Revisiting the multiple roles of T-cadherin in health and disease.

As a non-canonical member of cadherin superfamily, T-cadherin was initially described as a molecule involved in homophilic recognition in the nervous and vascular systems. The ensuing decades clearly demonstrated that T-cadherin is a remarkably multifunctional molecule. It was validated as a bona fide receptor for both: LDL exerting adverse atherogenic action and adiponectin mediating many protective metabolic and cardiovascular effects. Motivated by the latest progress and accumulated data unmasking important roles of T-cadherin in blood vessel function and tissue regeneration, here we revisit the original function of T-cadherin as a guidance receptor for the growing axons and blood vessels, consider the recent data on T-cadherin-induced exosomes' biogenesis and their role in myocardial regeneration and revascularization. The review expands upon T-cadherin contribution to mesenchymal stem/stromal cell compartment in adipose tissue. We also dwell upon T-cadherin polymorphisms (SNP) and their possible therapeutic applications. Furthermore, we scrutinize the molecular hub of insulin and adiponectin receptors (AdipoR1 and AdipoR2) conveying signals to their downstream targets in quest for defining a putative place of T-cadherin in this molecular circuitry.

Secretome of Multipotent Mesenchymal Stromal Cells as a Promising Treatment and for Rehabilitation of Patients with the Novel Coronaviral Infection.

As a rule, coronavirus infections are mild in healthy adults and do not require special approaches to treatment. However, highly pathogenic strains, particularly the recently isolated SARS-CoV2, which causes COVID-19 infection, in about 15% of cases lead to severe complications, including acute respiratory distress syndrome, which causes high patient mortality. In addition, a common complication of COVID-19 is the development of pulmonary fibrosis. Why is the novel coronavirus so pathogenic? What new treatments can be proposed to speed up the recovery and subsequent rehabilitation of the organism? In 2020, over 34 000 scientific articles were published on the structure, distribution, pathogenesis, and possible approaches to the treatment of infection caused by the novel SARS-CoV2 coronavirus. However, there are still no definitive answers to these questions, while the number of the diseased is increasing daily. One of the comprehensive approaches to the treatment of the consequences of the infection is the use of multipotent human mesenchymal stromal cells and products of their secretion (secretome). Acting at several stages of the development of the infection, the components of the secretome can suppress the interaction of the virus with endothelial cells, regulate inflammation, and stimulate lung tissue regeneration, preventing the development of fibrosis. The results of basic and clinical research on this topic are summarized, including our own experimental data, indicating that cell therapy approaches can be successfully applied to treat patients with COVID-19.

Urokinase receptor deficiency results in EGFR-mediated failure to transmit signals for cell survival and neurite formation in mouse neuroblastoma cells.

Urokinase-type plasminogen activator uPA and its receptor (uPAR) are the central players in extracellular matrix proteolysis, which facilitates cancer invasion and metastasis. EGFR is one of the important components of uPAR interactome. uPAR/EGFR interaction controls signaling pathways that regulate cell survival, proliferation and migration. We have previously established that uPA binding to uPAR stimulates neurite elongation in neuroblastoma cells, while blocking uPA/uPAR interaction induces neurite branching and new neurite formation. Here we demonstrate that blocking the uPA binding to uPAR with anti-uPAR antibody decreases the level of pEGFR and its downstream pERK1/2, but does increase phosphorylation of Akt, p38 and c-Src Since long-term uPAR blocking results in a severe DNA damage, accompanied by PARP-1 proteolysis and Neuro2a cell death, we surmise that Akt, p38 and c-Src activation transmits a pro-apoptotic signal, rather than a survival. Serum deprivation resulting in enhanced neuritogenesis is accompanied by an upregulated uPAR mRNA expression, while EGFR mRNA remains unchanged. EGFR activation by EGF stimulates neurite growth only in uPAR-overexpressing cells but not in control or uPAR-deficient cells. In addition, AG1478-mediated inhibition of EGFR activity impedes neurite growth in control and uPAR-deficient cells, but not in uPAR-overexpressing cells. Altogether these data implicate uPAR as an important regulator of EGFR and ERK1/2 signaling, representing a novel mechanism which implicates urokinase system in neuroblastoma cell survival and differentiation.

Urokinase receptor regulates nerve regeneration through its interaction with α5ß1-integrin.

PURPOSE: Urokinase receptor (uPAR) promotes extracellular matrix proteolysis, regulates adhesion and cell migration, transduces intracellular signals through interactions with the lateral partners. The expression of uPAR and urokinase (uPA) is significantly upregulated in peripheral nerves after injury, however, little is known about uPAR function in nerve regeneration or the molecular mechanisms involved. The purpose of this study is to investigate the role of uPAR in nerve regeneration after traumatic injury of n. Peroneus communis in uPA-/-, uPAR-/- or control mice (WT) and in neuritogenesis in an in vitro Neuro 2A cell model. RESULTS: Electrophysiological analysis indicates that nerve recovery is significantly impaired in uPAR-/- mice, but not in uPA-/- mice. These data correlate with the reduced amount of NF200-positive axons in regenerating nerves from uPAR-/- mice compared to uPA-/- or control mice. There is an increase in uPAR expression and remarkable colocalization of uPAR with α5 and ß1 integrin in uPA-/- mice in recovering nerves, pointing to a potential link between uPAR and its lateral partner α5ß1-integrin. Using an in vitro model of neuritogenesis and α325 blocking peptide, which abrogates uPAR-α5ß1 interaction in Neuro 2A cells but has no effect on their function, we have further confirmed the significance of uPAR-α5ß1 interaction. CONCLUSION: Taken together, we report evidence pointing to an important role of uPAR, rather than uPA, in peripheral nerve recovery and neuritogenesis.

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.

[MOLECULAR AND CELLULAR MECHANISMS OF ANGIOGENESIS IN PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONS].

In normal conditions vascular system is in equilibrium, the processes of angiogenesis and vascular regression are precisely regulated. The mechanisms underlying the cardiovascular and cancer diseases are the insufficient or excessive angiogenesis, correspondingly. Understanding the mechanisms of angiogenesis is necessary for the development of new approaches to cure these diseases. The fundamental knowledge of the vascular growth and maturation mechanisms formed the basis for the strategy of «therapeutic angiogenesis¼, which is one of the rapidly developing technologies in regenerative medicine in the world. The strategy is based on the stimulation of blood vessel growth and remodeling in ischemic tissues via administration of recombinant angiogenic factors or genetic constructs for their expression. The same knowledge of the mechanisms of angiogenesis is necessary in the development of new drugs aimed at inhibiting the vascular growth in excessive or aberrant angiogenesis, which escapes the physiological control in various diseases. Herein we review the fundamental molecular and cellular mechanisms of regulation of blood vessel initiation, growth, and stabilization in normal and pathological conditions.

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.

[PARTICIPATION OF UROKINASE RECEPTOR AND ITS ENDOGENOUS LIGANDS IN BRAIN DEVELOPMENT AND FORMATION OF COGNITIVE FUNCTIONS].

Recently it has been found that the urokinase receptor (uPAR) and its ligands - urokinase (uPA) and SRPX2 protein play an important role in the development and functioning of the brain. There is a strong association between uPAR gene polymorphism and autism disorders in humans. Patients with autism, intractable lobe epilepsy, verbal dyspraxia and perisylvian polymicrogyria display significant changes in uPAR expression. Mice, lacking the uPAR gene develop epilepsy and demonstrate abnormal social behavior. uPA and SRPX2 protein, have been shown to be involved in pathological brain conditions such as autism, cognitive deficits and language disorders. Urokinase system that stimulates blood vessel growth as demonstrated before, also plays an important role in the regulation of the nerve growth via matrix remodeling and activation of neurotrophic and angiogenic factors. Moreover, the urokinase system also functions as a guidance system which determines the growth trajectory of the vessels' and nerves' in tissue regeneration. This review summarizes and integrates the results and recent progress in the field of uPAR and its endogenous ligands in brain development and cognitive functions.

Guidance Receptors in the Nervous and Cardiovascular Systems.

Blood vessels and nervous fibers grow in parallel, for they express similar receptors for chemokine substances. Recently, much attention is being given to studying guidance receptors and their ligands besides the growth factors, cytokines, and chemokines necessary to form structures in the nervous and vascular systems. Such guidance molecules determine trajectory for growing axons and vessels. Guidance molecules include Ephrins and their receptors, Neuropilins and Plexins as receptors for Semaphorins, Robos as receptors for Slit-proteins, and UNC5B receptors binding Netrins. Apart from these receptors and their ligands, urokinase and its receptor (uPAR) and T-cadherin are also classified as guidance molecules. The urokinase system mediates local proteolysis at the leading edge of cells, thereby providing directed migration. T-cadherin is a repellent molecule that regulates the direction of growing axons and blood vessels. Guidance receptors also play an important role in the diseases of the nervous and cardiovascular systems.

Detection of T-Cadherin Expression in Mouse Embryos.

The aim of the present study was to evaluate T-cadherin expression at the early developmental stages of the mouse embryo. Using in situ hybridization and immunofluorescent staining of whole embryos in combination with confocal microscopy, we found that T-cadherin expression is detected in the developing brain, starting with the E8.75 stage, and in the heart, starting with the E11.5 stage. These data suggest a possible involvement of T-cadherin in the formation of blood vessels during embryogenesis.

[Single Nucleotide Polymorphisms in T-Cadherin Gene (CDH13) Have Cumulative Effect on Body Mass in Patients With Ischemic Heart Disease].

BACKGROUND: Low adiponectin concentration observed in obese patients is associated with a high risk of metabolic disorders and cardiovascular diseases and could be related to single nucleotide polymorphisms (SNPs) in T-cadherin gene (CDH13). T-cadherin is a receptor for adiponectin and low-density lipoprotein. Aim of this study was to investigate association of CDH13 SNPs with the development of obesity in patients with ischemic heart disease (IHD). RESULTS: We established a statistically significant correlation between the number of minor alleles of rs11646213, rs4783244 and rs12444338 in CDH13 gene with body mass index: patients with smaller number of minor alleles tended to have normal body weight (odds ratio 3.03, 95% confidence interval 1.03-8.87). CONCLUSION: The obtained results are indicative of the cumulative effect of SNPs in CDH13 (rs11646213, rs4783244, rs12444338) on BMI in patients with IHD.

[Therapeutic Angiogenesis Using Growth Factors: Current State and Prospects for Development].

Therapeutic angiogenesis has been in use for treatment of ischemic diseases for about 15 years. During this period of successes and failures this field has accumulated a significant amount of published and ongoing surveys giving insights and raising new questions and problems. One of the most utilized methods for therapeutic angiogenesis suggests introduction of angiogenic growth factors (VEGF, bFGF, angiopoietin-1 etc.) into ischemic tissues. Still, there is a whole range of problems regarding the efficacy of therapeutic angiogenesis. These can be potentially circumvented by use of new delivery methods, development of combined approaches and use of more relevant pre-clinical animal models. Present review gives a brief analysis of crucial achievements and issues that has been recently raised in experimental and clinical studies focusing on therapeutic angiogenesis. Final part brings some possible directions for development that can give an opportunity to circumvent current obstacles and provide further development.

[UROKINASE SYSTEM INVOLVES IN VASCULAR CELLS MIGRATION AND REGULATES THE GROWTH AND BRANCHING OF CAPILLARIES].

Urokinase system representing urokinase-type plasminogen activator (urokinase, uPA) and urokinase re- ceptor (uPAR) plays an important regulatory role in the vascular wall and has the ability to run a proteolytic cascade, degradation of extracellular matrix and activate intracellular signaling in vascular cells. In this work, we have firstly shown a fundamental mechanism of urokinase system-dependent regulation of the trajectory of growth and branching of blood vessels what may be of particular importance in the growth of blood vessels in early embryogenesis and in adults during the repair/regeneration of tissues.

[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.

Mesenchymal stem cells in tissue growth and repair.

It has been established in the recent several decades that stem cells play a crucial role in tissue renewal and regeneration. Mesenchymal stem cells (MSCs) are part of the most important population of adult stem cells. These cells have hereby been identified for the very first time and subsequently isolated from bone marrow stroma. Bone marrow-derived MSCs have been believed to play the role of a source of cells for the renewal and repair of connective tissues, including bone, cartilage and adipose tissues. Cells similar to bone marrow-derived MSCs have now been identified in all postnatal tissues. Data on the distribution and function of MSCsin vivocollected using novel approaches pertaining to the identification of MSCsin situ, to their isolation from tissues, and finally to the determination of their biological properties have enabled successful revision of the role of MSCs in various organs and tissues. This review summarizes our own, as well as others', data concerning the role of MSCs in the regulation processes of tissue repair and regeneration. In our opinion, MSCs provide the connection between the blood-vascular, immune, endocrine, and nervous systems and tissue-specific stem cells in the body.

[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 hyperexpression of T-cadherin in comparison with the cells in the control. It is shown in experiments in vitro that the B16F10 cells with the hyperexpression of T-cadherin are less adaptive 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 hyperexpression of T-cadherin and the amount of the vessels growing to tumor cell masses formed by the cells with the hyperexpression of T-cadherin is significantly lower than the same index for the cells in the control. Thus, the hyperexpression 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 are demonstrated.

[Viability and angiogenic activity of mesenchymal stromal cells from adipose tissue and bone marrow in hypoxia and inflammation in vitro].

Adult progenitor stromal cells derived from adipose tissue (ADSC) and bone marrow (BMDSC) hold great promise for use in cell-based therapy of ischemic diseases. Both cell lines secrete a various number of angiogenic cytokines which are regulated by hypoxia and improve vascularization of ischemic tissues being injected in damaged muscle or intravenously. However, such factors as low oxygen level and inflammation may impair the viability and functional activity of these cells after delivery to the ischemic area. We directly compared the reactions of ADSCs and BMDSCs to hypoxic and inflammatory conditions in vitro. Cultured ADSCs and BMDSCs from Balb/c mice were cultivated for 48 h under 1% O2 (hypoxia), 20% O2 (normoxia) or in the presence of inflammatory cytokines. Cell viability analyzed by annexin V-PE binding and 7AAD storage (flow cytometry), and by quantitative TUNEL showed no decrease under hypoxic condition. But cell apoptotic rates significantly increased (up to 70 %) under inflammatory condition. Inflammatory cytokines did not stimulate gene expression of angiogenic growth factors. Otherwise, gene expression profiles of angiogenesis-related cytokines showed activation of pro-angiogenic and suppression of anti-angiogenic factors in the cells under hypoxic condition. In general this effect was higher for ADSCs than for BMDSCs. Using in vitro and in vivo models of angiogenesis we have demonstrated that incubation under hypoxic condition increases stromal cells ability to stimulate blood vessels growth.

[Mechanism of stimulation of angiogenesis in ischemic myocardium with the help of adipose tissue stromal cells].

Stromal cells from subcutaneous adipose tissue (adipose derived stromal cells - ASCs) are perspective for cell therapy of ischemic states because of ability to stimulate growth of vessels. For the elucidation of mechanisms of angiogenic action of ASCs we used the model of co-cultivation of ASCs with cells isolated from postnatal hearts (fraction of cardiomyocutes - CMC). CMC fraction contained mature cardiomyocytes, endothelial and progenitor cells. On the 2-nd day spontaneously beating colonies of CMC with growing from them CD31-positive capillary-like structures were formed in CMC culture. Observed structures were unstable and came apart after 5 days of cultivation. At co-cultivation of CMC with ASCs formation of stable ramified CD31-positive structures was observed. Using the method of co-cultivation of CMC with mitomycin C treated ASCs and the method of immune magnetic depletion for removal of epithelial cells from the CMC fraction we found that ASCs stimulates formation of capillary like structure at the account of secretion of angiogenic factors, stabilization of forming CD31-positive structures at the account of intercellular contacts and stimulation of endothelial differentiation of progenitor cells present in CMC fraction.