Lack of activity of the mitochondrial large-conductance calcium-regulated potassium channels in senescent vascular smooth muscle cells.

A limited number of studies have shown functional changes in mitochondrial ion channels in aging and senescent cells. We have identified, for the first time, mitochondrial large-conductance calcium-regulated potassium channels in human smooth muscle mitochondria. This channel, with a conductance of 273 pS, was regulated by calcium ions and membrane potential. Additionally, it was activated by the potassium channel opener NS11021 and blocked by paxilline. Importantly, we have shown that senescence of these cells induced by hydrogen peroxide treatment leads to the disappearance of potassium channel protein levels and channel activity measured by the single channel patch-clamp technique. Our data suggest that disturbances in the expression of mitochondrial large conductance calcium-regulated potassium channels may be hallmarks of cellular senescence and contribute to the misregulation of mitochondrial function in senescent cells.

Exploring Extracellular Vesicles of Probiotic Yeast as Carriers of Biologically Active Molecules Transferred to Human Intestinal Cells.

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos-e.g., proteins, RNAs, and lipids-that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces boulardii CNCM I-745. The size distribution and concentration of EVs in the liquid culture of yeast were estimated. Moreover, the vesicles of S. boulardii were tested for their cytotoxicity against three model human intestinal cell lines. This study did not show any significant negative effect of yeast EVs on these cells under tested conditions. In addition, EVs of S. boulardii were verified for their ability to internalize in vitro with human cells and transfer their cargo. The yeast vesicles were loaded with doxorubicin, an anticancer agent, and added to the cellular cultures. Subsequently, microscopic observations revealed that these EVs transferred the compound to human intestinal cell lines. A cytotoxicity test confirmed the activity of the transferred doxorubicin. Detailed information about the proteins present in EVs might be important in terms of exploring yeast EVs as carriers of active molecules. Thus, proteomic analysis of the EV content was also conducted within the present study, and it allowed the identification of 541 proteins after matching them to the Saccharomyces Genome Database (SGD). Altogether, this study provides strong evidence that the EVs of the probiotic CNCM I-745 strain could be considered a drug delivery system.

Fiji-Based Tool for Rapid and Unbiased Analysis of SA-ß-Gal Activity in Cultured Cells.

Normal cells under stressful conditions such as DNA damage or excessive mitogenic signaling may undergo senescence, which is associated with cell cycle arrest and induction of a proinflammatory phenotype. Accumulation of senescent cells may contribute to the shortening of the life span by accelerating aging and promoting chronic diseases. Cytochemical detection of the senescence-associated ß-galactosidase (SA-ß-gal) activity with 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-gal) is a widely recognised marker of cell senescence. However, its simplicity and cost effectiveness lead to limitations in quantification, which is usually limited to manual counting of the positive cells. In order to address those limitations, we developed a Fiji-based macro extension that performs automatic and unbiased analysis of the integrated density of SA-ß-gal specific signal. Our tool is not only faster than manual counting but also provides extra resolution compared to the manual methods. Our macro extension could be a valuable tool in any senescence research laboratory.

Exosomes derived from lung cancer cells: Isolation, characterization, and stability studies.

Recent advances in nanomedicine have paved the way for developing targeted drug delivery systems. Nanoscale exosomes are present in almost every body fluid and represent a novel mechanism of intercellular communication. Because of their membrane origin, they easily fuse with cells, acting as a natural delivery system and maintaining the bioactivity and immunotolerance of cells. To develop a reconstitutable exosome-based drug candidate for clinical applications, quality assurance by preserving its physical and biological properties during storage is necessary. Therefore, this study aimed to determine the best storage conditions for exosomes derived from lung cancer cells (A549). This study established that the phosphate-buffered saline buffer enriched with 25 mM trehalose is an optimal cryoprotectant for A549-derived exosomes stored at -80°C. Under these conditions, the concentration, size distribution, zeta potential, and total cargo protein levels of the preserved exosomes remained constant.

Unbiased proteomic analysis of extracellular vesicles secreted by senescent human vascular smooth muscle cells reveals their ability to modulate immune cell functions.

Atherosclerosis, a common age-related disease, is characterized by intense immunological activity. Atherosclerotic plaque is composed of endothelial cells, vascular smooth muscle cells (VSMCs), lipids and immune cells infiltrating from the blood. During progression of the disease, VSMCs undergo senescence within the plaque and secrete SASP (senescence-associated secretory phenotype) factors that can actively modulate plaque microenvironment. We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs). Based on unbiased proteomic analysis of VMSC-derived EVs and of the soluble fraction of SASP (sSASP), more than 900 proteins were identified in each of SASP compartments. Comparison of the composition of VMSC-derived EVs with the SASP atlas revealed several proteins, including Serpin Family F Member 1 (SERPINF1) and Thrombospondin 1 (THBS1), as commonly upregulated components of EVs secreted by senescent VSMCs and fibroblasts. Among soluble SASP factors, only Growth Differentiation Factor 15 (GDF15) was universally increased in the secretome of senescent VSMCs, fibroblasts, and epithelial cells. Bioinformatics analysis of EV proteins distinguished functionally organized protein networks involved in immune cell function regulation. Accordingly, EVs released by senescent VSMCs induced secretion of IL-17, INFγ, and IL-10 by T cells and of TNFα produced by monocytes. Moreover senEVs influenced differentiation of monocytes favoring mix M1/M2 polarization with proinflammatory characteristics. Altogether, our studies provide a complex, unbiased analysis of VSMC SASP and prove that EVs derived from senescent VSMCs influence the cytokine milieu by modulating immune cell activity. Our results strengthen the role of senescent cells as an important inducer of inflammation in atherosclerosis.