Shape Of Red Blood Cells Research Articles

A Human β-Spectrin Gene Promoter Directs High Level Expression in Erythroid but Not Muscle or Neural Cells

beta-Spectrin is an erythrocyte membrane protein that is defective in many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. It is expressed not only in erythroid tissues but also in muscle and brain. We wished to determine the regulatory elements that determine the tissue-specific expression of the beta-spectrin gene. We mapped the 5'-end of the beta-spectrin erythroid cDNA and cloned the 5'-flanking genomic DNA containing the putative beta-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, a beta-spectrin gene erythroid promoter with two binding sites for GATA-1 and one site for CACCC-related proteins was identified. All three binding sites were required for full promoter activity; one of the GATA-1 motifs and the CACCC-binding motif were essential for activity. The beta-spectrin gene promoter was able to be transactivated in heterologous cells by forced expression of GATA-1. In transgenic mice, a reporter gene directed by the beta-spectrin promoter was expressed in erythroid tissues at all stages of development. Only weak expression of the reporter gene was detected in muscle and brain tissue, suggesting that additional regulatory elements are required for high level expression of the beta-spectrin gene in these tissues.

Red blood cell shape as a function of medium’s ionic strength and pH

Glycocalyx, the characteristic first line of interaction between membrane and environment, can be visualized as a polyelectrolyte anchored to a bending-resistant matrix. This structure has an amazing resemblance with the ionized monolayers, in which, the cohesion among hydrocarbon chains is counteracted by the repulsion among similarly charged ionic heads, and thus the balance determines the curvature of the membrane. Likewise, it could be assumed that in biological membranes, repulsion among similarly charged groups in the glycocalyx could generate different curving trends. Hence, the factors directly influencing the electrostatic interaction among surface charged groups were studied, assessing the effect of the medium’s ionic strength (μ) and pH, in an extensive range of values around the physiological one. The results point out μ variations inducing different shapes, depending on whether μ values were lower or higher than the physiological ones; which could be explained by the polyelectrolyte theory. The occurrence of more invaginated shapes as the medium’s pH decreases, and the opposite event, when the pH increases, could be attributed to the coupling between the dissociation of the glycocalyx ionic groups and the H + concentration. The behavior of the cells with reduced surface charges (by neuraminidase degradation) supports the hypothesis that the observed μ and the pH effect on erythrocyte shape could be mediated by glycocalyx charged groups.

Amphiphile induced echinocyte-spheroechinocyte transformation of red blood cell shape.

A possible physical explanation of the echinocyte -spheroechinocyte red blood cell (RBC) shape transformation induced by the intercalation of amphiphilic molecules into the outer layer of the RBC plasma membrane bilayer is given. The stable RBC shape is determined by the minimization of the membrane elastic energy, consisting of the bilayer bending energy, the bilayer relative stretching energy and the skeleton shear elastic energy. It is shown that for a given relative cell volume the calculated number of echinocyte spicula increases while their size decreases as the number of the intercalated amphiphilic molecules in the outer layer of the cell membrane bilayer is increased, which is in agreement with experimental observations. Further, it is show that the equilibrium difference between the outer and the inner membrane leaflet areas of the stable RBC shapes increases if the amount of the intercalated amphiphiles is increased, thereby verifying theoretically the original bilayer couple hypothesis of Sheetz and Singer (1974) and Evans (1974).

Fundamental Examination of Cattle Red Blood Cells Damage with Ultrasound Exposure Microscopic System (UEMS)

In order to define the safety limit of the medical ultrasound equipment and to study the ultrasonic bioeffects, ultrasound exposure microscopic system (UEMS) was developed in our laboratory. We intended to observe the tissue damage caused by ultrasound exposure in real time. The change of size and shape of the cattle red blood cells was investigated experimentally with UEMS. It was observed that, at a driving frequency of 2.2 MHz, when the ultrasound intensity ISPTP was 28.4 W/cm2, the change of shape of the cattle red blood cells, due to the ultrasound exposure, occurred after 30 min of exposure. The change of size of the cattle red blood cells was investigated with a Coulter counter. The results, indicated that the size of the red blood cells after exposure to ultrasound was smaller than before exposure. Moreover, the results also indicated that, there was a tendency of the red blood cells to decrease in size after the ultrasound exposure when the ultrasound intensity was increased. At the two driving frequencies of 2.2 MHz and 0.55 MHz, in this study, the size of the cattle red blood cells decreased when the ultrasound intensities were increased.

Membrane skeleton and red blood cell vesiculation at low pH

Shapes of red blood cells at low pH were studied theoretically. It is assumed that the equilibrium shape of the red blood cell corresponds to the minimum of its membrane elastic energy which consists of the bending energy and relative stretching energy of the bilayer, the stretching energy of the skeleton and the interaction energy between the skeleton and the bilayer. It is shown that the aggregation of the skeleton at low pH can cause the red blood cell shape transformation from the stomatocytic shape to the cell shape composed of a spherical parent cell having the bilayer completely underlaid with the skeleton and spherical daughter vesicles without the skeleton.

Morphological Changes of Human Erythrocytes Induced by Cholesterol Sulphate

Morphological alterations of human erythrocytes induced by cholesterol sulphate (5-cholesten-3 beta-ol sulphate, CS) were studied. Influence of CS on red blood cell stability (in isotonic conditions) by simultaneous application of flow cytometry and scanning electron microscopy was studied. In isotonic medium CS induces erythrocyte size and shape changes in dose-and time-dependent manner. Incubation (in vitro) of erythrocytes with CS concentrations from 4 x 10(-5) mol/dm3 to 8 x 10(-5) mol/dm3 led to a progressive sphero-echinocitic shape transformation accompanied by a cell size decrease. In contrast to this, for CS content equal to 1 x 10(-5) mol/dm3 the maintenance of the normal biconcave shape of red blood cells was observed. The results suggest that CS, similarly to numerous evaginating amphiphilic agents, induces a transformation of the erythrocyte normal discoid shape to echinocytic form. This effect may be caused, at least partly, by an asymmetric expansion of the membrane lipid bilayer due to asymmetric distribution of CS incorporated into the membrane. The echinocytic shape transformation of erythrocytes indicated that CS intercalates in the outer hemileaflet of the lipid bilayer leading to membrane externalization.

Stability of spiculated red blood cells induced by intercalation of amphiphiles in cell membrane.

The stability of spiculated red blood cells, induced by intercalation of amphiphilic molecules into the cell membrane, is studied. It is assumed that the stable red blood cell shape corresponds to the minimum of its membrane elastic energy, which consists of the local and non-local bilayer bending energies and of the skeleton shear elastic energy. The cell, volume and the membrane area are kept constant. It is calculated that the number of spicules of the stable echinocytic shape is larger when the amphiphile concentration is higher, which is in agreement with experimental observations. Also, it is established that, in explaining the stability of the echinocytic shape of the red blood cell, it is necessary to include the membrane skeleton shear elasticity.

A model for red blood cell motion in glycocalyx-lined capillaries.

The interior surfaces of capillaries are lined with a layer (glycocalyx) of macromolecules bound or absorbed to the endothelium. Here, a theoretical model is used to analyze the effects of the glycocalyx on hematocrit and resistance to blood flow in capillaries. The glycocalyx is represented as a porous layer that resists penetration by red blood cells. Axisymmetric red blood cell shapes are assumed, and effects of cell membrane shear elasticity are included. Lubrication theory is used to compute the flow of plasma around the cell and within the glycocalyx. The effects of the glycocalyx on tube hematocrit (Fahraeus effect) and on flow resistance are predicted as functions of the width and hydraulic resistivity of the layer. A layer of width 1 micron and resistivity 10(8) dyn.s/cm4 leads to a relative apparent viscosity of approximately 10 in a 6-micron capillary at discharge hematocrit 45% and flow velocity of approximately 1 mm/s. This is consistent with experimental observations of increased flow resistance in microvessels in vivo, relative to glass tubes with the same diameters.

Red blood cell deformability in sepsis.

The microcirculatory disturbances in sepsis have prompted micropore bulk-filtration studies of red blood cell (RBC) mechanical behavior (i.e., deformability). However, these prior reports may not solely reflect RBC behavior because of possible white blood cell (WBC) occlusion of the filter pores. The present study was designed to examine RBC mechanical alterations in human and experimental sepsis using techniques that are not affected by WBC artifacts. RBC were obtained from adult patients with sepsis and from healthy control donors. RBC were also obtained from Swiss-albino rats in which experimental sepsis was induced via cecal ligation-puncture. Red cell mechanical behavior was tested using a computerized micropore filtration system (CTA) and a laser-diffraction shearing device (LORCA); the latter provides the extent of RBC deformation at various stresses and the time constant for RBC shape recovery. Salient findings include: (1) for human RBC, significantly decreased deformability at fluid shear stresses < 5 Pa (LORCA) yet no differences from control with the CTA; (2) for rat RBC in experimental sepsis, significant decreases of deformability and shape-recovery time constant (LORCA) but no differences with the CTA. We conclude that RBC deformability is reduced in sepsis but that micropore bulk-filtration methods may not be appropriate for detecting these changes.

Effect of free fatty acids on erythrocyte morphology and membrane fluidity.

Changes in red blood cell shape and membrane properties in response to the interaction with free fatty acids and their derivatives were studied by light scattering at small and large angles, light microscopy and fluorescence anisotropy. The influence of these agents depended on the end groups and increased with increasing chain length. The fatty acids exerted a biphasic effect on the cell size, shape and surface properties, and induced erythrocyte aggregation. After transient size alteration with a reduction in diameter, caused by low free fatty acid concentrations (up to 5-10 microM in the case of palmitic acid), fatty acids increased the erythrocyte diameter at higher concentrations (20-60 microM in the case of palmitic acid). The aliphatic aldehydes and methyl esters of fatty acids significantly decreased the cell diameter at the concentrations used. Changes in erythrocyte shape and size were accompanied by changes in membrane microviscosity. Palmitic acid decreased the rotational diffusion of the fluorescence probe incorporated into the membrane whereas methyl ester of palmitic acid and lauric aldehyde increased probe mobility. Also the erythrocyte modification by malondialdehyde influenced cell morphology and highly decreased membrane fluidity.

A possible physical mechanism of red blood cell vesiculation obtained by incubation at high pH

The membrane of human red blood cells is essentially composed of two parts, the lipid bilayer and the membrane skeleton that interacts with the lipid bilayer. The normal resting shape of the red blood cells at physiological pH 7.4 is the discocyte. However, at alkaline pH≃11 the shape of red blood cells is composed of a spherical parent cell and large spherical daughter vesicles. The daughter vesicles may be free or connected to the parent cell by a narrow neck. In this paper we show that the shapes of red blood cells at pH≃11 correspond to some of the calculated shapes of a closed lipid bilayer having an extreme area difference between the outer and the inner monolayer. Therefore, it is suggested that the observed shapes of the red blood cells at pH≃11 are a consequence of the abolishment of the skeleton–bilayer interactions at this pH.

Cellular determinants of low-shear blood viscosity

Low-shear viscometry is one of the methods commonly used to estimate the degree of red blood cell (RBC) aggregation in various bloods and RBC suspensions. However, it has been previously shown that alterations in RBC morphology and mechanical behavior can affect the low-shear apparent viscosity of RBC suspensions; RBC aggregation is also sensitive to these cellular factors. This study used heat treatment (48°C, 5 min), glutaraldehyde (0.005–0.02%) and hydrogen peroxide (1 mM) to modify cell geometry and deformability. Red blood cell aggregation was assessed via a Myrenne Aggregometer (“M” and “Ml” indexes), RBC suspension viscosity was measured using a Contraves LS-30 viscometer, and RBC shape response to fluid shear stresses ( i.e., deformability) was determined by ektacytometry (LORCA system). Our results indicate that low-shear apparent viscosity and related indexes may not always reflect changes of RBC aggregation if cellular properties are altered: for situations where RBC aggregation has been only moderately affected, cellular mechanical factors may be the major determinant of low-shear viscosity. These findings thus imply that in situations which may be associated alterations of RBC geometry and/or deformability, low-shear viscometry should not be the sole measurement technique used to assess RBC aggregation.

Determination of Red Blood Cell Shape Recovery Time Constant in a Couette System by the Analysis of Light Reflectance and Ektacytometry

Red blood cell (RBC) shape change under shear is generally reversible, with the time course of shape recovery a function of the elastic and viscous properties of the RBC membrane. RBC shape recovery can be investigated, using several different techni

Human red blood cell shape and volume are changed by physiological levels of hydrostatic pressure.

Application of hydrostatic pressure of several atmospheres (atm), such as that applied in diving or hyperbaric treatment, has been previously shown to induce the release of membrane components into the extracellular medium. As the shape of red blood cells (RBC) is sensitive to membrane composition, this might imply a subsequent change in RBC shape and volume. The present study demonstrates that application of hydrostatic pressure of up to 15 atm changes the shape of RBC from the normal discoids to stomatocytes (cup-shaped) and accordingly increases their volume. Changes in RBC shape and volume are known to impair physiological and cellular function. Thus, these changes might be pertinent to hemodynamic and physiological disorders observed in humans subjected to elevated pressure.

Determination of red blood cell shape recovery time constant in a couette system by the analysis of light reflectance and ektacytometry

Red blood cell (RBC) shape change under shear is generally reversible, with the time course of shape recovery a function of the elastic and viscous properties of the RBC membrane. RBC shape recovery can be investigated, using several different techniques, to provide information about the membrane material properties that are not directly accessible by frequently used methods to assess RBC deformability ( e.g., micropore filtration). In the present study, RBC shape recovery was studied in a Couette system after abrupt cessation of shear, either by analyzing the time course of laser light reflection or by serial measurements of elongation indexes from laser diffraction patterns. The time course of shape recovery monitored with both techniques can be described with an exponential equation. Calculated time constants for normal human RBC were 119 ± 17 msec and 97 ± 15 msec as measured by light reflection and ektacytometry, respectively. Treatment of RBC with glutaraldehyde resulted in dose-dependent decreases in the shape recovery time constant. Heat treatment (48 ° C, 20 min), which is known to increase mainly the shear elastic modulus of the membrane, decreased the time constant by 65%. In contrast, wheat germ agglutinin treatment increased the shape recovery time constant by 22%, presumably by increasing membrane surface viscosity. Our results indicate that the shape recovery time constant of RBC can be measured easily and accurately by computerized light reflection analysis.

A photometric method to analyze induced erythrocyte shape changes

In this paper, a photometric method was introduced to quantify biochemically-induced red blood cell (RBC) shape changes when no shear force was acting on the cells. To obtain the photometric RBC shape parameter (RF1), a monolayer of point-attached RBCs was prepared on the floor of a flat flow chamber and the transmission of light perpendicular to the monolayer plane was measured: 1) in phosphate buffered saline with 0.1% bovine serum albumin (PBS +) and 2) in PBS +, containing a shape changing compound (in both, the RBCs were not deformed due to shear flow). To normalize the data, a third transmission value at a shear stress of 3 Pa was measured in PBS + from the same RBC monolayer. To validate the photometric data, RF1 of RBCs exposed to shape changing agents was correlated by linear regression analysis with 1) data obtained with the tangent-counting technique (TC) and 2) the morphological index (MI). The coefficient of correlation was calculated at 0.95 for the TC data and 0.94 for the MI data, respectively. The sensitivity of the photometric method was tested with stomatocytogenic chlorpromazine (CP) and echinocytogenic sodium salicylate (SA). CP (2.5 μM) induced a significant decrease of RF1 to −0.045 (N = 6 donors, p < 0.01), whereas SA (2.5 mM) increased RF1 to +0.027 significantly (N = 6, donors, p < 0.01). Both the CP-induced and the SA-induced shape changes appeared less than 2 min after application of the shape changing agents, and changed gradually within another 30 min when the agent was present in PBS +, partly disappearing within about 2 min after reincubation of the shape transformed RBCs in PBS + not containing the agent.

Effects of alkanols, alkanediols and glycerol on red blood cell shape and hemolysis

The physicochemical effects of a series of alkanols, alkanediols and glycerol on erythrocyte shape and hemolysis at 4 and 20°C were examined. We calculated the dielectric constant of the incubation medium, D s, and the dielectric constant of the erythrocyte membrane D m in the presence of organic solutes. The ratio D s D m = −38.48 at 20°C defines the normal biconcave shape in a medium without hemolytic agents. A decrease in D s D m favors externalization or internalization with consequent hemolysis. Alkanols and alkanediols convert biconcave erythrocytes into echinocytes, which is accompanied by an increase in the projected surface area. Glycerol converts biconcave erythrocytes into stomatocytes, which was accompanied by a marginal decrease in the projected surface area. Progressive externalization in alkanols and alkanediols or internalization in glycerol resulted in a decrease in the projected surface area and the formation of smooth spheres. The degree of shape change induced was related to the degree of hemolysis and the ratio D s D m . A decrease in temperature reduced both the degree of shape change and hemolysis. Our results suggest that physicochemical toxicity may be a result of a temperature dependent hydrophobic interaction between the organic solutes and the membrane and is best interpreted by the ability of the solutes to change D s and D m. These results are discussed with respect to the physicochemical constants of the organic solutes.

Echinocytic transformation and aggregation of red cells in uremic patients.

Isolated rabbit lungs were perfused with washed and resuspended human red blood cells (RBCs) in the presence of drugs known to change the shape and deformability of RBCs. With sodium salicylate (0.5-2 g/l), which causes echinocytosis and increases RBC deformability, lung diffusing capacity for O2 (DLO2) increased by 21%. When chlorpromazine, which induces stomatocytosis and stiffens RBCs, was given (50 mg/l), DLO2 decreased by 18% under chlorpromazine. Comparative experiments with hemoglobin solutions did not reveal any effect of those two drugs either on DLO2 or on pulmonary arterial pressure, which indicates that the effects of sodium salicylate and chlorpromazine were due to changes in RBC shape and deformability. It is concluded that RBC shape and deformability affect pulmonary artery pressure and oxygen diffusing capacity, which may have an influence on oxygen transfer to tissue and hence be of clinical relevance.

Shape Response of Human Erythrocytes to Altered Cell pH

Alteration of red blood cell (RBC) pH produces stomatocytosis (at low pH) and echinocytosis (at high pH). Cell shrinkage potentiates high pH echinocytosis, but shrinkage alone does not cause echinocytosis. Mechanisms for these shape changes have not been described. In this study, measured dependence of RBC shape on cell pH was nonlinear, with a broad pH range in which normal discoid shape was maintained. Transbilayer distribution of phosphatidylcholine and phosphatidylserine, measured by back-extraction of radiolabeled lipid, was the same in control and altered pH cells. Possible roles of pH-titratable inner monolayer phospholipids were examined by assessing pH-dependent shape in cells in which their levels had been perturbed. In metabolically depleted cells and calcium-treated cells, which have altered levels of phosphatidic acid, phosphatidylinositol-4-phosphate, and/or phosphatidylinositol-4,5-bisphosphate, low cell pH was stomatocytogenic and high cell pH was echinocytogenic, as in control cells. Thus, neither change in membrane lipid asymmetry nor normal levels of the pH-titratable inner monolayer lipids is necessary for cell pH-mediated shape change.

Effect of RBC shape and deformability on pulmonary O2 diffusing capacity and resistance to flow in rabbit lungs

Isolated rabbit lungs were perfused with washed and resuspended human red blood cells (RBCs) in the presence of drugs known to change the shape and deformability of RBCs. With sodium salicylate (0.5-2 g/l), which causes echinocytosis and increases RBC deformability, lung diffusing capacity for O2 (DLO2) increased by 21%. When chlorpromazine, which induces stomatocytosis and stiffens RBCs, was given (50 mg/l), DLO2 decreased by 18%. With sodium salicylate, the mean pulmonary artery pressure dropped by 14% from control values, whereas it increased by 18% under chlorpromazine. Comparative experiments with hemoglobin solutions did not reveal any effect of those two drugs either on DLO2 or on pulmonary arterial pressure, which indicates that the effects of sodium salicylate and chlorpromazine were due to changes in RBC shape and deformability. It is concluded that RBC shape and deformability affect pulmonary artery pressure and oxygen diffusing capacity, which may have an influence on oxygen transfer to tissue and hence be of clinical relevance.