[Correlations between vessel stiffness and biomarkers of senescent cell in elderly patients].

Aim    To study the association between vascular wall stiffness and known markers for accumulation of senescent cells in blood, cells, and tissues of old patients.Material and methods    This study included male and female patients aged 65 years and older who were referred to an elective surgical intervention, that included a surgical incision in the area of the anterior abdominal wall or large joints and met the inclusion and exclusion criteria. For all patients, traditional cardiovascular (CV) risk factors and arterial wall stiffness (pulse wave velocity, PWV) were evaluated. Also, biomaterials (peripheral blood, skin, subcutaneous adipose tissue) were collected during the surgery and were used for isolation of several cell types and subsequent histological analysis to determine various markers of senescent cells.Results    The study included 80 patients aged 65 to 90 years. The correlation analysis identified the most significant indexes that reflected the accumulation of senescent cells at the systemic, tissue, and cellular levels (r>0.3, р<0.05) and showed positive and negative correlations with PWV. The following blood plasma factors were selected as the markers of ageing: insulin-like growth factor 1 (IGF-1), fibroblast growth factor 21 (FGF-21), and vascular endothelium adhesion molecule 1 (VCAM-1). A significant negative correlation between PWV and IGF-1 concentration was found. Among the tissue markers, P16INK, the key marker for tissue accumulation of senescent cells, predictably showed a positive correlation (r=0.394, p<0.05). A medium-strength correlation with parameters of the 96-h increment of mesenchymal stromal cells and fibroblasts and a weak correlation with IL-6 as a SASP (specific senescent-associated secretory phenotype) were noted. Results of the multifactorial linear regression analysis showed that the blood plasma marker, VCAM-1, and the cell marker, 96-h increment of fibroblasts, were associated with PWV regardless of the patient's age.Conclusion    Stiffness of great arteries as measured by PWV significantly correlates with a number of plasma, tissue, and cellular markers for accumulation of senescent cells. This fact suggests PWV as a candidate for inclusion in the panel of parameters for evaluation and monitoring of the biological age during the senolytic therapy.

Mechanisms of Endothelial-to-Mesenchymal Transition Induction by Extracellular Matrix Components in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis can be caused by different factors, including accumulation of pathological extracellular matrix (ECM) with abnormal composition, stiffness, and architecture in the lung tissue. We studied the effect of ECM produced by lung fibroblasts of healthy mice or mice with bleomycin-induced pulmonary fibrosis on the process of endothelialto- mesenchymal transition, one of the main sources of effector myofibroblasts in fibrosis progression. Despite stimulation of spontaneous and TGFß-1-induced differentiation of fibroblasts into myofibroblasts by fibrotic ECM, the appearance of α-SMA, the main marker of myofibroblasts, and its integration in stress fibrils in endotheliocytes were not observed under similar conditions. However, the expression of transcription factors SNAI1 and SNAI2/Slug and the production of components of fibrotic ECM (specific EDA-fibronectin splice form and collagen type I) were increased in endotheliocytes cultured on fibrotic ECM. Endothelium also demonstrated increased cell velocity in the models of directed cell migration. These data indicate activation of the intermediate state of the endothelial-to-mesenchymal transition in endotheliocytes upon contact with fibrotic, but not normal stromal matrix. In combination with the complex microenvironment that develops during fibrosis progression, it can lead to the replenishment of myofibroblasts pool from the resident endothelium.

European Biomedical Research Infrastructures and the Fight against COVID-19 Pandemic.

The study aims to assess the role of EU biomedical research infrastructures in the fight against the COVID-19 pandemic and to analyze their response to the challenges associated with the spread of the new pathogen. Materials and Methods: We analyzed the materials of the Seventh Framework Program for Research and Technological Development (FP7, 2007-2013) of the EU and the Eighth Framework Program "Horizon 2020" (FP8, 2014-2020), official reports of the European Strategic Forum on Research Infrastructures, expert reports, as well as documents of the European Commission, the COVID-19 Data Portal, and other relevant sources of information. Results: The analysis revealed that the mechanisms created within the united European research community provided for a flexible response to the emerging threat of COVID-19 as soon as January-May 2020. In particular, information channels were established to timely analyze the research results and coordinate the efforts in the fight against COVID-19. The biomedical infrastructures created in the EU and proved successful earlier have now been mobilized to search for ways of preventing and treating COVID-19. These mechanisms facilitated communication and data exchange between various research institutions and thus laid the ground for new achievements in this area. Conclusion: The decisions taken to combat the COVID-19 pandemic have convincingly illustrated that the EU research infrastructures, integrated into a united ecosystem, are highly adaptable and flexible, which allows to realign priorities in a short time and to create instruments that enable scientists to respond to new challenges.

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.

Biochemical Regulation of Regenerative Processes by Growth Factors and Cytokines: Basic Mechanisms and Relevance for Regenerative Medicine.

Regenerative medicine that had emerged as a scientific and medical discipline at end of 20th century uses cultured cells and tissue-engineered structures for transplantation into human body to restore lost or damaged organs. However, practical achievements in this field are far from the promising results obtained in laboratory experiments. Searching for new directions has made apparent that successful solution of practical problems is impossible without understanding the fundamental principles of the regulation of development, renewal, and regeneration of human tissues. These aspects have been extensively investigated by cell biologists, physiologists, and biochemists working in a specific research area often referred to as regenerative biology. It is known that during regeneration, growth factors, cytokines, and hormones act beyond the regulation of individual cell functions, but rather activate specific receptor systems and control pivotal tissue repair processes, including cell proliferation and differentiation. These events require numerous coordinated stimuli and, therefore, are practically irreproducible using single proteins or low-molecular-weight compounds, i.e., cannot be directed by applying classical pharmacological approaches. Our review summarizes current concepts on the regulatory mechanisms of renewal and regeneration of human tissues with special attention to certain general biological and evolutionary aspects. We focus on the biochemical regulatory mechanisms of regeneration, in particular, the role of growth factors and cytokines and their receptor systems. In a separate section, we discussed practical approaches for activating regeneration using small molecules and stem cell secretome containing a broad repertoire of growth factors, cytokines, peptides, and extracellular vesicles.

Cell Sheets of Mesenchymal Stromal Cells Effectively Stimulate Healing of Deep Soft Tissue Defects.

We used rat model of splinted defect of the skin and soft tissues to compare the efficiency f mesenchymal stromal cells applied in sheets or in suspension for the treatment of these injuries. Transplantation of mesenchymal stromal cells significantly accelerated wound healing in comparison with the control. In the group treated by application of mesenchymal stromal cell sheets, the defect was closed by day 28, in the group treated with cell suspension by day 35, and in the control group after 49 days. According to histological analysis of the tissue samples, the formation of the granulation tissue and fibrosis occurred earlier after application of mesenchymal stromal cells. Application of mesenchymal stromal cells in the form of cell sheets demonstrated high efficiency, which allowed us to consider this approach as a promising method of healing of skin and soft tissue injuries.

Extracellular Matrix in the Regulation of Stem Cell Differentiation.

Extracellular matrix (ECM) proteins fill the space between cells in multicellular organisms, contributing to the structure of organs and tissues. The mechanical properties of ECM are well studied. At present, the role of individual ECM components and the three-dimensional tissue-specific matrices in the regulation of cell functional activity, proliferation, migration, acquisition of a specialized phenotype and its maintenance is intensively studied. In this review, we described main ECM structural proteins, enzymes, and extracellular vesicles and present the data on the participation of ECM components in the regulation of stem cell differentiation and self-maintenance, as well as approaches to the modeling of stem cells microenvironment using decellularized ECM.

Application of rat cryptorchidism model for the evaluation of mesenchymal stromal cell secretome regenerative potential.

Male infertility represents a severe social and medical challenge. In recent years the progress in regenerative medicine promoted the development of novel options to overcome this medical condition. We are elaborating a promising approach to restore spermatogenesis using mesenchymal stromal cell (MSC) secretome components as a novel class of cell-free cell therapy medicinal products for regenerative medicine. However, the choice of the representative in vivo model of spermatogenesis failure to evaluate the effectiveness of regenerative drugs remains challenging. Using the rat model of bilateral abdominal cryptorchidism, we studied the contribution of MSC conditioned medium contained bioactive cell secreted products to the spermatogenesis recovery. The feasibility of this model to evaluate the drug-driven regenerative effects on spermatogenesis restoration after the injury was demonstrated. We revealed significant correlations between the extent of spermatogonial stem cell niche recovery, spermatozoa count and serum concentration of androgens as an indicator of Leydig cell function. The obtained results can be applied in preclinical studies to choose the proper criteria to appraise the specific activity of novel regenerative drugs developed for the treatment of non-obstructive spermatogenesis disorders.

Collagen-1 Membrane for Replacing the Bladder Wall.

We studied the possibility of using membrane fabricated from type 1 collagen isolated from cattle tissues (group 1) or porcine tissues (group 2) for replacement of the resected bladder wall defect in rabbits in order to retain functional volume of the organ. Satisfactory take of both types of collagen membranes with formation of competent anastomosis was observed. Histological studies revealed inflammatory process in the bladder wall at the site of contact with the implanted membrane (more pronounced in case of membranes from cattle tissues) that decreased by day 21 of the experiment. Bladder tissue ingrowth into the implant from was observed starting from day 14. The bladder capacity decreased in 7 days after surgery in both groups, presumably because of increasing tone of the organ wall resulting from surgical trauma and inflammation. In group 2, the bladder volume increased by day 14 after surgery and returned to normal by day 21, whereas in group 1 it remained below the control despite a trend to increase. These findings confirm good prospects of using collagen-1 membranes for plastic repair of the urinary bladder, the membranes from porcine collagen being more preferable.

[Urinary bladder substitution using combined membrane based on secretions of human mesenchymal stem cells and type I collagen].

AIM: Despite the widespread use of intestinal cystoplasty, urinary bladder substitution remains a challenging problem due to the complexity of operations and the potentially high risk of complications. A promising alternative may be bio-engineered collagen-based matrices containing stem cells or their secretions. MATERIAL AND METHODS: To evaluate the effectiveness of this bladder substitution modality, an experiment was conducted on 14 male rabbits. The animals underwent resection of urinary bladder, and the formed defect was substituted with a membrane of type I collagen (series 1, 5 rabbits) or a membrane of the same composition containing a conditioned medium with secretion of mesenchymal stem/stromal cells derived from human adipose tissue (series 2, 5 rabbits). In the comparison group (4 rabbits) resection of the bladder and the closure of the defect was carried out without bladder substitution (series 3). RESULTS: At 1 month after surgery, there was a complete epithelization of the inner surface of the implant, and body tissues replaced the collagen matrix. In series 1, the collagen implant was replaced mainly by connective tissue ingrown with occasional solitary smooth muscle cells. In series 2, the newly formed bladder wall contained numerous smooth muscle cells, growing into the collagen matrix and forming the muscular coat. In series 3, the muscular layer regeneration at the scar site was also noted, but it was less intense, which was confirmed by morphometry. In series 2, more active vascularization of the collagen implant occurred due to neo-angiogenesis, which was more intense than that in series 3, and especially in series 1. Functional studies revealed a reduced bladder functional capacity in series 1 and 3, while in series 2 it was close to normal. During filling cystometry, changes in intra-vesical pressure profile in series 2 were close to normal, while in series 1 and 3 infusion of a small volume of saline resulted in a marked increase in intra-vesical pressure, showing a reduced compliance of the reconstructed bladder. Discussion The study findings show that implants based on type I collagen can be effectively used to substitute a part of the urinary bladder wall, but bio-engineered collagen matrix grafts containing cell regeneration stimulants secreted by stem cells in their culture medium seem to be more promising.

[Therapeutic potential of human mesenchymal stromal cells secreted components: a problem with standartization].

Regenerative medicine approaches, such as replacement of damaged tissue by ex vivo manufactured constructions or stimulation of endogenous reparative and regenerative processes to treat different diseases, are actively developing. One of the major tools for regenerative medicine are stem and progenitor cells, including multipotent mesenchymal stem/stromal cells (MSC). Because the paracrine action of bioactive factors secreted by MSC is considered as a main mechanism underlying MSC regenerative effects, application of MSC extracellular secreted products could be a promising approach to stimulate tissue regeneration; it also has some advantages compared to the injection of the cells themselves. However, because of the complexity of composition and multiplicity of mechanisms of action distinguished the medicinal products based on bioactive factors secreted by human MSC from the most of pharmaceuticals, it is important to develop the approaches to their standardization and quality control. In the current study, based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for the complex of extracellular products secreted by human adipose-derived MSC on key indicators, such as "Identification", "Specific activity" and "Biological safety". Developed approaches were tested on the samples of conditioned media contained products secreted by MSC isolated from subcutaneous adipose tissue of 30 donors. This strategy for the standardization of innovative medicinal products and biomaterials based on the bioactive extracellular factors secreted by human MSC could be applicable for a wide range of bioactive complex products, produced using the different types of stem and progenitor cells.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells.

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53(-/-) mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53(-/-) xenografts in athymic mice. Growth of tumor xenografts of human HPV-16-associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53(-/-) and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53(-/-) tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P-glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models.

Effect of transauricular electroacupuncture on stress-induced changes in cognitive function and erosions of the gastric mucosa in rats.

Transauricular electroacupuncture prevented stress-induced increase in the degree of anxiety and suppression of exploratory activity in rats. This procedure significantly accelerated decision-making process during acute stress in both control and prestressed rats. Transauricular electroacupuncture decreased the number and area of erosions by producing a pronounced antistress effect at the central and peripheral levels.