Reactive oxygen species in the killing of Pseudomonas aeruginosa by human leukocytes

Pseudomonas aeruginosa (PA) infects hosts with compromised host defenses. An important defense mechanism is the generation of reactive oxygen species (ROS) by white blood cells (WBCs). What roles do ROS play in host defense against PA? Human WBCs killed PA in vitro, and they generated a respiratory burst as measured by the production of H₂O₂. ROS efficiently killed PA; in acellular assays, less than 10mm of H₂O₂ or OCl^- eliminated all bacteria in 90 min. However, WBCs with suppressed production of ROS (caused by hypoxia) killed PA normally. In addition, none of the antioxidants vitamin C, N-acetylcysteine, superoxide dismutase, or catalase affected PA killing by WBCs. Thus, PA stimulates WBCs to produce ROS, which can kill the bacteria, but disturbances of WBC ROS production do not interfere with the killing of PA. WBCs have robust, redundant mechanisms for PA elimination.     

Regulation of Active ICAM-4 on Normal and Sickle Cell Disease RBCs via AKAPs Is Revealed by AFM

Human healthy (wild-type (WT)) and homozygous sickle (SS) red blood cells (RBCs) express a large number of surface receptors that mediate cell adhesion between RBCs, and between RBCs and white blood cells, platelets, and the endothelium. In sickle cell disease (SCD), abnormal adhesion of RBCs to endothelial cells is mediated by the intercellular adhesion molecule-4 (ICAM-4), which appears on the RBC membrane and binds to the endothelial αvβ3 integrin. This is a key factor in the initiation of vaso-occlusive episodes, the hallmark of SCD. A better understanding of the mechanisms that control RBC adhesion to endothelium may lead to novel approaches to both prevention and treatment of vaso-occlusive episodes in SCD. One important mechanism of ICAM-4 activation occurs via the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA)-dependent signaling pathway. Here, we employed an in vitro technique called single-molecule force spectroscopy to study the effect of modulation of the cAMP-PKA-dependent pathway on ICAM-4 receptor activation. We quantified the frequency of active ICAM-4 receptors on WT-RBC and SS-RBC membranes, as well as the median unbinding force between ICAM-4 and αvβ3. We showed that the collective frequency of unbinding events in WT-RBCs is not significantly different from that of SS-RBCs. This result was confirmed by confocal microscopy experiments. In addition, we showed that incubation of normal RBCs and SS-RBCs with epinephrine, a catecholamine that binds to the β-adrenergic receptor and activates the cAMP-PKA-dependent pathway, caused a significant increase in the frequency of active ICAM-4 receptors in both normal RBCs and SS-RBCs. However, the unbinding force between ICAM-4 and the corresponding ligand αvβ3 remained the same. Furthermore, we demonstrated that forskolin, an adenylyl cyclase activator, significantly increased the frequency of ICAM-4 receptors in WT-RBCs and SS-RBCs, confirming that the activation of ICAM-4 is regulated by the cAMP-PKA pathway. Finally, we showed that A-kinase anchoring proteins play an essential role in ICAM-4 activation.     

Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

Background

In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown.

Results

To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2.

Conclusions

These findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.     

Redox-dependent impairment of vascular function in sickle cell disease

The vascular pathophysiology of sickle cell disease (SCD) is influenced by many factors, including adhesiveness of red and white blood cells to endothelium, increased coagulation, and homeostatic perturbation. The vascular endothelium is central to disease pathogenesis because it displays adhesion molecules for blood cells, balances procoagulant and anticoagulant properties of the vessel wall, and regulates vascular homeostasis by synthesizing vasoconstricting and vasodilating substances. The occurrence of intermittent vascular occlusion in SCD leads to reperfusion injury associated with granulocyte accumulation and enhanced production of reactive oxygen species. The participation of nitric oxide (NO) in oxidative reactions causes a reduction in NO bioavailability and contributes to vascular dysfunction in SCD. Therapeutic strategies designed to counteract endothelial, inflammatory, and oxidative abnormalities may reduce the frequency of hospitalization and blood transfusion, the incidence of pain, and the occurrence of acute chest syndrome and pulmonary hypertension in patients with SCD.     

Reactive oxygen species in vascular biology: implications in hypertension

Reactive oxygen species (ROS), including superoxide (·O₂^–), hydrogen peroxide (H₂O₂), and hydroxyl anion (OH-), and reactive nitrogen species, such as nitric oxide (NO) and peroxynitrite (ONOO^–), are biologically important O₂ derivatives that are increasingly recognized to be important in vascular biology through their oxidation/reduction (redox) potential. All vascular cell types (endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts) produce ROS, primarily via cell membrane-associated NAD(P)H oxidase. Reactive oxygen species regulate vascular function by modulating cell growth, apoptosis/anoikis, migration, inflammation, secretion, and extracellular matrix protein production. An imbalance in redox state where pro-oxidants overwhelm anti-oxidant capacity results in oxidative stress. Oxidative stress and associated oxidative damage are mediators of vascular injury and inflammation in many cardiovascular diseases, including hypertension, hyperlipidemia, and diabetes. Increased generation of ROS has been demonstrated in experimental and human hypertension. Anti-oxidants and agents that interrupt NAD(P)H oxidase-driven ·O₂^– production regress vascular remodeling, improve endothelial function, reduce inflammation, and decrease blood pressure in hypertensive models. This experimental evidence has evoked considerable interest because of the possibilities that therapies targeted against reactive oxygen intermediates, by decreasing generation of ROS and/or by increasing availability of antioxidants, may be useful in minimizing vascular injury and hypertensive end organ damage. The present chapter focuses on the importance of ROS in vascular biology and discusses the role of oxidative stress in vascular damage in hypertension.     

The Fucosylation Inhibitor, 2-Fluorofucose,Inhibits Vaso-Occlusion,Leukocyte-Endothelium Interactions and NF-?B Activation in Transgenic Sickle Mice

2-Fluorofucose (2FF) blocks the fucosylation and the tethering of sialyl-Lewis^x tetrasaccharide and structural variants on leukocytes and red blood cells to P- and E-selectins on activated endothelial cell surfaces. Because P- and E-selectin are required for vaso-occlusion in murine sickle cell disease (SCD), we investigated whether 2FF would inhibit vaso-occlusion in SCD mice. Microvascular stasis was measured in subcutaneous venules in NY1DD and HbSS-Townes SCD mice with dorsal skin-fold chambers after infusion of hemoglobin or exposure to hypoxia/reoxygenation. 2FF in drinking water or administered by gavage inhibited stasis in sickle mice in a dose-responsive manner. Significant inhibitory effects on stasis were seen 1 day post-treatment. 2FF treatment of SCD mice also significantly reduced leukocyte rolling and adhesion along the vessel walls of SCD mice and the static adhesion of neutrophils and sickle red blood cells isolated from 2FF-treated SCD mice to resting and activated endothelial cells. Total white blood cell counts increased in response to 2FF. NF-ĸB activation and VCAM-1 and E-selectin expression were inhibited in the livers of SCD mice consistent with an overall decrease in vascular inflammation and ischemia-reperfusion physiology. Pretreatment with 2FF completely eliminated heme-induced lethality in HbSS-Townes mice, consistent with the observed anti-inflammatory and anti-adhesive properties of 2FF in SCD mice. These data suggest that 2FF may be beneficial for preventing or treating vaso-occlusive crises in SCD patients.     

The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins

Hydrogen peroxide (H₂O₂) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H₂O₂ in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H₂O₂ is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H₂O₂ between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H₂O₂ permeation. The permeability coefficient of H₂O₂ through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10⁻⁴ and 5 × 10⁻³ cm s⁻¹, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H₂O₂ at 37 °C, on the other hand, was 1.6 × 10⁻³ cm s⁻¹. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H₂O₂. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H₂O₂ as normal human RBCs. Therefore, these results indicate that H₂O₂ does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.     

Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

Red blood cells (RBCs) are a major component of blood clots, which form physiologically as a response to injury or pathologically in thrombosis. The active participation of RBCs in thrombus solidification has been previously proposed but not yet experimentally proven. Holographic optical tweezers and single-cell force spectroscopy were used to study potential cell-cell adhesion between RBCs. Irreversible intercellular adhesion of RBCs could be induced by stimulation with lysophosphatidic acid (LPA), a compound known to be released by activated platelets. We identified Ca²⁺ as an essential player in the signaling cascade by directly inducing Ca²⁺ influx using A23187. Elevation of the internal Ca²⁺ concentration leads to an intercellular adhesion of RBCs similar to that induced by LPA stimulation. Using single-cell force spectroscopy, the adhesion of the RBCs was identified to be approximately 100 pN, a value large enough to be of significance inside a blood clot or in pathological situations like the vasco-occlusive crisis in sickle cell disease patients.     

Nitric oxide reduces sickle hemoglobin polymerization: potential role of nitric oxide-induced charge alteration in depolymerization

We previously demonstrated that inhaling nitric oxide (NO) increases the oxygen affinity of sickle red blood cells (RBCs) in patients with sickle cell disease (SCD). Our recent studies found that NO lowered the P₅₀ values of sickle hemoglobin (HbS) hemolysates but did not increase methemoglobin (metHb) levels, supporting the role of NO, but not metHb, in the oxygen affinity of HbS. Here we examine the mechanism by which NO increases HbS oxygen affinity. Because anti-sickling agents increase sickle RBC oxygen affinity, we first determined whether NO exhibits anti-sickling properties. The viscosity of HbS hemolysates, measured by falling ball assays, increased upon deoxygenation; NO treatment reduced the increment. Multiphoton microscopic analyses showed smaller HbS polymers in deoxygenated sickle RBCs and HbS hemolysates exposed to NO. These results suggest that NO inhibits HbS polymer formation and has anti-sickling properties. Furthermore, we found that HbS treated with NO exhibits an isoelectric point similar to that of HbA, suggesting that NO alters the electric charge of HbS. NO–HbS adducts had the same elution time as HbA upon high performance liquid chromatography analysis. This study demonstrates that NO may disrupt HbS polymers by abolishing the excess positive charge of HbS, resulting in increased oxygen affinity.     

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O₂^•-, H₂O₂, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death^1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response⁷. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O₂^•- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells⁸. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others^9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O₂^•- that are important in the host defense mechanism^11,12. Although paracrine-derived O₂^•- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca²⁺ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O₂^•- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O₂^•- lead to calcium signaling in adjacent endothelial cells.     

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids

Background

Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium.

Methods

RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500 kDa dextran with a flow chamber assay.

Results

Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction.

Conclusion

These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids.

General significance

Our findings should therefore be of great value in understanding abnormal RBC–EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.     

Sustained maternal smoking-associated changes in the physico-chemical properties of fetal RBC membranes might serve as early markers for vascular comorbidities

Maternal smoking-induced congenital heart and microvascular defects are closely associated with the impaired functioning of the in-utero feto-placental circulation system. Current groundbreaking facts revealed intimate crosstalk between circulating red blood cells (RBCs) and the vascular endothelium. Thus, RBCs have become the protagonists under varied pathological and adverse pro-oxidative cellular stress conditions. We isolated and screened fetal RBCs from the arterial cord blood of neonates, born to non-smoking (RBC-NS) and smoking mothers (RBC-S), assuming that parameters of fetal RBCs are blueprints of conditions experienced in-utero. Using atomic force microscopy and mass spectrometry-based shotgun lipidomics in the RBC-S population we revealed induced membrane stiffness, loss in intrinsic plastic activities and several abnormalities in their membrane-lipid composition, that could consequently result in perturbed hemodynamic flow movements. Altogether, these features are indicative of the outcome of neonatal microvascular complications and suggest unavailability for the potential rescue mechanism in cases of vascular endothelium impairment due to altered membrane integrity and rheological properties.     

Hydroxycarbamide Decreases Sickle Reticulocyte Adhesion to Resting Endothelium by Inhibiting Endothelial Lutheran/Basal Cell Adhesion Molecule (Lu/BCAM) through Phosphodiesterase 4A Activation

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α₄β₁ integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.     

Role of age and neuroinflammation in the mechanism of cognitive deficits in sickle cell disease

This study aims to determine whether sickle cell mice could recapitulate features of cognitive and neurobehavioral impairment observed in sickle cell patients and whether neuroinflammation could be a potential therapeutic target as in other non-sickle cell disease-related cognitive dysfunction. Cognitive (learning and memory) and behavioral (anxiety) deficits in 13- and later 6-month-old male Townes humanized sickle cell (SS) and matched control (AA) mice were evaluated using novel object recognition (NOR) and fear conditioning tests. Immunohistochemistry was performed to quantify peripheral immune cell (CD45⁺) and activated microglia (Iba1⁺) as markers of neuroinflammation in the dentate and peri-dentate gyrus areas. We evaluated cell fate by measuring 5''-bromodeoxyuridine and doublecortin fluorescence and phenotyped proliferating cells using either glial fibrillary acid protein (GFAP⁺), neuronal nuclei (NeuN⁺), CD45⁺, and Iba1⁺. In addition, Golgi-Cox staining was used to assess markers of neuroplasticity (dendritic spine density and morphology and density of dendrite arbors) on cortical and hippocampal pyramidal neurons. Compared to matched AA controls, 13-month-old SS mice showed significant evidence of cognitive and behavioral deficit on NOR and fear conditioning tests. Also, SS mice had significantly higher density of CD45⁺ and activated microglia cells (i.e. more evidence of neuroinflammation) in the dentate and peri-dentate gyrus area. Additionally, SS mice had significantly lower dendritic spine density, but a higher proportion of immature dendritic spines. Treatment of 13-month-old SS mice with minocycline resulted in improvement of cognitive and behavioral deficit compared to matched vehicle-treated SS mice. Also, treated SS mice had significantly fewer CD45⁺ and activated microglia cells (i.e. less evidence of neuroinflammation) in the dentate and peri-dentate gyrus, as well as a significant improvement in markers of neuroplasticity.Impact statementThis study provides crucial information that could be helpful in the development of new or repurposing of existing therapies for the treatment of cognitive deficit in individuals with sickle cell disease (SCD). Its impact is in demonstrating for the first time that neuroinflammation and along with abnormal neuroplasticity are among the underlying mechanism of cognitive and behavioral deficits in SCD and that drugs such as minocycline which targets these pathophysiological mechanisms could be repurposed for the treatment of this life altering complication of SCD.     

Mercury leads to abnormal red blood cell adhesion to laminin mediated by membrane sulfatides

Exposure to mercury is associated with numerous health problems, affecting different parts of the human body, including the nervous and cardiovascular systems in adults and children; however, the underlying mechanisms are yet to be fully elucidated. We investigated the role of membrane sulfatide on mercuric ion (Hg²⁺) mediated red blood cell (RBC) adhesion to a sub-endothelial matrix protein, laminin, using a microfluidic system that mimics microphysiological flow conditions. We exposed whole blood to mercury (HgCl₂), at a range of concentrations to mimic acute (high dose) and chronic (low dose) exposure, and examined RBC adhesion to immobilized laminin in microchannels at physiological flow conditions. Exposure of RBCs to both acute and chronic levels of Hg²⁺ resulted in elevated adhesive interactions between RBCs and laminin depending on the concentration of HgCl₂ and exposure duration. BCAM-Lu chimer significantly inhibited the adhesion of RBCs that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C, while it did not prevent the adhesion of 3 h and 24 h Hg²⁺-treated RBCs. Sulfatide significantly inhibited the adhesion of RBC that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C and 0.5 μM of HgCl₂ solution for 24 h at room temperature (RT). We demonstrated that RBC BCAM-Lu and RBC sulfatides bind to immobilized laminin, following exposure of RBCs to mercuric ions. The results of this study are significant considering the potential associations between sulfatides, red blood cells, mercury exposure, and cardiovascular diseases.     

Purinergic interplay between erythrocytes and platelets in diabetes-associated vascular dysfunction

Cardiovascular complications in diabetes are the leading causes for high morbidity and mortality. It has been shown that alteration of purinergic signaling contributes to diabetes-associated cardiovascular complications. Red blood cells (RBCs) and platelets play a fundamental role in regulation of oxygen transport and hemostasis, respectively. Of note, these cells undergo purinergic dysfunction in diabetes. Recent studies have established a novel function of RBCs as disease mediators for the development of endothelial dysfunction in type 2 diabetes (T2D). RBC-released ATP is defective in T2D, which has implication for induction of vascular dysfunction by dysregulating purinergic signaling. Platelets are hyperactive in diabetes. ADP-mediated P2Y₁ and P2Y₁₂ receptor activation contributes to platelet aggregation and targeting P2Y receptors particularly P2Y₁₂ receptor in platelets is effective for the treatment of cardiovascular events. In contrast to other P2Y₁₂ receptor antagonists, platelet-targeting drug ticagrelor has potential to initiate purinergic signaling in RBCs for the beneficial cardiovascular outcomes. It is increasingly clear that altered vascular purinergic signaling mediated by various nucleotides and nucleoside contributes to diabetes-associated vascular dysfunction. However, the contribution of complex purinergic networks between RBCs and platelets to the vascular dysfunction in diabetes remains unclear. This study discusses the possible interplay of RBCs and platelets via the purinergic network for diabetes-associated vascular dysfunction.     

Computational modeling of biomechanics and biorheology of heated red blood cells

Because of their compromised deformability, heat denatured erythrocytes have been used as labeled probes to visualize spleen tissue or to assess the ability of the spleen to retain stiff red blood cells (RBCs) for over three decades, e.g., see Looareesuwan et al. N. Engl. J. Med. (1987). Despite their good accessibility, it is still an open question how heated RBCs compare to certain diseased RBCs in terms of their biomechanical and biorheological responses, which may undermine their effective usage and even lead to misleading experimental observations. To help answering this question, we perform a systematic computational study of the hemorheological properties of heated RBCs with several physiologically relevant static and hemodynamic settings, including optical-tweezers test, relaxation of prestretched RBCs, RBC traversal through a capillary-like channel and a spleen-like slit, and a viscometric rheology test. We show that our in silico RBC models agree well with existing experiments. Moreover, under static tests, heated RBCs exhibit deformability deterioration comparable to certain disease-impaired RBCs such as those in malaria. For RBC traversal under confinement (through microchannel or slit), heated RBCs show prolonged transit time or retention depending on the level of confinement and heating procedure, suggesting that carefully heat-treated RBCs may be useful for studying splenic- or vaso-occlusion in vascular pathologies. For the rheology test, we expand the existing bulk viscosity data of heated RBCs to a wider range of shear rates (1–1000 s⁻¹) to represent most pathophysiological conditions in macro- or microcirculation. Although heated RBC suspension shows elevated viscosity comparable to certain diseased RBC suspensions under relatively high shear rates (100–1000 s⁻¹), they underestimate the elevated viscosity (e.g., in sickle cell anemia) at low shear rates (<10 s⁻¹). Our work provides mechanistic rationale for selective usage of heated RBC as a potentially useful model for studying the abnormal traversal dynamics and hemorheology in certain blood disorders.     

Blood Viscosity and the Expression of Inflammatory and Adhesion Markers in Homozygous Sickle Cell Disease Subjects with Chronic Leg Ulcers

Objective

To determine differences in TNF-α, IL-1β, IL-10, sICAM-1 concentrations, leg hypoxia and whole blood viscosity (WBV) at shear rates of 46 sec^-1 and 230 sec^-1 in persons with homozygous S sickle cell disease (SCD) with and without chronic leg ulceration and in AA genotype controls.

Design

& Methods: fifty-five age-matched participants were recruited into the study: 31 SS subjects without leg ulcers (SS_n), 24 SS subjects with leg ulcers (SS_u) and 18 AA controls. Haematological indices were measured using an AC.Tron Coulter Counter. Quantification of inflammatory, anti-inflammatory and adhesion molecules was performed by ELISA. Measurement of whole blood viscosity was done using a Wells Brookfield cone-plate viscometer. Quantification of microvascular tissue oxygenation was done by Visible Lightguide spectrophotometry.

Results

TNF-α and whole blood viscosity at 46 sec^-1 and 230 sec^-1 (1.75, 2.02 vs. 0.83, 1.26, p<0.05) were significantly greater in sickle cell disease subjects than in controls. There were no differences in plasma concentration of sICAM-1, IL-1β and IL-10 between SCD subjects and controls. IL-1β (median, IQR: 0.96, 1.7 vs. 0, 0.87; p<0.01) and sICAM-1 (226.5, 156.48 vs. 107.63, 121.5, p<0.005) were significantly greater in SS_u group compared with SS_n. However there were no differences in TNF-α (2, 3.98 vs. 0, 2.66) and IL-10 (13.34, 5.95 vs. 11.92, 2.99) concentrations between SS_u and SS_n. WBV in the SS_u group at 46 sec^-1 and at 230 Sec 1 were 1.9 (95%CI; 1.2, 3.1) and 2.3 (1.2, 4.4) times greater than in the SS_n group. There were no differences in the degree of tissue hypoxia as determined by lightguide spectrophotometry.

Conclusion

Inflammatory, adhesion markers and WBV may be associated with leg ulceration in sickle cell disease by way of inflammation-mediated vasoocclusion/vasoconstriction. Impaired skin oxygenation does not appear to be associated with chronic ulcers in these subjects with sickle cell disease.     

Endothelial cells in culture: An experimental model for the study of vascular dysfunctions

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [³H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor- and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selectin And vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also rn rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.Abbreviations AGEs advanced glycated end-products - ICAM intracellular adhesion molecule - IL-1 interleukin-1 - IFN- interferon- - MECIF monocyte-derived endothelial cell inhibitory factor - NO nitric oxide - PECAM-1 platelet-endothelial cell adhesion molecule-1 - RAGE receptor for advanced glycated end-products - RBCs red blood cells - TNF- tumor necrosis factor- - VCAM vascular adhesion molecule     

Effects of superoxide radicals on ACC synthase activity in chilling-stressed etiolated mungbean seedlings

The activity of 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase, ACS) and the concentrations of superoxide radical (O₂^−.) and hydrogen peroxide (H₂O₂) were measured in etiolated mungbean seedlings following their transfer to a growth chamber at 25°C after a 5-h-chilling treatment at 5°C. All of these variables increased dramatically after the transfer, and strong correlations were found between ACS activity and the concentrations of superoxide and H₂O₂. Exogenous applications of two generators of superoxide radicals, methylviologen (MV) and xanthine–xanthine oxidase (X–XOD), enhanced ACS activity in seedlings, but their effects were inhibited by exogenous applications of specific scavengers of O₂^−.. However, applications of H₂O₂ or specific H₂O₂-scavengers had no significant effects on seedlings ACS activity. The results indicate that O₂^−. was involved in the chilling-induced increases in ACS activity, but not H₂O₂. ACS activity peaked ca. 8 h after the transfer, and then declined, but the decline could be counteracted by exogenous applications of specific O₂^−. scavengers, this suggests that damage was caused by superoxide radicals influencing ACS activity in etiolated mungbean seedlings. Further analysis of changes in two key kinetic parameters of ACS activity—V _max (maximum velocity) and K _m (the Michaelis constant)—in the seedlings indicated that the presence of O₂^−. may reduce K _m, i.e. increase substrate (S-adenosyl methionine, SAM) affinity. That would be the main mechanism responsible for the observed chilling-induced increases in ACS activity in etiolated mungbean seedlings.