ИСТИНА

Vascular endothelial cells (ECs) make up the inner surface of arteries, veins, and capillaries and therefore interact directly with various components of the blood. The interaction between ECs and red blood cells (RBCs) is important because many hemorheological disorders are accompanied by increased adhesion of RBCs to vascular ECs. To date, the mechanisms of this pathology are not fully understood. This work was aimed at studying in vitro the effect of ECs on the microrheological properties of RBCs, in particular on spontaneous aggregation and shear deformation of the latter, using laser methods. The study of the mechanisms of adhesion and its quantitative characterization without damaging mechanical contact with the blood cells is of particular interest and novelty. We performed experiments with fresh blood samples drawn from the cubital vein of healthy volunteers. Human umbilical vein endothelial cells (HUVEC) were cultured and grown at the N.F. Gamaleya National Research Center for Epidemiology and Microbiology. Cells were placed inside microfluidic chips under flow or stopped flow conditions. Subsequently, tumor necrosis factor alpha (TNF) and adenosine diphosphate (ADP) activators were used to activate EC. In our experiments, we used laser aggregometry based on diffuse light scattering from whole blood samples, which makes it possible to obtain the critical shear stress (CSS) parameter characterizing the hydrodynamic strength of RBC aggregates. Analyzing the dependencies of the intensity of laser light scattered by the RBCs suspension at various shear stresses, we measured the CSS of the RBC aggregates in a microchannel coated with an EC layer. To assess the effect of the endothelium on the deformability of RBC, we used the laser ektacytometry method, which makes it possible to measure the elongation index of RBCs at different shear stresses. All measurements were taken with blood samples at 37°C. Similar microchannels without EC were used as controls. Experiments carried out using laser aggregometry showed a decrease in the CSS parameter in the presence of EC by about 40 ± 12% compared with the control. In the case of EC preactivation, the CSS parameter increased by approximately 16 ± 4% (TNF) and 50 ± 7% (ADP) compared with non-activated cells. Experiments performed using laser ectacytometry showed that the ability of erythrocytes to deform under shear stress somewhat decreases in the presence of EC (13 ± 5% lower than in control). We believe that this process is due to the adhesion of RBCs to the endothelial layer. Also, by capturing and manipulating single cells with laser tweezers in a microcuvette, we measured the strength of interaction between RBC and EC in a monolayer culture in various media using laser tweezers. In the first series of experiments, a solution of bovine fibrinogen in serum at various concentrations was used as a medium; in the second series of experiments, plasma (lithium heparin anticoagulant) was used with the addition of the amino acid L-arginine at various concentrations. The force of interaction between an RBC and EC is defined as the minimum force necessary for the free movement of an RBC along the surface of a monolayer of EC. Data were obtained indicating an increase in the force of interaction between RBC and endothelium with an increase in the concentration of fibrinogen in the medium and the subsequent release of this force to saturation. With an increase in the concentration of L-arginine in the medium, a slight decrease in the strength of interaction was found. Numerically, the interaction forces ranged from 1 to 4 pN.