Features of the Population of Mouse Peritoneal Macrophages Isolated after Stimulation with Concanavalin A and Thioglycolate.

Murine peritoneal macrophages isolated from the lavage fluid after administration of thioglycolate and concanavalin A are presented by two populations of cells of different diameters. Polarization of macrophages into a proinflammatory (M1) phenotype is accompanied by an increase in number of small cells. Macrophages obtained after administration of thioglycolate demonstrate higher tendency to anti-inflammatory (M2) phenotype, while macrophages isolated after administration of concanavalin A are committed in the proinflammatory direction. Lactate level is increased in M1 macrophages in comparison with M2 cells, which indicates predominance of glycolytic metabolism. Macrophages obtained after administration of concanavalin A have reduced mitochondrial potential, which reflects a tendency to apoptosis. Autophagy activation and inhibition neutralize the differences in pro- and anti-inflammatory properties of polarized macrophages obtained after thioglycolate administration, but have less pronounced effect on macrophages obtained after administration concanavalin A. Autophagy inhibitor increases mitochondrial potential in non-polarized macrophages obtained after administration of concanavalin A. These results demonstrate divergent properties of macrophages obtained after administration of glycolate and concanavalin A due to the difference in the mechanisms of experimental peritonitis.

Inhibitors of 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase (Statins) Suppress Differentiation and Reduce LPS/IFNγ-Induced Cytokine Production in Human Monocyte/Macrophage Culture.

We examined the effects of 72-h exposure to atorvastatin and rosuvastatin in concentrations of 2-10 nM on the cytokine expression in LPS/IFNγ-activated monocyte/macrophages derived from peripheral blood monocytes of healthy donors by culturing in the presence of GM-CSF. Pretreatment with statins was found to inhibit cytokine production in monocytes/macrophages after activation, while the level of cytokine mRNA in cells did not decrease. The number of cells containing active caspase-3 decreased in the culture. Culturing of monocytes/macrophages with statins was accompanied by changes in cell morphology and deceleration of cell growth. Cellular effects of "lipophilic" atorvastastin were observed at lower concentration compared to "hydrophilic" rosuvastatin.

[Study of the influence of etoxidol on expression of follistatin-like protein-1 (FSTL-1) in myocardium after experimental infarction in rats]. / Issledovanie vliianiia étoksidola na ékspressiiu follistatin-podobnogo belka-1 (FSTL-1) v miokarde posle éksperimental'nogo infarkta.

In heart attack, FSTL-1 is actively secreted by cardiomyocytes, accelerates growth of heart myofibrils and stimulates of vascular endothelial growth factor expression. The aim of this work was to investigate the effect of Etoxidol on synthesis of FSTL-1 in rats after myocardial infarction. The experiments were performed on Wistar rats weighing 250-350 g with simulated myocardial infarction or intact (group 5). Animals of control groups (groups 1, 2) were treated with saline for 7 and 14 days; Ethoxidol (24 mg/kg) was injected to animals of experimental groups (group 3, 4) (the daily dose was 6.36 mg/animal) for 6 or 14 days. The injection volume was 0.2 ml. At the beginning and at the end of the study plasma concentrations of FSTL-1 were determined by the ELISA method. Myocardial FSTL-1 gene expression was determined by real-time PCR. At the end of the experiments, the hearts were also used for histochemical analysis. To determine the size of the scar formed after the modeled heart attack, we used the classic Mallory staining method. The results show that the development of experimental acute myocardial infarction is accompanied by a significant increase in FSTL-1 expression in the heart, which was detected on the 7th day and stored increased by 14 days after a heart attack. After therapy with Ethoxidol, a tendency to a decrease in the expression of FSTL-1 by the 14th day was observed; it coincided with the dynamics of the plasma protein FSTL-1 level. It can be assumed that the downregulation trend in the FSTL-1 expression is associated with a more effective repair process after a heart attack, since FSTL-1 increases precisely in response to myocardial damage and decreases when the incentives for its expression from damaged heart tissue are reduced. Indirectly, this assumption is confirmed by the detected tendency to reduce the size of post-infarction fibrosis in the treatment with Ethoxidol. The results indicate the ability of Ethoxidol to influence FSTL-1 synthesis of in rats after myocardial infarction.

[Perspectives of cell therapy for myocardial infarction and heart failure based on cardiosphere cells].

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. In recent years, researchers are attracted to the use of cell therapy based on stem cell and progenitor cells, which has been a promising strategy for cardiac repair after injury. However, conducted research using intracoronary or intramyocardial transplantation of various types of stem/progenitor cells as a cell suspension showed modest efficiency. This is due to the low degree of integration and cell survival after transplantation. To overcome these limitations, the concept of the use of multicellular spheroids modeling the natural microenvironment of cells has been proposed, which allows maintaining their viability and therapeutic properties. It is of great interest to use so-called cardial spheroids (cardiospheres) spontaneously forming three-dimensional structures under low-adhesive conditions, consisting of a heterogeneous population of myocardial progenitor cells and extracellular matrix proteins. This review presents data on methods for creating cardiospheres, directed regulation of their properties and reparative potential, as well as the results of preclinical and clinical studies on their use for the treatment of heart diseases.

The Role of Urokinase, Tumor Necrosis Factor, and Matrix Metalloproteinase-9 in Monocyte Activation.

TNFα mediates the expression of MMP-9 in THP-1 monocytes induced by urokinase (uPA). Upregulation of MMP-9 caused by uPA and TNFα is suppressed by etanercept, a TNFα inhibitor. In addition, uPA stimulates TNFα mRNA expression. Both uPA and TNFα induce ROS generation in monocytes, while MMP-9 secretion induced by uPA and TNFα is inhibited by antioxidants. Inhibitors of NFκB, ligands of PPARα and PPARγ receptors, and SIRT1 activators negatively affect MMP-9 secretion induced by uPA. MMP-9 secretion during monocyte differentiation into macrophages is downregulated by etanercept and antioxidants. These factors as well as MMP inhibitor GM6001 reduce the number of macrophages attached to substrate during cell differentiation, indicating the role of urokinase, TNFα, and ROS in MMP expression in monocytes and MMP involvement in macrophage maturation.

Combined Action of GDNF and HGF Up-Regulates Axonal Growth by Increasing ERK1/2 Phosphorylation.

A stimulating effect of a combination of hepatocyte growth factor (HGF) and glial neurotrophic factor (GDNF) on the growth of neurites in the spinal ganglion model was demonstrated. The mechanism of neurite growth in the spinal ganglion model is associated with transactivation of HGF c-met receptor in the presence of both HGF and GDNF. The combination of HGF and GDNF significantly activated mitogenic signaling cascade mediated by protein kinases ERK1/2, which can be a mechanism for increasing the number of neurites. Our findings can be used for developing effective methods for restoring impaired peripheral nerve function after traumatic and ischemic injury using a combination of GDNF and HGF.

Urokinase Receptor Regulates Adhesion of Progenitor Cardiac Cells to Vitronectin.

Vitronectin, extracellular matrix protein, plays an important role in embryonic development and in organ and tissue reparation. A unique characteristic of vitronectin is specific binding of various biological molecules, including urokinase receptor (uPAR), extracellular matrix components, adhesion receptors, growth factors, thus supporting the modulation of cell behavior. Vitronectin is in fact not found in intact myocardium, while after infarction its level increases significantly, which correlates with accumulation of uPAR+ progenitor cardiac cells in the focus. The cells isolated from the heart of wild type mice are characterized by higher adhesion to vitronectin than progenitor cardiac cells from the myocardium of uPAR knockout mice. In addition, inhibition of urokinase receptor with specific antibodies on the surface of the progenitor cardiac cells of wild type mice leads to attenuation of their adhesive activity and flattening on vitronectin matrix, which can be important for their distribution in the postinfarction myocardium and realization of the reparative functions.

Application of Adeno-Associated Virus Vectors for Engineering SCF-Containing Extracellular Vesicles of Mesenchymal Stromal Cells.

Mesenchymal stromal cells from rat adipose tissue were transduced with adeno-associated viral (AAV) vector encoding stem cell factor SCF that stimulates proliferation of cardiac c-kit+ cells and improved cardiac function and survival of animals after myocardial infarction. Extracellular vesicles isolated from the medium conditioned by mesenchymal stromal cells by ultracentrifugation were characterized by Western blotting, transmission electron microscopy, nanoparticle tracking analysis, immunostaining, and mass spectrometry analysis. Using proteomic analysis, we identified transgenic SCF in extracellular vesicles released by AAV-modified mesenchymal stromal cells and detected some proteins specific of extracellular vesicles secreted by transduced cells. Extracellular vesicles from AAV-transduced mesenchymal stromal cells could be used for delivery of transgenic proteins as they were readily endocytosed by both cardiosphere-derived cells and cardiac-progenitor cells.

Modulation of the Inflammatory Status of Macrophages and Their Paracrine Effect on the Sensitivity of Adipocytes to Insulin with Sirtuin and PPARγ Receptor Activators.

We studied the effect of SIRT1 deacetylase and PPARγ receptor activators on proinflammatory (M1), anti-inflammatory (M2) polarization of RAW264.7 macrophages and their modulating effects on insulin sensitivity of adipocytes. In M1 macrophages, the expression of TNFα and CXCL9, secretion of CXCL11, ROS generation, and content of dendritic-like cells were elevated. In M2 macrophages, expression of IGF-1 and ALOX15 factors was enhanced. SIRT1 activator (DCHC) and PPARγ receptor ligand (rosiglitazone) reduced expression of inflammatory markers TNFα and CXCL9 and increased expression of IGF-1 and ALOX15. SIRT1 inhibitor Ex527 increased the proportion of dendritic cells in macrophage populations. The paracrine effect of M1-macrophage-conditioned media attenuated insulin-dependent phosphorylation of threonine (Thr308) in Akt kinase and enhanced phosphorylation of serine (Ser473). This effect was attenuated by DCHC and rosiglitazone.

Plasmid-based gene therapy with hepatocyte growth factor stimulates peripheral nerve regeneration after traumatic injury.

Peripheral nerve injury remains a common clinical problem with no satisfactory treatment options. Numerous studies have shown that hepatocyte growth factor (HGF) exerts neurotrophic effect in motor, sensory, and parasympathetic neurons in addition to mitogenic, morphogenic, angiogenic, antiapoptotic, antifibrotic, and anti-inflammatory effect on various tissues and cells. In our study we examined efficacy of gene therapy with HGF-bearing plasmid (pC4W-hHGF) to improve consequences of traumatic nerve injury in mice. Treatment by pC4W-hHGF led to restoration of nerve structure and functional recovery compared to similar parameters in control animals. Compound action potentials (CAP) in experimental groups treated with 100 or 200 µg of pC4W-hHGF demonstrated increased amplitude and latency decrease compared to spontaneous recovery control group. In HGF-treated mice histological analysis showed a three-fold increase in axon number in nerve portion located distal to the lesion site compared to control. Moreover, significant functional recovery of n. peroneus communis triggered by pC4W-hHGF gene therapy was observed using the footprints analysis. Obtained results provide evidence for plasmid-based HGF gene therapy as a potential treatment for traumatic injury of peripheral nerve.

Regulatory Effects of Urokinase on Mesenchymal Stromal Cell Migration, Proliferation, and Matrix Metalloproteinase Secretion.

We studied the effect of urokinase, its recombinant forms, and domain fragments on migration and proliferation of adipose tissue mesenchymal stromal cells (MSCs) and MMP secretion by these cells. Urokinase, but not its recombinant forms, slightly induced directed migration of MSCs. Spontaneous migration of MSCs increased under the action of urokinase or its isolated kringle domain. Migration induced by platelet-derived growth factor was inhibited by proteolytically inactive form of urokinase, the kringle domain, and blocking antibody to urokinase receptor. Urokinase, its proteolytically inactive form, and kringle domain produced no effect on MSC proliferation. In contrast to platelet-derived growth factor, all urokinase forms induced secretion of MMP-9 by MSCs.

ISOLATION AND CHARACTERIZATION OF CARDIAC PROGENITOR CELLS OBTAINING FROM MYOCARDIAL RIGHT ATRIAL APPENDAGE TISSUE.

Resident stem cells of the heart are denoted as heterogeneous population of immature cells, which reside in the myocardium and characterized by their ability to self-renewal and are multipotent differentiation capacity into cardiomyocyte-like and vascular like cells. CSCs were originally isolated directly by long enzymatic digestion of heart tissue and selection using stem cell markers. However, long exposure to enzymatic digestion and small myocardial sample size can affect the possibility of obtaining a significant amount of viable cells. To avoid these problems, we developed a method consisting of growing of the CPC in explant culture and subsequent immunomagnetic selection.

Urokinase stimulates production of matrix metalloproteinase-9 in fibroblasts with involvement of reactive oxygen species.

In cultured fibroblasts, urokinase stimulated expression of MMP-9 and generation of ROS, while antioxidant ebselen abolished the stimulating effect of urokinase on MMP-9 expression. sTNF-α produced similar and more pronounced stimulating effect. The data showed that urokinase could regulate MMP-9 expression via ROS generation in fibroblasts, which can play an important role in stimulation of their migration and development of constrictor (negative) vascular remodeling due to thickening of the adventitia.

Proteolytically inactive recombinant forms of urokinase suppress migration of endothelial cells.

Proteolytically inactive recombinant forms of urokinase (uPAHQ and amino-terminal fragment) inhibit spontaneous migration of endothelial cells; amino-terminal fragment also suppresses angiogenesis stimulated by basic fibroblast growth factor in vitro. These findings suggest the possibility of using synthesized proteolytically inactive recombinant forms of urokinase for the regulation of endothelial cell migration and suppression of neoangiogenesis.

[Recombinant human alpha-fetoprotein as a regulator of adipose tissue stromal cell activity].

Recombinant human alpha-fetoprotein (rhAFP) expressed in yeast system as a glycoprotein, was isolated and purified to 98% by multistep method. The testing of the rhAFP in the culture of adipose tissue stromal cells (hASC) has revealed its ability to enhance hASC proliferation and migration as well as vascular endothelial growth factor production, with no significant influence on cell invasion and matrix metalloproteinase-2 and -9 secretion. It has been also estimated that rhAFP is internalized in hASC via clathrin-dependent mechanism. A study in the murine experimental model of hindlimb ischemia has shown the capability of rhAFP to enhance blood flow recovery. These data suggest that rhAFP is a promising agent for enhancement of the hASC regenerative ability.

[Principles of constructing synbiotic preparations, and evaluation of their clinical effectiveness].

The problem of constructing means of correction of gastrointestinal microflora is utterly important. The most promising direction here seems to be construction of synbiotics containing a multiprobiotic complex of bifidobacteria and lactobacilli, modeling the microbiocenosis of a certain age group, inulin, as a prebiotic factor and a vitamin and mineral complex. The aim of the study was to evaluate the biologically active food supplement "Normospectrum" vs. the commercial preparation "Bifidumbacterin", its ability to correct the intestinal microflora and the functional condition of the gastrointestinal tract in children. "Normospectrum" was well tolerated, favored regression of the main clinical manifestations, and had positive effect on the intestinal microbiocenosis, increasing the proportion of bifidobacteria, lactobacilli and escherichiae with full enzymatic activity, and lowering the proportion of conditionally pathogenic bacteria and fungi in the intestinal tract. The effects of "Normospectrum" on the functional disturbances of the gastrointestinal tract and the intestinal microflora were much more profound than those of "Bifidumbacterin". Good tolerance and positive effects of the new probiotic preparation "Normospectrum" on the intestinal microbiosis allows recommending it for broad clinical application in children when correction of dysbiotic shifts in the intestinal microflora is necessary.

[Experience of pediatric application of bifidumbacterin multi, a biologically active food supplement].

The article is dedicated to a clinical trial of Bifidumbacterin Multi-1, a biologically active food supplement (BAFS) for children aged 0 to 3 years. The study showed that application of BAFS in children with intestinal dysbiosis helped to cope with the symptoms of alimentary disturbances, promoted improvement of body mass index, increase of hemoglobin end erythrocyte level, suppression of zymotic and putrefactive dyspepsia, decrease of inflammation of the intestinal mucosa, elimination of bibidobacterial deficit, and reduction of conditionally pathogenic microflora. Bifidumbacterin Multi-1 was well tolerated and highly effective according to clinical and laboratory data. This preparation may be recommended as a component of complex therapy in children aged 0 to 3 years old.