Endothelialization of Polycaprolactone Vascular Graft under the Action of Locally Applied Vascular Endothelial Growth Factor.

We have previously developed a polycaprolactone (PCL) vascular graft with incorporated vascular endothelial growth factor (VEGF). Functioning of the PCL/VEGF graft in rat circulatory system over 1, 3 and 6 months after implantation into abdominal aorta was tested. Graft patency and formation of vascular wall elements were assessed histologically and by immunofluorescence staining for von Willebrand factor, CD31, CD34, and collagens I and IV and DAPI staining. Local application of VEGF promoted endothelialization and improved patency of the graft. The wall of the PCL/VEGF graft underwent remodeling due to active cellular infiltration and the extracellular matrix deposition.

Influence of visceral obesity on the secretion of adipokines with epicardial adipocytes in patients with coronary heart disease.

AIM: To study adipokine-cytokine profile of epicardial adipocytes (EAT) and subcutaneous adipose tissue (SAT) in conjunction with the area of visceral adipose tissue (VAT), biochemical and clinical characteristics of patients with coronary heart disease. MATERIALS AND METHODS: Examined 84 patients (70 men and 14 women) with coronary artery disease. In fact the presence of visceral obesity (VO) the patients were divided into two groups. Patients VO the sampling of adipocytes of EAT and SAT, with subsequent cultivation and evaluation of adipokine and provospalitelna activity. Carried out the determination of carbohydrate and lipid metabolism, adipokine and pro-inflammatory status in the blood serum. RESULTS: It was found that adipokine-cytokine profile of adipocytes of EAT and SAT differ. Adipocytes art of the disease on the background characterized by an increase IL-1, TNF-α, leptin-adiponectin relationships and a decrease in the content of protective factors: adiponectin and anti-inflammatory cytokine IL-10. While the SAT adipocytes was characterized by a decrease in the concentration of soluble receptor for leptin and the more pronounced leptinresistance, and the increase in proinflammatory cytokines was offset by the increase in the concentration of IL-10. The presence associated with multi-vessel coronary bed lesion, multifocal atherosclerosis, insulin resistance, atherogenic dyslipidemia, an imbalance of adipokines and markers of inflammation. So the value of the square VAT determined higher concentrations of leptin, TNF-α in adipocytes and serum, lipid and carbohydrate metabolism and a lower content of soluble receptor for leptin. CONCLUSION: Thus, the disease on the background of the status of the adipocytes of EAT characterized as a "metabolic inflammation", and may indicate the direct involvement of adipocytes in the pathogenesis of coronary artery disease, due to the formation of adipokine imbalance and the activation of proinflammatory reactions.

A study of biocatalysts based on glucose oxidase.

During this work, we studied the possibility of glucose oxidase (GOx) covalent immobilization on a modified inorganic support. A series of GOx-based biocatalysts was synthesized by crosslinking the enzyme to a surface of modified silica or alumina. Polyelectrolyte layers were used as modifiers for the silica and alumina surfaces. These layers promote tight binding of the GOx to the support. The biocatalyst's activity and stability were studied using an oxidation reaction of d-glucose to d-gluconic acid. It was found that GOx immobilized on the modified SiO2 using glutardialdehyde as a crosslinking agent was the most active and stable catalytic system, showing an 85% yield of gluconic acid. A study of the synthesized biocatalyst structure using FTIR spectroscopy showed that the enzyme was covalently crosslinked to the surface of an inorganic support modified with chitosan and glutardialdehyde. In the case of SiO2, the quantity of the immobilized enzyme was higher than in the case of Al2O3.

Adipokine and Cytokine Profiles of Epicardial and Subcutaneous Adipose Tissue in Patients with Coronary Heart Disease.

The content of adipokines, pro- and anti-inflammatory cytokines were studied in adipocytes isolated from epicardial and subcutaneous adipose tissue of 24 coronary heart disease patients. The content of leptin and soluble leptin receptor in adipocytes of epicardial adipose tissue was higher by 28.6 and 56.9% and the level of adiponectin was lower by 33% than in adipocytes of the subcutaneous fat. In culture of epicardial adipocytes, the levels of proinflammatory cytokines TNF-α and IL-1 were higher. Subcutaneous adipose tissue adipocytes were characterized by higher levels of anti-inflammatory cytokines IL-10 and FGF-ß. In epicardial adipocytes of coronary heart disease patients, the concentrations of leptin, TNF-α, and IL-1 were higher, while the levels of defense regulatory molecules (adiponectin, IL-10, and FGF-ß) were lower than in subcutaneous adipocytes.

HUMAN KARYOTYPE ANALYSIS IN DIAGNOSIS VERIFICATION.

When analyzing a patient's karyotype using classic cytogenetic tools, clinical cytogeneticists frequently face a problem of whether the observed morphological variant of a chromosome is the norm or pathology. Here we present three cases, when the use of additional approaches allowed us to accurately and reliably describe the chromosomal abnormalities and to provide a substantiated medical and genetic prognosis. Translocations were preliminary diagnosed in the first two patients. This opinion was subsequently challenged, as these patients were the carriers of rare variants of normal chromosome polymorphisms (21pstkstkpss and 20cenh+). Thus, these diagnostic measures helped the wife of the first patient to maintain the pregnancy, whereas the second patient was referred for IVF. In the third case, the preliminary diagnosis trisomy of chromosome 22 has not been confirmed. This patient turned out to be a carrier of a supernumerary marker chromosome invdup(15)(q13), which offers a much more favorable medical prognosis.

Problems and prospects of investigating monocyte subsets during the development of inflammation-associated diseases.

In this review, we present information about a heterogeneity of monocyte subsets based on their unique functional and phenotypic properties. Here we also discuss the search of an optimal technique for the isolation of monocyte subsets as well as the origin of monocyte subsets and their role in inflammation.

[Proliferative and secretory activity of human umbilical vein endothelial cells cultured under varying degrees of hypoxia].

In this study we examined the impact of 3-day hypoxia of varying degrees on the viability, proliferative and secretory activity of endothelial cells in human umbilical vein (HUVEC). The gas mixture of the three components (%) was used: 1) 10 O2, 5 CO2, 85 Ar; 2) 5 O2, 5 CO2, 90 Ar and 3) 1 O2, 5 CO2, 94 Ar. The HUVEC, cultivated in CO2-incubator under conditions of atmospheric oxygen (21% O2) were the controls. Comprehensive assessment of the results after has shown that 3-day HUVEC cultivating in the presence of 1% O2 led to pathological activation of endotheliocytes: increased NO synthesis combined with the marked secretion of endothelin-1, IL-6, IL-8 and TNF-alpha, sVCAM-1, sE-cadherin and of sE-selectin, VEGF-A and bFGF, and slow proliferation. When HUVEC were cultivated at 10% O2 and 5% O2, the level of basal secretion of the substances listed above was the least against the background of increased proliferative activity. The results showing the changes in the secretory activity of endothelial cells when cultivated under the conditions of atmospheric oxygen levels have demonstrate HUVEC activation, because the secretion of NO, IL-6, IL-8 and von Willebrand factor after 3 days of cultivation in 21% 02 exceeded that in the case of 10 and 5% O2. Thus, a gaseous medium with reduced oxygen content of up to 5% provides more physiological conditions for HUVEC cultivation. The maximum proliferative activity of HUVEC with minimal basal secretion proved such a composition to be comfortable. Increasing the oxygen content to the atmospheric level leads to the activation of endotheliocytes with signs of endothelial dysfunction, and the critical reduction in oxygen to 1% causes the development of endothelial dysfunction and reduces the proliferative potential.

New Tissue-Engineered Vascular Matrix Based on Regenerated Silk Fibroin: in vitro Study.

The aim of the study was to make a vascular patch based on regenerated silk fibroin (SF) and study its physical and mechanical characteristics, biocompatibility and matrix properties in comparison with polyhydroxybutyrate/valerate/polycaprolactone with incorporated vascular endothelial growth factor (PHBV/PCL/VEGF) and commercial bovine xenopericardium (XP) flap in experiments in vitro. Materials and Methods: Tissue-engineered matrices were produced by electrospinning. The surface structure, physical and mechanical characteristics, hemocompatibility (erythrocyte hemolysis, aggregation, adhesion and activation of platelets after contact with the material) and matrix properties of vascular patches (adhesion, viability, metabolic activity of EA.hy926 cells on the material) were studied. Results: The surface of SF-based matrices and PHBV/PCL/VEGF-based tissue engineered patches had a porous and fibrous structure compared to a denser and more uniform XP flap. The physical and mechanical characteristics of SF matrices were close to those of native vessels. Along with this, tissue-engineered patches demonstrated high hemocompatible properties, which do not differ from those for commercial XP flap. Adhesion, viability, and metabolic activity of EA.hy926 endothelial cells also corresponded to the previously developed PHBV/PCL/VEGF matrix and XP flap, which indicates the nontoxicity and biocompatibility of SF matrices. Conclusion: Matrices produced from regenerated SF demonstrated satisfactory results, comparable to those for PHBV/PCL/VEGF and commercial XP flap, and in the case of platelet adhesion and activation, they outperformed these patches. In total, SF can be defined as material having sufficient biological compatibility, which makes it possible to consider a tissue-engineered matrix made from it as promising for implantation into the vascular wall.

[Postoperative dynamic changes in matrix metalloproteinase levels in patients with coronary artery bypass graft procedure complications].

The purpose of this study was to evaluate diagnostic and prognostic value of matrix metalloproteinases in postoperative complication development after on-pump coronary artery bypass grafting. 29 coronary artery disease patients who had undergone on-pump coronary artery bypass grafting were examined, 4 of those had complicated systemic inflammatory response and 5 of those had isolated renal failure. Serum matrix metalloproteinase/tissue inhibitor of matrix metalloproteinase concentrations were measured before surgery, at day 1 and day 7 after surgery. Postoperative period was found to be characterized by higher levels of serum matrix metalloproteinases (MMP-9, proMMP-1) and lower levels of matrix metalloproteinase 1 tissue inhibitor. Complicated systemic inflammatory response was associated with higher levels of MMP-9, MMP-3 and proMMP-1, on particular, at day 7.

Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis.

The aim of the study was to assess the suitability of endothelial colony-forming cells in the development of tissue engineering constructs based on the study of the gene expression profile compared to mature endothelial cells. Materials and Methods: In the experiment, we used the endothelial colony-forming cells (ECFC) obtained from the peripheral blood of patients who underwent percutaneous coronary intervention. The cells were isolated on a Histopaque 1077 density gradient (Sigma-Aldrich, USA), and then cultured in EGM-2MV culture medium (Lonza, Switzerland). A commercial culture of primary human coronary artery endothelial cells (HCAEC) was used as a control. The cells were unfrozen and cultured according to the manufacturer's recommendations in MesoEndo Cell Growth Medium (Cell Applications, USA).The experiment was carried out in specialized µ-Luer plates in the perfusion system (IBIDI, Germany), which provided a continuous unidirectional flow of the culture medium with a shear stress of 5 dyn/cm2. Control plates were cultured under standard conditions for a similar period of time. Total RNA was isolated from cell samples. The expression of the genes NOTCH4, NRP2, PLAT, PLAU, NOTCH1, FLT1, COL4A2, CD34, SERPINE1, HEY2, MKI67, KLF4, LYVE1, FLT4 was assessed using a quantitative real-time polymerase chain reaction. The expression of the genes was calculated by the ΔCt method and expressed on a logarithmic (log10) scale as a fold change relating to the control samples. Results: In mature endothelial cells HCAEC when exposed to a laminar flow, only the transcription factor KLF4 and venous differentiation NRP2 marker values increased significantly. ECFC showed statistically significant growth in KLF4, NRP2, CD34, and LYVE1, as well as PLAU expression decrease. In addition, we observed the overexpression of FLT4, LYVE1, NOTCH4, and NRP2 in ECFC in relation to HCAEC and HEY2 hypoexpression. CD34 overexpression characteristic of progenitor cells was also found. An increase in COL4A2 expression associated with type IV collagen synthesis was a characteristic feature of ECFC. Conclusion: The gene expression profile of endothelial colony-forming cells is quite close to that of primary endothelial cells of the human coronary artery, and thus, the cells obtained from patients' peripheral blood can be used to develop personalized tissue-engineered constructs.

[Effect of radio frequency discharge plasma on surface properties and biocompatibility of polycaprolactone matrices].

Surface modification of bioresorbable polymer material (polycaprolactone, PCL) with abnormal glow discharge, initiated during radio-frequency magnetron sputtering of a hydroxyapatite target was investigated. Plasma treatment resulted in an increase of surface roughness of PCL, crystallite size, the surface free energy and hydrophilicity. Increased treatment time (30, 60, 150 seconds) provoked the polymer surface saturation with the sputtering target ions (calcium, phosphorus). The assessment of plasma exposure of PCL surface on bone marrow multipotent mesenchymal stromal cells behavior (BM MSCs) has been performed. Modification of the polymer surface with the abnormal glow discharge stimulated adhesion and subsequent proliferation of BM MSCs; thus, maximum values were achieved with the surface treatment for 60 s. This type of plasma modification did not affect cell viability (apoptosis, necrosis). Thus, the surface modification with abnormal glow discharge, initiated during radio-frequency magnetron sputtering of a hydroxyapatite target, appear to be a promising method of surface modification of bioresorbable polymer material (PCL) for tissue engineering.

[Effectiveness in culturing amniocytes for prenatal diagnosis of chromosomal diseases]. / Effektivnost' kul'tivirovaniia amniotsitov dlia prenatal'noi diagnostiki khromosomnykh boleznei.

Cytogenetic studies of 150 cell samples were performed. The fetus karyotype was established in 121 cases. Efficiency of the analysis differed significantly, depending on methods of cell cultivation and preparation of chromosome plates: for the in situ method it was 50%; for the trypsin method, 82%; and for the pipette method, 99%. Analysis of 63 samples demonstrated that when the pipette method is used, the results are available as early as within the first week of cultivation; this method is reliable for revealing karyotypic mosaicism in individual cell colonies, can be used successfully from the 16th to the 26th week of pregnancy, and provides a high level of G-staining in prometaphase chromosomes. Comparison of the three methods of prenatal genetics provides unambiguous evidence in favor of the pipette method.

[Genome mapping of the silver fox. I. Determination of the chromosomal location of 8 fox genes and the search for homologous regions on fox and human chromosomes]. / Kartirovanie genoma serebristo-chernoi lisitsy. Soobshchenie I. Opredelenie khromosomnoi lokalizatsii vos'mi genov lisitsy i poisk gomeologichnykh raionov khromosom lisitsy i cheloveka.

Twenty-three silver fox-Chinese hamster somatic cell hybrids were analysed for the expression of fox enzyme loci and the segregation of fox chromosomes. This analysis made it possible to assign the gene PGD to chromosome 2, MDH2 to chromosome 3. NP to chromosome 10. APRT, ENO1, PGM1 to chromosome 12, MDH1 and IDH1 to chromosome 16. Possible use of the above-mentioned clone panel for fox gene mapping is analysed. An attempt to reveal homologous regions on fox and human chromosomes was made by comparative analysis of prometaphase fox and human chromosomes containing the homologous genes. The means and perspectives of verification of the hypothesis proposed are discussed.

[Creation of a clone panel of fox x Chinese hamster somatic cell hybrids and chromosome mapping of genes for LDHA, LDHB, GPI, ESD, G6PD, HPRT, alpha-GALA in the silver fox]. / Sozdanie paneli klonov gibridov somaticheskikh kletok lisitsa x kitaiskii khomiachok i opredelenie khromosomnoi lokalizatsii genov LDHA, LDHB, GPI, ESD, G6PD, HPRT, alpha-GALA u serebristo-chernoi lisitsy.

A clone panel of fox-hamster somatic cell hybrids which can be used for fox gene mapping was set up. Analysis of patterns of chromosome-enzyme segregation made it possible to assign gene GPI to chromosome 1, LDHA to chromosome 11, LDHB to chromosome 8, ESD to chromosome 6 and G6PD, HPRT, alpha-GALA to chromosome X.

[Genome mapping in silver fox. Syntenic genes in Carnivora]. / Kartirovanie genoma serebristo-chernoi lisitsy. Sintennye otnosheniia genov u khishchnykh.

Hamster X fox somatic cell hybrids segregating individual fox chromosomes in different combinations were used to assign seven structural loci to fox chromosomes. The gene for ME1 was mapped on the VFU1 chromosome, the genes for ADK and PP being located on the VFU4 chromosome. The gene for GSR was assigned to the VFU7 chromosome and the genes for MPI and COT1 were assigned to the VFU15 chromosome. Localization of these genes enhances the established fox genetic map and extends the known syntenic homologies between the fox and other mammalian. The comparison of data on gene mapping has provided basis for suggestion that there are significant differences in rates of karyotypic evolution in many mammalian taxa.

[Clinical and molecular cytogenetic analysis of a rare case of mosaicism for partial monosomy 3p and partial trisomy 10q in human]. / Klinicheskii i molekuliarno-tsitogeneticheskii analiz redkogo sluchaia mozaitsizma po chastichnoi monosomii 3p i chastichnoi trisomii 10q u cheloveka.

The results of comprehensive clinical examination and molecular cytogenetic analysis of a patient carrying chromosome 3p+ in 69% of the peripheral blood lymphocytes are presented. Using microdissection of the metaphase chromosomes followed by DOP-PCR, a DNA library specific for the abnormal chromosome was obtained. By fluorescence in situ hybridization (FISH) of this DNA library with chromosomes from the patient and a healthy donor, the aberrant chromosome was identified as der(3)t(3;10)(3p25;q24.3). Since this chromosome was present in only a proportion of patient's cells studied and no chromosome aberrations were revealed in cells of his parents, the der(3)t(3;10) is suggested to appear de novo. The cells carrying der(3)t(3;10) are monosomic for a proportion of 3p25 and trisomic for 10q24.3-->qter. The developmental malformations revealed in the patient, such as the specific features of facial skeleton, mental retardation, microcephaly, and others are similar to those described previously in patients with partial 3p monosomy and 10q trisomy.

[Mapping of the silver fox genome. III. Determination of the chromosomal localization of the GOT2, AK1, ALDOC, ACP1, ITPA, PGP and BLVR genes]. / Kartirovanie genoma serebristo-chernoi lisitsy. Soobshchenie III. Opredelenie khromosomnoi lokalizatsii genov GOT2, AK1, ALDOC, ACP1, ITPA, PGP and BLVR.

Evidence is presented for the chromosome localization of seven silver fox genes obtained with the help of panel of fox x Chinese hamster somatic cell hybrids. Thus, the AK1, GOT2 and ALDOC are assigned to chromosome VFU2, PGP to chromosome VFU3, BLVR to chromosome VFU5, ACP1 to chromosome VFU8 and ITPA to chromosome VFU14. The genetic map of 29 fox genes is compared with those reported for man and other animals. The results obtained support and extend our previous suggestion that formation of the Canidae branch of the Carnivora phylogenetic tree was associated with great increase in the rate of reorganization of the ancestral karyotype.

[Rapid karyotyping of mammalian cells]. / Bystroe kariotipirovanie kletok mlekopitaiushchikh.

The use of "pipette" method ensures rapid preparation of standardized whole metaphase spreads. Experiments with human, murine, Chinese hamster, American mink, green African monkey, dog, and vole cells demonstrated that G-banded whole metaphase spreads can be obtained in less than two hours after the beginning of work with cell or tissue culture. Due to that, it became possible to start karyotyping of animal tissue explants, as well as fetal cells present in human amniotic fluid, on day 3 to 4 after their receiving.

[Reverse in situ hybridization of DNA probes of anomalous chromosomes in diagnosing chromosome pathologies]. / Obratnaia in situ gibridizatsiia DNK-zondov anomal'nykh khromosom v diagnostike khromosomnykh patologii.

The results of analysis of congenital chromosomal pathologies and chromosomal rearrangements upon the occurrence of haematological diseases, which was involved constructing DNA libraries of abnormal chromosomes and subsequent reverse CISS hybridization have been considered. High effectiveness of this approach for analysis of chromosomal translocations, deletions of chromosomal regions, minor extra chromosomes, and large marker chromosomes with complex organization was shown. The possibility of implementation of this approach and its large-scale application in medical and genetic studies of congenital developmental pathologies and chromosomal diagnostics of haematological diseases has been discussed.

[Mutagenic activity of synthetic interferon inducers in laboratory mice]. / Issledovanie mutagennoi aktivnosti sinteticheskikh induktorov interferona na laboratornykh myshakh.

The mutagenic activity of poly(I) . poly(C) and poly(G) . poly(C) complexes in somatic and generative cells of mice was tested. Both preparations exhibited mutagenic activity in both test-systems. The poly(I) . poly(C) complex in the three doses tested was found to be more active both in bone marrow cells and in sex cells of male mice. The maximum effectiveness of this complex was demonstrated upon treatment of postmeiotic cells of males in an intermediate dose of 2.5 mg/kg bw. The rate of induced dominant lethals was 19.1%. In bone marrow metaphases the subtoxic dose of this complex increased 10-fold the control value of structural aberrations. The activity of poly(G) . poly(C) was found to be slightly lower.