Stem-Cell-Derived Extracellular Vesicles: Unlocking New Possibilities for Treating Diminished Ovarian Reserve and Premature Ovarian Insufficiency.

Despite advancements in assisted reproductive technology (ART), achieving successful pregnancy rates remains challenging. Diminished ovarian reserve and premature ovarian insufficiency hinder IVF success-about 20% of in vitro fertilization (IVF) patients face a poor prognosis due to a low response, leading to higher cancellations and reduced birth rates. In an attempt to address the issue of premature ovarian insufficiency (POI), we conducted systematic PubMed and Web of Science research, using keywords "stem cells", "extracellular vesicles", "premature ovarian insufficiency", "diminished ovarian reserve" and "exosomes". Amid the complex ovarian dynamics and challenges like POI, stem cell therapy and particularly the use of extracellular vesicles (EVs), a great potential is shown. EVs trigger paracrine mechanisms via microRNAs and bioactive molecules, suppressing apoptosis, stimulating angiogenesis and activating latent regenerative potential. Key microRNAs influence estrogen secretion, proliferation and apoptosis resistance. Extracellular vesicles present a lot of possibilities for treating infertility, and understanding their molecular mechanisms is crucial for maximizing EVs' therapeutic potential in addressing ovarian disorders and promoting reproductive health.

Phytohormones Affect Differentiation Status of Human Skin Fibroblasts via UPR Activation.

Normalization of secretory activity and differentiation status of mesenchymal cells, including fibroblasts, is an important biomedical problem. One of the possible solutions is modulation of unfolded protein response (UPR) activated during fibroblast differentiation. Here, we investigated the effect of phytohormones on the secretory activity and differentiation of cultured human skin fibroblasts. Based on the analysis of expression of genes encoding UPR markers, abscisic acid (ABA) upregulated expression of the GRP78 and ATF4 genes, while gibberellic acid (GA) upregulated expression of CHOP. Evaluation of the biosynthetic activity of fibroblasts showed that ABA promoted secretion and synthesis of procollagen I and synthesis of fibronectin, as well as the total production of collagen and non-collagen proteins of the extracellular matrix (ECM). ABA also stimulated the synthesis of smooth muscle actin α (α-SMA), which is the marker of myofibroblasts, and increased the number of myofibroblasts in the cell population. On the contrary, GA increased the level of fibronectin secretion, but reduced procollagen I synthesis and the total production of the ECM collagen proteins. GA downregulated the synthesis of α-SMA and decreased the number of myofibroblasts in the cell population. Our results suggest that phytohormones modulate the biosynthetic activity of fibroblasts and affect their differentiation status.

Changes of discharge properties of neurons from dorsomedial hypothalamic nuclei during aging in rats.

The hypothalamus is a vital brain center that is participated in the integration of the endocrine and nervous systems and control of the homeostasis and aging. Spontaneous firing activity from single neurons of the dorsomedial hypothalamic nucleus (DMN) was studied extracellularly in vivo in urethane-anaesthetized rats. The discharge patterns of the majority of DMN neurons were irregular, including periods of relatively stable activity interrupted by pauses. Based on the features of interval interspike histogram, we have selected neurons with an irregular arrhythmic activity (50% in young, 46% in adult and 44% in aged rats), with a constant rhythmic activity (18% of neurons in young, 19% in adult and 23% in aged rats), with a wide interspike interval distribution (22% in young, 26% in adult and 25% in aged rats) and cells with bursts of two or three spikes (10% in young, 9% in adult and 8% in aged rats). The firing rate of DMN neurons was 2.5 ± 0.12 Hz in young and 2.4 ± 0.21 Hz in adult rats and significantly decreased to 1.8 ± 0.17 Hz in aged rats.

Expression of calbindin and calretinin in the dorsomedial and ventromedial hypothalamic nuclei during aging.

The hypothalamus is involved in the regulation of rhythms, autonomic, endocrine, and behavioral functions and may also participate in aging development and control. The aim of this work was to study the expression of calbindin (CB) and calretinin (CR) in the ventromedial (VMH) and dorsomedial (DMH) hypothalamic nuclei in young and old rats of both sexes by immunohistochemistry and western blotting. In young animals, the largest number of CB-immunoreactive (IR) neurons was detected in the ventral part of DMH (DMHv) and smaller percentage was found in its dorsal part (DMHd), in the dorsomedial part of the VMH (VMHdm) and in the ventrolateral part of the VMH (VMHvl). In aged animals, the percentage of CB-IR neurons significantly decreased in all studied nuclei, including DMHv, DMHd, VMHdm and VMHvl. CR-IR neurons were found in moderate number in the DMHv, DMHd, VMHdm and VMHvl of young rats. In aged rats, the percentage of CR-IR neurons significantly increased in the DMHv, DMHd, VMHdm and VMHvl. Less than one third of IR neurons colocalized CB and CR in young and aged rats. The expression of CB significantly decreased, and the expression of CR significantly increased in the DMH and VMH during aging by western blot analysis. Thus, there are opposite changes of the calcium-binding proteins expression in the hypothalamic nuclei involved in the metabolic and sexual regulation during aging.

In Vitro and in Vivo Analysis of Adhesive, Anti-Inflammatory, and Proangiogenic Properties of Novel 3D Printed Hyaluronic Acid Glycidyl Methacrylate Hydrogel Scaffolds for Tissue Engineering.

In this study, we prepared hydrogel scaffolds for tissue engineering by computer-assisted extrusion three-dimensional (3D) printing with photocured (λ = 445 nm) hyaluronic acid glycidyl methacrylate (HAGM). The developed product was compared with the polylactic-co-glycolic acid (PLGA) scaffolds generated by means of the original antisolvent 3D printing methodology. The cytotoxicity and cytocompatibility of the scaffolds were analyzed in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests, flow cytometry, and scanning electron microscopy. Anti-inflammatory and proangiogenic properties of the scaffolds were evaluated in the dorsal skinfold chamber mouse model by means of intravital fluorescence microscopy, histology, and immunohistochemistry throughout an observation period of 14 days. In vitro, none of the scaffolds revealed cytotoxicity on days 1, 2, and 5 after seeding with umbilical cord-derived multipotent stromal cells, and the primary cell adhesion to the surface of HAGM scaffolds was low. In vivo, implanted HAGM scaffolds showed enhanced vascularization and host tissue ingrowth, and the inflammatory response to them was less pronounced compared with PLGA scaffolds. The results indicate excellent biocompatibility and vascularization capacity of the developed 3D printed HAGM scaffolds and position them as strong candidates for advanced tissue engineering applications.

Macrophage Modification Strategies for Efficient Cell Therapy.

Macrophages, important cells of innate immunity, are known for their phagocytic activity, capability for antigen presentation, and flexible phenotypes. Macrophages are found in all tissues and therefore represent an attractive therapeutic target for the treatment of diseases of various etiology. Genetic programming of macrophages is an important issue of modern molecular and cellular medicine. The controllable activation of macrophages towards desirable phenotypes in vivo and in vitro will provide effective treatments for a number of inflammatory and proliferative diseases. This review is focused on the methods for specific alteration of gene expression in macrophages, including the controllable promotion of the desired M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes in certain pathologies or model systems. Here we review the strategies of target selection, the methods of vector delivery, and the gene editing approaches used for modification of macrophages.

Molecular mechanisms of splenectomy-induced hepatocyte proliferation.

Functional and anatomical connection between the liver and the spleen is most clearly manifested in various pathological conditions of the liver (cirrhosis, hepatitis). The mechanisms of the interaction between the two organs are still poorly understood, as there have been practically no studies on the influence exerted by the spleen on the normal liver. Mature male Sprague-Dawley rats of 250-260 g body weight, 3 months old, were splenectomized. The highest numbers of Ki67+ hepatocytes in the liver of splenectomized rats were observed at 24 h after the surgery, simultaneously with the highest index of Ki67-positive hepatocytes. After surgical removal of the spleen, expression of certain genes in the liver tissues increased. A number of genes were upregulated in the liver at a single time point of 24 h, including Ccne1, Egf, Tnfa, Il6, Hgf, Met, Tgfb1r2 and Nos2. The expression of Ccnd1, Tgfb1, Tgfb1r1 and Il10 in the liver was upregulated over the course of 3 days after splenectomy. Monitoring of the liver macrophage populations in splenectomized animals revealed a statistically significant increase in the proportion of CD68-positive cells in the liver (as compared with sham-operated controls) detectable at 24 h and 48 h after the surgery. The difference in the liver content of CD68-positive cells between splenectomized and sham-operated animals evened out by day 3 after the surgery. No alterations in the liver content of CD163-positive cells were observed in the experiments. A decrease in the proportion of CD206-positive liver macrophages was observed at 48 h after splenectomy. The splenectomy-induced hepatocyte proliferation is described by us for the first time. Mechanistically, the effect is apparently induced by the removal of spleen as a major source of Tgfb1 (hepatocyte growth inhibitor) and subsequently supported by activation of proliferation factor-encoding genes in the liver.

Changes of nNOS expression in the tuberal hypothalamic nuclei during ageing.

The hypothalamus is the most important integrator of autonomic and endocrine regulation in the body and it also has a fundamental role in ageing development and lifespan control. In order to better understand the role of NO-ergic system in the hypothalamic regulation of ageing, the purpose of this study was to investigate the expression of neuronal nitric oxide synthase (nNOS) in the arcuate (ARC), ventromedial (VMH) and dorsomedial (DMH) hypothalamic nuclei in young (2-3-month-old) and old (24-month-old) male and female rats using immunohistochemistry and western blot analysis. In young animals, only single nNOS-immunoreactive (IR) neurons were detected in ARC, and nNOS-IR neurons were found in the VMH (19 ± 3.2% in females and 14.5 ± 2.6% in males) and DMH (17 ± 4.0% in females and 21 ± 2.8% in males). In aged animals, the number of nNOS-IR neurons increased in all studied nuclei, including ARC (36 ± 3.1% in females and 33.5 ± 3.7% in males), VMH (83 ± 4.3% in females and 58 ± 2.1% in males) and DMH (57 ± 1.9% in females and 54 ± 1.8% in males). The expression of nNOS also significantly increased in the ARC, VMH and DMH during ageing by western blot analysis. In conclusion, ageing is accompanied by increasing of nNOS expression in the hypothalamus and this process is related to regions involved in the control of feeding behavior.

Mitochondrial Calcium Uniporter Structure and Function in Different Types of Muscle Tissues in Health and Disease.

Calcium ions (Ca2+) influx to mitochondrial matrix is crucial for the life of a cell. Mitochondrial calcium uniporter (mtCU) is a protein complex which consists of the pore-forming subunit (MCU) and several regulatory subunits. MtCU is the main contributor to inward Ca2+ currents through the inner mitochondrial membrane. Extensive investigations of mtCU involvement into normal and pathological molecular pathways started from the moment of discovery of its molecular components. A crucial role of mtCU in the control of these pathways is now recognized in both health and disease. In particular, impairments of mtCU function have been demonstrated for cardiovascular and skeletal muscle-associated pathologies. This review summarizes the current state of knowledge on mtCU structure, regulation, and function in different types of muscle tissues in health and disease.

Phenotypical and Functional Polymorphism of Liver Resident Macrophages.

Liver diseases are one of the main causes of mortality. In this regard, the development of new ways of reparative processes stimulation is relevant. Macrophages play a leading role in the regulation of liver homeostasis in physiological conditions and in pathology. In this regard, the development of new liver treatment methods is impossible without taking into account this cell population. Resident macrophages of the liver, Kupffer cells, represent a unique cell population, first of all, due to their development. Most of the liver macrophages belong to the self-sustaining macrophage cell population, whose origin is not bone marrow. In addition, Kupffer cells are involved in such processes as regulation of hepatocyte proliferation and apoptosis, remodeling of the intercellular matrix, lipid metabolism, protective function, etc. Such a broad spectrum of liver macrophage functions indicates their high functional plasticity. The review summarizes recent data on the development, phenotypic and functional plasticity, and participation in the reparative processes of liver macrophages: resident macrophages (Kupffer cells) and bone marrow-derived macrophages.

Gestation age-associated dynamics of mitochondrial calcium uniporter subunits expression in feto-maternal complex at term and preterm delivery.

Calcium plays a role of universal cellular regulator in the living cell and one of the crucial regulators of proper fetal development during gestation. Mitochondria are important for intracellular calcium handling and signaling. Mitochondrial calcium uniporter (mtCU) is a multiprotein complex of the mitochondrial inner membrane responsible for the transport of calcium to the mitochondrial matrix. In the present study, we analyzed the expression level of mtCU components in two parts of the feto-maternal system - placenta and myometrium at full-term delivery and at preterm birth (PTB) on different stages: 22-27, 28-32, 33-36 weeks of gestation (n = 50). A gradual increase of mRNA expression and changes in protein content of MCU and MICU1 subunits were revealed in the placenta during gestation. We also observed slower depolarization rate of isolated placental mitochondria induced by Ca2+ titration at PTB. In myometrium at PTB relative gene expression level of MCU, MCUb and SMDT1 increased as compared to full-term pregnancy, but the tendency to gradual increase of MCU protein simultaneous with MCUb increase and MICU1 decline was shown in gestational dynamics. Changes observed in the present study might be considered both natural dynamics as well as possible pathological mechanisms underlying preterm birth.

Alterations in antioxidant system, mitochondrial biogenesis and autophagy in preeclamptic myometrium.

Preeclampsia is a pregnancy complication which causes significant maternal and fetal morbidity and mortality worldwide. Although intensive research has been performed in the last 40 years, the pathology of preeclampsia is still poorly understood. The present work is a comparative study of the myometrium of women with normal pregnancy, and those with late- and early-onset preeclampsia (n = 10 for each group). We observed significant changes in the levels of antioxidant enzymes, markers of mitochondrial biogenesis and autophagy proteins in preeclamptic myometrium. Levels of superoxide dismutase 1 and catalase were lower in both preeclamptic groups than the control group. In late-onset preeclampsia, expression levels of essential mitochondria-related proteins VDAC1, TFAM, hexokinase 1, PGC-1α and PGC-1ß, and autophagy marker LC3A, were significantly elevated. In the myometrium of the early-onset preeclampsia group OPA1 and Bcl-2 were up-regulated compared to those of the control (p < 0.05). These findings suggest that crucial molecular changes in the maternal myometrium occur with the development of preeclampsia.

Mitochondrial role in adaptive response to stress conditions in preeclampsia.

Preeclampsia (PE) is a pregnancy-specific syndrome, characterized in general by hypertension with proteinuria or other systemic disturbances. PE is the major cause of maternal and fetal morbidity and mortality worldwide. However, the etiology of PE still remains unclear. Our study involved 38 patients: 14 with uncomplicated pregnancy; 13 with early-onset PE (eoPE); and 11 with late-onset PE (loPE). We characterized the immunophenotype of cells isolated from the placenta and all biopsy samples were stained positive for Cytokeratin 7, SOX2, Nestin, Vimentin, and CD44. We obtained a significant increase in OPA1 mRNA and protein expression in the eoPE placentas. Moreover, TFAM expression was down-regulated in comparison to the control (p < 0.01). Mitochondrial DNA copy number in eoPE placentas was significantly higher than in samples from normal pregnancies. We observed an increase of maximum coupled state 3 respiration rate in mitochondria isolated from the placenta in the presence of complex I substrates in the eoPE group and an increase of P/O ratio, citrate synthase activity and decrease of Ca(2+)-induced depolarization rate in both PE groups. Our results suggest an essential role of mitochondrial activity changes in an adaptive response to the development of PE.

Ganglioside GD2 in reception and transduction of cell death signal in tumor cells.

BACKGROUND: Ganglioside GD2 is expressed on plasma membranes of various types of malignant cells. One of the most promising approaches for cancer immunotherapy is the treatment with monoclonal antibodies recognizing tumor-associated markers such as ganglioside GD2. It is considered that major mechanisms of anticancer activity of anti-GD2 antibodies are complement-dependent cytotoxicity and/or antibody-mediated cellular cytotoxicity. At the same time, several studies suggested that anti-GD2 antibodies are capable of direct induction of cell death of number of tumor cell lines, but it has not been investigated in details. In this study we investigated the functional role of ganglioside GD2 in the induction of cell death of multiple tumor cell lines by using GD2-specific monoclonal antibodies. METHODS: Expression of GD2 on different tumor cell lines was analyzed by flow cytometry using anti-GD2 antibodies. By using HPTLC followed by densitometric analysis we measured the amount of ganglioside GD2 in total ganglioside fractions isolated from tumor cell lines. An MTT assay was performed to assess viability of GD2-positive and -negative tumor cell lines treated with anti-GD2 mAbs. Cross-reactivity of anti-GD2 mAbs with other gangliosides or other surface molecules was investigated by ELISA and flow cytometry. Inhibition of GD2 expression was achieved by using of inhibitor for ganglioside synthesis PDMP and/or siRNA for GM2/GD2 and GD3 synthases. RESULTS: Anti-GD2 mAbs effectively induced non-classical cell death that combined features of both apoptosis and necrosis in GD2-positive tumor cells and did not affect GD2-negative tumors. Anti-GD2 mAbs directly induced cell death, which included alteration of mitochondrial membrane potential, induction of apoptotic volume decrease and cell membrane permeability. This cytotoxic effect was mediated exclusively by specific binding of anti-GD2 antibodies with ganglioside GD2 but not with other molecules. Moreover, the level of GD2 expression correlated with susceptibility of tumor cell lines to cytotoxic effect of anti-GD2 antibodies. CONCLUSIONS: Results of this study demonstrate that anti-GD2 antibodies not only passively bind to the surface of tumor cells but also directly induce rapid cell death after the incubation with GD2-positive tumor cells. These results suggest a new role of GD2 as a receptor that actively transduces death signal in malignant cells.