Adenosine signaling inhibits erythropoiesis and promotes myeloid differentiation.

Intracellular uptake of adenosine is essential for optimal erythroid commitment and differentiation of hematopoietic progenitor cells. The role of adenosine signaling is well documented in the regulation of blood flow, cell proliferation, apoptosis, and stem cell regeneration. However, the role of adenosine signaling in hematopoiesis remains unclear. In this study, we show that adenosine signaling inhibits the proliferation of erythroid precursors by activating the p53 pathway and hampers the terminal erythroid maturation. Furthermore, we demonstrate that the activation of specific adenosine receptors promotes myelopoiesis. Overall, our findings indicate that extracellular adenosine could be a new player in the regulation of hematopoiesis.

Lipid profiles in French West Indies sickle cell disease cohorts, and their general population.

BACKGROUND: The pathophysiology of sickle cell disease (SCD) and the variability of its clinical expression remain not fully understood, whether within or between different SCD genotypes. Recent studies have reported associations between lipid levels and several SCD complications. If lipid levels have been previously described as low in sickle cell anemia (SCA), few data have been provided for sickle cell SC disease (SCC). We designed our epidemiological study to isolate lipid levels and profiles by genotype in Guadeloupian cohorts of SCA and SCC adult patients, at steady state. We compared SCD lipid levels with those of the Guadeloupian general population (GGP), and analyzed potential associations between lipid levels and SCD complications (vaso-occlusive crises, acute chest syndrome and osteonecrosis). METHODS: Lipids, apolipoproteins, biological variables and anthropometric evaluation, were collected at steady state from medical files for 62 SCC and 97 SCA adult patients. Clinical SCD complications were collected from the clinical files. Analysis was conducted by genotype for all variables. RESULTS: Different SCC and SCA lipid profiles, both distinct from their GGP's, were identified. Compared to SCC and GGP, higher triglyceride (TG) levels were observed in SCA patients, independent of hydroxyurea, hemolysis, gender, age, body mass index (BMI), abdominal obesity and clinical nutritional status. Our survey highlights also subsequent anthropometrical phenotypes, with an over-representation of abdominal obesity with normal BMI in SCA patients, and affecting almost exclusively females in both genotypes. Moreover, more frequent positive history of acute chest syndrome (ACS) was observed in SCA patients with TG level higher than 1.50 g/l, and of osteonecrosis in SCC patients having non high-density lipoprotein-cholesterol level (Non HDL-C) higher than 1.30 g/l. CONCLUSIONS: This study reveals that SCA and SCC patients exhibit distinct lipid profiles and suggests that high TG and Non HDL-C levels are associated with past histories of ACS and osteonecrosis in SCA and SCC patients, respectively.

Exacerbation of oxidative stress during sickle vaso-occlusive crisis is associated with decreased anti-band 3 autoantibodies rate and increased red blood cell-derived microparticle level: a prospective study.

Painful vaso-occlusive crisis, a hallmark of sickle cell anaemia, results from complex, incompletely understood mechanisms. Red blood cell (RBC) damage caused by continuous endogenous and exogenous oxidative stress may precipitate the occurrence of vaso-occlusive crises. In order to gain insight into the relevance of oxidative stress in vaso-occlusive crisis occurrence, we prospectively compared the expression levels of various oxidative markers in 32 adults with sickle cell anaemia during vaso-occlusive crisis and steady-state conditions. Compared to steady-state condition, plasma levels of free haem, advanced oxidation protein products and myeloperoxidase, RBC caspase-3 activity, as well as the concentrations of total, neutrophil- and RBC-derived microparticles were increased during vaso-occlusive crises, whereas the reduced glutathione content was decreased in RBCs. In addition, natural anti-band 3 autoantibodies levels decreased during crisis and were negatively correlated with the rise in plasma advanced oxidation protein products and RBC caspase-3 activity. These data showed an exacerbation of the oxidative stress during vaso-occlusive crises in sickle cell anaemia patients and strongly suggest that the higher concentration of harmful circulating RBC-derived microparticles and the reduced anti-band 3 autoantibodies levels may be both related to the recruitment of oxidized band 3 into membrane aggregates.

Quantification of pain in sickle mice using facial expressions and body measurements.

Pain is a hallmark feature of sickle cell disease (SCD). Subjects typically quantify pain by themselves, which can be biased by other factors leading to overtreatment or under-treatment. Reliable and accurate quantification of pain, in real time, might enable to provide appropriate levels of analgesic treatment. The mouse grimace scale (MGS), a standardized behavioral coding system with high accuracy and reliability has been used to quantify varied types of pain. We hypothesized that addition of the objective parameters of body length and back curvature will strengthen the reproducibility of MGS. We examined MGS scores and body length and back curvature of transgenic BERK sickle and control mice following cold treatment or following treatment with analgesic cannabinoid CP55,940. We observed that sickle mice demonstrated decreased length and increased back curvature in response to cold. These observations correlate with changes in facial expression for the MGS score. CP55,940 treatment of sickle mice showed an increase in body length and a decrease in back curvature concordant with MGS scores indicative of an analgesic effect. Thus, body parameters combined with facial expressions may provide a quantifiable unbiased method for objective measure of pain in SCD.

High red blood cell nitric oxide synthase activation is not associated with improved vascular function and red blood cell deformability in sickle cell anaemia.

Human red blood cells (RBC) express an active and functional endothelial-like nitric oxide (NO) synthase (RBC-NOS). We report studies on RBC-NOS activity in sickle cell anaemia (SCA), a genetic disease characterized by decreased RBC deformability and vascular dysfunction. Total RBC-NOS content was not significantly different in SCA patients compared to healthy controls; however, using phosphorylated RBC-NOS-Ser(1177) as a marker, RBC-NOS activation was higher in SCA patients as a consequence of the greater activation of Akt (phosphorylated Akt-Ser(473) ). The higher RBC-NOS activation in SCA led to higher levels of S-nitrosylated α- and ß-spectrins, and greater RBC nitrite and nitrotyrosine levels compared to healthy controls. Plasma nitrite content was not different between the two groups. Laser Doppler flowmetric experiments demonstrated blunted microcirculatory NO-dependent response under hyperthermia in SCA patients. RBC deformability, measured by ektacytometry, was reduced in SCA in contrast to healthy individuals, and pre-shearing RBC in vitro did not improve deformability despite an increase of RBC-NOS activation. RBC-NOS activation is high in freshly drawn blood from SCA patients, resulting in high amounts of NO produced by RBC. However, this does not result in improved RBC deformability and vascular function: higher RBC-NO is not sufficient to counterbalance the enhanced oxidative stress in SCA.

Haemolysis and abnormal haemorheology in sickle cell anaemia.

Although pulmonary hypertension, leg ulcers, priapism, stroke and glomerulopathy in sickle cell anaemia (SCA) result from the adverse effects of chronic haemolysis on vascular function (haemolytic phenotype), osteonecrosis, acute chest syndrome and painful vaso-occlusive crises are caused by abnormal vascular cell adhesion and increased blood viscosity (viscosity-vaso-occlusion phenotype). However, this model with two sub-phenotypes does not take into account the haemorheological dimension. We tested the relationships between the biological parameters reflecting the haemolytic rate (haemolytic component) and red blood cell (RBC) rheological characteristics in 97 adults with SCA. No significant difference in the proportion of patients with low or high haemolytic component in the low and high blood viscosity groups was observed. The RBC elongation index (i.e. deformability) was negatively correlated with the haemolytic component. The RBC aggregates strength (i.e. RBC aggregates robustness) was negatively correlated with RBC elongation index. Sickle RBCs with high density had lower elongation index and higher aggregates strength. In conclusion, (i) the 'haemolytic' phenotype is characterized by decreased RBC deformability and increased RBC aggregates strength and (ii) the viscosity-vaso-occlusive phenotype is characterized by increased RBC deformability but not always by increased blood viscosity. α-thalassaemia modulates the haemorheological properties but other factors seem to be involved.

Hematologic and hemorheological determinants of resting and exercise-induced hemoglobin oxygen desaturation in children with sickle cell disease.

The aim of the study was to determine the factors associated with resting and exercise-induced hemoglobin oxygen desaturation. The well-established six-minute walk test was conducted in 107 sickle cell children (50 with sickle hemoglobin C disease and 57 with sickle cell anemia) at steady state. Hemoglobin oxygen saturation was measured before and immediately after the six-minute walk test. Blood samples were obtained on the same day to measure hematologic and hemorheological parameters. Exercise-induced hemoglobin oxygen desaturation was defined as a drop in hemoglobin oxygen saturation of 3% or more at the end of the six-minute walk test compared to resting levels. No children with sickle hemoglobin C disease, but approximately 50% of children with sickle cell anemia showed mild or moderate oxygen desaturation at rest, which was independently associated with the percentage of reticulocytes. Exercise-induced hemoglobin oxygen desaturation was observed in 18% of children with sickle hemoglobin C disease and 34% of children with sickle cell anemia, and was independently associated with the six-minute walk test, acute chest syndrome rate and the strength of red blood cell aggregates in children with sickle cell anemia. No association was found in children with sickle hemoglobin C disease between exercise-induced hemoglobin oxygen desaturation and the measured parameters. Hemoglobin oxygen desaturation at rest was common in children with sickle cell anemia but not in children with sickle hemoglobin C disease, and was mainly associated with greater hemolysis. Physiological strain during exercise and red blood cell aggregation properties may predict the occurrence of exercise-induced hemoglobin oxygen desaturation in children with sickle cell anemia.

Effect of inositol hexaphosphate-loaded red blood cells (RBCs) on the rheology of sickle RBCs.

BACKGROUND: The recent in vitro demonstration that inositol hexaphosphate-loaded red blood cells (IHP-RBCs) may reduce the risks of sickling of sickle RBCs (SS RBCs) exposed to hypoxia make these modified RBCs potentially useful in transfused sickle cell anemia (SCA) patients. STUDY DESIGN AND METHODS: Hemorheologic properties of IHP-RBCs, normal RBCs (AA RBCs), SS RBCs, SS RBCs plus AA RBCs, and SS RBCs plus IHP-RBCs were compared under normoxia and/or after hypoxic challenges. RESULTS: Although IHP-RBCs have reduced deformability compared with SS RBCs or AA RBCs, IHP-RBCs exhibited lower aggregability than AA RBCs and SS RBCs and, when mixed with SS RBCs, the aggregation level was below the one of SS RBCs alone or SS RBCs plus AA RBCs. Blood viscosity of SS RBC plus IHP-RBC suspension was lower than the viscosity of SS RBCs alone and greater than viscosity of SS RBCs plus AA RBCs. The hypoxic challenge was detrimental for deformability and viscosity of SS RBCs alone or SS plus AA RBC suspension but not for SS plus IHP-RBC suspension. CONCLUSION: Our results support the fact that IHP-RBCs could be useful in SCA by decreasing RBC aggregation and blunting the adverse effects of hypoxia on RBC deformability and blood viscosity.

Hemorheological risk factors of acute chest syndrome and painful vaso-occlusive crisis in children with sickle cell disease.

BACKGROUND: Little is known about the effects of blood rheology on the occurrence of acute chest syndrome and painful vaso-occlusive crises in children with sickle cell anemia and hemoglobin SC disease. DESIGN AND METHODS: To address this issue, steady-state hemorheological profiles (blood viscosity, red blood cell deformability, aggregation properties) and hematologic parameters were assessed in 44 children with sickle cell anemia and 49 children with hemoglobin SC disease (8-16 years old) followed since birth. Clinical charts were retrospectively reviewed to determine prior acute chest syndrome or vaso-occlusive episodes, and rates of these complications were calculated. RESULTS: Multivariate analysis revealed that: 1) a higher steady-state blood viscosity was associated with a higher rate of vaso-occlusive crises in children with sickle cell anemia, but not in children with hemoglobin SC disease; 2) a higher steady-state red blood cell disaggregation threshold was associated with previous history of acute chest syndrome in children with hemoglobin SC disease and boys with sickle cell anemia. CONCLUSIONS: Our results indicate for the first time that the red blood cell aggregation properties may play a role in the pathophysiology of acute chest syndrome in children with hemoglobin SC disease and boys with sickle cell anemia. In addition, whereas greater blood viscosity is associated with a higher rate of vaso-occlusive crises in children with sickle cell anemia, no association was found in children with hemoglobin SC disease, underscoring differences in the etiology of vaso-occlusive crises between sickle cell anemia and hemoglobin SC disease.

Hemorheological alterations, decreased cerebral microvascular oxygenation and cerebral vasomotion compensation in sickle cell patients.

Sickle cell anemia (SS) is characterized by a reduced cerebral microvascular oxygen saturation (cerebral TOI), which is not associated with hemoglobin concentration. Cerebral TOI has never been studied in sickle cell-hemoglobin C disease (SC). We focused on the relationships between hemorheological alterations and cerebral TOI in sickle cell patients with no cerebral vasculopathy and on the usefulness of TOI variability to assess the cerebral vasomotion activity. The blood rheological profile, the level of cerebral TOI (spatial resolved spectroscopy) and the cerebral TOI variability, which reflects vasomotion activity, were compared between 20 healthy subjects (AA), 21 SC patients, and 21 SS patients. Cerebral TOI exhibited the following order: AA > SC > SS. The low cerebral TOI in SS patients was related to red blood cell aggregation and deformability properties. The cerebral TOI variability of SS and SC patients was increased above healthy values and vasomotion activity was negatively associated with the reduced cerebral TOI in SS patients. We demonstrated that (1) blood rheology could be involved in the reduced cerebral TOI in SS patients but not in SC patients; (2) vasomotion activity is increased in SS and SC patients to compensate for the reduced cerebral TOI.

Extracellular Vesicles in Sickle Cell Disease: A Promising Tool.

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide. It is characterized by an impairment of shear stress-mediated vasodilation, a pro-coagulant, and a pro-adhesive state orchestrated among others by the depletion of the vasodilator nitric oxide, by the increased phosphatidylserine exposure and tissue factor expression, and by the increased interactions of erythrocytes with endothelial cells that mediate the overexpression of adhesion molecules such as VCAM-1, respectively. Extracellular vesicles (EVs) have been shown to be novel actors involved in SCD pathophysiological processes. Medium-sized EVs, also called microparticles, which exhibit increased plasma levels in this pathology, were shown to induce the activation of endothelial cells, thereby increasing neutrophil adhesion, a key process potentially leading to the main complication associated with SCD, vaso-occlusive crises (VOCs). Small-sized EVs, also named exosomes, which have also been reported to be overrepresented in SCD, were shown to potentiate interactions between erythrocytes and platelets, and to trigger endothelial monolayer disruption, two processes also known to favor the occurrence of VOCs. In this review we provide an overview of the current knowledge about EVs concentration and role in SCD.

Down-regulation of MyoD by calpain 3 promotes generation of reserve cells in C2C12 myoblasts.

Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated "reserve cells" are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

Laminin-111: a potential therapeutic agent for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) still needs effective treatments, and myoblast transplantation (MT) is considered as an approach to repair damaged skeletal muscles. DMD is due to the complete loss of dystrophin from muscles. The lack of link between the contracting apparatus and the extracellular matrix leads to frequent damage to the sarcolemma triggering muscle fiber necrosis. Laminins are major proteins in the extracellular matrix. Laminin-111 is normally present in skeletal and cardiac muscles in mice and humans but only during embryonic development. In this study, we showed that intramuscular injection of laminin-111 increased muscle strength and resistance in mdx mice. We also used laminin-111 as a coadjuvant in MT, and we showed this protein decreased considerably the repetitive cycles of degeneration, inflammatory reaction, and regeneration. Moreover, MT is significantly improved. To explain the improvement, we confirmed with the same myoblast cell batch that laminin-111 improves proliferation and drastically increases migration in vitro. These results are extremely important because DMD could be treated only by the injection of a recombinant protein, a simple and safe therapy to prevent loss of muscle function. Moreover, the improvement in MT would be significant to treat the muscles of DMD patients who are already weak.

Heme Causes Pain in Sickle Mice via Toll-Like Receptor 4-Mediated Reactive Oxygen Species- and Endoplasmic Reticulum Stress-Induced Glial Activation.

Aims: Lifelong pain is a hallmark feature of sickle cell disease (SCD). How sickle pathobiology evokes pain remains unknown. We hypothesize that increased cell-free heme due to ongoing hemolysis activates toll-like receptor 4 (TLR4), leading to the formation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Together, these processes lead to spinal microglial activation and neuroinflammation, culminating in acute and chronic pain. Results: Spinal heme levels, TLR4 transcripts, oxidative stress, and ER stress were significantly higher in sickle mice than controls. In vitro, TLR4 inhibition in spinal cord microglial cells attenuated heme-induced ROS and ER stress. Heme treatment led to a time-dependent increase in the characteristic features of sickle pain (mechanical and thermal hyperalgesia) in both sickle and control mice; this effect was absent in TLR4-knockout sickle and control mice. TLR4 deletion in sickle mice attenuated chronic and hypoxia/reoxygenation (H/R)-evoked acute hyperalgesia. Sickle mice treated with the TLR4 inhibitor resatorvid; selective small-molecule inhibitor of TLR4 (TAK242) had significantly reduced chronic hyperalgesia and had less severe H/R-evoked acute pain with quicker recovery. Notably, reducing ER stress with salubrinal ameliorated chronic hyperalgesia in sickle mice. Innovation: Our findings demonstrate the causal role of free heme in the genesis of acute and chronic sickle pain and suggest that TLR4 and/or ER stress are novel therapeutic targets for treating pain in SCD. Conclusion: Heme-induced microglial activation via TLR4 in the central nervous system contributes to the initiation and maintenance of sickle pain via ER stress in SCD. Antioxid. Redox Signal. 34, 279-293.

Microfluidics in Sickle Cell Disease Research: State of the Art and a Perspective Beyond the Flow Problem.

Sickle cell disease (SCD) is the monogenic hemoglobinopathy where mutated sickle hemoglobin molecules polymerize to form long fibers under deoxygenated state and deform red blood cells (RBCs) into predominantly sickle form. Sickled RBCs stick to the vascular bed and obstruct blood flow in extreme conditions, leading to acute painful vaso-occlusion crises (VOCs) - the leading cause of mortality in SCD. Being a blood disorder of deformed RBCs, SCD manifests a wide-range of organ-specific clinical complications of life (in addition to chronic pain) such as stroke, acute chest syndrome (ACS) and pulmonary hypertension in the lung, nephropathy, auto-splenectomy, and splenomegaly, hand-foot syndrome, leg ulcer, stress erythropoiesis, osteonecrosis and osteoporosis. The physiological inception for VOC was initially thought to be only a fluid flow problem in microvascular space originated from increased viscosity due to aggregates of sickled RBCs; however, over the last three decades, multiple molecular and cellular mechanisms have been identified that aid the VOC in vivo. Activation of adhesion molecules in vascular endothelium and on RBC membranes, activated neutrophils and platelets, increased viscosity of the blood, and fluid physics driving sickled and deformed RBCs to the vascular wall (known as margination of flow) - all of these come together to orchestrate VOC. Microfluidic technology in sickle research was primarily adopted to benefit from mimicking the microvascular network to observe RBC flow under low oxygen conditions as models of VOC. However, over the last decade, microfluidics has evolved as a valuable tool to extract biophysical characteristics of sickle red cells, measure deformability of sickle red cells under simulated oxygen gradient and shear, drug testing, in vitro models of intercellular interaction on endothelialized or adhesion molecule-functionalized channels to understand adhesion in sickle microenvironment, characterizing biomechanics and microrheology, biomarker identification, and last but not least, for developing point-of-care diagnostic technologies for low resource setting. Several of these platforms have already demonstrated true potential to be translated from bench to bedside. Emerging microfluidics-based technologies for studying heterotypic cell-cell interactions, organ-on-chip application and drug dosage screening can be employed to sickle research field due to their wide-ranging advantages.