V. Yu. Sysoeva

T-cadherin suppresses the cell proliferation of mouse melanoma B16F10 and tumor angiogenesis in the model of the chorioallantoic membrane

The influence of T-cadherin on the pigmentation and proliferation of mouse melanoma B16F10 cells in vitro and on the growth and neovascularization of tumor cell masses formed by the B16F10 cells in a model of the chorioallantoic membrane of a chicken embryo is studied. It is found that the proliferative activity of the cells decreases in the cell culture of mouse melanoma upon the overexpression of T-cadherin in comparison with the cells in the control. It is shown in experiments in vitro that the B16F10 cells with the overexpression of T-cadherin are rarely settling are to the chorioallantoic membrane than the control cells. In addition, it is found that the control cells of mouse melanoma form tumors with area more 0.1 mm2 more often than the cells with the overexpression of T-cadherin and the amount of the vessels growing to tumor cell masses formed by the cells with the overexpression of T-cadherin is significantly lower than the same index for the cells in the control. Thus, the overexpression of T-cadherin in the B16F10 cells suppresses the proliferation of these cells in vitro and the growth of the tumor masses formed by melanoma cells on the chorioallantoic membrane and their neovascularization in vivo.

Increased expression of uPA, uPAR, and PAI-1 in psoriatic skin and in basal cell carcinomas

There is substantial evidence implicating the urokinase system in tissue remodeling during neo-vascularization, inflammation, tumor invasion, and metastasis. Regulated degradation of the extracellular matrix at the leading edge of migrating cells, mediated by uPA and uPAR, is required for tissue remodeling, invasiveness, and angiogenesis. Psoriasis and basal cell carcinoma (BCC) are the most common skin diseases. Pathogenesis of both of them is associated with keratinocyte hyperproliferation, inflammatory cell migration, and angiogenesis—processes in which the plasminogen system (uPA, uPAR, tPA, and PAI-1) plays a crucial role. In the present study, the comparative analysis of uPA, uPAR, tPA, and PAI-1 expression in the normal skin, in the biopsies of patients with psoriasis vulgaris, and BCC was carried out. uPA, uPAR, and PAI-1 expression was up-regulated in the epidermis of psoriatic skin and in tumor cells in BCC. Increased uPAR expression was detected in the derma of psoriatic lesions and in the stroma surrounding tumor cells in BCC. Increased expression of uPA in epidermal cells in psoriasis and in tumor cells in BCC suggests an important role of the uPA system for aggressively proliferating and invading cells of epidermal origin. A possible activation of the stroma, as a result of uPA–uPAR interaction between tumor cells and the surrounding stroma, is suggested.

The role of urokinase in vascular cell migration and in regulation of growth and branching of capillaries

The urokinase system, represented by a plasminogen activator of urokinase type (urokinase, uPA), urokinase receptor (uPAR), and inhibitors of plasminogen activator (PAI-1 and PAI-2), plays an important role in the regulation of vascular wall functioning. Urokinase signaling initiates proteolytic cascade and degradation of the extracellular matrix; and also activates intracellular signaling in vascular cells. This study is the first to reveal a urokinase-mediated fundamental mechanism that regulates the growth trajectory and branching morphogenesis of blood vessels. This mechanism may be of particular importance during vessel growth in early embryogenesis and in the adult during tissue regeneration.

Isolation and characterization of cardiac progenitor cells from myocardial right atrial appendage tissue

Resident cardiac stem cells, known as “cardiogenic progenitor cells” (CPCs), are a heterogeneous population of immature cells residing in the myocardium and capable of self-renewal and differentiation into cardiomyocyte-like and vascular-like cells. CPCs are usually isolated by long enzymatic digestion of heart tissue and selection with stem cell markers. However, long exposure to enzymatic digestion and the small size of a myocardial sample significantly hinder acquiring a large number of viable cells. To avoid these problems, we developed a method based on CPC growth ex vivo and subsequent immunomagnetic selection.

Migration properties of adipose-tissue-derived mesenchymal stromal cells cocultured with activated monocytes in vitro

Mesenchymal stromal cells (MSCs) are a promising tool in regenerative medicine. MSC migration to damaged inflammatory sites (homing) is essential for tissue repair. We have studied the migration properties of adipose-tissue-derived MSCs (AT-MSC) after their cocultivation with activated monocytes from the THP-1 cell line. We observed the increased migration rate of AT-MSC in vitro with the lack of chemoattractant gradient and to the platelet-derived growth factor (PDGF BB), which is a well-known chemoattractant for cells of mesenchymal origin. Moreover, the rate of directional AT-MSC migration through fibronectin was also increased. We demonstrated that signaling via PDGFR-β activated through the binding of integrin receptors with an extracellular matrix is a possible mechanism for stimulation of cellular migration under simulated inflammatory conditions.

Calcium-induced calcium release mediates all-or-nothing responses of mesenchymal stromal cells to noradrenaline

By using Ca2+ imaging and Fluo-4 dye, we examined the capability of certain agonists of G-protein coupled receptors to stimulate Ca2+ signaling in cultured mesenchymal stromal cells (MSC) derived from the human adipose tissue. In particular, a small subpopulation (∼5%) MSC was found to respond to noradrenaline with Ca2+ transients. The all-or-nothing fashion was characteristic of adrenergic Ca2+ signaling in MSC, that is, while at low concentrations noradrenaline stimulated undetectable Ca2+ transients, virtually maximal responses were elicited by this agonist at any concentration above the threshold of 100–200 nM. In some experiments, MSC were loaded with the photosensitive Ca2+ chelator NP-EGTA to produce local or global jumps in cytosolic Ca2+ concentration by virtue of Ca2+ uncaging. Global uncaging eliciting a high enough Ca2+ jump triggered a Ca2+ transient in the MSC cytoplasm, which was similar to a noradrenaline response kinetically and by magnitude. When Ca2+ uncaging was produced locally, it initiated a Ca2+ signal that traveled along a cell with a speed that exceeded an expected one by two orders of magnitude, should Ca2+ signal transfer be mediated merely by passive Ca2+ diffusion in the presence of Ca2+ buffer. These findings implicated Ca2+-induced Ca2+ release (CICR) as a mechanism amplifying local Ca2+ signals in MSC. Of Ca2+ targets involved in CICR, the ryanodine receptor and IP3 receptor are only known. The inhibitory analysis revealed IP3 receptors to be principally responsible for CICR in MSC, whereas a contribution of ryanodine receptors was negligible. Altogether, our results suggest that an initial noradrenaline-dependent rise in cytosolic Ca2+ stimulates, should it reach the threshold level, IP3 receptors, thereby triggering an avalanche-like Ca2+ release from Ca2+ stores and underlying the all-or-nothing dependence of cellular responses on the agonist concentration.

Centrosome and Golgi Complex during Differentiation of Hepatocytes in Early Postnatal Development of Mice

The structure and functional activity of the centrosome was analyzed in hepatocytes of 5-day old mice, as well as the lengths of Golgi complex cisternae. In the early postnatal development of mice, the liver was represented by two types of hepatocytes: in the first type hepatocytes, the centrosome was active as a microtubule organizing center, while in the second type hepatocytes, it was inactive. It was proposed that during ontogenesis the centrosome is inactivated as a microtubule organizing center and activated as an organizing center of intermediate filaments characteristic for differentiated hepatocytes of adult liver. Morphometry of the Golgi complex has shown that Golgi cisternae in the cell center area of early postnatal hepatocytes were longer than in the adult hepatocytes and comparable to those in G1-phase hepatocytes of regenerating liver. The possibility of relations between the differences in the Golgi complex morphology and ontogenetic changes in the functional activity of centrosomes is discussed.

Three-dimensional model of biomatrix as a method of studying blood vessels and nerve growth in tissue engineering structures

The method involves isolating and culturing ex vivo cultures of mouse dorsal root ganglia and abdominal aorta in a three-dimensional gel (Matrigel). Unlike other explant cultures, this technique allows to study the physiological and biochemical processes in the tissue explants of dorsal root ganglia and abdominal aorta in three-dimensional space but in contrast to a two-dimensional adhesive culture. Administration of the tested substances to the Matrigel allows analyzing their effects on the growth of blood vessels and neurites from the explants for 21 days. The developed method can be applied in modern cardiological and neurobiological research and explores how new therapeutic agents can accelerate the regeneration of blood vessels and nerves.

Identification of TREK-2 K+ channels in human mesenchymal stromal cells

The maintenance of pluripotency of mesenchymal stromal cells (MSCs), their proliferation and initiation of differentiation may critically depend on functional expression of ion channels. Despite such a possibility, mechanisms of electrogenesis in MSCs remain poorly understood. In particular, little is known about a variety of ion channels active in resting MSCs or activated upon MSC stimulation. Here we aimed at uncovering ion channels operating in MSCs, including those being active at rest, using the patch clamp technique and inhibitory analysis. In trying to evaluate a contribution of anion channels in MSC resting potential, we employed a number of diverse inhibitors of anion channels and transporters, including niflumic acid (NFA). Basically, NFA caused hyperpolarization of MSCs that was accompanied by a marked increase in ion conductance of their plasma membranes. The blockage of Cl− channels could not underlie such a NFA effect, given that cells dialyzed with a CsCl solution were weakly or negligibly sensitive to this blocker. This and other findings indicated that NFA affected the MSC ion permeability not by targeting Cl− channels but by stimulating K+ channels. NFA-activated K+ current was TEA and diltiazem blockable, and K+ channels involved were potentiated from outside by solution acidification and Cu2+ ions. Taken together, the data obtained implicated two-pore domain K+ channels of the TREK-2 subtype in mediating stimulatory effects of NFA on MSCs. The notable inference from our work is that TREK-2 channels should be expressed and functional virtually in every MSC, given that all cells examined by us (n > 100) similarly responded to NFA by increasing their TREK-2-like K+ conductance.

Left-ventricular heart aneurism as a new source of resident cardiac stem cells

In the past few years, it has been established that the heart contains a reservoir of stem and progenitor cells. These cells are able to differentiate in cardiomyogenic, endothelial, and smooth muscle lineages in vitro and in vivo and, following injection into an infarcted myocardium, these cells prevent the remodeling of the left ventricle and improve cardiac function. The aim of the present study was the immunophenotypic characterization of resident stem cells in the tissue of a chronic left aneurism, an analysis of their localization, and distribution in various aneurism areas.Fifteen samples of aneurism tissue obtained surgically were analyzed. The number of cells positive for c-kit stem-cell marker was about 3327 ± 234 cells per 1 cm3 tissue. Single cells were found in fibrous, muscle, and adipose parts of aneurism tissue. Most of the cells were revealed in fibrous tissue near large vessels; however, the coexpression of c-kit and markers of endothelial cells was not observed. C-kit-positive cells did not express CD34 and CD45 markers and, thereby, were not of hematopoietic origin. The majority of c-kit-positive cells expressed MDR1, another stem-cell marker. These c-kit-positive cells did not proliferate, since they did not express Ki67. About 20% of these cells expressed an inhibitor of cyclin-dependent kinase p21cip/Waf1. C-kit cells did not express markers of cardiomyocytes (Nkx 2.5, Gata 4, Mef2c, α-actinin), endothelium (Ets 1, CD105, wW), and smooth muscle cells (Gata 6, SMA). Single-cell clusters made up of 70–100 c-kit-positive cells (in cell niches) were observed; however, no markers of cardiomyogenic differentiation were expressed in these cells. Thus, aneurism tissue of the left ventricle contains c-kit-positive cells that, are most likely resident cardiac stem cells; however, their regenerative capacity should be further explored.