Role of cytoskeleton and deformability in laminin-mediated cell rolling

Recent mathematical models show that molecular events that mediate rolling interactions also have an impact on the stochastic features of rolling. In spherical cells, statistical fluctuations in cell displacement were shown to be an indication that only a few adhesion bonds are involved in rolling interactions. In this study, we investigated whether cell shape and cell deformability could also modulate the stochastic features of rolling. As an experimental model we considered the flow-initiated rolling of MCF-10 breast epithelial cells on laminin. The dynamic adhesion of MCF-10A cells to laminin, which involves integrin alpha 6 beta 4, occurs slow enough to allow for an accurate determination of the trajectories of rolling cells. The data from high-magnification videomicroscopy showed that cell shape, cell deformability, and the level of fluid shear stress were all strong determinants of the rolling velocity and the extent of fluctuations in the trajectory of rolling cells. MCF-10A cells with large surface projections rolled faster and wobbled more extensively than spherical cells under the same flow conditions. The extent of wobbling decreased and the variation of rolling velocity increased with increasing fluid shear stress. MCF-10A cells treated with cytochalasin B, which increased cell deformability and caused extensive blebbing without significantly altering surface expression of laminin integrins, reduced mean rolling velocity and increased its variance. Because leukocytes change shape as they roll in postcapillary blood venules at high shear rates, results indicate the need for further expanding the present biophysical models of rolling to the case of deformable cells.     

Electrical impedance assays of blood cells

In this review, the capability of electrical impedance spectroscopy analysis of blood cells, especially for red blood cells is presented, highlighting its large area of related biomedical relevance. The method is briefly introduced and basic theoretical aspects are discussed by considering both phenomenological (e.g. equivalent circuit) and microscopic approaches. The latter include a comparative analysis of the relevance of considering real shape (consistent with microscopic observations) versus spheroidal approximations (prolate and oblate spheroids) with the same surface and volume concentration. We show that while ellipsoidal approximation is fairly good for randomly oriented cells, it is quite poor whenever oriented cells are measured. The voluminous literature on the electrical analysis of blood cells is reviewed to stress the most promising biomedical applications of the method either per se or in combination with complementary e.g. (micro) fluidic approaches.     

Impedimetric characterization of human blood using three-electrode based ECIS devices

In this review, the capability of electrical impedance spectroscopy analysis of blood cells, especially for red blood cells is presented, highlighting its large area of related biomedical relevance. The method is briefly introduced and basic theoretical aspects are discussed by considering both phenomenological (e.g. equivalent circuit) and microscopic approaches. The latter include a comparative analysis of the relevance of considering real shape (consistent with microscopic observations) versus spheroidal approximations (prolate and oblate spheroids) with the same surface and volume concentration. We show that while ellipsoidal approximation is fairly good for randomly oriented cells, it is quite poor whenever oriented cells are measured. The voluminous literature on the electrical analysis of blood cells is reviewed to stress the most promising biomedical applications of the method either per se or in combination with complementary e.g. (micro) fluidic approaches.     

Effects of calcium and A23187 on deformability and volume of human red blood cells

Human red blood cells treated in vitro with Ca2+ plus A23187 in low K+ medium exhibited significantly decreased cell volume and deformability, the latter determined by ektacytometry. These effects of Ca2+ plus A23187 were prevented in the presence of high K+ medium. Increased K+ permeability mediated by increased intracellular Ca2+ (Gardos effect) was apparently responsible for decreased cell volume and deformability in low K+ medium. Although it is commonly accepted that Ca2+ accumulation and/or ATP depletion per se cause decreased red blood cell deformability, the present results demonstrate that acutely induced changes in red blood cell volume as promoted by Ca2+ are a more important determinant of red blood cell deformability.     

Spin label study of erythrocyte deformability I. Electron spin resonance spectral change under shear flow

A spin labeling method in electron spin resonance spectroscopy (ESR) is applied for the first time to study the deformability of human red blood cells (RBC). ESR measurements of a RBC suspension incubated with a fatty acid spin label were performed, using a narrow-gap flat ESR sample cell under various flow shear stresses (tau). Remarkable changes were observed in ESR spectra with tau, indicating that RBC are oriented in such a way that the greater part of the membrane surface is aligned parallel to the ESR cell walls. The diamide-treated, hardened RBC, in which the biconcave discoid shape remains intact under no shear stress, exhibit a smaller ESR spectral change with tau than the intact, demonstrating that the present method can be used to assess the deformation of RBC occurring with flow orientation. In particular, the relative amplitude of an ESR difference spectrum may be used as a measure of the elongation of RBC. The conclusion is further supported by experiments using glutaraldehyde-treated or heat-denatured RBC. All these ESR results are in good agreement with the corresponding results obtained by several different methods. The present spin labeling technique is thus proven to be applicable for evaluating RBC deformability.     

Scanning electron microscope study of the splenic red pulp in relation to the sequestration of immature and abnormal red cells

Spleens from normal, healthy cats, dogs and rabbits were perfused with Ringer solution until only a few red cells remained. After fixation of the intact organ, small pieces of tissue were dried by a camphene method and examined under the scanning electron microscope. In all three species the red cells remaining in the spleen were either reticulocytes, spiculated cells, or cells of tear-drop shape and they were found adhering to macrophages and reticulum cells throughout the red pulp. Elongated masses were found on the sinusal surface of fenestrated endothelium (only in dog and rabbit); some of these appeared to be cells of tear-drop shape emerging from the cords into the sinus. This may perhaps denote a pitting process, as suggested by others, but it cannot be a unique function of fenestrated endothelium for red cells of similar shape were found elsewhere in the pulp. In all three species the network of reticulum fibres presents a very large contact surface area for blood cells and it seems likely that increased cell stickiness, rather than decreased deformability, leads to the trapping of immature red cells in the spleen.     

Separation and characterization of red blood cells with different membrane deformability using steric field-flow fractionation

Human red blood cells were treated in different ways to alter their membrane deformability, and the hydrodynamic behavior of these altered cells was studied using the steric field-flow fractionation (FFF) technique. The relationships between cell retention in the FFF channel, flow-rate of the carrier fluid and the applied field strength were studied for normal and glutaraldehyde-fixed human red cells, and separation conditions were optimized. The effect of flow-induced hydrodynamic lift forces on red cell retention in the steric FFF channel was studied, and the results suggest that the membrane deformability of the red cell is an important factor contributing to the lift force, besides other previously described effects due to density and flow velocity. Using steric FFF, a mixture of normal and glutaraldehyde-fixed human red cells was completely separated with a resolution twice that found in published d ata from gel permeation, another hydrodynamic separation technique. Partial loss of membrane deformability, induced by different degrees of glutaraldehyde-fixation, by diamide, or by a thermal treatment, has also been studied. Steric FFF is thus shown to have potential for rapid separation and differentiation of red cells with different density and membrane deformability, conditions known to be associated with, e.g., cell senescence and certain hematological diseases.     

Parallel microchannel-based measurements of individual erythrocyte areas and volumes

We describe a microchannel device which utilizes a novel approach to obtain area and volume measurements on many individual red blood cells. Red cells are aspirated into the microchannels much as a single red blood cell is aspirated into a micropipette. Inasmuch as there are thousands of identical microchannels with defined geometry, data for many individual red cells can be rapidly acquired, and the fundamental heterogeneity of cell membrane biophysics can be analyzed. Fluorescent labels can be used to quantify red cell surface and cytosolic features of interest simultaneously with the measurement of area and volume for a given cell. Experiments that demonstrate and evaluate the microchannel measuring capabilities are presented and potential improvements and extensions are discussed.     

Effects of simulated microgravity (HDT) on blood fluidity.

Exposures to microgravity and head-down tilt (HDT) produce similar changes in body fluid. This causes an increase in hematocrit that significantly affects hemorheological values. Lack of physical stimulation under bed rest conditions and the relative immobility of the crew during spaceflight also affects the blood fluidity. A group of six healthy male subjects participated as volunteers, and blood samples were collected 10 days before, on day 2 and day 9, and 2 days after the HDT phase. Blood rheology was quantified by plasma viscometry, red cell aggregability, and red cell deformability. A reduced red cell deformability, an indication of the diminished quality of the red blood cells, was measured under HDT conditions that finally led to the so-called "space flight anemia." Enhanced red cell membrane fragility induced by diminished physical activity and an increase in hemoglobin concentration are responsible for this effect. Plasma viscosity is reduced as a result of diminished plasma proteins. However, despite the reduction in plasma proteins, including fibrinogen, alpha 2-macroglobulin, and immunoglobulin M, red cell aggregation was enhanced, principally because of the increase in hematocrit. Our results of hemorheological alterations under HDT conditions may help to elucidate the formerly documented hematologic changes during spaceflight.     

Determination of erythrocyte transit times through micropores. I--Basic operational principles

To study the transit times of each red blood cell passing through cylindrical micropores and in order to evaluate sub-population of cells with regard to their deformability, we have developed a new system called the cell transit time analyser (CTTA). By using an AC voltage (100 KHz) across a special filter, we measure the electrical conductance change produced by the cells passing through the pores under a known driving pressure. This computer based device provides the distribution of transit times tau for 2000 cells in 1 minute and as a result the mean transit time [tau]. Experiments with red cells were designed to evaluate the flow behavior of both normal cells and cells whose mechanical properties were artificially altered. Cell volume was changed by use of non-isotonic media. Cell shape and cell volume were modified by varying the pH of the suspending buffer. Results of these experiments are: 1) a skew distribution of transit times towards high tau values for both control cells and artificially altered cells is observed: 2) [tau] is minimum for isotonic conditions and increases sharply for either hypotonic or hypertonic media: 3) [tau] is minimum at physiological pH and increases for either acid or alcaline changes of pH.     

Red cell mechanics: what changes are needed to adversely affect in vivo circulation

The ability of red cells to deform is essential to allow their circulation. However the degree of rheological abnormality which can be tolerated before flow is impaired is not so clear. Red cell rheology has been characterised in a number of physiological, pathological and genetic conditions, and some inferences can be drawn. In vivo aging causes a small loss of cell deformability attributable to increased membrane and internal viscosity; volume and surface area are also lost. These changes cannot be sufficient to cause cellular removal, since the cells sampled had continued to circulate. In sickle cell disease, the oxygenated blood contains dense cells that are more severely abnormal than dense, aged cells from normal individuals. Melanesian ovalocytes have comparable rigidity to dense SS cells, but this condition has no marked circulatory pathology. Thus circulatory problems in SS disease probably stem from deoxygenation-induced sickling which causes extreme loss of deformability, rather than from the abnormal cells in oxygenated blood. In falciparum malaria, immature parasites cause appreciable loss of deformability but continue to circulate. Maturation of the parasites causes much greater rheological changes, including attachment to vascular endothelium, and the cells cease to circulate. In summary, quite marked changes in cell mechanics can occur without loss of ability to circulate. It thus seems that slight rheological alterations reported in some clinical studies are unlikely to cause appreciable flow disruption.     

Effects of nitric oxide and prostacyclin on deformability and aggregability of red blood cells of rats ex vivo and in vitro.

Although many diseases of the heart and circulatory system have been linked with insufficient deformability and increased aggregability of red blood cells, there are only a few drugs which can modulate these biological functions of erythrocytes. Here, we show evidences that iloprost, stable prostacyclin analogue and SIN-1, active metabolite of molsidomine which spontaneously releases NO, may be sufficient pharmacological tools for modulating red blood cell deformability and aggregability. Deformability of red blood cells was measured by shear stress laser diffractometer (Rheodyn SSD) and expressed in percent of red blood cell deformability index (DI). MA-1 (Myrenne) erythrocyte aggregometer was used for photometric measurements of aggregability in arbitrary units (MEA) of mean extent of aggregation. Experiments were carried out on rats ex vivo and in vitro using whole rat blood or isolated erythrocytes. Ex vivo SIN-1 (infusion 2 mg/kg/min i.v.) and iloprost (bolus injection 10 microg/kg i.v.) significantly improved erythrocyte deformability and aggregability at 5-15 min after administration. L-NAME (10 mg/kg i.v.)- inhibitor of nitric oxide synthase, and aspirin (1 mg/kg i.v.) caused worsening of deformability of erythrocytes in experiments ex vivo. Studies in vitro also revealed improvement of red blood cell deformability and aggregability by SIN-1 (3 microM, 15 min incubation at 22 degrees C) or iloprost (1 microM, 15 min incubation at 22 degrees C) and this phenomenon appeared not only in whole blood but also in isolated red cells. It is concluded that NO- and prostacyclin-induced improvement of red blood cell deformability and aggregability results from direct action of these compounds on erythrocytes. NO-donors and iloprost could be useful in the treatment of disorders of blood fluidity.     

Relations among variations in human red cell volume,density, membrane area,hemoglobin content and cation content

It is shown how variations in different properties of red cells can be inter-related provided relations exist among these properties at the single cell level. On the basis of the cell density dependence on cell volume and hemoglobin content, and the assumed volume dependence on red cell cation and hemoglobin content, nine relations among the variations in red cell volume, density, membrane area, hemoglobin content and cation content, and their correlations are derived. Values of seven correlation coefficients are theoretically predicted and are shown to be consistent with the experiments performed by density fractionated red blood cells. The cell volume dependence on cation and hemoglobin content obtained from relations among variations is compared with the predictions obtained by the existing model about the osmotic behavior of the red blood cell. Furthermore, it is shown that data on the variations of the red cell properties indicate the existence of the relation among cation content, hemoglobin content, and membrane area at the level of a single cell.     

Effect of temperature on the deformability of a lamprey, Entosphenus japonicus, and Pacific salmon, Oncorhynchus keta, red blood cells, studied using a modified filtration method

The rates of filtration through Nuclepore filters (5 or 8 μm) of blood from lampreys and Pacific salmon have been studied using a method which visualizes the flow pattern. From these measurements, passage times for single red blood cells have been calculated and serve as an index of their deformability. The deformability increases as temperature is raised in vitro , but even at 5°C the passage time of lamprey blood is relatively rapid. The increase in deformability with a rise in temperature is small relative to that found in other fish such as yellowtail and carp.
The distribution of red cell volumes has shown the presence of a secondary peak for salmon blood taken during surgery which is reduced following recovery, the main peak being at a lower volume.     

Protection of erythrocytes from sub-hemolytic mechanical damage by nitric oxide mediated inhibition of potassium leakage

The effects of mechanical stress on red blood cell (RBC) deformability were evaluated by subjecting cells to a uniform fluid shear stress of 120 Pa for 15-120 seconds at 37 degrees C. This level of stress induced significant impairment of RBC deformability as assessed by ektacytometry, with the degree of impairment independent of extracellular calcium concentration. Inhibition of RBC nitric oxide (NO) synthesis by a competitive inhibitor of NO synthases (N-omega-nitro-L-arginine methyl ester, L-NAME) had no effect on deformability after exposure to mechanical stress. The NO donor sodium nitroprusside (SNP) prevented the deterioration of RBC deformability in a dose-dependent manner with 10(-4) M being the most effective concentration. A similar protective effect by the non-selective potassium channel blocker, tetraethylammonium chloride (TEA) suggests that the effect of NO might be mediated by the inhibition of potassium leakage from RBC. These results suggest that NO may prevent mechanical deterioration of RBC exposed to high shear stresses. While RBC are not exposed to such high levels of shear stresses for prolonged periods under normal circulatory conditions, comparable levels of mechanical stress can be encountered under certain situations (i.e., artificial organs, extracorporeal circulation) and may result in subhemolytic damage and hemorheological alterations.     

Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability

The mechanics of leukocyte (white blood cell; WBC) deformation and adhesion to endothelial cells (EC) has been investigated using a novel in vitro side-view flow assay. HL-60 cell rolling adhesion to surface-immobilized P-selectin was used to model the WBC-EC adhesion process. Changes in flow shear stress, cell deformability, or substrate ligand strength resulted in significant changes in the characteristic adhesion binding time, cell-surface contact and cell rolling velocity. A 2-D model indicated that cell-substrate contact area under a high wall shear stress (20 dyn/cm2) could be nearly twice of that under a low stress (0.5 dyn/cm2) due to shear flow-induced cell deformation. An increase in contact area resulted in more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy that inputs to a cell decreased due to a flattened cell shape. The model also predicted a plateau of WBC rolling velocity as flow shear stresses further increased. Both experimental and computational studies have described how WBC deformation influences the WBC-EC adhesion process in shear flow.     

Viscoelastic properties of the human red blood cell membrane. II. Area and volume of individual red cells entering a micropipette.

Previous work demonstrated that human red cells can be drawn into cylindrical glass micropipettes of internal diameter approximately 2.0 mum without lysing. For pipettes of less than approximately 2.9 mum inside diameter, the red cell must become less spherical, that is, reduce its volume-to-area ratio. In this work measurements were made from 16-mm film records that allowed the determination of the cellular area and volume of individual erythrocytes as they were drawn into a 2.0-mum pipette with negative pressures. The results showed that the total surface area of the membrane remains constant and that the cell endures the passage into the pipette by losing volume. The volume loss was interpreted to be due to cell water and solute loss when the membrane is under stress. The loss of cell volume, rather than the stretching of the membrane, adds confirmation that although it is very deformable, the membrane is very resistant to two-dimensional strain.     

Erythrocyte adenosine triphosphate depletion during voluntary hyperventilation

Chronic hypophosphatemia in humans is associated with a slow depletion of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes, combined with shape alteration, impaired deformability, and viability of the cells. Likewise, incubation of erythrocytes in alkaline solution is associated with ATP depletion. Since in hyperventilation both hypophosphatemia and alkalosis are present, we have investigated red cell organic phosphates, shape, deformability, and osmotic fragility before, during, and after 20 min of voluntary hyperventilation. On the average, red cell ATP decreased by 42%, the blood pH increased by 0.2 units, and plasma inorganic phosphorus decreased by 46% compared with the initial values. Red cell 2,3-DPG, shape, deformability, and osmotic fragility remained unchanged. After the end of hyperventilation ATP increased rapidly to control values in parallel with the normalization of the blood pH, whereas inorganic plasma phosphorus remained at the low level observed during hyperventilation. It is concluded that the combined effects of hypophosphatemia and alkalosis in acute hyperventilation lead to an isolated fall of red cell ATP, which occurs as rapid as after total inhibition of red cell glycolysis in vitro.     

Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways

To investigate the mechanism of theregulation of human red blood cell deformability, we examined thedeformability under mechanical stress. Washed human red blood cellswere rapidly injected through a fine needle, and their filterabilitywas measured using a nickel mesh filter. The decrease in filterabilityshowed a V-shaped curve depending on the extracellularCa²⁺ concentration; the maximumdecrease was achieved at ~50 µM. The decreased filterability wasaccompanied by no change in cell morphology and cell volume, indicatingthat the decrease in filterability can be ascribed to alterations ofthe membrane properties. Ca²⁺entry blockers (nifedipine and felodipine) inhibited the impairment offilterability under mechanical stress. ProstaglandinsE₁ and E₂, epinephrine, andpentoxifylline, which are thought to modulate the intracellularadenosine 3',5'-cyclic monophosphate (cAMP) level of redblood cells, improved or worsened the impaired filterability accordingto their expected actions on the cAMP level of the cells. These resultsstrongly suggest that the membrane properties regulating red blood celldeformability are affected by the signal transduction system, includingCa²⁺-dependent and cAMP-mediatedsignaling pathways.