Microrheological disorders in patients with polycythemia vera suffered acute ischemic stroke.

The relevance of the study is determined by the fact that the combination of cerebrovascular disorders and myeloproliferative diseases requires the search for a predictive biomarker to improve outcomes. The aim of this article was to explore the meanings of microrheological disorders in patients with polycythemia vera (PV) who suffered an acute ischemic stroke (AIS). The study was carried out at the Research center of Neurology. We studied microrheological properties in 181 patients (aged 42-75 years). From the AIS developed in 68 (38%) patients with PV; 59 (32%) patients with AIS were without PV; 54 (30%) patients with PV did not suffer AIS. Microrheological disorders, first of all, the red blood cells (RBC) deformability correlated to AIS severity and its features in comorbid patients. The RBC deformability was dependent on the allelic load of the V617F mutation in the JAK2 gene. Additionally, it was found that RBC deformability perform diagnostic value in the acute phase of ischemic stroke as well as get predictive value for thrombotic complications development within 2 years after AIS in such patients. We suppose that in patients with PV an ischemic stroke and thrombosis would directly depend on the success of PV treatment. In turn, RBC deformability is applicable for some predictive models to late thrombosis development.

Acute Cycling Exercise Induces Changes in Red Blood Cell Deformability and Membrane Lipid Remodeling.

Here we describe the effects of a controlled, 30 min, high-intensity cycling test on blood rheology and the metabolic profiles of red blood cells (RBCs) and plasma from well-trained males. RBCs demonstrated decreased deformability and trended toward increased generation of microparticles after the test. Meanwhile, metabolomics and lipidomics highlighted oxidative stress and activation of membrane lipid remodeling mechanisms in order to cope with altered properties of circulation resulting from physical exertion during the cycling test. Of note, intermediates from coenzyme A (CoA) synthesis for conjugation to fatty acyl chains, in parallel with reversible conversion of carnitine and acylcarnitines, emerged as metabolites that significantly correlate with RBC deformability and the generation of microparticles during exercise. Taken together, we propose that RBC membrane remodeling and repair plays an active role in the physiologic response to exercise by altering RBC properties.

Characterization of hereditary red blood cell membranopathies using combined targeted next-generation sequencing and osmotic gradient ektacytometry.

Hereditary red blood cell (RBC) membranopathies are characterized by mutations in genes encoding skeletal proteins that alter the membrane complex structure. Hereditary spherocytosis (HS) is the most common inherited RBC membranopathy leading to hereditary hemolytic anemia with a worldwide distribution and an estimated prevalence, in Europe, of about 1:2000 individuals. The recent availability of targeted next generation sequencing (t-NGS) and its combination with RBC deformability measured with a laser-assisted optical rotational ektacytometer (LoRRca) has demonstrated to be the most powerful contribution to lower the percentage of hereditary hemolytic anemia undiagnosed cases. In order to know the kind and frequency of RBC membrane mutations in our geographical area (Catalonia) and to better understand their pathophysiology, 42 unrelated, non-transfusion-dependent (NTD) patients with hereditary hemolytic anemia have been studied by combining t-NGS and LoRRca. The osmoscan module of LoRRca provides three rheological profiles that reflect the maximal deformability (EImax), osmotic fragility (Omin), and hydration state (Ohyper) of RBCs and contribute to a better understanding of the contribution RBC rheology to the severity of anemia. From the 42 patients studied, 37 were suspected to be a RBC membrane defect due to phenotypic characteristics and abnormal RBC morphology and, from these, in 31 patients (83.8% of cases) the mutation was identified by t-NGS. No definite diagnosis was achieved in 11 patients (26.2% of cases), including 6 out of 37 cases, with suspected membranopathy, and 5 with unclassifiable HHA. In all these undiagnosed patients, the existence of hemoglobinopathy and/or enzymopathy was ruled out by conventional methods.

Methodological aspects of oxygen gradient ektacytometry in sickle cell disease: Effects of sample storage on outcome parameters in distinct patient subgroups.

Sickle cell disease (SCD) is a genetic disorder characterized by the production of an abnormal hemoglobin (Hb), which, under deoxygenation, may polymerize and cause a mechanical distortion of red blood cell (RBC) into a crescent-like shape. Recently a method, using ektacytometry principle, has been developed to assess RBC deformability as a function of oxygen tension (pO2) and is called oxygen gradient ektacytometry (oxygenscan). However, standardization of this test is needed to properly assess the tendency of sickling of RBCs under deoxygenation and to allow comparisons between different laboratories. The study compared the oxygenscan responses during blood storage between distinct populations of SCD patients. Blood from 40 non-transfused homozygous SCD patients (HbSS), 16 chronically transfused HbSS patients, and 14 individuals with compound heterozygous hemoglobin SC disease (HbSC) at steady-state was collected in EDTA tubes. Measurements were performed within 4 hours after collection and after 24 hours of storage at 4°C. We showed that storage affected the minimum RBC deformability reached during deoxygenation (EImin) in both non-transfused HbSS and HbSC patients and the maximum RBC deformability (EImax) measured before deoxygenation (i.e., in normoxia) in the three groups. In contrast, the tendency of RBCs to sickle under deoxygenation (i.e., the point of sickling; PoS) remained rather stable between the two time of measurements. Collectively, since the time between blood sampling and analysis affects some key oxygen gradient ektacytometry-derived parameters we recommend that each laboratory performs oxygenscan measurements at a standardized time point.

Phototherapy decreases red blood cell deformability in patients with psoriasis.

BACKGROUND: Alternations in erythrocyte deformability (ED), namley, the ability of erythrocytes to change shape under flow in the microcirculation, can contribute to cardiovascular diseases. Psoriasis, a systemic inflammatory skin disorder, is associated with an increased cardiovascular risk. The effect of psoriasis and psoriasis treatment on ED was only scarcely evaluated. OBJECTIVE: To evaluate ED changes in psoriasis patients following narrow band-ultraviolet B (NB-UVB) treatment. METHODS: Erythrocyte deformability was determined using a computerized cell flow properties analyzer in 9 patients with psoriasis before and after a course of Goeckerman regimen. ED was quantified using two parameters: average elongation ratio (AER) in the cell population, and the fraction of low deformable cells (% LDFC). RESULTS: All 9 patients showed decreased ED (i.e. impaired deformability) following NB-UVB treatment. There was a significant (p = 0.003) decrease in AER after treatment (AER±SD; 1.58±0.06) compared to the starting values (1.69±0.1). Additionally, there was a significant (p = 0.002) increase in the fraction of low deformable cells (% LDFC±SD; 60.00±9.05) compared to their fraction before treatment (34.86±11.44). CONCLUSIONS: The decreased ED observed following phototherapy could have clinical influences on psoriasis patients, and may partially explain why phototherapy does not decrease the cardiovascular risk in psoriasis compared to other treatments.

Red blood cell deformability is reduced in homozygous sickle cell disease patients with leg ulcers.

Previous reports differ as to whether a decreased elongation index (EI), a proxy for red blood cell (RBC) deformability, is associated with leg ulcers (LU) in people with homozygous sickle cell disease (SCD). We sought to determine whether erythrocyte deformability (ED) and haematological indices were associated with the presence of LU in patients with SCD. The study design was cross-sectional. Twenty-seven patients with LU and 23 with no history of ulceration were recruited into the study. A laser assisted rotational red cell analyzer was used in the determination of the EI. Haematological indices were determined using a CELL-DYN Ruby haematology analyzer. Data were normally distributed and presented as means±SD. Two-sample t-test was used to test for associations between haemorheological variables in SCD patients with and without LU. Statistical significance was taken as p < 0.05. The EI was significantly lower in the group with ulcers (0.30±0.07 vs. 0.35±0.07, p = 0.02). Haematological indices were comparable in patients with and without LU. Erythrocyte deformability, but not haematological indices, was associated with LU in patients with SCD.

Impaired erythrocyte deformability in transgenic HO-1G143H mutant mice.

To investigate the potential effects of variation of HO-1 activity on hemorheology, this study compared the hemorheological properties between transgenic HO-1G143H mutant mice and wild-type (WT) control mice. Fresh blood samples were obtained from mice via the ocular venous sinus. The whole blood viscosity was measured using a cone-plate viscometer. Erythrocyte deformability and aggregation was measured using ektacytometry. The elongation index was significantly reduced in the HO-1G143H mutant mice compared to the WT mice at the shear rates of 600, 800, and 1,000 s(-1). The integrated elongation index was decreased in the HO-1G143H mutant mice compared to the WT mice. There was no statistically significant difference between the HO-1G143H mutant mice and the WT mice in terms of whole blood viscosity, aggregation index, amplitude of aggregation, and aggregation half time. The present study demonstrated that a reduction in HO-1 activity results in an impaired erythrocyte deformability. Although the mechanism underlying this effect remains unclear, our study brings to light the participation of HO-1 in the variations of hemorheology.

Erythrocyte deformability and nitric oxide mobilization under pannexin-1 and PKC dependence.

The erythrocyte adenosine triphosphate (ATP) is utilised for protein phosphorylation and exported through the pannexin 1 hemichannel (Px1) in the microcirculation. The physiological stimuli for ATP release are dependent of blood shear rate level and of the tissue oxygen content. The deoxygenated and oxygenated states of haemoglobin are respectively bound and unbound to N terminal domain of the protein band 3 of the erythrocyte membrane in dependence of its degree of phosphorylation. The protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) contribute to the phosphorylation degree of band 3 and are modulated by protein kinase C (PKC). Chelerythrine (Che) is a competitive inhibitor of ATP for PKC and a negative modulator of erythrocyte deformability. The aim of this study was to assess the mobilization of nitric oxide (NO) in erythrocyte in absence and presence of Che and Px1 inhibitor (carbenoxolone). Erythrocyte deformability was evaluated in presence of carbenoxolone (Carb). Regarding the effects observed in the erythrocyte by presence of Che or Carb, the values of efflux of NO and the concentration of nitrosogluthatione are similar and with no changes in relation to their absence. Px1inhibition by Carb 10 µM ameliorates the erythrocyte deformability at a shear force of 0.6 and 1.2 Pa. The PKC inhibitor shows similar effects to the Carb on the mobilization of nitric oxide in erythrocyte. The blockage of ATP release by Carb from erythrocytes suggests a possible benefit to develop in ischemia reperfusion or in inflammatory response where will be needed to rescue the excess of NO present and ameliorate the red blood cell deformability at low shear rates.

Effects of detyrosinated tubulin on Na+,K+-ATPase activity and erythrocyte function in hypertensive subjects.

Formation of tubulin/Na(+),K(+)-ATPase (NKA) complex in erythrocytes of hypertensive subjects results in a 50% reduction in NKA activity. We demonstrate here that detyrosinated tubulin, which is increased in hypertensive erythrocytes membranes, enhances the inhibitory effect of acetylated tubulin on NKA activity. Moreover, we report a reduced content and activity of the enzyme tubulin tyrosine ligase in erythrocytes of hypertensive subjects. Such alterations are related to changes in erythrocyte deformability. Our findings indicate that the detyrosination/tyrosination cycle of tubulin is important in regulation of NKA activity, and that abnormalities in this cycle are involved in hypertension development.

SOD2 deficiency in hematopoietic cells in mice results in reduced red blood cell deformability and increased heme degradation.

Among the three types of super oxide dismutases (SODs) known, SOD2 deficiency is lethal in neonatal mice owing to cardiomyopathy caused by severe oxidative damage. SOD2 is found in red blood cell (RBC) precursors, but not in mature RBCs. To investigate the potential damage to mature RBCs resulting from SOD2 deficiency in precursor cells, we studied RBCs from mice in which fetal liver stem cells deficient in SOD2 were capable of efficiently rescuing lethally irradiated host animals. These transplanted animals lack SOD2 only in hematopoietically generated cells and live longer than SOD2 knockouts. In these mice, approximately 2.8% of their total RBCs in circulation are iron-laden reticulocytes, with numerous siderocytic granules and increased protein oxidation similar to that seen in sideroblastic anemia. We have studied the RBC deformability and oxidative stress in these animals and the control group by measuring them with a microfluidic ektacytometer and assaying fluorescent heme degradation products with a fluorimeter, respectively. In addition, the rate of hemoglobin oxidation in RBCs from these mice and the control group were measured spectrophotometrically. The results show that RBCs from these SOD2-deficient mice have reduced deformability, increased heme degradation products, and an increased rate of hemoglobin oxidation compared with control animals, indicative of increased RBC oxidative stress.

Erythrocyte remodeling in Plasmodium berghei infection: the contribution of SEP family members.

The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine-rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep-coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein-protein interactions.

Hereditary spherocytosis.

Hereditary spherocytosis is a common hemolytic disorder characterized by a defect or deficiency in one or more of the proteins composing red blood cell membrane. As a result, red blood cells have an abnormal shape, higher metabolic requirements, and are prematurely trapped and destroyed in the spleen. Hereditary spherocytosis, including the very mild or subclinical forms, is the most common cause of non-immune hemolytic anemia among people of Northern European ancestry, with a prevalence of approximately 1 in 2000. However very mild forms of the disease may be much more common. Hereditary spherocytosis is inherited in a dominant fashion in 75% of cases, whereas the remaining are truly recessive cases and de novo mutations. This review reports current concepts on red cell membrane structure and it will attempt to clarify molecular defects leading to spherocyte and their consequences.

Rac GTPases in erythroid biology.

Rac1 and Rac2 GTPases, members of the Rho GTPases family, control actin organization and play distinct and overlapping roles in hematopoietic and mature blood cells of all lineages. Here, we review our findings on the role of Rac GTPases in erythroid cells, by using conditional gene-targeting in mice. Rac1 and Rac2 deficiency causes anemia with reticulocytosis, indicating decreased red blood cell (RBC) survival, altered actin assembly in the erythrocyte membrane skeleton and decreased RBC deformability. On the other hand, Rac1(-/-); Rac2(-/-) megakaryocyte-erythrocyte progenitors demonstrate decreased proliferation in the bone marrow, but increased survival and proliferation in the spleen, indicating that stress erythropoiesis circumvents Rac GTPases deficiency. Further elucidation of the signaling pathways controlled by Rac GTPases in erythroid cells may reveal potential therapeutic targets for diseases characterized by hemolytic anemia and erythropoiesis disorders.

Maintenance of red blood cell integrity by AMP-activated protein kinase alpha1 catalytic subunit.

AMP-activated protein kinase (AMPK) plays a pivotal role in regulating cellular energy metabolism. We previously showed that AMPKalpha1-/- mice develop moderate anemia associated with splenomegaly and high reticulocytosis. Here, we report that splenectomy of AMPKalpha1-/- mice worsened anemia supporting evidence that AMPKalpha1-/- mice developed a compensatory response through extramedullary erythropoiesis in the spleen. Transplantation of bone marrow from AMPKalpha1-/- mice into wild-type recipients recapitulated the hematologic phenotype. Further, AMPKalpha1-/- red blood cells (RBC) showed less deformability in response to shear stress limiting their membrane flexibility. Thus, our results highlight the crucial role of AMPK to preserve RBC integrity.

Impaired erythrocyte filterability of spontaneously hypertensive rats: investigation by nickel filtration technique.

BACKGROUND: Deformability of erythrocytes plays a key role in the impairment of the microcirculation in hypertension. However, erythrocyte deformability in spontaneously hypertensive rats (SHR) during development of hypertension has not been fully investigated so far. METHODS AND RESULTS: Erythrocyte filterability (whole cell deformability) was investigated in relation to blood pressure measured by the tail-cuff method in SHR and age-matched Wistar-Kyoto rats (WKY), using a highly sensitive and reproducible nickel mesh filtration technique. Impaired erythrocyte filterability was marked (37.0+/-17.5%) in prehypertensive young SHR (7 weeks of age) and sustained (51.6+/-13.3%) in hypertensive mature SHR (18 weeks of age), when compared with that of age-matched WKY (62.1+/-7.2% in 7 weeks of age, P<0.005, and 71.1+/-3.9% in 18 weeks of age, P<0.005, respectively). This impairment in SHR could not be explained by the mean corpuscular volume or mean corpuscular hemoglobin concentration of erythrocytes, but the erythrocyte count was significantly (P<0.005) greater in SHR than in the age-matched WKY. CONCLUSIONS: Although the precise mechanisms remain to be elucidated, markedly impaired erythrocyte filterability in SHR is considered to contribute to the development and maintenance of genetic hypertension. (Circ J 2010; 74: 129 - 136).

Sickle cell anaemia: haemorheological aspects.

The maintenance of erythrocyte shape and membrane integrity is bound to the modification of deformability and/or permeability. Usually, this features are not investigated with normal laboratory tests. The membrane stiffness, the cell geometry, and the viscoelasticity components are influencing factors on survival and functionality of the erythrocytes. Only few studies have analyzed the viscoelastic characteristics of red blood cells, even less are the studies on patients affected by sickle cell disease (SCD), a pathology characterized by acute and chronic impairment of cell flexibility due to the formation of intracellular sickle haemoglobin (Hb S) polymers. A critical point of SCD is represented by the rheologic alterations of sickle cells determined by the transition from sol to gel of haemoglobin producing a dramatic change in cell viscosity and viscoelastic properties. We have investigated the behaviour of the blood in SCD, from an original rheological point of view, by evaluating the viscoelastic properties of sickle cells in oscillating harmonic sinusoidal mode. A comparison between patients with different severity of the disease, with transfusion dependence (TD) or without transfusion dependence (NTD), has been carried out. This study has confirmed the rheologic impairment of SC blood. The TD patients showed a minor heterogeneneity of rheologic behaviour in comparison with NTD patients, because of the normalizing effect of transfusion. The analysis of viscoelastic properties might be an additional useful tool for monitoring transfusional and pharmacological treatments.

Effect of asymmetric dimethylarginine on atherogenesis and erythrocyte deformability in apolipoprotein E deficient mice.

Previous investigations have shown that the level of asymmetric dimethylarginine (ADMA) was increased in hypercholesterolemic animal and humans, and the decreased erythrocyte deformability has been suggested to be a factor contributing to atherogenesis. In the present study, we investigated the effect of ADMA, endogenous or exogenous, on atherogenesis and erythrocyte deformability in apolipoprotein E deficient (ApoE-/-) mice. On a regular chow diet, ApoE-/- mice or C57BL/6 J mice at 12 weeks of age were treated with ADMA (5 mg/kg/day) for 4 weeks. Atherosclerotic lesion area, erythrocyte deformability, plasma lipids and asymmetric dimethylarginine (ADMA) level were determined. Plasma concentrations of triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), total cholesterol (TC), ADMA, and atherosclerotic lesion area were significantly increased, and the level of plasma high-density lipoprotein-cholesterol (HDL-C), erythrocyte deformability in ApoE-/- mice were markedly decreased compared with that of C57BL/6J mice (P<0.05 or P<0.01). Exogenous ADMA treatment increased the plasma TG level, produced atherosclerotic lesions, and decreased erythrocyte deformability in C57BL/6J mice (P<0.05 or P<0.01). Treatment with exogenous ADMA further increased the plasma TG level and lesion areas, and decreased erythrocyte deformability in ApoE-/- mice. In vitro, exogenous ADMA caused a decrease of erythrocyte deformability in a concentration-dependent manner, and the effect of ADMA was reversed by L-arginine. The present results suggest that endogenous ADMA is an important contributor to the development of atherosclerosis and that reduction of erythrocyte deformability and impaired endothelial function induced by ADMA may be an important factor facilitating atherosclerotic lesions.

Effects of TFAR19 gene on the in vivo biorheological properties and pathogenicity of mouse erythroleukemia cell line MEL.

After injecting VP16, MEL cells and MEL-TF19 cells into the body of mice, with those injected with the same dose of saline as the control group, we observed the mice for their blood pictures, histological changes of the liver and spleen, and the hemorheological indexes within 4 weeks. The results indicated that after injecting MEL cells, the mice entered into a pathological status similar to erythroleukemia, which had the following exhibitions: the tissue structures of the liver and spleen were damaged, a mass of proerythroblasts, basophil erythroblasts and polychromatophilic erythroblasts could be observed on the smears of the bone marrow and spleen, and the deformability and orientation ability of erythrocytes were both depressed. The pathogenicity of MEL-TF19 cells carrying TFAR19 gene was obviously lower than that of MEL cells, and the MEL-TF19 cells even lost their faintish pathogenicity under the apoptosis-inducing effect of the chemotherapeutic reagent. The outcome from the animal experiments suggests that the TFAR19 gene suppresses the pathogenicity of MEL cells to the mice, and the effect may be better exerted with the synergy of the chemotherapeutic reagent.

Hemorheologic profile in systemic sclerosis: role of NOS3 -786T > C and 894G >T polymorphisms in modulating both the hemorheologic parameters and the susceptibility to the disease.

OBJECTIVE: Microvascular disorders are relevant in systemic sclerosis (SSc). Hyperviscosity, due to alterations of blood cells and plasma components, may play a role in the pathogenesis of microcirculatory disorders. An impaired availability of nitric oxide, related to polymorphisms in NOS3, the gene for endothelial cell nitric oxide synthase, might influence erythrocyte deformability. We undertook this study to investigate the hemorheologic profile in SSc and the role of NOS3 polymorphisms in modulating the hemorheologic status of SSc patients. METHODS: We studied 113 consecutive SSc patients (75 with limited cutaneous SSc [lcSSc] and 38 with diffuse cutaneous SSc [dcSSc]) and 113 healthy controls. The hemorheologic profile was obtained by assessing whole blood viscosity (WBV; at shear rates of 0.512 and 94.5 seconds(-1)), plasma viscosity (PLV; at a shear rate of 94.5 seconds(-1)), and erythrocyte deformability index (DI). We determined NOS3 polymorphisms by molecular analysis. RESULTS: A marked alteration of hemorheologic parameters was found both in patients with lcSSc and in those with dcSSc compared with controls (P < 0.0001). In multivariate analysis, rheologic variables were significantly associated with the disease (for WBV at a shear rate of 94.5 seconds(-1), odds ratio [OR] 5.4, 95% confidence interval [95% CI] 1.4-19.9, P = 0.01; for PLV, OR 2.8, 95% CI 1.2-6.5, P = 0.01; for DI, OR 3.9, 95% CI 1.4-10.8, P = 0.007), and NOS3 -786C and 894T alleles significantly affected the DI (for -786C allele, OR 2.3, 95% CI 1.01-5.4, P = 0.04; for 894T allele, OR 2.2, 95% CI 1.01-4.8, P = 0.04). The simultaneous presence of the -786C and 894T alleles represented a susceptibility factor for SSc (OR 2.8, 95% CI 1.4-5.7, P = 0.004). CONCLUSION: Our findings document an altered rheologic profile in SSc and demonstrate a relationship between this alteration and NOS3 polymorphisms, thus shedding light on a potential novel mechanism influencing the microcirculation in this disease.