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Pliyev B.K.,Moscow State University | Antonova O.A.,Institute of Experimental Cardiology | Menshikov M.,Moscow State University
Molecular Immunology | Year: 2011

The mechanisms underlying migration of neutrophils across endothelium are not completely understood. The urokinase-type plasminogen activator receptor (uPAR) plays a key role in neutrophil adhesion and migration. In the present study, we addressed whether uPAR regulates neutrophil transendothelial migration. We first showed that siRNA-mediated knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) did not affect neutrophil migration across HUVEC monolayers indicating that endothelial uPAR does not regulate neutrophil transmigration. In contrast, the transmigration was significantly inhibited by Fab' fragment of anti-uPAR monoclonal antibody and proteolytically inactive urokinase (uPA), whereas inhibition of proteolytical activity of endogenous uPA (with amiloride or plasminogen activator inhibitor-1) did not affect the transmigration. Both the anti-uPAR Fab' fragment and proteolytically inactive uPA did not exert significant effects upon the transmigration conducted in the presence of F(ab') 2 fragment of blocking antibody to integrin Mac-1 indicating that uPAR regulates Mac-1-dependent transmigration. Mac-1-dependent, but not Mac-1-independent, transmigration was significantly reduced in the presence of N-acetyl-d-glucosamine and d-mannose, the saccharides that disrupt uPAR/Mac-1 association, but was unaffected in the presence of control saccharides (d-sorbitol and sucrose). We conclude that physical association of uPAR with Mac-1 mediates the regulatory effect of uPAR over the transmigration. Finally, we provide evidence that the functional cooperation between uPAR and Mac-1 is essential at both adhesion and diapedesis steps of neutrophil migration across endothelium. Thus, uPAR expressed on neutrophil plasma membrane regulates transendothelial migration independently of uPA proteolytical activity and acting as a cofactor for integrin Mac-1. © 2011 Elsevier Ltd.

Pliyev B.K.,Moscow State University | Menshikov M.Y.,Institute of Experimental Cardiology
Inflammation | Year: 2010

Soluble form of the urokinase-type plasminogen activator receptor (suPAR) is markedly increased in biological fluids during different inflammatory conditions. It has previously been observed that the highest suPAR concentrations in inflammatory exudates tend to be associated with the presence of high number of neutrophils. Guided by this observation and our recent finding that activated neutrophils release suPAR we investigated whether neutrophils can be a source of suPAR during the inflammatory response in vivo. To address this question we conducted the comparative analysis of neutrophils isolated from the paired samples of synovial fluid (SF) and peripheral blood (PB) of rheumatoid arthritis patients. Freshly isolated SF neutrophils released significantly (p < 0.01) higher amounts of suPAR compared with PB neutrophils. We demonstrated that neutrophils from both sources release predominantly the truncated D2D3 form of suPAR. Migration of formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney (HEK) 293 cells toward the supernatants harvested from in vitro cultured SF neutrophils was significantly diminished when D2D3 form of suPAR was immunodepleted from the supernatants. Taken together, these data demonstrate that neutrophils, first, contribute to or are responsible for the generation of the increased suPAR levels during the inflammatory response and, second, release the chemotactically active form of suPAR that might be involved in the recruitment of formyl peptide receptors-expressing leukocytes into the inflamed tissues. © 2009 Springer Science+Business Media, LLC.

Aliev M.K.,Institute of Experimental Cardiology | Tikhonov A.N.,Moscow State University
Molecular and Cellular Biochemistry | Year: 2011

Using a Monte Carlo simulation technique, we have modeled 3D diffusion of low molecular weight metabolites inside a skeletal muscle cell. The following structural elements are considered: (i) a regular lattice of actin and myosin filaments inside a myofibril, (ii) the membranes of sarcoplasmic reticulum and mitochondria surrounding the myofibrils, (iii) a set of myofibrils inside a skeletal muscle cell encircled by the outer cell membrane, and (iv) an additional set of regular intracellular structures (" macrocompartments") embedded into the cell interior. The macrocompartments are considered to simulate diffusion restrictions because of hypothetical cylindrical structures (16-22 μm in diameter) suggested earlier (de Graaf et al. Biophys J 78: 1657-1664, 2000). This model allowed us to calculate the apparent coefficients of particle diffusion in the radial and axial directions, D⊥ app and DII app, respectively. Particle movements in the axial direction are considered, at first approximation, as unrestricted diffusion (DII app = const). The apparent coefficient of radial diffusion, D⊥ app, decreases with time because of particle collisions with myofilaments and other rigid obstacles. Results of our random walk simulations are in fairly good agreement with experimental data on NMR measurements of restricted radial diffusion of phosphocreatine in white and red skeletal muscles of goldfish (Kinsey et al. NMR Biomed 12:1-7, 1999). Particle reflections from the low-permeable borders of macrocompartments (efficient diameter, d MC eff≈ 9.2-10.4 μm) are the prerequisite for agreeing theoretical and experimental data. The low-permeable coverage of hypothetical macrocompartments (99.8% of coverage) provides the main contribution to time-dependent decrease in D⊥ app.

Tkachuk V.A.,Moscow State University | Vorotnikov A.V.,Institute of Experimental Cardiology
Diabetes Mellitus | Year: 2014

Insulin resistance (IR) is a phenomenon associated with an impaired ability of insulin to stimulate glucose uptake by target cells and to reduce the blood glucose level. A response increase in insulin secretion by the pancreas and hyperinsulinemia are compensatory reactions of the body. The development of IR leads to the inability of target cells to respond to insulin that results in developing type 2 diabetes mellitus (T2DM) and metabolic syndrome. For this reason, the metabolic syndrome is defined in practice as a combination of IR with one or more pathologies such as T2DM, arterial hypertension, dyslipidemia, abdominal obesity, non-alcoholic fatty liver disease, and some others. However, a combination of high blood glucose and insulin levels always serves as its physiological criterion. IR should be considered as a systemic failure of the endocrine regulation in the body. Physiological causes of IR are diverse. The main ones are nutritional overload and accumulation of certain lipids and their metabolites in cells, low physical activity, chronic inflammation and stress of various nature, including oxidative and endoplasmic reticulum stress (impairment of damaged protein degradation in the cell). Recent studies have demonstrated that these physiological mechanisms likely act through a single intracellular scenario. This is the impairment of signal transduction from the insulin receptor to its targets via the negative feedback mechanism in intracellular insulin-dependent signaling cascades. This review describes the physiological and intracellular mechanisms of insulin action and focuses on their abnormalities upon IR development. Finally, feasible trends in early molecular diagnosis and therapy of IR are discussed.

Maksimenko A.V.,Institute of Experimental Cardiology
Oxidative Medicine and Cellular Longevity | Year: 2016

Undiminishing actuality of enzyme modification for therapeutic purposes has been confirmed by application of modified enzymes in clinical practice and numerous research data on them. Intravenous injection of the superoxide dismutase-chondroitin sulfate-catalase (SOD-CHS-CAT) conjugate in preventive and medicative regimes in rats with endotoxin shock induced with a lipopolysaccharide bolus has demonstrated that antioxidant agents not only effectively prevent damage caused by oxidative stress (as believed previously) but also can be used for antioxidative stress therapy. The results obtained emphasize the importance of investigation into the pathogenesis of vascular damage and the role of oxidative stress in it. The effects of intravenous medicative injection of SOD-CHS-CAT in a rat model of endotoxin shock have demonstrated a variety in the activity of this conjugate in addition to prevention of NO conversion in peroxynitrite upon interaction with O 2 - superoxide radical. Together with the literature data, these findings offer a prospect for the study of NO-independent therapeutic effects of SOD-CHS-CAT, implying the importance of a better insight into the mechanisms of the conjugate activity in modeled cardiovascular damage involving vasoactive agents other than NO. © 2016 Alexander V. Maksimenko.

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