Научная статья на тему 'INTERACTION OF ERYTHROCYTES WITH ENDOTHELIUM IN MICROFLUIDIC CHANNELS STUDIED BY OPTICAL TECHNIQUES'

INTERACTION OF ERYTHROCYTES WITH ENDOTHELIUM IN MICROFLUIDIC CHANNELS STUDIED BY OPTICAL TECHNIQUES Текст научной статьи по специальности «Медицинские технологии»

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Текст научной работы на тему «INTERACTION OF ERYTHROCYTES WITH ENDOTHELIUM IN MICROFLUIDIC CHANNELS STUDIED BY OPTICAL TECHNIQUES»

INTERACTION OF ERYTHROCYTES WITH ENDOTHELIUM IN MICROFLUIDIC CHANNELS STUDIED

BY OPTICAL TECHNIQUES

A.E. LUGOVTSOV1, P.B. ERMOLINSKY1, M.K. MAKSIMOV1, L.I. DYACHUK2, OLGA N. SCHEGLOVITOVA3, A.V. PRIEZZHEV1

1Faculty of Physics, Moscow State University named after M. V. Lomonosov, Russian Federation 2Medical Research and Educational Center, M.V. Lomonosov Moscow State University, Russian Federation 3N.F. Gamaleya National Research Center for Epidemiology and Microbiology, Russian Federation

[email protected]

ABSTRACT

In this work, the intercellular interaction of erythrocytes and endothelial cells was studied in vitro using optical methods. Using laser tweezers, the adhesion forces of erythrocytes to the endothelium were measured, as well as the aggregation forces of erythrocytes upon addition of various compounds (L-Arginine, L-NAME) affecting the interaction of these cells to blood plasma or whole blood. The aggregation of erythrocytes in the blood flow in the presence of endothelial cells was studied using the laser aggregometry technique. The results demonstrate a decrease in the aggregation forces of erythrocytes in the presence of the endothelium upon stimulation of nitric oxide release. In the flow, erythrocyte aggregation also decreases in the presence of endothelial cells. The adhesion forces of erythrocytes to the endothelium do not change or decrease slightly (in some cases) with an increase in the concentration of nitric oxide.

Blood microbiology is essentially determined by the properties of blood plasma and the interaction between blood cells, i.e., the aggregation of red blood cells (RBCs), the interaction between different blood cells and endothelium, etc. [1]. Endothelial cells do not only act as an insulating layer between blood and tissues, but also play an important role in controlling the blood flow and influence blood cell properties, e.g., RBCs aggregation. RBCs can reversibly interact with each other under low shear stress forces forming linear and more complex structures. RBCs aggregation is mainly responsible for the non-Newtonian behavior of blood and regulates the microcirculation of blood in human body.

Blood microrheology and its microcirculation are determined by the composition of blood plasma, deformability and interaction of blood cells, in particular, aggregation of erythrocytes and interaction between blood cells and endothelium. Endothelial cells (EC) covering the walls of blood vessels play an important role in regulating blood flow and also affect the properties of blood cells through the signaling molecules of gas transmitters secreted by them, in particular, nitric oxide (NO). The aim of the work is to measure the force of interaction between single erythrocytes and EC, as well as aggregation of erythrocytes in whole blood in microchannels at different concentrations of fibrinogen and gas transmitter NO.

In the work, laser tweezers were used, allowing manipulation of individual cells without mechanical contact, as well as measuring the interaction forces of erythrocytes and EC in vitro. The ability of erythrocytes to aggregate in microchannels was measured using the diffuse light scattering method on whole blood. EC were obtained from the human umbilical vein (HUVEC) and grown at 37 degrees Celsius in a carbon dioxide environment on round glass slides to form a monolayer of cells, which were placed in blood plasma with a fibrinogen concentration of 0 - 8 mg / ml. To stimulate NO production, EC were incubated with a solution of L-arginine in various concentrations of 0 - 1000 ^M.

One of the goal of this work was to investigate the interaction between endothelium cells in a monolayer and RBCs of healthy volunteers at different concentrations of fibrinogen at the level of individual cells in vitro. Laser tweezers were used to manipulate individual cells without mechanical contact, as well as to measure the forces of their interaction [2].

Blood for the study was drawn from the cubital veins of four healthy donors.

Laser tweezers is device that allows trapping and manipulating by dielectric particles using a highly focused laser beam. This makes it possible to measure the forces of intercellular interaction. In this work, dual-channel laser tweezers based on a Nd:YAG laser (1064 nm) were used [3]. This method was performed

to measure the forces of interaction between two single erythrocytes, as well as the forces of interaction between one erythrocyte and a monolayer of endothelial cells. The aggregation force is understood as the minimum force required to prevent aggregation of a pair of partially overlapping erythrocytes, and the disaggregation force is understood as the minimum force required to separate such a pair. In order to minimize errors caused by individual characteristics of cells and donors, the data were normalized to the average control value for each donor.

In this work, the laser erythrocyte aggregometer RheoScan-AnD300 (RheoMediTech, Korea) was used to measure aggregation parameters of erythrocytes in whole blood samples. The laser beam falls on a cuvette filled with whole blood, in the microchannel of which a flow is created. The intensity of backscattered light is detected. The smaller the size of the scattering particles (erythrocyte aggregates) and their number, the higher the detected signal. The pressure difference between the ends of the cuvette microchannel decreases monotonically over the measurement time (1-2 minutes), and at a certain point in time, a dynamic equilibrium is observed between the processes of aggregation and disaggregation of erythrocytes. The shear stress in the cuvette, calculated for this moment, is called the critical shear stress. This parameter characterizes the hydrodynamic strength of the aggregates.

Figure 1. The dependences of normalized RBC aggregation forces on L-arginine concentration in cuvettes with EC monolayer. Each point in the figure is one measurement on one pair of RBCs. The box corresponds to the first (Q1) and to the third quartile (Q3) of the data with a median line. The whiskers are standard deviation with the mean in the

middle.

It was shown that the interaction force between RBCs and endothelial cells increases with fibrinogen concentration and reaches the saturation level of 4 pN at the concentration of 4 mg/ml. This force value is comparable with the interaction forces between individual RBCs in autological plasma. Incubation of endothelial cells with L-arginine leads to a decrease in the force of erythrocyte aggregation at L-arginine concentrations up to 100 ^M and its growth with a subsequent increase in concentration (fig. 1). The laser tweezers and laser aggregometry technique can be used to assess the interaction of erythrocytes and endothelial cells, as well as the aggregation properties of erythrocytes in microchannels with the endothelium. The presented results are important for understanding the influence of vascular endothelium on microrheology and blood microcirculation.

This work was supported by the Russian Science Foundation (Grant No. 23-45-00027).

REFERENCES

[1] Furst E.M., Squires T.M. "Microrheology" Oxford University Press, United Kingdom (2017).

[2] Kapkov A.A., Semenov A.N., Ermolinskiy P.B., Lugovtsov A.E., Priezzhev A.V. "Forces of RBC interaction with single endothelial cells in stationary conditions: Measurements with laser tweezers". Journal of Innovative Optical Health Sciences. 14(5), 2142005 (2021).

[3] A.Y. Maklygin et al. Measurement of interaction forces between red blood cells in aggregates by optical tweezers // Quantum Electron, vol. 42, № 6, pp. 500-504, (2012).

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