Features of biophysical properties in blood cells from the elderly under mechanical stress applied in vitro have been studied. This stress was reported to trigger the signaling pathway engaged by purinergic receptor activation. The stiffness of the cell surfaces of red blood cells, granulocytes, lymphocytes, and platelets decreased, and the surface potential became more positive. Our findings add to the knowledge of the effect of mechanical deformation on leukocytes and platelets, which are the key regulators of homeostatic processes in the microvasculature, and on red blood cells involved in the regulation of vascular tone in arterioles and tissue oxygenation in the elderl

Skorkina, M. Yu.

Sladkova, E. A.

DSpace at Belgorod State University

ISSN 0006-3509, Biophysics, 2020, Vol. 65, No. 6, pp. 948–950. © Pleiades Publishing, Inc., 2020.Russian Text © The Author(s), 2020, published in Biofizika, 2020, Vol. 65, No. 6, pp. 1114–1117.CELL BIOPHYSICSChanges of the Biophysical Properties of Blood Corpuscles from the Elderly under Mechanical Stress in vitroE. A. Sladkovaa, * and M. Yu. SkorkinaaaBelgorod National Research University, Belgorod, 308015 Russia*e-mail: sladkova@bsu.edu.ruReceived March 5, 2020; revised April 21, 2020; accepted May 20, 2020Abstract—Features of biophysical properties in blood cells from the elderly under mechanical stress appliedin vitro have been studied. This stress was reported to trigger the signaling pathway engaged by purinergicreceptor activation. The stiffness of the cell surfaces of red blood cells, granulocytes, lymphocytes, and plate-lets decreased, and the surface potential became more positive. Our findings add to the knowledge of theeffect of mechanical deformation on leukocytes and platelets, which are the key regulators of homeostaticprocesses in the microvasculature, and on red blood cells involved in the regulation of vascular tone in arte-rioles and tissue oxygenation in the elderly.Keywords: mechanical stress, purinergic signaling system, surface potential, Young’s modulusDOI: 10.1134/S0006350920060196In the greatest majority of cases, the aging of thebody is accompanied by the development of cardio-vascular diseases. These diseases are caused mainly bymolecular and cellular changes in the endothelium [1]and by changing rheological indices of blood [2].These disturbances may be associated with changes inthe functioning of purinergic receptors in blood cor-puscles and endothelium cells. Several studies provideevidence for a significant role of purinergic signalingin the development of various cardiovascular condi-tions: myocardium infarction, cardiac failure, hyper-tension, stroke, and clotting [3]. In addition, elderlyand senile people are at greater risk of infectious dis-eases, caused primarily by weakening of the immunesystem. The purinergic regulation of immunocompe-tent cells mediating inflammatory responses in thebody plays a significant role in mechanisms of thesenescence of the immune system [4].It has been shown that erythrocytes that are under-going mechanical deformation release ATP molecules[5], thereby triggering paracrine and autocrine signal-ing pathways [6]. In the intercellular space, the ATPmolecules are degraded to ADP, AMP, and adenosine[7], which are agonists of the wide range of purinergicreceptors present on the surface of blood corpuscles.Therefore, this study was dedicated to the biophys-ical features of the response of blood corpuscles fromelderly people to mechanical stress in vitro.MATERIALS AND METHODSExperiments were conducted with blood samplesfrom elderly volunteers (60 to 75 years, n = 30), whowere undergoing standard medical examinations inthe regional hospital of Belgorod. Samples were takenfrom the cubital vein by skilled personnel.Mechanical stress was modeled in vitro asdescribed in [8]. The model provides conditions closeto the physiological parameters of microvasculature[9]. Whole blood (0.5 mL) was loaded into a dispos-able tuberculin syringe and air bubbles were carefullyremoved. The cell suspension was passed through a25-mm disposable needle. For the injection, thesyringe was installed vertically and a 1-kg weight wasplaced onto the upper end of the syringe plunger. Theblood was sprayed into 2-mL centrifuge tubes. Thedistance from the needle tip to the bottom was 15 mm,and the tangential stress of the wall was within6600 dyn/cm2. The mean velocity in the needlereached 5100 cm/s and the travel time of corpuscleswas 0.6 ms. Hence, cells were exposed to relativelyhigh tension within a short time. Experimental bloodsamples were exposed to mechanical stress and controlones remained intact.Concentrations of high-energy compounds inmechanical stress in vitro were assayed colorimetri-cally [10] on a KFK-3 photometer (Sergiev PosadOptical-Mechanical Plant, Russia). The concentra-tions were calculated from the difference between theoptical densities in the tube where phosphate bondswere hydrolyzed and in the control tube, which wasnot subjected to hydrolysis. The standard curve wasAbbreviation: AFM, atomic force microscopy.948CHANGES OF THE BIOPHYSICAL PROPERTIES OF BLOOD CORPUSCLES 949Table 1. The surface potential (mV) of blood corpuscles* Differences are significant according to the Student’s t test atp < 0.05.Cell population Control ExperimentErythrocytes –15.2 ± 0.8 –10.7 ± 1.2*Granulocytes –25.8 ± 1.2 –17.5 ± 0.6*Lymphocytes –39.6 ± 0.2 –28.7 ± 0.3*Platelets –16.2 ± 1.3 –7.4 ± 1.1*Table 2. The Young modulus (nN) of blood corpuscles* Differences are significant according to the Student’s t test atp < 0.05.Cell population Control, mV Experiment, mVErythrocytes 4.2 ± 0.01 3.3 ± 0.01*Granulocytes 6.3 ± 0.01 5.1 ± 0.01*Lymphocytes 4.7 ± 0.02 3.6 ± 0.01*Platelets 4.5 ± 0.03 2.7 ± 0.02*drawn by using solutions with phosphate concentra-tions (State Standard Sample 77912000) from 50 to500 μg/mL at 50-μg/mL intervals. The measurementswere done in triplicate for each sample.Whole blood was separated into erythrocyte, leu-kocyte, and platelet concentrates by centrifuging at1500 rpm for 5 min. Plasma enriched with platelets wascollected and additionally centrifuged at 2500 rpm for15 min; in this way, a pure platelet suspension wasobtained. The leukocyte ring was collected into a sep-arate tube. The leukocyte suspension was separatedinto granulocytes and lymphocytes with an EasySepMagnet and an EasySep/EasySep Direct HumanTotal Lymphocyte Isolation Kit (StemCell Technolo-gies, United States).The biophysical properties of blood corpuscleswere examined with an INTEGRA Vita atomic forcemicroscope (an assembly based on the Olympus IX71inverted optical microscope, NT-MDT Company,Zelenograd, Russia). The electrical properties of theerythrocyte, granulocyte, lymphocyte, and plateletmembranes were assessed by measuring the surfacepotentials with the AFM in the Kelvin probe mode.The preparation of the cell suspension for measuringthe surface potential and the measurement followedthe method reported in [11]. Use was made of cantile-vers with conducting titanium coating of theNSG03/TiN series (Nanoworld, United States).Twenty cells of each population were scanned in eachsample, and the scans were processed with Nova soft-ware (NT MDT, Russia).Cell surface stiffness was assessed from the Youngmodulus numerical data. The method for recordingBIOPHYSICS  Vol. 65  No. 6  2020Young modulus involves the measurement of samplesurface deformation in its interaction with the AFMprobe tip. We employed modified AFM probes madeby us from polymeric microspheres with the cornerradius 5 μm [12]. The Young modulus was measuredwith the AFM INTEGRA VITA in the force spectros-copy mode as described in [13]. Twenty cells of eachpopulation were scanned in each sample.The experimental results were processed by varia-tion statistics methods. The significance of differencesbetween experimental and control samples wasassessed by the Student’s t test at p < 0.05 and the nor-mal distribution. The mean values M and standarderrors of the mean m are presented.RESULTS AND DISCUSSIONThe concentration of high-energy compounds inblood in experiments with modeled shearing deforma-tion of blood cells was 0.021 ± 0.001 μmol/L, 2.6 timeshigher than the control value (0.008 ± 0.001).Mechanical stress was shown to alter the biophysi-cal properties of blood corpuscles. In particular, thesurface potentials of erythrocytes, granulocytes, lym-phocytes, and platelets became more positive. As indi-cated in Table 1, it increased with reference to the con-trol by 30, 32.2, 27.5, and 54.3%, respectively (p <0.05).Mechanical stress decreased the Young modulus ofblood corpuscles from elderly people: by 22% in eryth-rocytes (p < 0.05), 19% in granulocytes (p < 0.05), 23%in lymphocytes (p < 0.05), and 40% in platelets ((p <0.05) compared to the control values (Table 2).Our experiments reproduced the in vitro mechani-cal stress conditions that had been reported to approx-imate the physiological parameters of the microcircu-latory bloodstream and favor the activation of puriner-gic signaling in blood corpuscles [8, 9, 14, 15]. The2.6-fold increase in the concentration of high-energycompounds noted in blood samples exposed tomechanical stress may be related to the excretion ofATP molecules to the intercellular space by erythro-cytes in response to the force action of shearing plasmalayers [1].The effect of mechanical stress manifested itself aschanges in biochemical properties of blood corpusclesfrom the peripheral blood of elderly people. In partic-ular, the increase in membrane charge and lower stiff-ness of erythrocytes may be associated with the effectsof high-energy compounds, that is, ATP and its deriv-atives, on P2Y13 receptors on the cell surface mediatedby changes in the action of ion channels selective forCa2+ [16]. The activation of these receptors on theerythrocyte membrane may be accompanied bychanges not only in their functions but also in cellmorphology, ion composition in the cytoplasm, andthe state of cytoskeletal proteins [17]. A similar mech-anism involving the effect of high-energy compounds950 SLADKOVA, SKORKINAmay alter the biophysical parameters of lymphocytes.The lymphocyte surface houses receptors of the P2Xfamily, which are membrane ion channels [18] thatcontrol the cytosolic Ca2+ concentration [19].Elevated concentrations of high-energy moleculeswere also accompanied by lower stiffness and a highersurface potential of granulocyte plasmalemma. It hadbeen shown that the presence of receptors for purines(of which P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 arepredominant [20]) on the granulocyte surface wasessential for cell orientation and movement inresponse to chemotactic stimuli [19, 21]. The detec-tion of the increase in the platelet surface charge,probably by means of binding of high-energy com-pound metabolites to P2Y receptors of platelets [20], isan important point in our study. Platelets are known tobe involved in the formation of neutrophil extracellu-lar traps. An increase in the surface potential of bothgranulocytes and platelets may deter the interactionbetween their surfaces, thereby weakening the inflam-matory response of immunocompetent cells in theblood of the elderly.Thus, our model study of mechanical stress in vitrohas demonstrated an elevation in the concentration ofhigh-energy compounds in blood from elderly people.Along with it, mechanical stress results in a decrease inthe Young modulus and increase in plasmalemmacharge in all of the four populations of blood corpus-cles. The experimental results contribute substantiallyto the knowledge of the effect of mechanical deforma-tion on leukocytes and platelets, which are the key reg-ulator of homeostasis in the vasculature, and on eryth-rocytes, which are involved in the regulation of rheo-logical parameters and oxygen supply to tissues.Further study of blood corpuscle morphology andfunctional parameters under shear deformation ofmembranes may help us with the correction of patho-logical conditions (immune dysfunction, microcircu-lation disturbance, and progression of cardiovasculardiseases) that accompany aging.FUNDINGThis work was supported by the Russian Science Foun-dation, Studies initiated by young scientists program 2018–2020 (agreement 18-75-00041).COMPLIANCE WITH ETHICAL STANDARDSConflict of InterestsThe authors declare that they have no conflict of inter-est.Statement on the Welfare of AnimalsAll manipulations with humans met the ethical stan-dards of the Declaration of Helsinki, 1964, as amended bythe 52nd WMA General Assembly, Edinburgh, Scotland,October 2000. Informed consent was obtained from eachparticipant of the study.REFERENCES1. A. Olivieri, M. Pala, and F.Gandini, PLoS One 8 (7),34 (2013).2. E. V. Uzikova, M. Yu. Miloradov, and V. N. Levin,Yaroslav. Ped. Vestn. 3 (3), 108 (2011).3. G. Burnstock, Keio J. Med. 62, 63 (2013).4. D. Erlinge and G. Burnstock, Purinergic Signalling 1,1 (2008).5. R. Sprung, R. Sprague, and D. Spence, Anal. Chem. 74(10), 2274 (2002).6. A. Baroja-Mazo, H. Barbera-Gremades, andP. Pelegrini, Biochim. Biophys. Acta 1828 (1), 79(2013).7. G. G. Yegutkin, Biochim. Biophys. Acta 1783 (5), 673(2008).8. T. Oonishi, K. Sakashita, and N. Uyesaka, Am. J.Physiol. Soc. 273, 1828 (1997).9. A. Trautmann, Sci. Signal. 2 (56), pe6 (2009).10. T. L. Aleinikova and G. V. Rubtsova, Guidelines forPractical Course in iochemistry (Vysshaya Shkola, Mos-cow, 1988) [in Russian].11. E. A. Sladkova and M. Yu. Skorkina, Biophysics (Mos-cow) 59 (2), 254 (2014).12. M. Yu. Skorkina, M. Z. Fedorova, E. A. Sladkova, andN. A. Zabinyakov, RF Patent 2466401, 2012.13. M. Yu. Skorkina, M. Z. Fedorova, A. V. Murav’ev,et al., Klet. Tekhnol. Biol. Med. 3, 172 (2012).14. M. Ellworth, C. Ellis, and D. Goldman, Physiology 24,107 (2009).15. J. Evans, W. Gratzer, N. Mohandas, K. Parker, andJ. Sleep, Biophys. J. 94 (10), 4134 (2008).16. D. H. Lee, K. S. Park, I. D. Kong, et al., BMC Immu-nol. 7, 22 (2006).17. L. Wang, G. Olivercrona, and M. Gotberg, Circ. Res.96 (2), 189 (2005).18. R. A. North, Philos. Trans. R. Soc. Lond. B 371 (1700),20150427 (2016).19. W. G. Junger, Nat. Rev. Immunol. 11 (3), 201 (2011).20. E. R. Lazarowski, Purinergic Signalling 8, 359 (2012).21. M. D. Ita and M. H. Vargas, Life Sci. 145 (5), 85(2016).Translated by V. GulevichBIOPHYSICS  Vol. 65  No. 6  2020

Changes of the biophysical properties of blood corpuscles from the elderly under mechanical stress in vitro

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Changes of the biophysical properties of blood corpuscles from the elderly under mechanical stress in vitro

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