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Glaucoma is one of the main causes of irreversible blindness worldwide. Up to 76% of the glaucoma cases are accompanied with complicated cataract. The issue of cataract treatment in glaucoma patients is a difficult task for any surgeon, for the surgical procedure itself can result in a number of complications. One of them is the loss of endothelial cells in the cornea. A decrease in the endothelial cell density in such patients occurs due to long-term use of various hypotensive drops, due to variations of intraocular pressure, as well as due to the surgical interventions themselves. Up to 16.9% of cataract removal cases with a background of glaucoma are accompanied by pronounced post-operative corneal swelling, which leads to an increased risk of losing corneal endothelial cells. The perspective branch of surgical treatment for cataract and glaucoma is the development of a unified algorithm taking into account the individual characteristics of the patient, such as the eye lens clouding, the glaucoma stage, the intraocular pressure, the past surgeries, the hypotensive therapy and the density of corneal endothelial cells.

Burn injury and its consequences are an important medical and social problem, given the high mortality and disability rates. The leading cause of death in patients with extensive and deep burns is burn septicotoxemia.Material and methods. The material was the heart muscle of the anterior, posterior and lateral walls of the left ventricle of those who died from burn disease during septicotoxemia. The control group included cases of male mortality as a result of “acute coronary death”. The study of the ultrastructure of endotheliocytes of blood capillaries of the myocardium has been carried out.Results and discussion. Ultrastructural analysis of endotheliocytes of blood capillaries of the myocardium showed that in burn septicotoxemia, intracellular degradation processes are observed in the form of swelling of the cytoplasm and organelles, a decrease in the concentration of vesicular structures, as well as activation of autophagy due to the appearance of autophagosomes and autolysosomes in the cytoplasm. Activation of autophagy should be considered as a naturally developing compensatory reaction aimed at preserving the cellular pool of endotheliocytes of the blood capillaries of the myocardium in response to the alterative effect caused by burn septicotoxemia.Conclusion. The obtained data can serve as a justification for the manifestation of left ventricular contractile insufficiency, which is a consequence of endothelial dysfunction and microcirculatory disorders in the myocardium in burn septicotoxemia.

Obesity and diabetes mellitus are known to lead to the development of metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). The mechanisms of programmed cell death are actively involved in maintaining cellular homeostasis along development of NAFLD. Proteins of the BCL-2 family are key regulators of physiological and pathological apoptosis. Homozygous males of BKS.Cg-Dock7mLeprdb/+/+/J mice (db/db mice) are characterized by progressive obesity and the development of type 2 diabetes mellitus (DM2) with severe hyperglycemia at 4–8 weeks and organ lesions at 8–10 weeks of age. The aim of this research was to study the expression of molecular cell regulators of apoptosis in liver cells of db/db mice males at different stages of obesity and diabetes development (at the age of 10 and 18 weeks). Immunohistochemical analysis (using the indirect avidin-biotin peroxidase method) and morphometric evaluation of the expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bad in liver cells of studied animals at different stages of obesity and DM2 were carried out. An excess of the value of the Bcl-2 protein staining area over the Bad protein staining area was revealed in the liver of 10-week-old animals. The Bcl-2/Bad expression area ratio in 10-week-old animals was twice as high as in 18-week-old animals, which indicates the presence of conditions for blocking apoptosis in the liver of younger db/db mice. At the 18th week of life, db/db mice displayed an almost threefold increase in the expression area of the Bad protein against the background of an unchanged expression of the Bcl-2 protein. The decrease in the Bcl-2/Bad staining area ratio in 18-week-old animals was due to the increase in the Bad expression area, which indicates the absence of antiapoptotic cell protection and creates conditions for activation of the mitochondrial pathway of apoptosis in the liver of male db/db mice with pronounced signs of obesity and DM2.

Development of angiogenesis depends on the functional state of endothelial cells, as well as on the balanced secretion of cytokines, growth factors and chemokines by endothelial cells and cells of microenvironment. Macrophages represent an essential component of the microenvironment and take part in the formation of blood vessels both due to the production of cytokines and due to contact interactions with endothelial cells. VEGF is among the most important cytokines that control angiogenesis at all its stages. Currently, the role of VEGF in the intercellular interactions of endothelial cells and macrophages is not well described. The aim of our study was to investigate the effect of VEGF deprivation using monoclonal antibodies on angiogenesis under conditions of co-cultivation of endothelium and macrophages. Materials and methods: monoclonal antibodies to VEGF-A were used for VEGF deprivation in monoculture of endothelial cells and in co-culture of endothelial cells with macrophages. The IL-1β, IL-6 and TNFα cytokines were used as inducers. When VEGF-A was removed from the medium, endothelial cells show plasticity and form longer vessels, they modify the expression of VEGF receptors. Macrophages regulate endothelial cell activity through the secretion of cytokines, including VEGF, and through contact interactions with endothelial cells. THP-1 cells increase the sensitivity of endothelial cells to VEGF by stimulating the VEGFR1 and VEGFR3 expression, this effect is VEGF-A-independent. The IL-1β, IL-6, TNFa cytokines independently stimulate non-branching angiogenesis, increasing the length of the vessels. At the same time, IL-ip increases the VEGFR1 expression on the surface of endothelial cells. In contrast, IL-6 and TNFα decrease it, thereby regulating the sensitivity of endothelial cells to VEGF. The effects of these cytokines are not dependent on VEGF-A. The IL-1β, IL-6, TNFα cytokines promote acquisition of anti-angiogenic properties by THP-1 cells that is independent on VEGF-A, as well as on expression of its receptors by endothelial cells. Thus, VEGF is an important, but not the sole factor controlling angiogenesis. Under conditions of VEGF-A deficiency, either endothelial cells or microenvironment cells are able to compensate for its functional load due to the production of other growth factors.

Regulation of angiogenesis in the utero-placental bed determines adequate trophoblast invasion, placenta formation and development, as well as successful course of pregnancy. Natural killer (NK) cells, macrophages and trophoblast have the most significant effect on angiogenesis. To date, the functions of cells participating in placenta formation have been described in detail, both individually (in vitrо) and in tissues (in situ). However, no models have yet been created that reflect the interactions of NK cells, trophoblast and endothelium during angiogenesis. It remains unclear, how each cell population contributes to placental angiogenesis regulation, and to the cross-regulation of participating cell functions. Therefore, the aim of this research was to study contact and distant effects of NK cells upon formation of tube-like structures through co-culture of endothelial and trophoblast cells influenced by various cytokines (bFGF, VEGF, PlGF, TGF-β, IL-8, IFNγ and IL-1β). Introduction of NK cells to the co-culture of endothelial and trophoblast cells under conditions of both contact and distance-dependent culturing did not change the length of tube-like structures formed by endothelial cells. During contact-dependent culturing of NK cells with co-culture of endothelial and trophoblast cells in presence of IL-1β, the length of tubule-like structures remained unchanged, compared with the length of tube-like structures formed under the same culturing conditions, but without the cytokine added. During distant culturing of NK cells with co-culture of endothelial and trophoblast cells in the presence of IL-1β, the length of tube-like structures increased as compared with those formed under the same culturing conditions but without the cytokine. During contact-dependent (but not distant) culturing of NK cells with the co-culture of endothelial and trophoblast cells in the presence of VEGF, the length of tube-like structures was greater than those formed under the same culturing conditions but without the cytokine. When used in a three-component cell system, the pro-inflammatory cytokine IFNγhad no effect upon angiogenesis. During distant (but not contact-dependent) culturing of NK cells with co-culture of endothelial and trophoblast cells in the presence of TGF-β, the length of tube-like structures was less than the length of tube-like structures formed under the same culturing conditions but without the cytokine. Under conditions of distant culturing, TGF-βtriggered a signal in NK cells that inhibited angiogenesis. Decreased length of tube-like structures under conditions of a three-component cell co-culture in the presence of the following pro-angiogenic factors was revealed: IL-8, PlGF (during contact-dependent culturing only) and bFGF (during both contact-dependent and distant culturing). Thus, the effects of cytokines upon angiogenesis in a three-component co-culture (NK cells, trophoblast and endothelium) differed from those revealed previously in single-component (endothelium only) and two-component (co-culture of endothelium and trophoblast) cell models. The results of these experiments indicated that regulation of placental cell interactions involved both cellular contacts and effects produced by cytokines.

Tissue engineering is a promising area for the production of small-diameter vascular grafts. In recent years, a number of strategies have been developed to make the polymer surfaces of vascular prostheses capable to selectively adhesion of endothelial cells. The arginine–glycine–aspartic acid (RGD) sequence (a cell adhesion site that is present on many extracellular matrix proteins) is the promising target for modification. The efficiency of attachment of endothelial cells can be influenced both by the structure of RGD peptide and the extent of linker group.Aim: to determine the optimal method for modification of non-woven matrices of polyhydroxybutyrate/ valerate and polycaprolactone (PHBV/PCL) by RGD-peptides leading to the increasing of adhesion, viability and proliferation of endothelial cells.Materials and methods. Electrospinning was used to produce 4 mm diameter tubular polymer matrices from PHBV/PCL. Modification of surface of polymer scaffolds was performed using 4,7,10-trioxa-1,13-tridekandiamin, hexamethylenediamine, glutaraldehyde, ninhydrin, ascorbic acid, a cyclic peptide c [RGDFK], RGDK, AhRGD. The quality of modification was assessed by ninhydrin test and determination of arginine-containing peptide. The structure of the surface of matrices before and after modification was studied by scanning electron microscopy. Adhesion, viability and proliferation of Human umbilical vein (HUVEC) endothelial cells cultured for 7 days on the surface of matrices in the presence of RGD and without one were examined using fluorescence and laser scanning microscopy after the cells were pre-stained with fluorescent nuclear dyes (ethidium bromide and Hoechst 33342), and also by special kits for proliferation assessment (Click-iTTM Plus EdU Alexa FluorTM 488 Imaging Kit).Results. RGD peptides bound to the matrix surface via a long linker (4,7,10-trioxa-1,13-tridecanediamine) were characterized by the increased bioavailability and activity. High level of cell adhesion, viability and proliferation were noted on the surface of RGDK and c[RGDFK] modified matrices, whereas their paired analogues with a short linker (hexamethylenediamine) showed low results of cellular viability even against satisfactory cell adhesion.Discussion. Non-woven matrices based on PHBV/PCL and modified using 4,7,10-trioxa-1,13-tridecanediamine showed better results in case of adhesion of HUVEC and subsequent preservation of cell viability and proliferation. RGD-containing peptides of RGDK and c [RGDFK] were more tropic to endothelial cell receptors.

Aim. To identify death subroutine of endothelial cells upon the exposure to calcium phosphate bions (CPB) and to define whether CPB induce specific molecular response. Materials and Methods. Immortalized murine lymphatic endothelial cells (2H-11, 85-90 % confluence) were exposed to either magnesium phosphate bions (MPB), spherical calcium phosphate bions (SCPB), or needle-shaped calcium phosphate bions (NCPB) for 24 hours with the subsequent protein extraction using radioimmunoprecipitation assay (RIPA) buffer followed by Western blotting to: 1) apoptosis effector proteins (cleaved caspase-3 (cCasp-3), cleaved poly (ADP-ribose) polymerase (cParp-1)); 2) intrinsic apoptosis proteins ((X-linked inhibitor of apoptosis protein (Xiap), survivin, plasminogen activator inhibitor 1 (Pai-1), HtrA2/Omi, cytochromec, p53 upregulated modulator of apoptosis (Puma)); 3) proteins relate to central signaling pathways (phosphorylated extracellular signal-regulated kinase (pErk), phosphorylated mitogen-activated proteinkinase (pMapk), phosphorylated focal adhesion kinase (pFak), phosphorylated nuclear factor kappa-light-chain-enhancer of activated Bcells(pNF-kB), Itch, and Gli). Results were assessed by chemiluminescence detection and using the standard ImageJ algorithm for analysis of chemiluminescent gels. Results. We found increased levels of cleaved effector caspase 3 and its cleaved substrate, cPARP-1, in endothelial cells exposed to SCPB or NCPB as compared to those exposed to MPB or control phosphate buffered saline. Further, we documented decreased level of Xiap, a key inhibitor of intrinsic apoptosis, in endothelial cells exposed to SCPB or NCPB. In contrast, survivin, an other major inhibitor of intrinsic apoptosis, and anti-apoptotic protein Pai-1 were abated upon the SCPB or NCPB exposure, possibly due to the activation of negative feedback loop. In addition, we did not detect any significant changes in master regulators of cell signaling pathways after exposure to CPB. Conclusion. CPB induce intrinsic apoptosis in endothelial cells but do not cause any specific molecular signaling response.

Aim. To evaluate the potential synergistic effects of basic fi broblast growth factor (bFGF) and stromal cell-derived factor-1α (SDF-1α) complemented with VEGF compared to VEGF alone when being added into poly(3-hydroxybutyrateco-3-hydroxyvalerate) / poly(ε-caprolactone) vascular grafts implanted into rat abdominal aorta for 3, 6, or 12 months.Materials and methods. Utilizing emulsion electrospinning, we fabricated one-layer vascular grafts with either VEGF, bFGF, or SDF-1α, and two-layer vascular grafts with VEGF incorporated into the inner layer and bFGF and SDF-1α incorporated into the outer layer following structural evaluation, tensile testing, and in vivo testing using a rat abdominal aorta replacement model.Results. Grafts containing all three growth factors had a 100% primary patency rate. The combination of bFGF, SDF-1α and VEGF improved morphology and mechanical properties of the grafts. Furthermore, such combination of the bioactive factors promoted endothelialization compared to VEGF alone. In addition, bFGF induced a rapid formation of a SMC layer.Conclusion. Taking together, these fi ndings show that the incorporation of bFGF and SDF-1α into the vascular grafts in combination with VEGF enhances vascular tissue regeneration and provides a higher primary patency rate.

Study aim. To investigate whether the shape of calcium phosphate bions (CPB) affects their endothelial toxicity via evaluating the cytokine secretion profile of endothelial cells upon the exposure to either spherical or spindle-shaped CPB.Material and methods. For the experiments, we used an immortalized human vein endothelial cell line EA.hy 926. Cells were seeded into 6-well plates (3*105 cells) with the further: 1) addition of 100 |jL either spherical CPB, spindle-shaped CPB, or 1x phosphate buffered saline (PBS) upon 1 h following culture for 24 h (non-confluent cell culture); 2) culture for 44 h and subsequent addition of 100 jL either spherical CPB, spindle-shaped CPB, or PBS following culture for 4 h (confluent cell culture). Upon the collection of cell culture supernatant (n=11 wells per group), the levels of proatherosclerotic cytokines (interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12, IL-23, tumor necrosis factor (TNF)-a, interferon (IFN)-y, and soluble vascular cell adhesion molecule (sVCAM)-1) were measured utilizing an enzyme-linked immunosorbent assay.Results. In a non-confluent cell culture, exposure to spindleshaped CPB increased the secretion of several proatherosclerotic cytokines (IL-1 ß, IL-10, IL-12, IL-23, IFN-y) compared to either spherical CPB-treated or control cells. In a confluent cell culture, exposure to either of CPB types decreased the release of IL-1 ß, IL- 10, and IFN-y; however, their concentration was still higher upon the exposure to spindle-shaped CPB in comparison with exposure to spherical CPB. Discriminant analysis and principal component analysis demonstrated that the cytokine secretion profile of spindle-shaped CPB-treated endothelial cells significantly differed from those of either spherical CPB-treated or control cells.Conclusion. Spindle-shaped CPB induce the secretion of proatherosclerotic cytokines by endothelial cells compared to spherical CPB; this suggests higher endothelial toxicity of spindleshaped CPB.

There is a growing body of data about the cytopathic effect of bacterial arginine deiminase on human endothelial cells, but the precise mechanisms of endothelial dysfunction caused by the activity of the enzyme remain poorly understood. Activity of arginine deiminase causes arginine depletion in the microenvironment of the host organism cells. In view that arginylation of beta-actin regulates actin cytoskeleton structure and cell motility, we proposed that the cytopathic effect of arginine deiminase may be associated with disruption of actin in the cytoskeleton of endothelial cells. The aim of this study was to investigate the effect of arginine deiminase from S. pyogenes on migration and actin cytoskeleton structure of the human endothelial cells, line EA.hy926. The supernatant of sonicated S. pyogenes M49-16, its isogenic mutant with a deletion of the arginine deiminase gene (S. pyogenes M49-16delAD), supernatant of sonicated S. pyogenes M22, and arginine deiminase isolated from the latter strain were used. The effect of bacterial factors on migration activity of endothelial cells was studied in the model of "wound healing" in vitro. To analyze the influence of bacterial factors on the actin cytoskeleton structure, cells were stained with phalloidin-rhodamine. It was shown that supernatants of destroyed S. pyogenes, as well as arginine deiminase significantly reduced the migration activity of endothelial cells and altered the structure of their actin cytoskeleton. The supernatants of destroyed S. pyogenes M49-16delAD with deleted gene of arginine deiminase showed a significantly reduced ability to suppress cell migration as compared with the supernatant of sonicated S. pyogenes M49-16. No significant differences were revealed in the structure of actin filaments in cells cultured in the presence of supernatants of destroyed S. pyogenes M19-16, and cells cultured in the presence of isogenic mutant S. pyogenes M4916delAD. Adding exogenous arginine to the cells cultured with supernatants of destroyed S. pyogenes did not restore their migratory activity and the structure of their actin cytoskeleton. However, if arginine deficiency caused by the activity of arginine deiminase was compensated, endothelial cells migration activity was restored, and the structure of actin cytoskeleton was recovered. A decrease of migration activity of endothelial cells under the influence of streptococcal arginine deiminase was due to the disruption of actin cytoskeleton structure.

Aim. To study changes in the glycoproteins content in the glycocalyx of endothelial cells under the influence of magnesium orotate.Material and methods. Endotheliocytes of cell culture EA.hy 926 were examined before and after addition of the magnesium orotate to the culture medium. Intimal endothelium of internal carotid arteries, fragments of which were obtained by sequential execution of bilateral resection of the arteries before and after course of treatment with magnesium orotate was also investigated. Anthony’ method (1931), designed for the study of glycoproteins in the bacteria capsule and adapted by L.V. Didenko (2013) for eukaryotes was used to detect glycoproteins. The scanning electron microscopy with simultaneous x-ray microanalysis was applied.Results. Total number of Cu2+-signals per one scanning field in the mapping of samples for Cu2+ as the indirect index of proteoglycan content in EA.hy 926 endothelial cell culture before incubation with magnesium orotate was 6928±124, and after incubation with magnesium orotate – 7592±131. Number of Cu2+-signals on the surface of the internal carotid artery intima before treatment with magnesium orotate was 5015±407, and after treatment – 6100±152 per one scanning field.Conclusion. A significant increase in the content of glycoproteins on the surface of EA.hy926 endothelial cell culture (+10%) and internal carotid artery intima (+22%) under the influence of magnesium orotate was found.

Purpose. To determine the long-term status of the corneal endothelium after anterior radial keratotomy. Patients and methods. The main group consisted of 69 patients (104 eyes) who underwent radial or radial-tangential keratotomy to correct myopia and myopic astigmatism an average of 23,1±2,8 years ago (range 16 to 30 years). The average age of patients was 53,8±5,7 years (range 39 to 65 years). The number of keratotomy scars ranged from 4 to 14. Repeated keratotomy was performed on 8 eyes. The control group consisted of 56 patients (110 eyes) aged from 39 to 64 years(average 54,2±4,3 years) with refractive-axial myopia from medium to high stage, whom potentially could be done anterior keratotomy in the 90th of the last century. Patients with concomitant pathology and ocular trauma that could lead to loss ofendothelial cells were excluded from both groups. Corneal endothelium cell densit y, polimegatizm, pleomorphism in addition to standard eye examination were performed to all patients with the help of contactless endothelial microscope «EM-3000» company Tomey (Japan). Results. In the control group endothelial cell densit y averaged 2734,8±121,2 cells /mm2, in the main group — 2134,2±299,1 cells/mm2 (p

Arginine deiminase is a bacterial enzyme that hydrolyses arginine with citrulline and ammonia formation. In recent years, increasing evidence is reported about in vitro and in vivo anti-angiogenic action of arginine deiminase from Mycoplasma spp. Our studies have shown that arginine deiminase from Streptococcus pyogenes M22 exerts similar effects, i.e., inhibits proliferation and other endothelial cell functions related to angiogenesis. To confirm a leading role of arginine deiminase, as a factor responsible for the anti-proliferative effect, we have constructed an isogenic S. pyogenes M49-16 mutant unable to express arginine deiminase. A comparative analysis of anti-proliferative activity of original S. pyogenes M49-16 strain and its isogenic mutant with arginine deiminase gene deletion (M49-16delAD) was performed, using an endothelial EA.hy926 cell line. The bacterial supernatantes obtained by sonication of S. pyogenes M49-16 and M49-16delAD were tested. The ability of S. pyogenes M49-16 and M49-16delAD supernatantes to hydrolyze arginine was assessed. Moreover, we compared effects of the Streptococcus supernatantes upon proliferative activity of endothelial cells and their distribution through the cell cycle phases.Supernatantes from original S. pyogenes 49-16 strain were shown to inhibit endothelial cell proliferation to a significant degree (down to 50% of controls). This effect was due to its arginine hydrolyzing activity, i. e. addition of exogenous arginine to the medium resulted into recovery of the cell proliferation levels. The supernatante from S. pyogenes M49-16delAD showed a lower ability to hydrolyze arginine as compared to the supernatante of original strain. Culturing of endothelial cells supplied with S. pyogenes M49-16delAD supernatantes resulted into reduction of their proliferative activity by 10% of control values. Analysis of the cell cycle distribution was concordant with these results. S. pyogenes M49-16 supernatante caused a decrease in S-phase cell fraction by 20% against controls. With a supernatants from S. pyogenes M49-16delAD, such drop in DNA-synthesizing cell ratio was significantly weaker (by only 5% of the control). These results reveal new pathogenetic mechanisms of endothelial dysfunction during streptococcal infection and suggest anti-angiogenic potential of streptococcal arginine deiminase.

Regulation of angiogenesis in the utero-placental bed determines adequate trophoblast invasion, placenta formation and development, as well as successful course of pregnancy. Natural killer (NK) cells, macrophages and trophoblast have the most significant effect on angiogenesis. To date, the functions of cells participating in placenta formation have been described in detail, both individually (in vitrо) and in tissues (in situ). However, no models have yet been created that reflect the interactions of NK cells, trophoblast and endothelium during angiogenesis. It remains unclear, how each cell population contributes to placental angiogenesis regulation, and to the cross-regulation of participating cell functions. Therefore, the aim of this research was to study contact and distant effects of NK cells upon formation of tube-like structures through co-culture of endothelial and trophoblast cells influenced by various cytokines (bFGF, VEGF, PlGF, TGFβ, IL-8, IFNγ and IL-1β). Introduction of NK cells to the co-culture of endothelial and trophoblast cells under conditions of both contact and distance-dependent culturing did not change the length of tube-like structures formed by endothelial cells. During contact-dependent culturing of NK cells with co-culture of endothelial and trophoblast cells in presence of IL-1β, the length of tubule-like structures remained unchanged, compared with the length of tube-like structures formed under the same culturing conditions, but without the cytokine added. During distant culturing of NK cells with co-culture of endothelial and trophoblast cells in the presence of IL-1β, the length of tube-like structures increased as compared with those formed under the same culturing conditions but without the cytokine. During contact-dependent (but not distant) culturing of NK cells with the co-culture of endothelial and trophoblast cells in the presence of VEGF, the length of tube-like structures was greater than those formed under the same culturing conditions but without the cytokine. When used in a three-component cell system, the pro-inflammatory cytokine IFNγ had no effect upon angiogenesis. During distant (but not contact-dependent) culturing of NK cells with co-culture of endothelial and trophoblast cells in the presence of TGFβ, the length of tube-like structures was less than the length of tube-like structures formed under the same culturing conditions but without the cytokine. Under conditions of distant culturing, TGFβ triggered a signal in NK cells that inhibited angiogenesis. Decreased length of tube-like structures under conditions of a three-component cell co-culture in the presence of the following pro-angiogenic factors was revealed: IL-8, PlGF (during contact-dependent culturing only) and bFGF (during both contact-dependent and distant culturing). Thus, the effects of cytokines upon angiogenesis in a three-component co-culture (NK cells, trophoblast and endothelium) differed from those revealed previously in single-component (endothelium only) and two-component (co-culture of endothelium and trophoblast) cell models. The results of these experiments indicated that regulation of placental cell interactions involved both cellular contacts and effects produced by cytokines.

The article examines the potential role of brain mechanical damage as a trigger for the development of neurodegenerative changes. Attention is paid to dysfunction of the neurovascular unit, and disruption of the functional and compensatory capabilities of blood flow. The importance of microhemorrhages that occur in the acute period of injury and the formation of first focal and then diffuse neuroinflammation is emphasized. The importance of mitochondrial dysfunction was separately determined as a significant factor in increasing the risk of developing Alzheimer's disease (AD) in patients after traumatic brain injury (TBI). In TBI, there is a decrease in the expression of tight junction (TC) proteins of endothelial cells, such as occludin, claudin, JP, which leads to increased permeability of the blood-brain barrier. TBI, provoking endothelial dysfunction, contributes to the development of metabolic disorders of β-amyloid and tau protein, which in turn leads to worsening vascular damage, resulting in a vicious circle that can ultimately lead to the development of AD and dementia. Age-related changes in cerebral arteries, which impair perivascular transport of interstitial fluid, are currently considered as an important part of the «amyloid cascade», especially against the background of genetically mediated disorders of glial membranes associated with defective aquaporin-4 (encoded by the APOE4 ). Studies in animal models of TBI have revealed an increase in tau protein immunoreactivity and its phosphorylation, which correlates with the severity of injury. A comprehensive analysis of research results shows that the cascade of reactions triggered by TBI includes all the main elements of the pathogenesis of AD: disorders of energy metabolism, microcirculation and clearance of cerebral metabolic products. This leads to a disruption in the metabolism of amyloid protein and its accumulation in brain tissue with the subsequent development of tauopathy. Cerebrolysin, by modulating the permeability of the blood-brain barrier, blocks the development of neuroinflammation, reduces the accumulation of pathological forms of proteins and may be slow down the progression of neurodegeneration.

Purpose: This study evaluates the long-term results of surgical treatment of patients with Fuchs' endothelial corneal dystrophy and cataract., Material and Methods: The study included 24 patients (24 eyes) with primary Fuchs' endothelial corneal dystrophy and cataract, who underwent cataract phacoemulsification with IOL implantation and of Descemet's membrane endothelial keratoplasty with a semicircular graft (hemi-DMEK). The effect of treatment was assessed by best corrected visual acuity (BCVA), central corneal thickness (CCT) and endothelial cell density (ECD)., Results: In total, surgical treatment involved 14 donor corneas that were divided in half during the preparation and isolation of the Descemet's membrane (DM). By month 12 after the surgery an increase in visual functions and graft transparency were observed in 23 patients (23 eyes) out of 24. Repeated keratoplasty was required in one case due to fibrosis of the posterior layers of recipient's corneal stroma. At 12 months postoperatively, the study group showed an increase in BCVA from 0.16±0.1 to 0.75±20, a decrease in CCT from 650.9±4.5 μm to 519.6±43.9, and a decreased in ECD from 2850.5±84.7 cells/mm 2 up to 1285.5±277.2 cells/mm 2 . Thus, the loss of endothelial cells at one year after surgery amounted to 54.9%., Conclusions: The developed method for transplantation of a semicircular DM fragment provides a tissue-saving approach to endothelial keratoplasty, and considering the high percentage of transparent engraftment of grafts and complete visual rehabilitation, it can be recommended in the treatment of patients with cataract and Fuchs' endothelial corneal dystrophy.

Background: Extracellular vesicles are surrounded by a phospholipid bilayer, carrying various active biomolecules and participating in many physiological and pathological processes, including infectious ones., Objective: To research the role of exosomes in intercellular interactions in the pathogenesis of various types of lung damage in fatal cases of COVID-19., Material and Methods: We conducted a clinical and morphological analysis of 118 fatal cases caused by coronavirus infection in Moscow. We selected 32 cases with morphological signs of various types of lung lesions for immunohistochemical reaction (IHC) with antibodies against tetraspanin proteins (CD63, CD81), which are involved in the assembly of exosomes, as well as with antibodies against viral proteins: nucleocapsid and spike protein. We determined the main producing cells of extracellular vesicles and cells containing viral proteins, carried out their comparison and quantitative analysis., Results: IHC reaction with antibodies against CD63 showed cytoplasmic granular uniform and subapical staining of cells, as well as granular extracellular staining. We determined similar staining using antibodies against viral proteins. Extracellular vesicles were found in the same cells as viral proteins. The main producing cells of vesicles and cells containing viral proteins were found to be macrophages, type II pneumocytes, and endothelial cells., Conclusion: Taking into account the results of the literature, the localization of viral proteins and extracellular vesicles in the same cells indicates the key role of vesicles in the pathogenesis of various forms of lung damage by the SARS-CoV-2 virus, in the dissemination of the pathogen in the organism, which leads to interaction with the adaptive immune system and the formation of immunity.

New coronavirus infection is registered less frequently in children than in adults. Among all patients with COVID-19, the share of children is 8.6%. Clinical practice shows that in children, COVID-19 can be severe and even fatal. Articles have been published reflecting the clinical manifestations of Long Covid in children, while data on pathomorphological examination of the lungs during long-term COVID-19 in children are not available in the literature. On the basis of the Department of Pathological Anatomy with a course of Forensic Medicine and the Pathological-Anatomical Department of the Clinic of St. Petersburg State Pediatric Medical University, an analysis of medical documentation was carried out, autopsy materials were selected from 3 observations of the death of children from COVID-19. The selection criterion was the duration of the disease. A histological examination using standard methods and IHC analysis using antibodies to the nucleocapsid of SARS-Cov-2, CD95, CD31 were carried out on the lung tissue of 3 children aged 2 months to 2 years who died from a new coronavirus infection. Microscopically, all three patients showed microvessels damage, their thrombosis, angiogenesis, as well as signs of diffuse alveolar damage The combination of expression of the SARS-CoV-2 nucleocapsid and the apoptosis marker on the vascular endothelium of the MCR is of interest., Conclusion: The data obtained indicate infection with coronavirus and death of endothelial cells due to apoptosis. Endothelial damage in the microvessels of the lungs is the initiating factor in the development of capillary-alveolar block, tissue hypoxia, and disseminated intravascular coagulation syndrome, leading in some cases to respiratory/multiple organ failure and death.

Deep anterior lamellar keratoplasty or penetrating keratoplasty are currently considered the optimal methods of surgical treatment of stromal dystrophies and corneal degeneration. Despite certain advantages and benefits of these methods, they also have significant limitations: involvement of superficial corneal layers in the surgery, need for suturing, development of post-keratoplasty astigmatism etc., Purpose: This study aimed to test and describe the new method of closed sutureless keratoplasty (intracorneal selective stromal transplantation), which was indicated in isolated dystrophic and degenerative pathology of the stroma., Material and Methods: Intracorneal selective stromal transplantation was performed in a 62-year-old patient with stromal degeneration and intact corneal layers between the altered stroma and the Descemet's membrane posteriorly, and the Bowman's layer anteriorly. The patient also had immature senile cataract. Corneal stroma was removed and replaced with a graft in the optical center of the lens, while the endothelium, the Descemet's membrane and the Bowman's layer remained intact., Results: The proposed technique of intracorneal selective stromal transplantation makes it possible to replace only the pathologically altered stroma through closed surgical approach, without affecting the anterior and posterior surfaces of the cornea. Best-corrected visual acuity has increased in the patient from 0.01 to 0.6, while mean endothelial cell density has not changed in the course of 24-months follow-up., Conclusion: The proposed keratoplasty method can be used in patients with dystrophy or degeneration of the corneal stroma and preserved endothelial cells, intact Descemet's membrane and Bowman layer. Since the superficial corneal layers are not involved during the surgery, intracorneal selective stromal transplantation combined the advantages of both deep anterior lamellar keratoplasty and endothelial keratoplasty. The biologically favorable result in this first clinical case allows a preliminary conclusion on the technical possibility and functional effectiveness of the proposed method, but further long-term observation and more clinical cases are required.

Background: Early diagnosis of stroke, its treatment and rehabilitation are considered priorities in the state health care policy. Pharmacological intervention is used as a strategy to protect the neurons from the ischemic penumbra. Aim: To evaluate changes of functional state and levels of endothelial progenitor cells (EPC) in the circulation in ischemic stroke patients under administration of citicoline.Materials and methods: We assessed 11 patients in their early rehabilitation period of ischemic stroke (9 men and 2 women, mean age 58.4 ± 9.2 years). Patients from group 1 (n = 7) were given citicoline at dose 2000 mg/daily for 14 days, whereas patients from group 2 (n = 4) received regular treatment. Two healthy volunteers served as a control group. Circulatory EPC phenotype was assessed by multicolor laser flow cytometry and monoclonal antibody assay. Stroke severity and functional abilities were measured with NIHSS, Rankin scale and Rivermead mobility index.Results: Patients with ischemic stroke had decreased levels of early EPC, compared to healthy subjects. After 7 days of treatment in the group 1, there was a significant increase of circulatory EPC expressing VEGFR2+ up to 25.5 ± 20.9%, as well as an increase of early EPC numbers (CD117+ / CD133+) up to 18.8 ± 14.6%, compared to their respective baseline levels (21.9 ± 19.2 and 9.4 ± 5.4%) and compared to respective parameters in the group 2 (8.1 ± 2.7 and 5.5 ± 3.4%). In the group 1, severity of stroke and functional ability at baseline and at day 7 were as follows: NIHSS scores, 5.8 ± 4.9 and 3.5 ± 0.7, Rankin scale scores, 2.7 ± 1.3 and 2.3 ± 0.5, Rivermead mobility index, 8.8 ± 4.9 and 9.4 ± 4.8, respectively. In the group 2 corresponding parameters scored as 4.5 ± 2.3 and 3.3 ± 0.5, 2.5 ± 0.6 and 2.5 ± 0.4, 12.5 ± 1.3 and 12.7 ± 0.7, respectively.Conclusion: Treatment with citicoline in acute ischemic stroke promoted almost a 2-fold increase in early EPC levels. A decrease in neurologic deficiency and an improvement of functional status of patients with ischemic stroke were observed.

Aim. Study the effect of components of destroyed streptococci on human blood monocyte functions related to processes of trans-endothelial migration in vitro. Materials and methods. Mononuclear leukocytes, isolated from blood of healthy donors, endothelial cells of EA.hy 926 line and supernatant of ultrasound disintegrated Streptococcus pyogenes (DSS) were the objects of the study. Evaluation of adhesion and monocyte migration, level of expression of adhesion molecules and phosphokinases on monocytes was carried out by flow cytometry using monoclonal antibodies. Cytokine concentration was determined by using standard commercial test systems in enzyme immunoassay. Results. Under the effect of DSS, expression of adhesion molecules CD162 and CD11b, as well as phospho-p38 MAPK changed, IL-6 and IL-8 secretion induction took place. DSS caused enhancement of migration and adhesive activity of monocytes, however, inhibited intensity of trans-endothelial migration. Conclusion. Products of destroyed streptococci have a multi-directional effect on human blood monocytes, that could be explained by the presence of components with varying biological activity in DSS.

This review deals with the relationship between red blood cells and nitric oxide, the role of erythrocytes as participants of vasoregulation. There were the results of studies on the physiological origin of vasoactive NO under hypoxic conditions, sources of nitric oxide associated with erythrocytes. The data of the NO effect on the erythrocyte deformability have been analyzed. The hypothesis of insufficient NO bioavailability during blood storage caused by degenerative changes in red blood cells (increased levels of free hemoglobin and microparticles from erythrocytes) was presented. The results of evaluation of hemoglobin associated with NO by means of Raman-spectroscopy were given. Prospects have been identified in the diagnosis and therapy related to the definition and changes in levels of erythrocyte-derived NO and ATP.

Complicated systemic inflammatory response (SIR) often determines the outcome in patients after cardiac surgery. Systemic endothelial activation plays the most important role in SIR pathogenesis. We have studied the impact of mechanical myocardial injury products, LPS and their combination on human umbilical vein endothelial cells (HUVEC). We have found that HUVEC increase the production of proinflammatory cytokines in response tocardiomyocyte cytosolic fraction responsible for mechanical injury modeling. 2% cytosolic fraction containing 0.204 ng/mL of Hsp70 was a greater stimulus for endothelial cells to produce IL-6 and IL8 than moderateendotoxin concentrations.

Abstract. We demonstrated that TNFα, IFNγ and IL-4 induce different patterns of endothelial cells activation. The greatest alterations were observed in TNFα threated endothelial cells. Under the influence of this cytokine there was a statistically significant elevation of IL-8 and MCP-1 production by endothelial cells, of THP-1 adhesion and of migration toward endothelial cells monolayer. At the same time we had not observed any immunophenotype alteration of THP-1 cocultured with endothelial cells treated with TNFα.IFNγ и IL-4 induced statistically significant inhibition of THP-1 adhesion. IL-4 treated endothelial cells have elevated levels of IL-8 and MCP-1 production and stimulated THP-1 migrations. IFNγ showed inhibition of IL-8 and MCP-1 spontaneous production and THP-1migration.Incubation of THP-1 with untreated and IFNγ treated endothelial cells led to significant elevation of HLA-DR expression. Expression of CD68 was significantly higher in case of THP-1 cocultured with IL-4 treated endothelial cells. There was no any correlation between immunophenotype alteration THP-1cells and others parameters alteration.

Abstract. In present article, essentially new methods are described for evaluation of adhesion of peripheral blood mononuclears to human endothelial cell line Ea.Hy926. These methods are suitable for both experimental purposes and research in clinical immunology, including laboratory diagnostics of immune deficiencies, infectious diseases, complicated pregnancy, and other kinds of clinical disorders.

Abstract. Vascular endothelial cells (VECs) are able to induce HIV1 replication in СD4+ Tcells, thus determining their resistance for apoptosis. The revealed facts are specific to EC/Tcell culture and are stipulated by the cellcell interactions between the VECs and HIV1 producing cells, thus being a consequence of activation of ‘productively infected’ T cells.

Abstract. Placental cell populations produce a great variety of angiogenic factors and cytokines than control angiogenesis in placenta. Functional regulation of endothelial cells proceeds via modulation of endothelial cell receptors for endogenous angiogenic and apoptotic signals. Endothelial phenotype alteration during normal pregnancy and in cases of preclampsia is not well understood. The goal of this investigation was to evaluate altered expression of angiogenic and cytokine receptors at EA.hy926 endothelial cells under the influence of placental tissue supernatants. Normal placental tissue supernatants from 1st and 3rd trimesters, and pre-eclamptic placental tissue supernatants (3rd trimester) stimulated angiogenic and cytokine receptors expression by the cultured endothelial cells, as compared with their background expression. Tissue supernatants from placental samples of 3rd trimester caused a decreased expression of angiogenic and cytokine receptors by endothelial cells, thus reflecting maturation of placental vascular system at these terms. Supernatants from preeclamptic placental tissue induced an increase of CD119 expression, in comparison with normal placental supernatants from the 3rd trimester. This finding suggests that IFNγ may be a factor of endothelial activation in pre-eclampsia. The study was supported by grants ГК №02.740.11.0711, НШ-3594.2010.7., and МД-150.2011.7.

Abstract. Leukocyte recruitment to placental tissue is an important factor of its development. In this respect, adhesion molecules at the endothelial cell surface represent a key determining factor of leukocyte adhesion and their trans-endothelial migration. The goal of investigation was to evaluate changed expression of adhesion molecules on the endothelial cells induced by supernates of placental tissue cultures. Placental tissue supernatants produced by the first- and third-trimester placental tissue from normal pregnancy, as well as from women with gestosis, induced higher expression of CD31, CD9, CD62E, CD62P, CD34, CD54, CD51/61, CD49d and integrin β7 expression by endothelial cells, as compared with their baseline levels. However, the supernates from pre-eclamptic placental tissue (3rd trimester) caused an increased CD9 expression by endothelial cells, as compared with effects of placental supernates from eclampsia-free cases. Our data contribute to understanding a possible role of endothelial cell adhesion molecules in recruitment of leukocytes to placental tissue and possible participation of adhesion molecules in pathogenesis of pre-eclampsia. The work was supported by a grant from Russian Ministry of Education and Science ГК №02.740.11.0711 and Presidential grant № НШ-3594.2010.7 and МД-150.2011.7. (Med. Immunol., 2011, vol. 13, N 6, pp 589-596)

Abstract. Adhesion to blood vessel endothelium is an important stage of leukocyte migration. This study was aimed for studying the effects of supernates from ultrasound-destroyed Streptococcus pyogenes (SUDS) upon adhesive properties and adherence degree of THP-1 myelomonocytic leukemia cells to EA.hy926 endothelial cells. After 24-hour incubation of THP-1 cells with SUDS, a reduction in CD49d expression levels on these cells is observed. Plastic adherence of endothelial cells was reduced in presence of SUDS, whereas similarly treated THP-1 cells become more adherent to plastic surfaces. A 24-hour pre-incubation of THP-1 cells with SUDS resulted in reduction of their adherence to endothelium, either with SUDS-treated, or non-treated endothelium cultures. Prolonged incubation of endothelial cells with SUDS caused an increase in adherence of intact THP-1 cells to endothelium. Hence, the adhesion properties of endothelial and THP-1 cells are changed in different ways under the influence of streptococcal components. Such variability may be due to involvement of different cellular receptors for monocytes and endothelial cells during recognition of bacterial components. (Med. Immunol., 2011, vol. 13, n 6, pp 581-588)

The paper considers the consequences and causes of magnesium deficiency in patients with cardiovascular diseases. The features of magnesium metabolism in atherosclerosis, hypertension, and heart failure are described. Prospects of magnesium therapy in cardiology practice are discussed.

Endothelial cells (ECs), which form the inner surface of the blood vessels, contact the blood, withstand mechanical pressure, and demonstrate heterogeneous reactions to exogenous and endogenous stimuli. ECs have unique properties in accordance with their niches and play an important role in regulating vascular homeostasis. Endothelial cells may undergo a dynamic phenotypic switch in terms of its heterogeneity, which may lead to endothelial dysfunction and a number of associated pathologies. Endothelial-mesenchymal transition (EndMT) is one of the possible molecular and cellular mechanisms of this kind. EndMT is characterized by phenotypic changes in ECs through which endothelial cells acquire new properties, i.e., start producing mesenchymal markers such as alpha-SMA and vimentin, change morphology, and become able to migrate. EndMT is a complex biological process that can be induced by inflammation, hypoxia, or oxidative stress and be involved in pathogenesis of cardiovascular disease. This review describes the key markers, inhibitors, and inducers of endothelial-mesenchymal transition and overall state-of-the-art of EndMT in cardiovascular diseases.

Using neuromorphometry analysis differences in the effects of nootropic drugs on morphology and function of neurons and endothelial cells of hippocampus, content of RNA, content of apoptotic and destructive neurons were examined in white rats with chronic alloxan-induced hyperglycemia. It ha s been found that diabetes in rats is accompanied by specific morphological and functional changes and activation of apoptosis in neurons of the CA1-area in hi ppocampus, which may be related to disturbance of local blood flow due to endothelial damage. N-carbamoyl-methyl-4-phenyl-2-pyrrolidone (entrop), N-phenylacetyl-L-prolylglycine (noopept), pramiracetam, cerebrocurin and citicoline show protective effects on neurons and endothelial cells, which are much larger in force than effect s of ginkgo biloba extract, piracetam and pentoxifylline. This protective activity is characterized by reducing the number of apoptotic and dest ructive neurons in hippocampal CA1-area, increasing the density of functioning nerve and endothelial cells, activation of RNA biosynthesis in the neurocytes and endo-thelial cells

Aim      To evaluate the endothelial function using an Endo-PAT2000 instrument before and after mental stress tests in patients with Tacotsubo syndrome (TS) during acute and long-term periods and to compare the obtained results with laboratory markers of endothelial dysfunction (ED).Material and methods  This study included 45 patients with TS (mean age, 63.5±13.7 years) and 40 healthy volunteers (control group, CG). All patients of the main group during the acute period (first 7-14 days) and long-term period (at 1 and 2 years), as well as CG subjects, underwent evaluation of the endothelial function with an Endo-PAT 2000 instrument, and the reactive hyperemia index (RHI) was determined before and after mental stress tests. Also, concentrations of endothelin 1 (ET-1) and numbers of circulating endothelial cells (CEC) were measured after a two-year follow-up of TS patients.Results During the acute period of disease, all TS patients (n=45) had ED: RHI was below the threshold level of 1.67; furthermore, 42 (93.3 %) patients retained a lower RHI following mental stress. At one year (n=40), 16 (40%) patients showed ED at rest along with a significantly increased mean RHI. Mental stress produced at one year was associated with ED in 28 (70 %) TS patients. At two years (n=44), resting RHI was lower than normal in 19 (43.2 %) patients. Mental stress tests performed at two years were associated with ED in 29 (65.9 %) patients (RHI ≤1.67). Only 10% of CG subjects had a lower-than-normal RHI, which was significantly less than in the main group of TS patients during the acute and long-term periods (p<0.05). Mean values of laboratory markers for ED also were significantly different between TS patients during the long-term period of disease (n=41) and CG subjects (n=40; p<0.01).Conclusion      During acute and long-term periods of disease, most of TS patients had impaired vascular reactivity both at rest and during mental stress. The laboratory markers of ED, ET-1 and CEC can be used in clinical practice for evaluation of the risk for TS.

Aim. Cytostatic drugs used in the treatment of hemopoietic malignancies affect cells of other organs and systems, vascular endothelium, in particular. This leads to cardiovascular complications. We studied effects of some drugs and their combinations on cultured human endothelial cells (EC). Material and methods. Primary culture of umbilical human vein EC was used in the study. Cytosinearabinoside (cytozar, alexan, cytarabin) and daunorubicin were added in concentration from 1 ng/ml to 1 mg/ml; the action of the drugs was assessed by morphology of the cells, their viability, inclusion of bromodesoxyuridine by means of cloning, immunohistochemistry and flow cytofluorimetry. Results. It is shown that in a wide range of concentrations cytosinearabinoside and daunorubicine cause specific structural-functional changes in the cells related to cytostatic and cytotoxic effects. Conclusion. The data of the study confirm that cardiovascular complications in the course of acute leukemia treatment are resultant from systemic damage to vascular endothelium.

Objective: Evaluation of structural and immunohistochemical features of cornea in Fuchs endothelial corneal dystrophy (FECD) and bullous keratopathy (BK)., Material and Methods: Group 1 - 44 patients (46 eyes) with FECD, group 2 - 42 patients (42 eyes) with BK. All patients underwent keratoplasty. Preoperative anterior segment optical coherence tomography (AS-OCT, RTVue-100, Optovue, USA) was performed. Endothelium-Descemet membrane (EDM) complexes, corneal buttons were obtained intraoperatively. Morphological (H&E staining) and immunohistochemical (primary antibodies to pancytokeratin, vimentin, fibronectin) studies were performed at the light microscope level (Leica DM-2500, Leica Application Suite V4.8, Leica Microsystems, Switzerland)., Results: A direct correlation is found between the results of DM analysis in vivo with OCT and ex vivo with light microscopy. DM thickness (AS-OCT) was significantly greater in FECD (23.0 [19.0; 27.0] μm), than in BK (13.0 [12.0; 14.0] μm). Morphological study of EDM and corneal buttons showed similar difference in DM thickness: 17.9 [16.1; 20.0] μm in FECD and 11.9 [11.3; 13.0] μm in BK. Irregular optical density of stroma is a feature of edema and local fibrosis. In FECD and BK pancytokeratin is expressed in epithelial and endothelial cells, vimentin - in keratocytes, macrophages and vascular endothelium, fibronectin - in DM. In FECD, vimentin is expressed in endothelial cells., Conclusion: FECD and BK are associated with different DM' and endothelium' abnormalities, which lead to similar changes of stroma and epithelium. AS-OCT is a useful method of FECD and BK in vivo diagnostics and the selection of treatment option.

Intrastromal corneal ring segments (ICRS) of in various shapes are used to correct structural changes in the cornea and improve visual acuity in keratoconus (KC). The most serious complications of the technology infection, vascular ingrowth, and extrusion. In this study a 44-year-old patient underwent deep anterior lamellar keratoplasty (DALK) 5 years after implantation of ICRS in the shape of an incomplete ring to treat its extrusion of up to 30%, vascular ingrowth into the cornea and corneal syndrome. The following DALK technique was used: excision of the free part of the corneal segment, removal of the remaining part of the corneal segment, excision of 80% of thickness of the corneal block with a 8.5-mm trepan, pneumatic dissection of the stroma down to the Descemet membrane (DM), removal of the remaining stroma on the viscoelastic pillow, laying and suture fixation of the donor graft. There were no complications during the postoperative period, the graft remained transparent for up to 10 months. Uncorrected visual acuity (UCVA) increased from 0.01 to 0.4, best corrected visual acuity (BCVA) increased from 0.3 to 0.7. The number of endothelial cells decreased from 2980 to 2670 cells/mm 2 (10.4%). This clinical case demonstrates that DALK surgery can be a reliable method of patient rehabilitation in cases of extrusion of ICRS in the shape of an incomplete ring and vascular ingrowth.

The study presents a study of the expression of CD68+ and CD163+ in the bladder as a marker of cancer in age-related aspects. As part of the work performed, 35 patients with bladder cancer with histological verification of the disease aged 36 to 70 years were studied. The role of CD68+ and CD163+ cells in bladder reactions can be interpreted as an indicator of diagnostic parameters of the activity of tissue macrophages and endothelial cells in cancer. Their progression during oncogenesis is especially pronounced in the group of middle-aged patients. In old age, there is a decrease in these parameters, which indicates in favor of a decrease in the activity of the development of oncological processes with age. The combination of morphological and immunological atypism leads to a violation of the functional specialization of the organ.

In vitro blood-brain barrier (BBB) modeling with the use of the brain endothelial cells grown on a transwell membrane is widely used to investigate BBB disorders and factors intended to ameliorate these pathologies. Endothelial cells, due to tight junction proteins, ensure selective permeability for a number of substances. The low integrity (i.e., high permeability) of the BBB model, as compared to the physiological one, complicates evaluation of the effects caused by different agents. Thus, the selection of conditions to improve barrier integrity is an essential task. In this study, mouse brain endothelial cells bEnd.3 are used in experiments on transwell modeling. To determine which factors enhance BBB integrity, the effects of the cultivation medium, the number of cells during seeding, the state of the transwell membrane, and cultivation in the presence or in the absence of primary mouse neurons and matrigel as a matrix on the passage of a fluorescent label through the cell monolayer were assessed. The effect of fetal bovine serum on the tight junction protein claudin-5 was analyzed by immunocytochemistry. The obtained cultivation parameter data facilitate the solution to the problem of low integrity of the BBB transwell model and bring the model closer to the physiologically relevant indicators.

DNA-methyltransferases catalyze DNA methylation in the CpG sites, which play an important role in the maintenance of genome stability. The association between DNA methylation and genotoxic stress resulting in the action of various clastogens has been shown. Genotoxic stress is one of the triggers of endothelial dysfunction. In this study, the transcription of DNMT1, DNMT3A and DNMT3B genes in coronary (HCAEC) and internal thoracic (HITAEC) artery endothelial cells exposed to alkylating mutagen mitomycin C was studied using quantitative polymerase chain reaction. In HCAEC exposed to mitomycin C, DNMT1 transcription is 1.7-fold higher compared to the unexposed control. After elimination of the mutagen from the cultures followed by 24-hours of cultivation, a 2-fold increase of transcription of DNMT3B in HCAEC exposed to mitomycin C compared to the control was observed. At the same time, no changes in transcription of the studied DNA-methyltransferases were found in HITAEC exposed to the mutagen. Thus, increased transcription of DNA-methyltransferase may be a possible molecular mechanism underlying endothelial dysfunction in response to mutagenic load in an in vitro experiment.

Background: Cancer-associated fibroblasts (CAFs) are a heterogeneous cell population in the tumor stroma and have important prognostic and clinical significance for solid tumors, including colorectal cancer. The identification of CAF presents difficulties due to the lack of a unique diagnostic marker., Objective: Detection of CAF by multiplex immunohistochemical staining and assessment of their colocalization., Material and Methods: For multiplex IHC staining specimens of 10 colon adenocarcinomas without neoadjuvant treatment were selected. We used «OPAL 7-COLOR MANUAL IHC KIT» (Akoya Biosciences, USA) with five antibodies (FAP, PDGFRβ, CD31, POD, PCK) for staining and Mantra 2 Quantitative Pathology Imaging System (Akoya Biosciences, USA) for evaluation of results., Results: CD31 and CAF markers (FAP, PDGFRβ, POD) are expressed fundamentally in different cells ( p <0.0001) in all areas of the tumor (apical, central, invasive margin). Pairs FAP+PDGFRβ in all zones demonstrated significantly higher ( p <0.0001) square of tandem staining. It shows that these markers are expressed in the same stromal cells (probably CAF). In pair FAP+POD significant colocalization ( p =0.011) was detected only in apical zone. We connect this finding rather with active proliferation of population of young fibroblasts in zones of ulceration and granulations than with CAF., Conclusion: We evaluated co-localization of CAF markers (FAP, PDGFRβ, POD) and endothelial cells (CD31) in different zones of colorectal carcinomas. We showed colocalization of CAF markers for pairs FAP+PDGFRβ in all tumor zones and for pair FAP+POD in apical zone.

Objective: To estimate the efficiency of stimulation of reparative processes in patients with diabetes mellitus type 2 and trophic ulcers of the lower limbs., Material and Methods: There were 94 patients with diabetes mellitus type 2 complicated by trophic ulcers of the lower limbs. Mean age of patients was 67.8±8.6 years. The main group consisted of 50 (53.2%) patients who underwent stimulation of reparative processes, control group - 44 (46.8%) patients with standard treatment and hydrocolloid dressings. Efficiency of treatment was analyzed every 5 days considering wound healing and cytological data., Results: We found significant reduction of neurotrophic ulcer volume by 29.9% after 10 days. Positive macroscopic data were accompanied by reduction of white blood cells count up to 40-50% and increase in the number of histiocytes and endothelial cells. Among patients with ischemic ulcers, we obtained significant reduction of ulcerative defect only after 15-20 days in 3 (60%) cases. Overall efficiency of original management way 96%, mean duration of treatment - 37.1±5.9 days. In the control group, overall efficiency of treatment was 70.5%, duration of treatment - 45.9±6.1 days., Conclusion: The original method was valuable to improve the outcomes in patients with diabetes mellitus type 2 and trophic ulcers of the lower limbs. The best results were obtained for neurotrophic ulcers. In case of ischemic ulcers, these technologies should be combined with measures on improvement of pulsatile blood flow and microcirculation. This method does not require hospitalization and reduces treatment cost.

Secondary decompensation of corneal endothelium, including transplanted, is a frequent long-term complication of glaucoma drainage surgery. According to literature data, after implantation of a glaucoma drainage device into the anterior chamber, the speed of endothelial cells density (ECD) loss increases significantly., Purpose: To study the possibility of performing modified Descemet membrane endothelial keratoplasty (DMEK) with maximum graft diameter, and to assess its short-term effectiveness in the treatment of bullous keratopathy (BK) in the presence of a glaucoma drainage device in the anterior chamber., Material and Methods: Modified surgery 3 / 4 -DMEK was performed in 9 eyes of 7 patients with BK and Ahmed glaucoma drainage using graft of maximum diameter (11±1 mm). Average follow-up time was 15.3±2.5 months (12 to 18 months)., Results: No intraoperative or postoperative complications were observed. Mean ECD after 12 months amounted to 1664±346 cells/mm 2 , which corresponds to ECD reduction of 34±9.6% (24 to 49%) in comparison to preoperative values. Mean best corrected visual acuity 12 months after surgery increased from 0.04±0.03 (0.01 to 0.1) to 0.3±0.2 (0.04 to 0.7). The last follow-up examination revealed all corneas to have preserved transparency and stable pachymetry measurements (from 488 to 548 µm)., Conclusion: Performing 3 / 4 -DMEK with a mega graft for treatment of BK after drainage surgery is technically possible. Absence of Descemet membrane (DM) donor and preservation of DM recipient in the drainage tube plane eliminates its contact with the endothelium. Maximum graft diameter should in the long term help prolong the effectiveness of the surgery.

Objective: To compare the neointima structure in conduits for coronary bypass grafting, bioprosthetic heart valves, tissue-engineered vascular grafts, and metal stents., Material and Methods: The objects of the study were the fragments of the human internal thoracic artery, experimental biodegradable vascular prostheses, leaflets of xenopericardial bioprostheses of heart valves, and fragments of stented vessels. Tissue samples were fixed in formalin and post-fixed in osmium tetroxide. After dehydration and epoxy resin embedding, the samples were ground and polished. Samples were counterstained with uranyl acetate and lead citrate and visualized by means of backscattered scanning electron microscopy., Results: Neointimal pattern in all samples was similar. Neointima was comprised of endothelial cells, smooth muscle cells, fibroblasts, and the extracellular matrix. Endothelial cells showed significant diversity both between different elements of the circulatory system and within the same tissue, having either elongated or polygonal shape. Adhesion of leukocytes testified to the endothelial cell activation. In the absence of inflammation in the superficial layer of the neointima, the arrangement of smooth muscle cells and extracellular matrix fibers was parallel to the endothelium. Clusters of foam cells were frequently detected around the neointimal layers with solid inclusions (metal stents or calcium deposits). Thickening of the neointima was accompanied by the presence of capillaries and capillary-like structures., Conclusion: Neointima formation is a typical response to the damage inflicted to the elements of the circulatory system. Neointima underwent a constant remodeling characterized by an altered cellular composition, macrophage invasion, neovascularization, and calcification.

Extracellular vesicles (EVs) are spherical structures of cell membrane origin, ranging in the size from 40 nm to 5000 nm. They are involved in the horizontal transfer of many proteins and microRNAs. The mechanisms EV internalization include clathrin-dependent endocytosis, caveolin-dependent endocytosis, raft-mediated endocytosis, and macropinocytosis. Type 2 diabetes mellitus (T2DM) is a common group of metabolic disorders in adults; the incidence and prevalence increase in parallel with the obesity epidemic. Since adipose tissue plays a crucial role in the development of insulin resistance, EVs secreted by adipose tissue can be a kind of information transmitter in this process. EVs of adipocytic origin are predominantly absorbed by tissue macrophages, adipocytes themselves, hepatocytes, and skeletal muscles. This contributes to the M1 polarization of macrophages, a decrease in glucose uptake by hepatocytes and myocytes due to the transfer of functionally active microRNAs by these EVs, which affect carbohydrate and lipid metabolism. Patients with T2DM and impaired glucose tolerance have significantly higher levels of CD235a-positive (erythrocyte) EVs, as well as a tendency to increase CD68-positive (leukocyte) and CD62p-positive (platelets/endothelial cells) EVs. The levels of CD31+/CD146-positive BB (endothelial cells) were comparable between diabetic and euglycemic patients. EVs from diabetic patients were preferably internalized by monocytes (mainly classical and intermediate monocyte fractions and to a lesser extent by non-classical monocyte fractions) and B cells compared to euglycemic patients. Internalization of EVs from patients with T2DM by monocytes leads to decreased apoptosis, changes in differentiation, and suppression of reactions controlling oxidative stress in monocytes. Thus, insulin resistance increases secretion of EVs, which are preferentially internalized by monocytes and influence their function. EVs are considered as sources of promising clinical markers of insulin resistance, complications of diabetes mellitus (endothelial dysfunction, retinopathy, nephropathy, neuropathy), and markers of EVs can also be used to monitor the effectiveness of therapy for these complications.

Introduction: Influenza A virus infection can lead to endothelial dysfunction (ED), including apoptosis of endothelial cells and modulation of endothelial factor activities. Affected biochemical factors may include those playing important roles in vascular homeostasis. However, the effect of this pathogen on the expression pattern of key endothelial factors is still unknown.The aim of this work was to study the expression of endothelial nitric oxide synthase (eNOS) and plasminogen activator inhibitor-1 (PAI-1, serpin E1) in the EA.hy926 endothelial cells., Research Objectives: to assess expression of eNOS and PAI-1 in endothelial cells infected with influenza virus A(H1N1)pdm09, and to identify homologous fragments in structure of viral proteins and endothelial factors., Material and Methods: Cells were infected with influenza virus A/St. Petersburg/48/16 (H1N1)pdm09 and analyzed in dynamics in 6, 12, 18, 24, 48, and 72 hrs post infection (hpi). Detection of endothelial factors expression levels was performed by immunocytochemical method (ICC) using antibodies for eNOS and PAI-1 while quantitative assessment of expression levels was carried out by program Nis-Elements F3.2 («Nikon», Japan). The search for homologous sequences between viral proteins and eNOS and PAI-1 was performed by computer comparison. Sequences were analyzed as fragments 12 amino acid residues (aar) in length., Results and Discussion: eNOS expression in infected cells had decreased to 7.9% by 6 hpi (control was taken as 100%) to 3.3% at 72 hpi. PAI-1 expression varied significantly over the course of the experiment: by 6 hpi it had decreased to 49.6%, and to 43.2% by 12 hpi. Later PAI-1 levels were: 116.3% (18 hpi); 18.9% (24 hpi); 23.5% (48 hpi), and 35% (72 hpi)., Conclusion: These results indicate that influenza A infection of endothelial cells causes a significant decrease in eNOS expression, while modulating PAI-1 one. The described phenomenon can be used in the further development of directions of pathogenetic therapy of vascular complications of infection caused by this pathogen.

The article discusses modern views on the metabolic characteristics of adipose tissue in patients with non-alcoholic fatty liver disease (NAFLD). An association has been shown between NAFLD and metabolic risk factors such as dyslipidemia, hyperglycemia, and visceral obesity. The analysis of modern literature on adipose tissue as an endocrine organ is carried out. The recently revealed physiological and pathophysiological properties of adipokines are discussed. It has been documented that adiponectin counteracts the effect of angiotensin II on endothelial cells and prevents their apoptosis by increasing the association between eNOS and heat shock protein. Adiponectin can function as a negative regulator of angiogenesis. It inhibits the proliferation and migration of endothelial cells and markedly inhibits the growth of new blood vessels. The ratio of adiponectin to leptin is a biomarker of adipose tissue dysfunction and correlates better and more accurately with insulin resistance than adiponectin or leptin. The researchers concluded that fatty tissue dysfunction caused by low adiponectin levels may contribute to oxidative stress and inflammation. Medications aimed at synthesizing adiponectin will be useful in the treatment of NAFLD, obesity, diabetes, and cardiovascular disease.