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.

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.

[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.

Integral and Local Methods for the Evaluation of the Hemostasiological Profile in Sheep at Various Stages of Implantation of a Biodegradable Vascular Graft.

The aim of this study was to evaluate the efficiency of local and integral methods of the assessment of the hemostasiological profile in sheep at various stages of implantation of a biodegradable vascular graft. Materials and Methods: The object of the study was the whole blood of sheep collected at the stage of premedication, during the intraoperative period, and in the early postoperative period. Thromboelastography was used to assess the kinetics of clot formation and changes in its viscoelastic properties in whole blood samples. The thrombin generation test was performed in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) with the assessment of quantitative and temporal parameters. The platelet factor 4 concentration in PRP and PPP was measured by the enzyme immunoassay. The functional activity of platelets in PPP was assessed with inductors and without additional stimulation. Prothrombin complex activity, APTT values, thrombin time, fibrinogen concentration, antithrombin III and protein C activity, soluble fibrin monomer complexes, and fibrinolysis were determined in blood plasma. Results: Multidirectional changes in the hemostasiological profile at various stages of vascular prosthesis implantation have been revealed. On the one hand, it is an increased prothrombogenic status, on the other hand, it is the development of hypocoagulation. Shortening of the R (blood coagulation time) and K (clot formation time) intervals and an increase in the angle parameter and maximum amplitude on the thromboelastogram in all the studied periods relative to the reference values, a significant increase in platelet factor 4 in PRP and increased platelet aggregation testified in favor of hypercoagulation. However, the quantitative parameters of the thrombin generation test and a number of coagulogram indicators pointed to hypocoagulation in the intraoperative and early postoperative periods. Conclusion: The comparative analysis of local tests characterizing the state of hemostasis and indicators of integral methods demonstrated the advantages of the latter in assessing thrombotic risks during implantation of vascular grafts. Local tests are not sufficient to assess the dynamics of the coagulation process in real time and are not always sensitive to hypercoagulation. The use of integral methods will help to fill these gaps, make a timely diagnosis of hypercoagulability and minimize the risks associated with the implantation of vascular grafts in future.

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.

Comparative Analysis of the Hemostasiological Profile in Sheep and Patients with Cardiovascular Pathology as the Basis for Predicting Thrombotic Risks During Preclinical Tests of Vascular Prostheses.

The aim of the investigation was to study the details of hemostasiological profile in sheep and patients with coronary heart disease (CHD) and to find the possibility of predicting thrombotic risks during preclinical tests of vascular prostheses on a large laboratory animal model. Materials and Methods: The functional activity of platelets was measured in platelet-rich plasma with inductors: ADP, epinephrine, collagen. Prothrombin activity, international normalized ratio, activated partial thromboplastin time (APTT), thrombin time, fibrinogen concentration, antithrombin III and protein C activity, fibrinolysis were determined in blood plasma. Changes in clot formation and viscoelastic properties of clots were assessed using thromboelastography. Results: Significant differences were found in the hemostasiological profile of sheep and CHD patients. Sheep platelets had increased response to ADP induction and practically no response to epinephrine induction; collagen-induced aggregation was comparable in the study groups. Coagulation hemostasis of sheep was characterized by increased activity of the prothrombin complex, shortened thrombin time, while APTT and fibrinogen values remained comparable. At the same time, sheep exhibited a significant decrease in the activity of anticoagulant and fibrinolytic systems as compared to CHD patients. When assessing dynamic changes in clot formation, it was observed that initiation phase was faster in animals, while clot density exceeded that in patients. Conclusion: The hemostasiological profile of sheep is characterized by the increased speed of thrombus formation, greater strength of the formed clot, and lower lysis ability as compared to CHD patients. The revealed details of the hemostasiological profile of sheep can be potential targets for therapy with antithrombotic drugs that minimize thrombotic risks in preclinical testing of vascular prostheses.

A Technology for Anti-Thrombogenic Drug Coating of Small-Diameter Biodegradable Vascular Prostheses.

The aim of the study was to develop a technology for anti-thrombogenic drug coating of biodegradable porous scaffolds and to evaluate the physicomechanical and hemocompatible properties of functionally active vascular prostheses with and without a drug coating. MATERIALS AND METHODS: Vascular prostheses from polyhydroxybutyrate/valerate and polycaprolactone with the incorporated vascular endothelial growth factor, the main fibroblast growth factor, and the chemoattractant SDF-1α were made by emulsion electrospinning. Additional surface modification of the prostheses was carried out by forming a hydrogel coating of polyvinylpyrrolidone capable of binding drugs as a result of complexation. Unfractionated heparin and iloprost were used as anti-thrombogenic drugs. RESULTS: We show that after the modification of vascular prostheses with heparin and iloprost, a 5.8-fold increase in the Young's modulus value was noted, which indicated a greater stiffness of these grafts compared to the unmodified controls. Platelet aggregation on the surface of heparin + iloprost coated vascular prostheses was 3.3 times less than that with the unmodified controls, and 1.8 times less compared to intact platelet-rich plasma. The surface of vascular prostheses with heparin and iloprost was resistant to adhesion of platelets and blood proteins. CONCLUSION: Drug (unfractionated heparin and iloprost) coating of the surface of biodegradable prostheses significantly improved the anti-thrombogenic properties of these grafts but contributed to the increased stiffness of the prostheses.

Regulation of c-Fos gene expression in the rat olfactory bulb during olfactory learning.

An immunohistochemical method was used to study the expression of transcription factor c-Fos in the mid part of the main olfactory bulbs in 18-day-old rats after training to seek their mothers using an olfactory orientation cue (propionic acid) in a Y-maze. On the next day, rat pups demonstrated a significant preference for the propionic acid odor in behavioral tests, as compared with control animals trained to the skill without the olfactory orientation cue and pups familiarized with the odor without forming any association between it and any kind of reinforcement. The propionic acid odor evoked an insignificant increase in c-Fos expression, predominantly in the granular layer of the dorsomedial area of the olfactory bulb. Training in a maze lacking the odor signal evoked diffuse activation of c-Fos, in both the mitral and granular layers of all areas of the olfactory bulb. Training with the olfactory orientation cue was also accompanied by an increase in c-Fos expression to a level significantly exceeding that seen after odor-free training, in the granular layer of the dorsomedial area, i.e., the area in which the unreinforced odor acting alone induced insignificant activation of expression. These data provide evidence that, at the level of the regulation of neuronal transcription, associative learning is manifest as summation of the effects of a new neutral stimulus and excitation initiated by the motivational state. In the olfactory bulb, this summation is seen in areas on which non-specific centrifugal excitation and specific afferentation from the signal odor converge.

[Regulation of c-Fos expression in the rat olfactory bulbs during olfactory learning].

The distribution of c-Fos-immunopositive neurons was examined in the mitral/tufted and granular cell layers in the medium part of the main olfactory bulbs of 18-day-old rats after they had been trained for propionic acid vapour-guided search for dam in the Y-maze. On the next day these pups exhibited a strong preference for the propionic acid odor as compared to the control pups trained for this task without the odor cue and odor-familiarized pups exposed to propionic acid as a novel neutral stimulus. Exposure to propionic acid produced a moderate activation of c-Fos expression, mainly in the granular layer of the dorsomedial part of the bulb. Training in the Y-maze devoid of odor cues resulted in diffuse increase in the number of c-Fos-positive neurons both in the mitral and granular cell layers in all parts of the olfactory bulb. Maze training with the odor cue produced activation of c-Fos expression (which significantly exceeded the non-odor Y-maze group) in the dorsomedial olfactory bulb. These data suggest that associative olfactory conditioning results in activation of c-Fos expression that combines the effect of diffuse motivational excitation and specific olfactory input to the neurons which process odor cues.

[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.

The neurotropic activity of a structural analog of ACTH(5-7).

The tripeptide glutamyl-arginyl-proline (ERP) mimicking some structural features of the N-terminal fragment of adrenocorticotropic hormone, ACTH(5-7), has been synthesized. In contrast with ACTH fragments, ERP (0.015-0.15 mg/kg; intraperitoneal injection) hindered the formation of food-reinforced task in adult albino male rats. Besides, ERP caused a rapid inhibition of the previously formed conditioned task. Similar doses of ERP changed orientative-trying reaction and emotional behavior in rats in the 'open field' test. ERP at a dose of 0.15 mg/kg administered prior to the injection of 0.5 mg/kg ACTH(1-39) prevented increases in locomotor activity normally induced by this hormone. Thus, ERP disrupts learning, impairs performance of acquired task and blocks behavioral effects of ACTH and its fragments.

Entry of the synthetic ACTH(4-10) analogue into the rat brain following intravenous injection.

In view of known central effects of N-terminal ACTH fragments, a possibility of their entry into the brain was studied. Rat blood and brain extracts after intravenous injection of the tritiated synthetic ACTH(4-10) analogue, Met-Glu-His-Phe-Pro-Gly-Pro, were subjected to a high-performance liquid chromatographic analysis. At two time points the labelled peptide was detected in brain extracts. The brain to blood ratios of peptide content in brain and blood were found to be significantly higher than those calculated for a distribution of labelled bovine serum albumin in rat brain capillaries and blood. This strongly suggests that this peptide penetrates into the brain tissues, its quantity not exceeding 0.01% of dose injected. Peptide diffusion through the vascular epithelium of brain capillaries could account for the data obtained.

[New approaches to the diagnosis and treatment of inflammatory diseases of the adnexa uteri]. / Novye podkhody v diagnostike i lechenii vospalitel'nykh zabolevanii pridatkov matki.

The paper summarizes follow-ups of 2500 patients with inflammatory diseases of the uterine appendages, provides evidence for the benefits of laparoscopic diagnosis and treatment of all types of this condition, which makes it possible to verify the diagnosis and perform less traumatic organ-sparing operations. To use combined antiinflammatory antibiotic therapy in view of the etiology of inflammation, as well as immunomodulators is the requisite condition for treating acute types of a disease. The new approaches to the diagnosis and treatment of inflammatory diseases of the uterine appendages substantially improved therapeutical outcomes and maintained female reproductive function.

[Specificity of hippocampal field CA3 neuronal responses to repetitive single stimuli]. / Spetsifika otvetov neironov polia CA3 gippokampa krolika na povtoriaiushchiesia odinochnye stimuly.

It has been shown on alert rabbits that responses of field CA3 units to stimuli eliciting (single tones) and not eliciting (single flashes) an activation reaction are substantially different. 75% of units responsive to a flash exhibited: less pronounced tonic extinctive responses, stable structurized phasic reactions and the dynamics of responses in the form of changes in their types. The system of field CA3 units "triggered" by single stimulation is under control of the sensorimotor cortical area. Modulating cortical influences enhance the revealed tendencies of responses of the examined neurones to a single photic stimulus.

[Possible mechanisms of the divergent effects of ACTH4--10 and its analog containing the D-isomer of phenylalanine on behavior]. / Vozmozhnye mekhanizmy raznonapravlennogo deistviia AKTG4--10 i ego analoga, soderzhashchego D-izomer fenilalanina, na povedenie.

The effect of two oligopeptides--ACTG4-10 and hexopeptide met-glu-his-D-phen-lis-L-phen (Dphen-GP) on memorizing the situation and on orienting-investigating reaction was studied in albino rats by the method of elaboration of food-procuring habit in T-maze and by the method of "open field". It was shown that these peptides in a dose of 15 mcg/kg with certain periods of administration, have an opposite effect on maze learning but a similar effect on memorizing in the "open field". ACTG4-10 slightly increases motor activity in the "open field", whereas Dphen-GP decreases it considerably. It is suggested that ACTG4-10 improves the formation of trace processes independently of the sign of reinforcement, whereas Dphen-GP selectively enhances defensive reaction and memorizing, connected with negative reinforcement.

[Disorders in the interferon system in patients with virus-associated and chlamydial infections]. / Narusheniia v sisteme interferona u patsientov s virusassotsiirovannymi i khlamidiinymi infektsiiami.

A more than 80% incidence of herpes simplex virus, cytomegalovirus, and chlamydial infection was revealed in 58 patients with various gynecological diseases. In 50% cases the infection was mixed. An appreciable reduction of the gamma-interferon system was detected in 20 patients, which was caused by a concomitant infectious process, possibly provoking the development of the underlying disease. Treatment of such patients should include antiviral and antichlamydial therapy, as well as correction of defects in interferon production.

[Secretory IgA in amniotic fluid]. / Sekretornyi IgA v okolopolodnykh vodakh

Immunoglobulin composition was studied in the amniotic fluid (AF) and in the urine of newborn babies. IgA was revealed with the aid of anti-secretory component of the serum in 50 of 57 AF samples; in the urine of babies in the course of the first 24 hours after birth it was revealed in 2 of the 21 cases only. This fact and also the detection of the secretory IgA in the AF at the early periods of pregnancy permit to suppose the synthesis of IgA or of its C-fragment in the amniotic membranes.

[The immune status of patients with acute inflammatory diseases of adnexa uteri associated with different combinations of microorganisms]. / Issledovanie immunnogo statusa u bol'nykh ostrymi vospalitel'nymi zabolevaniiami pridatkov matki, assotsiirovannykh s raznymi kombinatsiiami mikroorganizmov.

The immune status of 130 patients with acute inflammatory diseases of uterine appendages was studied. As etiologically associated infective agents detected in these patients were opportunistic microorganisms (group 1), Neisseria gonorrhoeae in combination with opportunistic microorganisms (group 2) and Chlamydia trachomatis in combination with opportunistic microorganisms (group 3). In all three groups of patients the response of T lymphocytes to PHA and hyperactivation of the B-cell element of immunity (an increase in the relative number of B-lymphocytes and the content of IgA and IgM in the serum) was found to be suppressed. In addition, in groups 1 and 3 an increase, and in group 2 a decrease in IgG were detected. In the mucus of the cervical canal in the patients of groups 1, 2 and 3 IgM was detected, while in patients of groups 2 and 3 a decrease in sigA was established.