Особенности цитограммы и цитокинового профиля жидкости бронхоальвеолярного лаважа при экспериментальном метаболическом синдроме

The aim of the study was to identify the features of the cellular composition and cytokine profile of bronchoalveolar lavage fluid in rats in a model of diet-induced metabolic syndrome. Materials and methods. In an experiment on animals (rats), a model of metabolic syndrome (MS) induced by a high-fat and high-carbohydrate diet was reproduced. To assess the viability of the reproduced model, biochemical and morphometric methods were used, such as measurement of body weight, specific gravity of liver and visceral fat, and blood pressure, determination of glucose concentration in the blood (including a glucose tolerance test), as well as determination of blood lipid parameters. To assess the intensity of the inflammatory response in the blood, the concentration of total protein, the total number of leukocytes, and the levels of immunocytokines (interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)α, monocyte chemoattractant protein (MCP)-1) were determined. Open bronchoalveolar lavage was performed on the isolated heart – lung complex. The concentration of protein, immunocytokines (IL-6, IL-10, TNFα, MCP-1), the total number of leukocytes, and the ratio of their morphological types were determined in the bronchoalveolar lavage fluid (BALF). Results. In animals with MS, an increase in the total number of leukocytes in the blood due to granulocytes and a rise in the concentration of protein, TNFα, and IL-10 were revealed compared with the parameters in the controls. BALF analysis revealed an increase in the concentration of protein, the total number of leukocytes, and the absolute number of alveolar macrophages, neutrophil granulocytes, and lymphocytes. The levels of IL-6 and MCP-1 were more than 1.5 times higher. Conclusion. Changes in the qualitative and quantitative parameters of BALF are inflammatory in nature and are formed during a systemic inflammatory response accompanying metabolic disorders in modeling MS in rats in the experiment

SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

SOX9 is upregulated in the majority of pancreatic ductal adenocarcinoma cases. It is hypothesized that the increased expression of SOX9 is necessary for the formation and maintenance of tumor phenotypes in pancreatic cancer cells. In our research, we studied six pancreatic cancer cell lines, which displayed varying levels of differentiation and a range of oncogenic mutations. We chose the method of downregulation of SOX9 expression via siRNA transfection as the main method for investigating the functional role of the SOX9 factor in pancreatic cancer cells. We discovered that the downregulation of SOX9 expression in the cell lines leads to cell-line-specific changes in the expression levels of epithelial and mesenchymal protein markers. Additionally, the downregulation of SOX9 expression had a specific effect on the expression of pancreatic developmental master genes. SOX9 downregulation had the greatest effect on the expression levels of the protein regulators of cell proliferation. In three of the four cell lines studied, the transfection of siSOX9 led to a significant decrease in proliferative activity and to the activation of proapoptotic caspases in transfected cells. The acquired results demonstrate that the SOX9 protein exerts its multiple functions as a pleiotropic regulator of differentiation and a potential promoter of tumor growth in a cell-specific manner in pancreatic cancer cells

Presentation1_Declined adipogenic potential of senescent MSCs due to shift in insulin signaling and altered exosome cargo.pptx

Multipotent mesenchymal stromal cells (MSCs) maintain cellular homeostasis and regulate tissue renewal and repair both by differentiating into mesodermal lineage, e.g., adipocytes, or managing the functions of differentiated cells. Insulin is a key physiological inducer of MSC differentiation into adipocytes, and disturbances in MSC insulin sensitivity could negatively affect adipose tissue renewal. During aging, regulation and renewal of adipose tissue cells may be disrupted due to the altered insulin signaling and differentiation potential of senescent MSCs, promoting the development of serious metabolic diseases, including metabolic syndrome and obesity. However, the potential mechanisms mediating the dysfunction of adipose-derived senescent MSC remains unclear. We explored whether aging could affect the adipogenic potential of human adipose tissue-derived MSCs regulated by insulin. Age-associated senescent MSCs (isolated from donors older than 65 years) and MSCs in replicative senescence (long-term culture) were treated by insulin to induce adipogenic differentiation, and the efficiency of the process was compared to MSCs from young donors. Insulin-dependent signaling pathways were explored in these cells. We also analyzed the involvement of extracellular vesicles secreted by MSCs (MSC-EVs) into the regulation of adipogenic differentiation and insulin signaling of control and senescent cells. Also the microRNA profiles of MSC-EVs from aged and young donors were compared using targeted PCR arrays. Both replicatively and chronologically senescent MSCs showed a noticeably decreased adipogenic potential. This was associated with insulin resistance of MSCs from aged donors caused by the increase in the basal level of activation of crucial insulin-dependent intracellular effectors ERK1/2 and Akt. To assess the impact of the paracrine cross-talk of MSCs, we analyzed microRNAs profile differences in MSC-EVs and revealed that senescent MSCs produced EVs with increased content of miRNAs targeting components of insulin-dependent signaling cascade PTEN, MAPK1, GAREM1 and some other targets. We also confirmed these data by differentiation of control MSCs in the presence of EVs from senescent cells and vice versa. Thus, aging attenuated the adipogenic potential of MSCs due to autocrine or paracrine-dependent induction of insulin resistance associated with the specific changes in MSC-EV cargo.</p

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

Summary Myeloperoxidase (MPO) is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist). In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca2+ through enhancement of store-operated Ca2+ entry (SOCE). Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations

Novel Immortalized Human Multipotent Mesenchymal Stromal Cell Line for Studying Hormonal Signaling

Multipotent mesenchymal stromal cells (MSCs) integrate hormone and neuromediator signaling to coordinate tissue homeostasis, tissue renewal and regeneration. To facilitate the investigation of MSC biology, stable immortalized cell lines are created (e.g., commercially available ASC52telo). However, the ASC52telo cell line has an impaired adipogenic ability and a depressed response to hormones, including 5-HT, GABA, glutamate, noradrenaline, PTH and insulin compared to primary cells. This markedly reduces the potential of the ASC52telo cell line in studying the mechanisms of hormonal control of MSC’s physiology. Here, we have established a novel immortalized culture of adipose tissue-derived MSCs via forced telomerase expression after lentiviral transduction. These immortalized cell cultures demonstrate high proliferative potential (up to 40 passages), delayed senescence, as well as preserved primary culture-like functional activity (sensitivity to hormones, ability to hormonal sensitization and differentiation) and immunophenotype up to 17–26 passages. Meanwhile, primary adipose tissue-derived MSCs usually irreversibly lose their properties by 8–10 passages. Observed characteristics of reported immortalized human MSC cultures make them a feasible model for studying molecular mechanisms, which regulate the functional activities of these cells, especially when primary cultures or commercially available cell lines are not appropriate