TGFß1 Regulates Cellular Composition of In Vitro Cardiac Perivascular Niche Based on Cardiospheres.

The cardiac perivascular niche is a cellular microenvironment of a blood vessel. The principles of niche regulation are still poorly understood. We studied the effect of TGFß1 on cells forming the cardiac perivascular niche using 3D cell culture (cardiospheres). Cardiospheres contained progenitor (c-Kit), endothelial (CD31), and mural (αSMA) cells, basement membrane proteins (laminin) and extracellular matrix proteins (collagen I, fibronectin). TGFß1 treatment decreased the length of CD31+ microvasculature, VE cadherin protein level, and proportion of NG2+ cells, and increased proportion of αSMA+ cells and transgelin/SM22α protein level. We supposed that this effect is related to the stabilizing function of TGFß1 on vascular cells: decreased endothelial cell proliferation, as shown for HUVEC, and activation of mural cell differentiation.

Culturing of Cardiac Cells in 3D Spheroids Modulates Their Expression Profile and Increases Secretion of Proangiogenic Growth Factors.

We studied the effect of 3D-culturing of cells in the form of cardiospheres on the expression of genes encoding vascular progenitor cell markers and angiogenesis regulators and on the production of proangiogenic factors. Cardiospheres were obtained by culturing mouse cardiac explants followed by self-assembly on poly-D-lysine. Gene expression was assessed by real-time PCR, and the production of proangiogenic factors was assessed by Microarray analysis of the cell secretome. It was found that cells in the cardiospheres in comparison with 2D-culture of cardiosphere-forming cells demonstrated increased expression of vascular progenitor cell markers (Pdgfrα, Kit, and Vegfr1) and angiogenesis regulatory factors (Vegf, Fgf2, and Angpt1), as well as an enhanced secretion of proangiogenic factors (ANGPT1, VEGF, CXCL16, and PIGF-2). Thus, culturing of cells in the form of cardiospheres can be considered as a basis for developing approaches to increasing their angiogenic activity and regenerative properties.

Deficiency of Urokinase-Type Plasminogen Activator Receptor Is Associated with the Development of Perivascular Fibrosis in Mouse Heart.

It was suggested that the urokinase system plays a certain role in the regulation of activity of the endothelial-mesenchymal transition and in the development of perivascular fibrosis. Urokinase (uPA), the key component of the urokinase system, is a serine protease that binds to its receptor on the cell surface (uPAR) and affects the cell microenvironment components through the formation of plasmin, remodeling of the extracellular matrix, release of growth factors, and initiation of intracellular signals. The heart of PLAUR gene knockout C57BL/129 (uPAR-/-) mice showed signs of vasculopathy: reduced number of capillaries/arterioles, signs of endothelial-mesenchymal transition in endothelial cells, vascular wall remodeling, and deposition of extracellular matrix components. These changes were combined with enhanced expression of urokinase and active forms of TGF-ß1. Apparently, uPAR is a part of a multicomponent system that provides multifaceted regulatory effects on the components of forming vessels and vascular wall cells, which allows considering it as a possible target for targeted antifibrotic therapy.

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.

Hypoxia - as a Possible Regulator of the Activity of Epicardial Mesothelial Cells After Myocardial Infarction.

Aim      To study the effect of hypoxia on the activity of epithelial-mesenchymal transition (EMT) in epicardial cells, which provides formation of a specialized microenvironment.Material and methods   This study used a model of experimental myocardial infarction created by ligation of the anterior descendent coronary artery. The activity of epicardial cells after a hypoxic exposure was studied with the hypoxia marker, pimonidazole, bromodeoxyuridine, immunofluorescent staining of heart cryosections, and in vitro mesothelial cell culture.Results The undamaged heart maintained the quiescent condition of mesothelial cells and low levels of their proliferation, extracellular matrix protein production, and of the EMT activity. Acute ischemic injury induced moderate hypoxia in the epicardial/subepicardial region. This caused a global rearrangement of this region due to the initiation of EMT in cells, changes in the cell composition, and accumulation of extracellular matrix proteins. We found that the initiation of EMT in mesothelial cells may result in the formation of smooth muscle cell precursors, fibroblasts, and a population of Sca-1+ cardiac progenitor cells, which may both participate in construction of new blood vessels and serve as a mesenchymal link for the paracrine support of microenvironmental cells. In in vitro experiments, we showed that 72­h hypoxia facilitated activation of EMT regulatory genes, induced dissembling of intercellular contacts, cell uncoupling, and increased cell plasticity.Conclusion      The epicardium of an adult heart serves as a "reparative reserve" that can be reactivated by a hypoxic exposure. This creates a basis for an approach to influence the epicardium to modulate its activity for regulating reparative processes.

Transforming Growth Factor Beta-1 (TGF-ß1) Regulates Assembly of Cardiac Spheroids.

Cells of all tissues in human body interact with their neighboring cells and components of the extracellular matrix thereby creating a unique 3D microenvironment. These interactions are realized through a complex network of biochemical and mechanical signals that are important in maintaining normal cellular homeostasis. Numerous attempts have been undertaken during the last two decades to develop 3D models for studying their properties and understanding the mechanisms of regulation of cell microenvironment in vivo. Cardiac spheroids (cardiospheres) are one these models of cardiac microenvironment. In this study we demonstrate that unique microenvironment formed in cardiospheres consists of stem/progenitor and mesenchymal cells surrounded by extracellular matrix proteins synthesized by these cells. TGF-ß1 participates in the regulation of contraction of cells forming cardiospheres, promotes activation of the epithelial-mesenchymal transition and self-organization of cells, which leads to the formation of larger spheroids. Thereby, the effect of TGF-ß1 on the cells of cardiospheres can serve as a model for studying the mechanisms of regulation of cardiac microenvironment.

Transforming Growth Factor Beta (TGF-ß1) Induces Pro-Reparative Phenotypic Changes in Epicardial Cells in Mice.

We evaluated the content of active form of TGF-ß1 in the intact and post-infarction heart and the effect of this factor on the properties of epicardial cells. During the acute stage after myocardial infarction, the production of TGF-ß1 in the heart increased, which closely correlated with activation of epicardial cells (appearance of a pool of Wt1+ epicardial cells entering the epithelial-mesenchymal transition). The role of TGF-ß1 as the factor of epicardial activation was confirmed by the results of in vitro experiments: addition of recombinant TGF-ß1 to cultured epicardial cells led to enhanced expression of genes of epithelial-mesenchymal transition and phenotypic transformation of these cells leading to the appearance of cells with markers of smooth muscle cells and fibroblasts. Our findings suggest that the regulatory axis "TGF-ß1-epicardium cells" can be considered as an important link of the post-infarction reparative process and adaptive response during heart remodeling after myocardial infarction and as the target for therapeutic interventions.

[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.

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.

[Epicardial Transplantation of Cardiac Progenitor Cells Based Cells Sheets is More Promising Method for Stimulation of Myocardial Regeneration, Than Conventional Cell Injections].

Today, transplantation of stem / progenitor cells is a promising approach for the treatment of heart diseases. The therapeutic potential of transplanted cells directly depends on the method of delivery to the myocardium, which determines their regenerative properties. It is important for the development of effective methods of cell therapy. In this paper, we performed a comparative study of efficacy of cardiac progenitor cell (CPC) transplantation by intramyocardial needle injections and by tissue engineering constructs (TEC) - "cell sheets" consisting of cells and their extracellular matrix. It has been shown, that transplantation of TEC in comparison with the intramyocardial delivery provides more extensive distribution and retains more proliferating cellular elements in the damaged myocardium, attenuates the negative cardiac remodeling of the left ventricle and promotes its vascularization.

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.

Combination of Mesenchymal Stromal Cells and Cardiac Stem Cells in a Multilayer Cell Construct Promotes Activation of Notch Signaling and Initiation of Endothelial Differentiation.

We showed the possibility of generating combined tissue-engineered cell consisting of layers of rat cardiac stem cells and mesenchymal stromal cells from the adipose tissue. Cell-cell interaction within the cell sheet promoted proliferation of cardiac stem cells, expression of endothelial differentiation marker ETS1, and Notch signaling activation. The obtained results provide new insights into possible mechanisms of stimulation of endogenous regeneration processes after myocardial damage and demonstrate potential of cell-based cardiomyoplasty with the use of these combined cell sheets.

C-Kit Cardiac Progenitor Cell Based Cell Sheet Improves Vascularization and Attenuates Cardiac Remodeling following Myocardial Infarction in Rats.

The adult heart contains small populations of multipotent cardiac progenitor cells (CPC) that present a convenient and efficient resource for treatment of myocardial infarction. Several clinical studies of direct CPC delivery by injection have already been performed but showed low engraftment rate that limited beneficial effects of procedure. «Cell sheet¼ technology has been developed to facilitate longer retention of grafted cells and show new directions for cell-based therapy using this strategy. In this study we hypothesized that СPC-based cell sheet transplantation could improve regeneration after myocardial infarction. We demonstrated that c-kit+ CPC were able to form cell sheets on temperature-responsive surfaces. Cell sheet represented a well-organized structure, in which CPC survived, retained ability to proliferate, expressed progenitor cell marker Gata-4 formed connexin-43+ gap junctions, and were surrounded by significant amount of extracellular matrix proteins. Transplantation of cell sheets after myocardial infarction resulted in CPC engraftment as well as their proliferation, migration, and differentiation; cell sheets also stimulated neovascularization and cardiomyocyte proliferation in underlining myocardium and ameliorated left ventricular remodeling. Obtained data strongly supported potential use of CPC sheet transplantation for repair of damaged heart.

Notch signal pathway - therapeutic target for regulation of reparative processes in the heart.

Notch signaling pathway is a universal regulator of cell fate in embryogenesis and in maintaining the cell homeostasis of adult tissue. Through local cell-cell interactions, he controls neighboring cells behavior and determines their capacity for self-renewal, growth, survival, differentiation, and apoptosis. Recent studies have shown that the control of regenerative processes in the heart is also carried out with the participation of Notch system. At the heart of Notch regulates migration bone marrow progenitors and stimulates the proliferation of cardiomyocytes, cardiac progenitor cell activity, limits cardiomyocyte hypertrophy and fibrosis progression and stimulates angiogenesis. Notch signaling pathway may be regarded as a very promising target for the development of drugs for the stimulation of regeneration in the myocardium.

Multifaced Roles of the Urokinase System in the Regulation of Stem Cell Niches.

Proliferation, subsequent migration to the damaged area, differentiation into appropriate cell types, and/or secretion of biologically active molecules and extracellular vesicles are important processes that underlie the involvement of stem/progenitor cells in the repair and regeneration of tissues and organs. All these functions are regulated through the interaction between stem cells and the microenvironment in the tissue cell niches that control these processes through direct cell-cell interactions, production of the extracellular matrix, release of extracellular vesicles, and secretion of growth factors, cytokines, chemokines, and proteases. One of the most important proteolytic systems involved in the regulation of cell migration and proliferation is the urokinase system represented by the urokinase plasminogen activator (uPA, urokinase), its receptor (uPAR), and inhibitors. This review addresses the issues of urokinase system involvement in the regulation of stem cell niches in various tissues and analyzes the possible effects of this system on the signaling pathways responsible for the proliferation, programmed cell death, phenotype modulation, and migration properties of stem cells.

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.

[Clinical characteristics of patients and number of stem cells in the myocardium of right atrial appendage in patients with ischemic heart disease].

In the last years stem cells (SC) have been identified in rodent and human hearts. These cells have ability to multilineage differentiation in vitro and in vivo and improve cardiac function. The development of new methods of isolation SC offers new approaches to cardiac regeneration. However, the question of how individual patient characteristics influence the number of SC remains unclear. In our study we aimed to define the correlation between patient characteristics and SC number. Our findings suggest that clinical characteristics and severity of the disease may affect the yield of SC in heart tissue. Our data contribute to the development of efficient methods for SC isolation for stem cell therapy.

[Resident cardiac stem cells].

The search for sources of stem/progenitor cells the use of which has a potential to affect course of ischemic heart disease and chronic heart failure is conducted nowadays in many countries. Resident cardiac stem cells (CSC) were revealed during recent years on the basis of expression of c-kit, sca-1, MDR1, and islet-1 markers. In vitro experiments demonstrated possibility of their differentiation into cardiomyocytes, smooth muscle cell and endothelial cells. Introduction of CSC in injured myocardium in animals facilitated its partial repair and short term improvement of cardiac function. This holds promise for the use of these cells in the future. In the review we have attempted to summarize literature data on resident CSC and their application for the treatment of heart diseases.