasmic Actin Isoforms Distribution in Endothelial Cells

Actin cytoskeleton is an essential component of living cells and plays a decisive role in many cellular processes. In mammals, β- and γ-actin are cytoplasmic actin isoforms in non-muscle cells. Despite minor differences in the amino acid sequence, β- and γ-actin localize in different cell structures and perform different functions. While cytoplasmic β-actin is involved in many intracellular processes including cell contraction, γ-actin is responsible for cell mobility and promotes tumor transformation. Numerous studies demonstrate that β- and γ-actin are spatially separated in the cytoplasm of fibroblasts and epithelial cells; this separation is functionally determined. The spatial location of β/γ-actin in endothelial cells is still a subject for discussion. Using super-resolution microscopy, we investigated the β/γ-actin colocalization in endotheliocytes and showed that the β/γ-actin colocalization degree varies widely between different parts of the marginal regions and near the cell nucleus. In the basal cytoplasm, β-actin predominates, while the ratio of isoforms evens out as it moves to the apical cytoplasm. Thus, our colocalization analysis suggests that β- and γ-actin are segregated in the endotheliocyte cytoplasm. The segregation is greatly enhanced during cell lamella activation in the nocodazole-induced endothelial barrier dysfunction, reflecting a different functional role of cytoplasmic actin isoforms in endothelial cells

The Centriolar Adjunct–Appearance and Disassembly in Spermiogenesis and the Potential Impact on Fertility

During spermiogenesis, the proximal centriole forms a special microtubular structure: the centriolar adjunct. This structure appears at the spermatid stage, which is characterized by a condensed chromatin nucleus. We showed that the centriolar adjunct disappears completely in mature porcine spermatozoa. In humans, the centriolar adjunct remnants are present in a fraction of mature spermatids. For the first time, the structure of the centriolar adjunct in the cell, and its consequent impact on fertility, were examined. Ultrastructural analysis using transmission electron microscopy was performed on near 2000 spermatozoa per person, in two patients with idiopathic male sterility (IMS) and five healthy fertile donors. We measured the average length of the “proximal centriole + centriolar adjunct„ complex in sections, where it had parallel orientation in the section plane, and found that it was significantly longer in the spermatozoa of IMS patients than in the spermatozoa of healthy donors. This difference was independent of chromatin condensation deficiency, which was also observed in the spermatozoa of IMS patients. We suggest that zygote arrest may be related to an incompletely disassembled centriolar adjunct in a mature spermatozoon. Therefore, centriolar adjunct length can be potentially used as a complementary criterion for the immaturity of spermatozoa in the diagnostics of IMS patients

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

Magnetocontrollability of Fe7C3@C superparamagnetic nanoparticles in living cells

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Protective effect of Growth Hormone-Releasing Hormone agonist in bacterial toxin-induced pulmonary barrier dysfunction

Rationale: Antibiotic treatment of patients infected with G(−) or G(+) bacteria promotes release of the toxins lipopolysaccharide (LPS) and pneumolysin (PLY) in their lungs. Growth Hormone-releasing Hormone (GHRH) agonist JI-34 protects human lung microvascular endothelial cells (HL-MVEC), expressing splice variant 1 (SV-1) of the receptor, from PLY-induced barrier dysfunction. We investigated whether JI-34 also blunts LPS-induced hyperpermeability. Since GHRH receptor (GHRH-R) signaling can potentially stimulate both cAMP-dependent barrier-protective pathways as well as barrier-disruptive protein kinase C pathways, we studied their interaction in GHRH agonist-treated HL-MVEC, in the presence of PLY, by means of siRNA-mediated protein kinase A (PKA) depletion. Methods: Barrier function measurements were done in HL-MVEC monolayers using Electrical Cell substrate Impedance Sensing (ECIS) and VE-cadherin expression by Western blotting. Capillary leak was assessed by Evans Blue dye (EBD) incorporation. Cytokine generation in broncho-alveolar lavage fluid (BALF) was measured by multiplex analysis. PKA and PKC-α activity were assessed by Western blotting. Results: GHRH agonist JI-34 significantly blunts LPS-induced barrier dysfunction, at least in part by preserving VE-cadherin expression, while not affecting inflammation. In addition to activating PKA, GHRH agonist also increases PKC-α activity in PLY-treated HL-MVEC. Treatment with PLY significantly decreases resistance in control siRNA-treated HL-MVEC, but does so even more in PKA-depleted monolayers. Pretreatment with GHRH agonist blunts PLY-induced permeability in control siRNA-treated HL-MVEC, but fails to improve barrier function in PKA-depleted PLY-treated monolayers. Conclusions: GHRH signaling in HL-MVEC protects from both LPS and PLY-mediated endothelial barrier dysfunction and concurrently induces a barrier-protective PKA-mediated and a barrier-disruptive PKC-α-induced pathway in the presence of PLY, the former of which dominates the latter

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