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

Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults

Background Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI <18·5 kg/m2) and obesity (BMI ≥30 kg/m2). For schoolaged children and adolescents, we report thinness (BMI <2 SD below the median of the WHO growth reference) and obesity (BMI >2 SD above the median). Findings From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and among boys in 15 countries (8%) with a posterior probability of at least 0·80, and increased among girls in 140 countries (70%) and boys in 137 countries (69%) with a posterior probability of at least 0·80. The countries with highest combined prevalence of thinness and obesity in school-aged children and adolescents in 2022 were in Polynesia and Micronesia and the Caribbean for both sexes, and Chile and Qatar for boys. Combined prevalence was also high in some countries in south Asia, such as India and Pakistan, where thinness remained prevalent despite having declined. In 2022, obesity in school-aged children and adolescents was more prevalent than thinness with a posterior probability of at least 0·80 among girls in 133 countries (67%) and boys in 125 countries (63%), whereas the converse was true in 35 countries (18%) and 42 countries (21%), respectively. In almost all countries for both adults and school-aged children and adolescents, the increases in double burden were driven by increases in obesity, and decreases in double burden by declining underweight or thinness. Interpretation The combined burden of underweight and obesity has increased in most countries, driven by an increase in obesity, while underweight and thinness remain prevalent in south Asia and parts of Africa. A healthy nutrition transition that enhances access to nutritious foods is needed to address the remaining burden of underweight while curbing and reversing the increase in obesit

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

International audienc

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