Red Blood Cell Homeostasis Research Articles

The Calcium Homeostasis of Human Red Blood Cells in Health and Disease: Interactions of PIEZO1, the Plasma Membrane Calcium Pump, and Gardos Channels.

Calcium ions mediate the volume homeostasis of human red blood cells (RBCs) in the circulation. The mechanism by which calcium ions affect RBC hydration states always follows the same sequence. Deformation of RBCs traversing capillaries briefly activates mechanosensitive PIEZO1 channels, allowing Ca2+ influx down its steep inward gradient transiently overcoming the calcium pump and elevating [Ca2+]i. Elevated [Ca2+]i activates the Ca2+-sensitive Gardos channels, inducing KCl loss and cell dehydration, a sequence operated with infinite variations in vivo and under experimental conditions. The selected health and disease themes for this review focus on landmark experimental results that led to the development of highly constrained models of the circulatory changes in RBCs homeostasis. Based on model predictions, a new perspective emerged, pointing to PIEZO1 dysfunction as the main trigger in the formation of the profoundly dehydrated irreversible sickle cells, the main pathogenic participants in vaso-occlusion, the root cause of sickle cell disease.

Anion exchanger1 (AE1/SLC4A1) function is impaired in red blood cells from prediabetic subjects: Potential benefits of finger lime (Citrus australasica, Faustrime cultivar) juice extract.

Prediabetes is a risk state that defines a high chance of developing diabetes and cardiovascular disease. Oxidative stress mediated by hyperglycemia-induced production of reactive species could play a crucial role in this context. In the present study, we investigated whether the anion exchange capability mediated by AE1 (SLC4A1), which is sensitive to oxidative stress, was altered in human red blood cells (RBCs) obtained from prediabetic volunteers. In addition, we assessed the precise composition of bioactive compounds and the potential benefits of finger lime juice extract (Citrus australasica, Faustrime cultivar) in counteracting oxidative stress-related functional alterations. Human RBCs from normal and prediabetic volunteers were incubated with 50 µg/mL juice extract for 2 h at 25°C. Juice extract restored alterations of the anion exchange capability mediated by AE1 and prevented the structural rearrangements of AE1 and α/β-spectrin in prediabetic RBCs. AE1 functional and structural alterations were not associated with an increase in lipid peroxidation or protein oxidation at the level of the plasma membrane. An increased production of intracellular ROS, which provoked the oxidation of hemoglobin to methemoglobin, both reverted by juice extract, was instead observed. Importantly, juice extract also induced a reduction in glycated hemoglobin levels in prediabetic RBCs. Finally, juice extract blunted the overactivation of the endogenous antioxidant enzymes catalase and superoxide dismutase and prevented glutathione depletion in prediabetic RBCs. These findings contribute to clarifying cellular and molecular mechanisms related to oxidative stress and glycation events that may influence RBC and systemic homeostasis in prediabetes, identify AE1 as a sensitive biomarker of RBC structural and function alterations in prediabetes and propose finger lime juice extract as a natural antioxidant for the treatment and/or prevention of the complications associated with the prediabetic condition.

#2505 PTEN ablation in osteocytes exacerbates anemia in mice with subtotal nephrectomy

Abstract Background and Aims The identification of novel roles for bone as a principal endocrine organ has grown in recent decades, being now recognized for its contribution to the regulation of numerous systemic functions. In previous studies, we observed that 5/6Nx mice with osteocytic deletion of phosphatase and tensin homolog (PTEN), exhibited higher bone volume in 5/6Nx mice, consistent with studies under normal renal conditions. However, we also noted a decrease in blood hemoglobin levels, prompting further exploration. This study aims to elucidate the causative actions by which specific PTEN deletion in osteocytes influences red blood cell (RBC) homeostasis and to identify its possible involvement in anemia development. Method To achieve this goal, we used mice with targeted PTEN deletion in osteocytes (PTEN Ocy-cKO) by crossing DMP1-Cre ± and PTENflox/flox mice, subjected to subtotal nephrectomy (5/6Nx) at 2 months of age. Two weeks post-surgery, bone and blood samples were collected for plasma biochemistry, blood count, RBC membrane integrity, and preliminary bone+bone marrow proteomic analysis. Cre-negative mice and animals with normal renal function were included as controls. Results Our findings revealed that PTEN Ocy-cKO mice, under both normal and reduced renal function conditions, showed higher bone volume than corresponding controls. In terms of blood homeostasis, PTEN Ocy-cKO mice with normal renal function showed splenomegaly and a lower number of circulating RBCs. Notably, PTEN Ocy-cKO mice undergoing 5/6Nx experienced a profound decrease in RBC number in only two weeks, independent of renal erythropoietin expression. Further exploration ruled out bone marrow failure as a cause, as the erythroid progenitor (CD45-CD71+Ter119+) population remained similar in both groups. Extramedullary hematopoiesis was observed in the spleen, in addition to increased circulating reticulocyte number in the PTEN Ocy-cKO mice. Finally, altered RBC membrane protein patterns in PTEN Ocy-cKO mice suggested reduced mechanical/osmotic resistance. Preliminary proteomic analysis identified downregulation of Slc4a1 (Band3) and Phosphofructokinase (Pfkm), whose deficiencies have been previously linked to anemia. Conclusion In conclusion, our results propose that osteocytes contribute to RBC homeostasis through a pathway downstream of PTEN, and open a new avenue for understanding anemia in chronic kidney disease and a potential target for future treatments.

Nuclear receptor Rev-erbα role in fine-tuning erythropoietin gene expression

AbstractThe regulation of red blood cell (RBC) homeostasis by erythropoietin (EPO) is critical for O2 transport and maintaining the adequate number of RBCs in vertebrates. Therefore, dysregulation in EPO synthesis results in disease conditions such as polycythemia in the case of excessive EPO production and anemia, which occurs when EPO is inadequately produced. EPO plays a crucial role in treating anemic patients; however, its overproduction can increase blood viscosity, potentially leading to fatal heart failure. Consequently, the identification of druggable transcription factors and their associated ligands capable of regulating EPO offers a promising therapeutic approach to address EPO-related disorders. This study unveils a novel regulatory mechanism involving 2 pivotal nuclear receptors (NRs), Rev-ERBA (Rev-erbα, is a truncation of reverse c-erbAa) and RAR-related orphan receptor A (RORα), in the control of EPO gene expression. Rev-erbα acts as a cell-intrinsic negative regulator, playing a vital role in maintaining erythropoiesis at the correct level. It accomplishes this by directly binding to newly identified response elements within the human and mouse EPO gene promoter, thereby repressing EPO production. These findings are further supported by the discovery that a Rev-erbα agonist (SR9011) effectively suppresses hypoxia-induced EPO expression in mice. In contrast, RORα functions as a positive regulator of EPO gene expression, also binding to the same response elements in the promoter to induce EPO production. Finally, the results of this study revealed that the 2 NRs, Rev-erbα and RORα, influence EPO synthesis in a negative and positive manner, respectively, suggesting that the modulating activity of these 2 NRs could provide a method to target disorders linked with EPO dysregulation.

PIEZO Ion Channels in Cardiovascular Functions and Diseases.

The cardiovascular system provides blood supply throughout the body and as such is perpetually applying mechanical forces to cells and tissues. Thus, this system is primed with mechanosensory structures that respond and adapt to changes in mechanical stimuli. Since their discovery in 2010, PIEZO ion channels have dominated the field of mechanobiology. These have been proposed as the long-sought-after mechanosensitive excitatory channels involved in touch and proprioception in mammals. However, more and more pieces of evidence point to the importance of PIEZO channels in cardiovascular activities and disease development. PIEZO channel-related cardiac functions include transducing hemodynamic forces in endothelial and vascular cells, red blood cell homeostasis, platelet aggregation, and arterial blood pressure regulation, among others. PIEZO channels contribute to pathological conditions including cardiac hypertrophy and pulmonary hypertension and congenital syndromes such as generalized lymphatic dysplasia and xerocytosis. In this review, we highlight recent advances in understanding the role of PIEZO channels in cardiovascular functions and diseases. Achievements in this quickly expanding field should open a new road for efficient control of PIEZO-related diseases in cardiovascular functions.

A new role for erythropoietin in the homeostasis of red blood cells

The regulation of red blood cell (RBC) homeostasis is widely assumed to rely on the control of cell production by erythropoietin (EPO) and the destruction of cells at a fixed, species-specific age. In this work, we show that such a regulatory mechanism would be a poor homeostatic solution to satisfy the changing needs of the body. Effective homeostatic control would require RBC lifespan to be variable and tightly regulated. We suggest that EPO may control RBC lifespan by determining CD47 expression in newly formed RBCs and SIRP-α expression in sinusoidal macrophages. EPO could also regulate the initiation and intensity of anti-RBC autoimmune responses that curtail RBC lifespan in some circumstances. These mechanisms would continuously modulate the rate of RBC destruction depending on oxygen availability. The control of RBC lifespan by EPO and autoimmunity emerges as a key mechanism in the homeostasis of RBCs.

Platelets mediate the clearance of senescent red blood cells by forming prophagocytic platelet-cell complexes

AbstractIn humans, ∼0.1% to 0.3% of circulating red blood cells (RBCs) are present as platelet-RBC (P-RBC) complexes, and it is 1% to 2% in mice. Excessive P-RBC complexes are found in diseases that compromise RBC health (eg, sickle cell disease and malaria) and contribute to pathogenesis. However, the physiological role of P-RBC complexes in healthy blood is unknown. As a result of damage accumulated over their lifetime, RBCs nearing senescence exhibit physiological and molecular changes akin to those in platelet-binding RBCs in sickle cell disease and malaria. Therefore, we hypothesized that RBCs nearing senescence are targets for platelet binding and P-RBC formation. Confirming this hypothesis, pulse-chase labeling studies in mice revealed an approximately tenfold increase in P-RBC complexes in the most chronologically aged RBC population compared with younger cells. When reintroduced into mice, these complexes were selectively cleared from the bloodstream (in preference to platelet-free RBC) through the reticuloendothelial system and erythrophagocytes in the spleen. As a corollary, patients without a spleen had higher levels of complexes in their bloodstream. When the platelet supply was artificially reduced in mice, fewer RBC complexes were formed, fewer erythrophagocytes were generated, and more senescent RBCs remained in circulation. Similar imbalances in complex levels and senescent RBC burden were observed in humans with immune thrombocytopenia (ITP). These findings indicate that platelets are important for binding and clearing senescent RBCs, and disruptions in platelet count or complex formation and clearance may negatively affect RBC homeostasis and may contribute to the known risk of thrombosis in ITP and after splenectomy.

Abstract P3032: 15( R )-lipoxin A 4 Contributes To Splenic Erythrophagocytosis Via Activation Of ALX/FPR2

Red blood cells (RBCs) are the most abundant cell type in the body and are traditionally appreciated for their role in respiration and gas exchange. Recently, however, RBCs have garnered new attention for their immunomodulatory effects which is primarily achieved via their interaction with resident macrophages in the spleen, known as red pulp macrophages. As RBCs enter the spleen, red pulp macrophages probe the cells for signs of mechanical and oxidative damage, as well as inflammatory stimuli bound to cell surface receptors (e.g., foreign pathogens, endogenous chemokines). Once recognized, red pulp macrophages prune damaged portions of RBC membranes or phagocytose the cell entirely, efficiently processing and recycling the cellular cargo of the RBC. In a healthy human, this process, termed erythrophagocytosis, accounts for the daily turnover of roughly 150-200 billion RBCs. Consequently, defects in the system can have wide-ranging health effects including anemia, impaired host defense, and even sepsis. Recent data suggest that arachidonic acid (AA)-derived prostaglandins promote erythrophagocytosis, yet the impact of other eicosanoid species on the process has not been well described. We performed targeted lipidomics analyses focused on the AA metabolome in two distinct models of erythrophagocytosis. First, mouse bone marrow-derived macrophages were incubated with freshly isolated RBCs; then, spleens from mice were collected following exogenous delivery of damaged RBCs. In both models, we found increased production of the 15( R )-epimer of lipoxin A 4 (15( R )-LXA 4 ), as well as its biosynthetic intermediate, 15-HETE. Similar to LXA 4 , 15( R )-LXA 4 has been shown to robustly promote the active resolution of inflammation by regulating leukocyte function via its interaction with the G protein-coupled receptor, ALX/FPR2. In mice genetically deficient in this receptor, we measured a significant decrease in splenic clearance of damaged RBCs. Together, these data suggest that 15( R )-LXA 4 activation of ALX/FPR2 on red pulp macrophages may be a critical aspect regulating erythrophagocytosis and maintaining RBC homeostasis.

Immune Mechanisms in Inflammatory Anemia.

Maintaining the correct number of healthy red blood cells (RBCs) is critical for proper oxygenation of tissues throughout the body. Therefore, RBC homeostasis is a tightly controlled balance between RBC production and RBC clearance, through the processes of erythropoiesis and macrophage hemophagocytosis, respectively. However, during the inflammation associated with infectious, autoimmune, or inflammatory diseases this homeostatic process is often dysregulated, leading to acute or chronic anemia. In each disease setting, multiple mechanisms typically contribute to the development of inflammatory anemia, impinging on both sides of the RBC production and RBC clearance equation. These mechanisms include both direct and indirect effects of inflammatory cytokines and innate sensing. Here, we focus on common innate and adaptive immune mechanisms that contribute to inflammatory anemias using examples from several diseases, including hemophagocytic lymphohistiocytosis/macrophage activation syndrome, severe malarial anemia during Plasmodium infection, and systemic lupus erythematosus, among others.

The circulatory dynamics of human red blood cell homeostasis: Oxy-deoxy and PIEZO1-triggered changes

The vital function of red blood cells (RBCs) is to mediate the transport of oxygen from lungs to tissues and of CO2 from tissues to lungs. The gas exchanges occur during capillary transits within fractions of a second. Each oxygenation-deoxygenation and deoxygenation-reoxygenation transition on hemoglobin triggers sharp changes in RBC pH, leading to downstream changes in ion fluxes, membrane potential, and cell volume. The dynamics of these changes during the variable periods between capillary transits in vivo remains a mystery inaccessible to study by current methodologies, a knowledge gap on a fundamental physiological process that is the focus of the present study. The use of a computational model of human RBC homeostasis of tested accreditation enabled a detailed investigation of the expected RBC changes during intercapillary transits, with results advancing novel insights and predictions. The predicted rates of relative RBC volume change on oxygenation-deoxygenation (oxy-deoxy) and deoxygenation-reoxygenation transitions were about 1.5%/min and −0.9%/min, respectively, far too slow to allow the cells to reach steady states in the intervals between capillary transits. The amplitude of the oxy-deoxy-reoxygenation volume fluctuations varied in proportion with the duration of the intercapillary transit intervals. Upon capillary entry, oxy-deoxy-induced changes occur concurrently with deformation-induced PIEZO1 channel activation, both processes affecting cell pH, membrane potential, and cell volume during intertransit periods. The model showed that the effects were strictly additive as expected from processes operating independently on the cell’s homeostatic fabric. Analysis of the mechanisms behind these predictions revealed, for the first time, the complex interactions between oxy-deoxy and ion transport processes that ensure the long-term homeostatic stability of RBCs for optimal gas transport in physiological conditions and how these may become altered in diseased states. Possible designs of microfluidic devices to test the model predictions are discussed.

Role of Piezo1 channel in cardiovascular diseases and its research progress in traditional Chinese medicine

Piezo1 channel is a mechanosensitive ion channel that opens in response to shear stress, tension, torsion and a series of mechanical stimulation and turns them into biological signals. It plays an important role in vascular development, vasoconstriction and vasodilation, formation of arterial and venous valves, blood pressure regulation and red blood cell homeostasis, and thus is closely related to cardiovascular system. Recent studies have shown that traditional Chinese medicine(TCM) compounds and their active components can affect Ca~(2+) influx and endothelial cell and platelet function by mediating Piezo1 channel, and regulate thrombosis and endothelial homeostasis, thereby treating cardiovascular diseases. This study mainly reviewed the role of Piezo1 channel in cardiovascular diseases and the prevention and treatment of cardiovascular diseases based on Piezo1 channel in TCM, in order to provide effective reference for the further research of Piezo1 channel in the field of TCM.

The chromatin-remodeling enzyme Smarca5 regulates erythrocyte aggregation via Keap1-Nrf2 signaling.

Although thrombosis has been extensively studied using various animal models, our understanding of the underlying mechanism remains elusive. Here, using zebrafish model, we demonstrated that smarca5-deficient red blood cells (RBCs) formed blood clots in the caudal vein plexus. We further used the anti-thrombosis drugs to treat smarca5zko1049a embryos and found that a thrombin inhibitor, argatroban, partially prevented blood clot formation in smarca5zko1049a. To explore the regulatory mechanism of smarca5 in RBC homeostasis, we profiled the chromatin accessibility landscape and transcriptome features in RBCs from smarca5zko1049a and their siblings and found that both the chromatin accessibility at the keap1a promoter and expression of keap1a were decreased. Keap1 is a suppressor protein of Nrf2, which is a major regulator of oxidative responses. We further identified that the expression of hmox1a, a downstream target of Keap1-Nrf2 signaling pathway, was markedly increased upon smarca5 deletion. Importantly, overexpression of keap1a or knockdown of hmox1a partially rescued the blood clot formation, suggesting that the disrupted Keap1-Nrf2 signaling is responsible for the RBC aggregation in smarca5 mutants. Together, our study using zebrafish smarca5 mutants characterizes a novel role for smarca5 in RBC aggregation, which may provide a new venous thrombosis animal model to support drug screening and pre-clinical therapeutic assessments to treat thrombosis.

Targeting Host Glycolysis as a Strategy for Antimalarial Development.

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase – a critical enzyme in glycolysis – and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.

P–430 Male fertility preservation: is there a role for cancer-induced inflammation that affects semen quality in oncological patients?

Abstract Study question What is the cause of semen quality impairment in oncological patients during fertility preservation programs? The cancer type and stadiation or the resulting inflammatory state? Summary answer The inflammatory state seems to be related to the decrease of sperm concentration, motility, morphology and viability due to the worsening of oxidative stress microenvironment. What is known already Fertility preservation acquired a great importance in the last decades due to increase survival of oncological patients, boost of diagnosis under 40 years and postponement of paternal age. At the time of cryopreservation, only one third of these males are normozoospermic. Tumor itself or other factors, added to psychological reasons, may be involved but there is no clear evidence. An imbalance of ROS (reactive oxygen species) in semen can compromise its quality. However, the correlation between cancer-related generalized stress state and fertility is poorly investigated. Inflammatory conditions induced by infections and pathologies, including cancer, increase ROS. Study design, size, duration Retrospective observational analysis was performed on 45 patients (29.0 ± 6.9 yrs) recruited during their fertility preservation program between 2016 and 2019 with written consent on use of their clinical data for research purpose. Patients presented several oncological diagnoses. Semen samples obtained from multiple collections (N = 58) were analyzed before applying standard freezing protocol. Data on semen parameters, inflammatory indices, hematological values and type/stage of tumors were collected. No exclusion criteria were applied. Participants/materials, setting, methods Routine semen analysis was performed according to the WHO standards. Sperm concentration and motility were evaluated on Makler Chamber, whereas eosin stain and Diff-quick slides were used for viability and morphology, respectively. Lymphoma was present in 72% of cases, leukemia in 8%, seminoma in 7% and other cancers in 13%. Correlations (Pearson/Spearman tests) among principal semen parameters and hematological values (leukocytes, erythrocytes, hemoglobin, RDW, albumin, etc.) were calculated with a P-value <0.05 considered statistically significant. Main results and the role of chance The majority of semen samples showed a severe impairment, with one or more parameters under lower reference limits (WHO): 48.3% had sperm concentration under 15 millions/ml, 43.1% had a progressive motility under 32%, 41.4% had viability under 58% and 91.4% had abnormal morphology (under 4%). The role of potential inflammatory state was analyzed by correlating semen parameters and some hematological values. No correlation was found with cancer type. Negative association resulted between progressive motility (%PR) and leukocytes (p = 0.041) or RDW% (p = 0.015), but positive one with albumin (p = 0.012). Even sperm count, total motility (%PR+NP) and morphology were significantly correlated to RDW% (p = 0.003, p = 0.032, p = 0.034, respectively). These findings suggest a possible role of inflammation and ROS related generation in semen quality impairment. Indeed, albumin exerts a protective action, but leukocytes are known to cause ROS increase. Cancer-induced oxidative stress state may alter red blood cells homeostasis and vitality and increase erythrocytes turnover resulting in high RDW values. It is likely semen is worse when blood values indicate more severe cancer-induced inflammatory condition. Limitations, reasons for caution Significant correlations with type/stage of cancer were not found due to small number of each diagnosis, in spite our study considered 3 years of patients inclusion. Moreover, we lack to analyze the same patient before the cancer onset to avoid the influence of inflammatory state generated by the tumor itself. Wider implications of the findings: Understanding the influence of cancer-induced inflammatory state on semen quality could increase the awareness that clinicians should direct patient to the fertility preservation as soon as possible, even if diagnosis is still ongoing. It should be evaluated whether offering specific treatments may reduce oxidative stress conditions. Trial registration number Not applicable

Effect of Media with Different Glycerol Concentrations on Sheep Red Blood Cells' Viability In Vitro.

Simple SummaryGlycerol is widely used as a feed supplement in ruminant nutrition. However, its administration at moderate and high doses results in an increase in plasma osmolality and in an alteration of red blood cell (RBC) indices. The present study aimed at further elucidating the effect of glycerol on RBCs’ functionality by evaluating the effect of different glycerol concentrations on RBC homeostasis under in vitro conditions. Obtained results showed that glycerol permeates the RBC membrane and leads to hemolysis when glycerol concentration exceeds 200 mg/dL. However, RBCs’ antioxidant defenses appear to protect cell membranes without causing an increase of oxidative stress markers. Moreover, no alteration in RBCs’ intracellular Ca2+ ion concentrations and metabolic activity were found. In conclusion, glycerol-based nutritional treatment should be designed in sheep to avoid exceeding 200 mg/dL glycerol circulating concentration to prevent RBC osmotic stress.The use of high doses of glycerol as a livestock feed supplement is followed by a rapid increase in plasma concentrations and consequently in plasma osmolality. Moreover, glycerol is a highly diffusible molecule that can readily permeate the red blood cell (RBC) membrane following a concentration gradient. A rise in glycerol plasma concentrations can thus alter RBC homeostasis. The present study aimed at investigating both glycerol osmotic effects on sheep RBCs and their oxidative response under in vitro conditions. Sheep blood samples were suspended in media supplemented with increasing glycerol concentrations (0, 25, 50, 100, 150, 200, 250, 300, 350, 400 mg/dL), which reflected those found in vivo in previous studies, and incubated at 37 °C for 4h. Thereafter, osmolality and hemolysis were determined in spent media, while cell extracts were used to assay intracellular concentration of glycerol, ATP, Ca2+ ions, oxidative stress markers and reactive oxygen species (ROS).The study confirmed that glycerol intracellular concentrations are directly related with its concentration in the incubation media, as well as hemolysis (p < 0.001) which increased significantly at glycerol concentrations higher form 200 mg/dL. ROS intracellular level increased at all glycerol concentration tested (p < 0.01) and total thiols decreased at the highest concentrations. However, RBCs proved to be able to cope by activating their antioxidant defense system. Superoxide dismutase activity indeed increased at the highest glycerol concentrations (p < 0.001), while total antioxidant capacity and malonyldialdehyde, a typical product of lipid peroxidation by ROS, did not show significant changes. Moreover, no alterations in intracellular Ca2+ ions and ATP concentrations were found. In conclusion, glycerol-induced hemolysis can be related to the induced osmotic stress. In sheep, nutritional treatments should be designed to avoid reaching glycerol circulating concentrations higher than 200 mg/dL.

PIEZO1 and the mechanism of the long circulatory longevity of human red blood cells

Human red blood cells (RBCs) have a circulatory lifespan of about four months. Under constant oxidative and mechanical stress, but devoid of organelles and deprived of biosynthetic capacity for protein renewal, RBCs undergo substantial homeostatic changes, progressive densification followed by late density reversal among others, changes assumed to have been harnessed by evolution to sustain the rheological competence of the RBCs for as long as possible. The unknown mechanisms by which this is achieved are the subject of this investigation. Each RBC traverses capillaries between 1000 and 2000 times per day, roughly one transit per minute. A dedicated Lifespan model of RBC homeostasis was developed as an extension of the RCM introduced in the previous paper to explore the cumulative patterns predicted for repetitive capillary transits over a standardized lifespan period of 120 days, using experimental data to constrain the range of acceptable model outcomes. Capillary transits were simulated by periods of elevated cell/medium volume ratios and by transient deformation-induced permeability changes attributed to PIEZO1 channel mediation as outlined in the previous paper. The first unexpected finding was that quantal density changes generated during single capillary transits cease accumulating after a few days and cannot account for the observed progressive densification of RBCs on their own, thus ruling out the quantal hypothesis. The second unexpected finding was that the documented patterns of RBC densification and late reversal could only be emulated by the implementation of a strict time-course of decay in the activities of the calcium and Na/K pumps, suggestive of a selective mechanism enabling the extended longevity of RBCs. The densification pattern over most of the circulatory lifespan was determined by calcium pump decay whereas late density reversal was shaped by the pattern of Na/K pump decay. A third finding was that both quantal changes and pump-decay regimes were necessary to account for the documented lifespan pattern, neither sufficient on their own. A fourth new finding revealed that RBCs exposed to levels of PIEZO1-medited calcium permeation above certain thresholds in the circulation could develop a pattern of early or late hyperdense collapse followed by delayed density reversal. When tested over much reduced lifespan periods the results reproduced the known circulatory fate of irreversible sickle cells, the cell subpopulation responsible for vaso-occlusion and for most of the clinical manifestations of sickle cell disease. Analysis of the results provided an insightful new understanding of the mechanisms driving the changes in RBC homeostasis during circulatory aging in health and disease.

Up-down biphasic volume response of human red blood cells to PIEZO1 activation during capillary transits.

In this paper we apply a novel JAVA version of a model on the homeostasis of human red blood cells (RBCs) to investigate the changes RBCs experience during single capillary transits. In the companion paper we apply a model extension to investigate the changes in RBC homeostasis over the approximately 200000 capillary transits during the ~120 days lifespan of the cells. These are topics inaccessible to direct experimentation but rendered mature for a computational modelling approach by the large body of recent and early experimental results which robustly constrain the range of parameter values and model outcomes, offering a unique opportunity for an in depth study of the mechanisms involved. Capillary transit times vary between 0.5 and 1.5s during which the red blood cells squeeze and deform in the capillary stream transiently opening stress-gated PIEZO1 channels allowing ion gradient dissipation and creating minuscule quantal changes in RBC ion contents and volume. Widely accepted views, based on the effects of experimental shear stress on human RBCs, suggested that quantal changes generated during capillary transits add up over time to develop the documented changes in RBC density and composition during their long circulatory lifespan, the quantal hypothesis. Applying the new red cell model (RCM) we investigated here the changes in homeostatic variables that may be expected during single capillary transits resulting from transient PIEZO1 channel activation. The predicted quantal volume changes were infinitesimal in magnitude, biphasic in nature, and essentially irreversible within inter-transit periods. A sub-second transient PIEZO1 activation triggered a sharp swelling peak followed by a much slower recovery period towards lower-than-baseline volumes. The peak response was caused by net CaCl2 and fluid gain via PIEZO1 channels driven by the steep electrochemical inward Ca2+ gradient. The ensuing dehydration followed a complex time-course with sequential, but partially overlapping contributions by KCl loss via Ca2+-activated Gardos channels, restorative Ca2+ extrusion by the plasma membrane calcium pump, and chloride efflux by the Jacobs-Steward mechanism. The change in relative cell volume predicted for single capillary transits was around 10-5, an infinitesimal volume change incompatible with a functional role in capillary flow. The biphasic response predicted by the RCM appears to conform to the quantal hypothesis, but whether its cumulative effects could account for the documented changes in density during RBC senescence required an investigation of the effects of myriad transits over the full four months circulatory lifespan of the cells, the subject of the next paper.

Prevalence and distribution of major β-thalassemia mutations and HbE/β-thalassemia variant in Nepalese ethnic groups

BackgroundBeta-thalassemia is a genetic disorder that is inherited in an autosomal recessive pattern. This genetic disease leads to a defective beta-globin hemoglobin chain causing partial or complete beta-globin chain synthesis loss. Beta-thalassemia major patients need a continuous blood transfusion and iron chelation to maintain the normal homeostasis of red blood cells (RBCs) and other systems in the body. Patients also require treatment procedures that are costly and tedious, resulting in a serious health burden for developing nations such as Nepal. MethodsA total of 61 individuals clinically diagnosed to have thalassemia were genotyped with multiplex amplification refractory mutation system–polymerase chain reaction (ARMS–PCR). Twenty-one major mutations were investigated using allele-specific primers grouped into six different panels. ResultsThe most common mutations found (23%) were IVS 1–5 (G-C) and Cd 26 (G-A) (HbE), followed by 619 deletion, Cd 8/9 (+G), Cd 16 (–C), Cd 41/42 (–TTCT), IVS 1–1 (G–T), Cd 19 (A-G), and Cd 17 (A-T) at 20%, 12%, 8%, 6%, 4%, 3%, and 1%, respectively. ConclusionThe results of this study revealed that Nepal's mutational profile is comparable to that of its neighboring countries, such as India and Myanmar. This study also showed that thalassemia could be detected across 17 Nepal's ethnic groups, especially those whose ancestors originated from India and Central Asia.

Red blood cell homeostasis in children and adults with and without asymptomatic malaria infection in Burkina Faso

Asymptomatic malaria infections may affect red blood cell (RBC) homeostasis. Reports indicate a role for chronic hemolysis and splenomegaly, however, the underlying processes are incompletely understood. New hematology analysers provide parameters for a more comprehensive analysis of RBC hemostasis. Complete blood counts were analysed in subjects from all age groups (n = 1118) living in a malaria hyperendemic area and cytokines and iron biomarkers were also measured. Subjects were divided into age groups (<2 years, 2-4, 5-14 and ≥15 years old) and clinical categories (smear-negative healthy subjects, asymptomatic malaria and clinical malaria). We found that hemoglobin levels were similar in smear-negative healthy children and asymptomatic malaria children but significantly lower in clinical malaria with a maximum difference of 2.2 g/dl in children <2 years decreasing to 0.1 g/dl in those aged ≥15 years. Delta-He, presenting different hemoglobinization of reticulocytes and RBC, levels were lower in asymptomatic and clinial malaria, indicating a recent effect of malaria on erythropoiesis. Reticulocyte counts and reticulocyte production index (RPI), indicating the erythropoietic capacity of the bone marrow, were higher in young children with malaria compared to smear-negative subjects. A negative correlation between reticulocyte counts and Hb levels was found in asymptomatic malaria (ρ = -0.32, p<0.001) unlike in clinical malaria (ρ = -0.008, p = 0.92). Free-Hb levels, indicating hemolysis, were only higher in clinical malaria. Phagocytozing monocytes, indicating erythophagocytosis, were highest in clinical malaria, followed by asymptomatic malaria and smear-negative subjects. Circulating cytokines and iron biomarkers (hepcidin, ferritin) showed similar patterns. Pro/anti-inflammatory (IL-6/IL-10) ratio was higher in clinical than asymptomatic malaria. Cytokine production capacity of ex-vivo whole blood stimulation with LPS was lower in children with asymptomatic malaria compared to smear-negative healthy children. Bone marrow response can compensate the increased red blood cell loss in asymptomatic malaria, unlike in clinical malaria, possibly because of limited level and length of inflammation. Trial registration: Prospective diagnostic study: ClinicalTrials.gov identifier: NCT02669823. Explorative cross-sectional field study: ClinicalTrials.gov identifier: NCT03176719.

Redox–Oligomeric State of Peroxiredoxin-2 and Glyceraldehyde-3-Phosphate Dehydrogenase in Obstructive Sleep Apnea Red Blood Cells under Positive Airway Pressure Therapy

In this study, we examined the effect of six months of positive airway pressure (PAP) therapy on Obstructive Sleep Apnea (OSA) red blood cell (RBC) proteome by two dimensional difference gel electrophoresis (2D-DIGE) - based proteomics followed by Western blotting (WB) validation. The discovered dysregulated proteins/proteoforms are associated with cell death, H2O2 catabolic/metabolic process, stress response, and protein oligomerization. Validation by nonreducing WB was performed for peroxiredoxin-2 (PRDX2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using antibodies against the sulfinylated/sulfonylated cysteine of these proteins to better evaluate their redox–oligomeric states under OSA and/or in response to PAP therapy. The results indicated that the redox–oligomeric state of GAPDH and PRDX2 involving overoxidation by sulfinic/sulfonic acids were differentially modulated in OSA RBC, which might be compromising RBC homeostasis. PAP therapy by restoring this modulation induced a higher oligomerization of overoxidized GAPDH and PRDX2 in some patients that could be associated with eryptosis and the chaperone “gain” of function, respectively. This varied response following PAP may result from the complex interplay between OSA and OSA metabolic comorbidity. Hence, information on the redox status of PRDX2 and GAPDH in RBC will help to better recognize OSA subtypes and predict the therapeutic response in these patients. GAPDH monomer combined with body mass index (BMI) and PRDX2 S-S dimer combined with homeostatic model assessment for insulin resistance (HOMA-IR) showed to be very promising biomarkers to predict OSA and OSA severity, respectively.