Sickle Cell Disease Erythrocytes Research Articles

Membrane bending and sphingomyelinase-associated, sulfatide-dependent hypoxic adhesion of sickle mature erythrocytes.

Abnormal erythrocyte adhesion owing to polymerization of sickle hemoglobin is central to the pathophysiology of sickle cell disease (SCD). Mature erythrocytes constitute >80% of all erythrocytes in SCD; however, the relative contributions of erythrocytes to acute and chronic vasculopathy in SCD are not well understood. Here, we showed that bending stress exerted on the erythrocyte plasma membrane by polymerization of sickle hemoglobin under hypoxia, enhances sulfatide-mediated abnormal mature erythrocyte adhesion. We hypothesized that sphingomyelinase (SMase) activity, which is upregulated by accumulated bending energy, leads to elevated membrane sulfatide availability, and thus, hypoxic mature erythrocyte adhesion. We found that mature erythrocyte adhesion to laminin in controlled microfluidic experiments is significantly greater under hypoxia than under normoxia (1856± 481 vs 78± 23, mean± SEM), whereas sickle reticulocyte (early erythrocyte) adhesion, high to begin with, does not change (1281± 299 vs 1258± 328, mean± SEM). We showed that greater mean accumulated bending energy of adhered mature erythrocytes was associated with higher acid SMase activity and increased mature erythrocyte adhesion (P= .022, for acid SMase activity and P= .002 for the increase in mature erythrocyte adhesion with hypoxia, N= 5). In addition, hypoxia results in sulfatide exposure of the erythrocyte membrane, and an increase in SMase, whereas anti-sulfatide inhibits enhanced adhesion of erythrocytes. These results suggest that the lipid components of the plasma membrane contribute to SCD complications. Therefore, sulfatide and the components of its upregulation pathway, particularly SMase, should be further explored as potential therapeutic targets for inhibiting sickle erythrocyte adhesion.

Proteomic and Lipidomic Investigation of an Ex-Vivo Human Model of Sickle Cell Disease Erythropoiesis and Patient Erythrocytes Reveals the Abnormal Localization and Dysfunction of the Proteasome, Disrupted Antioxidant Defenses and Altered Lipid Profiles

Introduction: The impact of the early expression of sickle hemoglobin (HbS) on erythroid differentiation remains unclear. We hypothesized that HbS already plays a detrimental role by altering the lipidome/proteome during sickle cell disease (SCD) erythropoiesis. Provided that in-vivo studies do not allow the dynamical follow-through of homogenous populations, are biased by non-cell autonomous interactions, and that bone marrow (BM) harvesting in SCD individuals is difficult to justify, we aimed to compare whole-cell/membrane proteomes and lipidomes in an ex-vivo human model of SCD and healthy erythropoiesis, as well as in mature peripheral blood (PB) erythrocytes. Methods: Healthy and SCD erythropoiesis was modeled ex-vivo in a 3-phase liquid system at ambient O2 tension starting with CD34+ hematopoietic stem cells (HSC) isolated either from healthy donor BM or GSCF-stimulated PB, and SCD patient apheresis products (Panel A). Whole cell protein extracts were obtained at 3 timepoints (day 7, 11, 18, i.e early-, late-erythroblast (EB) and reticulocyte stages). Membrane extracts were purified with a novel erythroid Cell-Surface-Capture method. Proteomes and lipidomes were characterized by quantitative LC-MS/MS and compared according to time-points and health vs. disease states in unbiased/unsupervised models. Multi-Omics Factor Analysis (MOFA) determined key modifiers of SCD erythropoiesis. Moreover, omic analyses were performed on mature healthy and SCD erythrocytes obtained from respective PB (after density-fractionation of light/dense cells for SCD). Proteasome subunits were quantified in healthy vs. SCD erythrocyte ghosts by WB. Proteasome activity was assayed in mature cells and in ex-vivo late-stage SCD erythroid cells under different L-glutamine levels. Results: First, we observed that CD34+ HSCs derived from BM vs. PB generate significantly divergent proteomes/lipidomes in ex-vivo erythropoiesis. Specifically, differences in glutathione and glucose pathway proteins were observed. Amongst them, alpha-enolase is 2-fold to 6-fold depleted in intermediate and reticulocyte stages respectively, when comparing PB vs BM-derived erythropoiesis. This finding is relevant in consideration of prior data on the regulation of HSC erythroid vs myeloid lineage commitment by glucose/glutamine metabolism. Second, we used SCD vs BM-derived erythropoiesis (reference healthy control) to capture the most significant modifications. Among those, the proteasome was progressively abnormally localized in SCD. We observed the depletion of core 20S subunits and sequestration of 19S regulators at the membrane (Panel B), which could be confirmed in dense SCD erythrocyte membranes. Also, we observed an inversion of proteasome activity between membrane and cytosolic extracts in light vs. dense SCD erythrocytes (increased membrane activity in dense SCD erythrocytes). Removing glutamine from culture in the ex-vivo model of SCD erythropoiesis significantly increased the membrane proteasome activity at the reticulocyte stage. Moreover, oxidative stress defenses and cholesterol biosynthesis were perturbed in SCD erythropoiesis, including the upregulation of Heme-oxygenase-1. Also, the antioxidant protein balance was abnormal in mature-dense SCD erythrocyte membranes where insoluble aggregates (i.e. actin/ankyrin + HbS + GAPDH) were sequestered. Additionally, increased GAPDH could be seen in cytoskeleton extracts. Third, we observed the erythroid lipidome to evolve harmoniously in SCD vs healthy erythropoiesis across time-points, while significant perturbations were seen once erythrocytes are in circulation. In fact, dense SCD erythrocytes had significantly abnormal levels of lysophosphatidylcholines (depleted), sphingomyelins (depleted) and triacylglycerols (enriched). Fourth, MOFA revealed the key contribution of heme synthesis, glycolysis and proteasome regulation to the differences in global phenotype between SCD and healthy erythropoiesis. Conclusion: Our study shows that a) ex-vivo human erythropoiesis models based on healthy PB vs BM-derived HSCs differ significantly on a lipidomic/proteomic level, highlighting the importance of source choice, b) HbS expression has an incremental proteomic impact during erythropoiesis and c) the lipidome is unaltered in SCD erythropoiesis but radically changes once erythrocytes are circulating. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Amiodarone improves anemia in a murine model of sickle cell disease and is associated with increased erythrocyte bis(monoacylglycerol) phosphate

Sickle cell disease (SCD) is associated with altered plasma and erythrocyte lipid profiles. In a previous study, SCD mice with deficiency of proprotein convertase subtilisin/kexin type 9 (PCSK9) were observed to have more severe anemia and increased sickling compared to control SCD mice. Although PCSK9 affects circulating low density lipoprotein (LDL) by regulation of the LDL receptor, the effect of PCSK9 on anemia was independent of LDL receptor expression. In the current study, erythrocyte metabolomics were performed and revealed altered erythrocyte lipid species between SCD mice with and without PCSK9. Of particular interest, the late endosome-specific lipid bis(mono)acylglycerol phosphate (BMP) 44:12 was markedly decreased in erythrocytes from SCD mice deficient in PCSK9 mice relative to control SCD mice. Incubation of sickle erythrocytes with a neutralizing antibody to BMP increased erythrocyte sickling in vitro. In vitro treatment of SCD erythrocytes with amiodarone (1.5 μM) or medroxyprogesterone (6.75 μM), two pharmacologic compounds known to increase BMP, resulted in reduced erythrocyte sickling. Treatment of SCD mice with amiodarone (10 mg/kg) for 2 weeks resulted in increased BMP, improvement in anemia with reduced reticulocytosis, and decreased ex vivo sickling. In conclusion, severity of anemia in SCD is improved with amiodarone treatment, an effect which may be mediated through increased erythrocyte BMP.

ATR1 Angiotensin II Receptor Reduces Hemoglobin S Polymerization, Phosphatidylserine Exposure, and Increases Deformability of Sickle Cell Disease Erythrocytes.

Angiotensin II (Ang II) regulates blood volume and stimulates erythropoiesis through AT1 (ATR1) and AT2 (ATR2) receptors, found in multiple tissues, including erythrocytes. Sickle cell disease (SCD) patients present altered Ang II levels. Hemoglobin S polymerization, deformability and phosphatidylserine translocation are important features of mature erythrocytes, therefore, our hypothesis is Ang II affects these parameters and, if it does, what would be the influence of AT1R and AT2R on these effects. A polymerization assay (PA), deformability, and annexin V binding were performed in SCD erythrocytes samples adding Ang II, ATR1 antagonist (losartan or eprosartan), and ATR2 antagonist (PD123319). Through the PA test, we observed a dose-dependent polymerization inhibition effect when comparing Ang II to control. Losartan did not affect the level or the rate of Ang II inhibition, while PD123319 showed an increased level of protection against polymerization, and eprosartan brought levels back to control. Ang II was able to reduce the translocation of phosphatidylserine from the inner to the outer leaflet, a marker of eryptosis, in the presence of PD123319. Also, ATR1 showed a positive effect increasing deformability. Our data shows that ATR1 is important for maintenance of erythrocyte physiological function in SCD and for prolonging its life.

Phenotypic analysis of erythrocytes in sickle cell disease using imaging flow cytometry.

The morphology and other phenotypic characteristics of erythrocytes in sickle cell disease (SCD) have been analyzed for decades in patient evaluation. This involves a variety of techniques, including microscopic analysis of stained blood films, flow cytometry, and cell counting. Here, we analyzed SCD blood using imaging flow cytometry (IFC), a technology that combines flow cytometry and microscopy to enable simultaneous rapid-throughput analysis of cellular morphology and cell-surface markers. With IFC, we were able to automate quantification of poikilocytes from SCD blood. An important subpopulation of poikilocytes represented dense cells, although these could not be distinguished from other poikilocytes without first centrifuging the blood through density gradients. In addition, CD71-positive RBCs from SCD patients had two subpopulations: one with high CD71 expression and a puckered morphology and another with lower CD71 expression and biconcave morphology and presumably representing a later stage of differentiation. Some RBCs with puckered morphologies that were strongly positive for DAPI and CD49d were in fact nucleated RBCs. IFC identified more phosphatidylserine-expressing red cells in SCD than did conventional flow cytometry and these could also be divided into two subpopulations. One population had diffuse PS expression and appeared to be composed primarily of RBC ghosts; the other had lower overall PS expression present in intense, punctate dots overlying Howell-Jolly bodies. This study demonstrates that IFC can rapidly reveal and quantify RBC features in SCD that require numerous tedious methods to identify conventionally. Thus, IFC is likely to be a useful technique for evaluating and monitoring SCD.

Cathepsin B, a Negative Regulator of Autophagy, Identified As a Novel Therapeutic Drug Target in Sickle Cell Disease

Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide. The disease is caused by a mutation of the beta-globin gene that results in polymerization of the sickle hemoglobin (HbS) when deoxygenated. Reactive oxygen species (ROS) induced hemolysis is a major critical event in SCD. We have previously shown that abnormal retention of mitochondria in the erythrocytes of both SCD patients and SCD mice is associated with elevated ROS levels and hemolysis (Exp. Hem.2017;50:46-52). The mechanism responsible for mitochondrial retention in SCD is unknown. Autophagy is one of the processes responsible for the elimination of mitochondria during erythroid differentiation. Autophagy is a conserved physiological process that promotes cellular homeostasis through the recycling of proteins, protein aggregates, and removal of damaged organelles. Hypothesis: In this study, we have investigated the hypothesis that autophagy pathway dysregulation is responsible for abnormal mitochondrial retention in SCD erythrocytes. We propose that pharmacological methods that address the abnormal activity of these genes may be therapeutic for SCD. Methods: The expression of autophagy pathway genes in reticulocytes of SCD patients and experimental SCD mice was compared with their respective controls using the RT2 Autophagy Profiler PCR Array. The expression of autophagy proteins was measured by Western blot. Cathepsin B (CTSB) activity was measured by a fluorometric assay using the substrate RR-AFC (amino-4-trifluoromethyl coumarin). Results: Six autophagy pathway genes were differentially expressed in SCD patients compared to control subjects. Expression of the autophagy pathway genes was also analyzed in SCD mice, normal AA mice, and phlebotomized anemic AA mice as a control for increased reticulocytosis. Eighteen genes were differentially expressed in SCD mice compared with control AA mice (p<0.05). Four genes were differentially expressed in SCD mice compared with control phlebotomized anemic AA mice (p<0.05). CTSB, a known negative regulator of autophagy (J Exp Med. 2016;213(10):2081-97), was over-expressed in the reticulocytes of both SCD patients and SCD mice. Expression of CTSB was 4.5 fold (p<0.007) higher in SCD mice compared to normal control AA mice and 8.6 fold (p<0.001) higher compared to phlebotomized AA anemic mice. We therefore focused our further analysis on CTSB. Western blot data confirmed that CTSB protein was overexpressed in red blood cells of SCD patients and experimental SCD mice (p<0.01) compared to respective controls. The level of CTSB enzyme activity was 3.3 fold higher in SCD mice compared to the control mice(p<0.01). To evaluate the functional significance of these differences in CTSB expression, CTSB inhibitor E64d was investigated in SCD mice. SCD mice treated with E64d (40mg/kg b.wt.) for two weeks showed a reduction in CTSB activity (SCD vehicle: 11.8±1.1 RFU/µg, n=3 vs SCD E64d treatment: 5 ± 0.1 RFU/µg, n=3, p<0.008) and in the proportion of reticulocytes that retained mitochondria (SCD vehicle: 45.2%±3%, n=3 vs. SCD E64d treatment: 32 % ± 1.9%, n=3, p<0.003). Reticulocytosis was also significantly reduced in SCD mice treated with the CTSB inhibitor (SCD vehicle: 47.2%±2.8%, n=3 vs. SCD E64d treatment: 35.9 % ± 3.9%, n=3, p<0.01). Conclusion: Our data shows that the multiple autophagy pathway gene expression is dysregulated in SCD. Among these genes, CTSB, a key negative regulator of autophagy, is overexpressed in the reticulocytes of both SCD patients and SCD mice. Pharmacological inhibition of CTSB activity partially reverses mitochondrial retention and decreases reticulocytosis in SCD mice, confirming that overexpression of CTSB contributes to SCD pathogenesis and is a potential therapeutic target. Disclosures No relevant conflicts of interest to declare.

Promising anti-sickling and fetal hemoglobin inducing effects of Boerhavia diffusa root extract on sickle cell erythrocytes

Sickle cell disease (SCD) is a genetic blood disorder and currently available drugs are ineffective and come with numerous side effects B. diffusa has been reported as an important herb in the ayurvedic texts. Although studies have shown immunomodulatory and anti-inflammation activities, the anti-sickling activity and fetal hemoglobin (HbF) inducing potential of Boerhavia diffusa have not yet been explored. Therefore, the present study was aimed to assess anti-sickling and HbF induction effects of B. diffusa aqueous root extract in sickle cell erythrocytes obtained from SCD patients. The in vitro anti-sickling activity of the extract was evaluated microscopically via the Emmel Test. The HbF inducing activity of the extract was determined on SCD erythrocytes through HPLC. The different doses and time-dependent treatments of the test extract and drug control (hydroxyurea) suggested that 30 mg/mL extract for 60 min exerts significant anti-sickling effect on SCD erythrocytes compared to the negative control (P < 0.0001). The anti-sickling activity of test extract was comparable to hydroxyurea. Moreover, the test extract showed significant increased HbF% in SCD patients’ erythrocytes at 30 mg/mL concentration for 120 min compared to negative control (P < 0.0001). The test extract showed similar HbF inducing potential when compared to hydroxyurea. In addition, the percentage of sickling was remarkably reduced with treatment of test extract in both severe and less severe SCD groups compared to negative control. The test extract and hydroxyurea were found to have similar anti-sickling effects irrespective of the severity of the disease. Overall, these results show for the first time the potent anti-sickling and HbF inducing activity of aqueous root extract of Boerhaviadiffusa, which may be used as an herb-based alternative therapy in SCD patients, warranting future in vivo studies.

Complement activation in sickle cell disease: Dependence on cell density, hemolysis and modulation by hydroxyurea therapy.

The complement system is an innate immune defense cascade that can cause tissue damage when inappropriately activated. Evidence for complement over activation has been reported in small cohorts of patients with sickle cell disease (SCD). However, the mechanism governing complement activation in SCD has not been elucidated. Here, we observe that the plasma concentration of sC5b-9, a reliable marker for terminal complement activation, is increased at steady state in 61% of untreated SCD patients. We show that greater complement activation in vitro is promoted by SCD erythrocytes compared to normal ones, although no significant differences were observed in the regulatory proteins CD35, CD55, and CD59 in whole blood. Complement activation is positively correlated with the percentage of dense sickle cells (DRBCs). The expression levels of CD35, CD55, and CD59 are reduced in DRBCs, suggesting inefficient regulation when cell density increases. Moreover, the surface expression of the complement regulator CD46 on granulocytes was inversely correlated with the plasma sC5b-9. We also show increased complement deposition in cultured human endothelial cells incubated with SCD serum, which is diminished by the addition of the heme scavenger hemopexin. Treatment of SCD patients with hydroxyurea produces substantial reductions in complement activation, measured by sC5b-9 concentration and upregulation of CD46, as well as decreased complement activation on RBCs in vitro. In conclusion, complement over activation is a common pathogenic event in SCD that is associated with formation of DRBCs and hemolysis. And, it affects red cells, leukocytes and endothelial cells. This complement over activation is partly alleviated by hydroxyurea therapy.

Fetal Hemoglobin Promotes P. Falciparum Growth in Sickle Cell Disease Erythrocytes

Introduction: We have demonstrated that sickle hemoglobin (HbS) polymerization in low oxygen (O2) is the main driver of sickle cell trait (AS) resistance to P.falciparum malaria. This suggests that homozygous sickle cell disease (SS) individuals should have even greater resistance to malaria. Instead SS individuals infected by P. falciparum often have increased malaria morbidity and mortality compared to individuals with normal hemoglobin (AA) or AS. The reasons for this paradox are poorly understood. We propose that fetal hemoglobin (HbF) inhibits polymerization thereby allowing parasite proliferation. To test this hypothesis the following experiments were performed. Methods: AA and SS erythrocytes were collected within two weeks of the assay date. P. falciparum 3D7 parasites were used for growth assays. Growth assays were performed at 1, 3, 5, 7.5, 10 and 16% O2 using synchronized schizonts obtained by magnet purification via the MACS system. Staging was performed via light microscopy analysis of May-Grünwald-Giemsa-stained cytospins. Parasite Multiplication Rate (PMR) was determined via flow cytometry using parasitemia at 64 and 16 hours post infection (hpi) as previously described. Results: In contrast to AS erythrocytes, SS RBCs contain varying amounts of HbF that differ by cell and by individual. In hypoxic SS RBCs with low (3.8%) HbF, P. falciparum parasites remain immature (early trophozoites) in 1, 3, and 5% O2 (Fig. 1) at 36 hpi, when they should have matured into schizonts. However, in SS RBCs with high (22.6%) HbF, 12, 16 and 27% of parasites matured into schizonts in 1, 3 and 5% O2 respectively compared to 0, 0, and 1% in the SS RBCs with 3.8% HbF. Preliminary data also demonstrate that the parasite multiplication rate (PMR), a surrogate of proliferation, in SS RBCs improves with increasing O2 and HbF. Using SS/Hereditary Persistence of Fetal Hemoglobin RBCs with 42% HbF, proliferation in 5% O2 or higher is exponential with a PMR of 2.6 or greater (Fig. 2). In RBCs isolated from SS patients treated with hydroxyurea with HbF of 26.4 or 21%, PMR is 2 or more in 7.5% O2 or greater. Conclusion: These data demonstrate that HbF promotes P. falciparum growth in SS erythrocytes. In addition, the data initiates a resolution of the malaria paradox. HbS in both hypoxic AS and SS surely inhibits P. falciparum growth due to HbS polymerization at low O2. But high levels of anti-sickling HbF observed in erythrocytes from many individuals with SS reverses this inhibition despite the increased content of HbS in SS individuals. Even small increases in P. falciparum proliferation within the broadly compromised SS host may enhance the malaria associated morbidity and mortality seen in this population. These data further suggest the importance of anti-malarial prophylaxis in patients with SS especially those treated with hydroxyurea or anti-sickling agents. Disclosures No relevant conflicts of interest to declare.

Editing a γ-Globin Repressor Binding Site Restores Fetal Hemoglobin Synthesis and Corrects the Phenotype of Sickle Cell Disease Erythrocytes

Sickle cell disease (SCD) is a severe, life-threatening disorder caused by a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) β-chain that causes Hb polymerization with consequent red blood cell (RBC) rigidity, anemia, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) decreases sickling and significantly reduces the clinical severity of SCD. HPFH mutations cluster at several loci in the promoters of the two γ-globin genes, HBG1 and HBG2, and, disrupt binding sites for transcriptional repressors (e.g., BCL11A and LRF), leading to elevated levels of fetal hemoglobin (HbF) representing up to 40% of total hemoglobin tetramers. In addition, SNPs at position -158 of both γ-globin promoters are associated with enhanced γ-globin expression and may identify a putative transcriptional repressor binding site. Here, we used the CRISPR/Cas9 system to mimic HPFH mutations in the HBG promoters by generating insertion and deletions (InDels) leading to disruption of known and putative binding sites for transcriptional repressors via non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) DNA repair mechanisms. Efficient editing of the LRF binding site (≥ 3 γ-globin promoters/cell in &gt;70% of cells) in SCD patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in a robust, virtually pancellular HbF reactivation and a concomitant reduction in βS-globin levels, recapitulating the phenotype of asymptomatic SCD-HPFH patients. Of note, in RBCs derived from edited HSPCs, HbF levels exceeded 40% of total Hb tetramers, suggesting that CRISPR/Cas9 mediated disruption of the LRF binding site is even more potent than naturally occurring HPFH point mutations in reactivating HbF expression. RBCs derived from edited HSPCs displayed HbF levels sufficient to substantially correct the SCD cell phenotype (~80% of non-sickling RBCs). Although larger deletions (in part generated likely by MMEJ) disrupt more efficiently the LRF binding site, shorter InDels mainly produced by NHEJ, the most active repair mechanism in bona fide hematopoietic stem cells (HSCs), were still able to induce a potent γ-globin reactivation and correct the SCD cell phenotype. Interestingly, upon targeting of the -158 site, we observed HBG de-repression, which, however, was mild, indicating that this region contains a sequence that moderately inhibits γ-globin expression in adult cells, consistently with the mild increase in HbF associated with the -158 SNP. To evaluate the efficiency and the safety of this approach in long-term repopulating HSCs, untreated and edited HSPCs were injected into NSG immunodeficient mice. 16 weeks after transplantation, the engraftment of control and HBG-edited cells was comparable, as analyzed in bone marrow, spleen, thymus and blood. Edited HSCs were capable to differentiate into multiple hematopoietic cell types, with no skewing towards any particular lineage. Importantly, xenotransplantation of HSPCs edited using the gRNAs targeting the LRF binding site demonstrated a high editing efficiency in long-term repopulating HSCs (up to 75%) and a modest reduction in the frequency of deletions likely generated by MMEJ. Overall, this study identifies the binding site for the LRF repressor as a novel, potent and safe target for a genome editing-based treatment of SCD. Disclosures El Nemer: Hemanext: Other: Other. Cavazzana:SmartImmune: Other: Founder.

Resolution of sickle cell disease–associated inflammation and tissue damage with 17R-resolvin D1

AbstractResolvins (Rvs), endogenous lipid mediators, play a key role in the resolution of inflammation. Sickle cell disease (SCD), a genetic disorder of hemoglobin, is characterized by inflammatory and vaso-occlusive pathologies. We document altered proresolving events following hypoxia/reperfusion in humanized SCD mice. We demonstrate novel protective actions of 17R-resolvin D1 (17R-RvD1; 7S, 8R, 17R-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) in reducing ex vivo human SCD blood leukocyte recruitment by microvascular endothelial cells and in vivo neutrophil adhesion and transmigration. In SCD mice exposed to hypoxia/reoxygenation, oral administration of 17R-RvD1 reduces systemic/local inflammation and vascular dysfunction in lung and kidney. The mechanism of action of 17R-RvD1 involves (1) enhancement of SCD erythrocytes and polymorphonuclear leukocyte efferocytosis, (2) blunting of NF-κB activation, and (3) a reduction in inflammatory cytokines, vascular activation markers, and E-selectin expression. Thus, 17R-RvD1 might represent a new therapeutic strategy for the inflammatory vasculopathy of SCD.

Microvasculature-on-a-chip for the long-term study of endothelial barrier dysfunction and microvascular obstruction in disease

Alterations in the mechanical properties of erythrocytes occurring in inflammatory and hematologic disorders such as sickle cell disease (SCD) and malaria often lead to increased endothelial permeability, haemolysis, and microvascular obstruction. However, the associations among these pathological phenomena remain unknown. Here, we report a perfusable, endothelialized microvasculature-on-a-chip featuring an interpenetrating-polymer-network hydrogel that recapitulates the stiffness of blood-vessel intima, basement membrane self-deposition and self-healing endothelial barrier function for longer than 1 month. The microsystem enables the real-time visualization, with high spatiotemporal resolution, of microvascular obstruction and endothelial permeability under physiological flow conditions. We found how extracellular heme, a hemolytic byproduct, induces delayed but reversible endothelial permeability in a dose-dependent manner, and demonstrate that endothelial interactions with SCD or malaria-infected erythrocytes cause reversible microchannel occlusion and increased in situ endothelial permeability. The microvasculature-on-a-chip enables mechanistic insight into the endothelial barrier dysfunction associated with SCD, malaria and other inflammatory and haematological diseases.

Artemisinin Therapy for Malaria in Hemoglobinopathies: A Systematic Review.

Artemisinin derivatives are widely used antimalarial drugs. There is some evidence from in vitro, animal and clinical studies that hemoglobinopathies may alter their disposition and antimalarial activity. This review assesses relevant data in α-thalassemia, sickle cell disease (SCD), β-thalassemia and hemoglobin E. There is no convincing evidence that the disposition of artemisinin drugs is affected by hemoglobinopathies. Although in vitro studies indicate that Plasmodium falciparum cultured in thalassemic erythrocytes is relatively resistant to the artemisinin derivatives, mean 50% inhibitory concentrations (IC50s) are much lower than in vivo plasma concentrations after recommended treatment doses. Since IC50s are not increased in P. falciparum cultures using SCD erythrocytes, delayed post-treatment parasite clearance in SCD may reflect hyposplenism. As there have been no clinical studies suggesting that hemoglobinopathies significantly attenuate the efficacy of artemisinin combination therapy (ACT) in uncomplicated malaria, recommended artemisinin doses as part of ACT remain appropriate in this patient group.

A microfluidic approach to study the effect of mechanical stress on erythrocytes in sickle cell disease.

The human red blood cell is a biconcave disc of 6-8 × 2 μm that is highly elastic. This capacity to deform enables it to stretch while circulating through narrow capillaries to ensure its main function of gas exchange. Red cell shape and deformability are altered in membrane disorders because of defects in skeletal or membrane proteins affecting protein-protein interactions. Red cell properties are also altered in other pathologies such as sickle cell disease. Sickle cell disease is a genetic hereditary disorder caused by a single point mutation in the β-globin gene generating sickle haemoglobin (HbS). Hypoxia drives HbS polymerisation that is responsible for red cell sickling and reduced deformability. The main clinical features of sickle cell disease are vaso-occlusive crises and haemolytic anaemia. Foetal haemoglobin (HbF) inhibits HbS polymerisation and positively impacts red cell survival in the circulation but the mechanism through which it exerts this action is not fully characterized. In this study, we designed a microfluidic biochip mimicking the dimensions of human capillaries to measure the impact of repeated mechanical stress on the survival of red cells at the single cell scale under controlled pressure. We show that mechanical stress is a critical parameter underlying intravascular haemolysis in sickle cell disease and that high intracellular levels of HbF protect against lysis. The biochip is a promising tool to address red cell deformability in pathological situations and to screen for molecules positively impacting this parameter in order to improve red cell survival in the circulation.

Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease

Sphingosine 1-phosphate (S1P) is a bioactive signaling lipid highly enriched in mature erythrocytes. Previous study has revealed that levels of S1P are significantly elevated in patients and mice with Sickle Cell Disease (SCD), a devastating and highly prevalent genetic hemolytic disorder that causes life-threatening hemolysis, tissue damage, and organ dysfunction with very limited treatment. Moreover, the activity of S1P generating enzyme-Sphingosine Kinase 1 (SphK1) is increased in human and mouse SCD erythrocytes, and inhibition of SphK1 decreased erythrocyte sickling. However, the structural and functional basis for the pathogenic nature of S1P in SCD remains obscure. Here, we report that increased erythrocyte S1P promotes pathogenic metabolic reprogramming coupled to increased channeling of glucose to glycolysis rather than through the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increasing deoxygenated sickle Hb (deoxyHbS), deoxyHbS polymerization, sickling, hemolysis and disease progression. S1P functioning intracellularly binds to deoxyHbS, facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the PPP. Extending from SCD, we unexpectedly found that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure of HbA complexed with S1P alone or in combination with 2,3-BPG at 1.9 Å resolution revealed the overall architecture and unique features of S1P-2,3-BPG-deoxyHbA complex. In the presence of 2,3-BPG, S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to a1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with b2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying pathogenic consequences of elevated S1P in SCD and its potential role in normal erythrocyte physiology. DisclosuresKato:Mast Therapeutics: Consultancy; Bayer: Research Funding.

Hypoxia-mediated impaired erythrocyte Lands’ Cycle is pathogenic for sickle cell disease

Although Lands’ cycle was discovered in 1958, its function and cellular regulation in membrane homeostasis under physiological and pathological conditions remain largely unknown. Nonbiased high throughput metabolomic profiling revealed that Lands’ cycle was impaired leading to significantly elevated erythrocyte membrane lysophosphatidylcholine (LysoPC) content and circulating and erythrocyte arachidonic acid (AA) in mice with sickle cell disease (SCD), a prevalent hemolytic genetic disorder. Correcting imbalanced Lands’ cycle by knockdown of phospholipase 2 (cPLA2) or overexpression of lysophosphatidycholine acyltransferase 1 (LPCAT1), two key enzymes of Lands’ cycle in hematopoietic stem cells, reduced elevated erythrocyte membrane LysoPC content and circulating AA levels and attenuated sickling, inflammation and tissue damage in SCD chimeras. Human translational studies validated SCD mouse findings and further demonstrated that imbalanced Lands’ cycle induced LysoPC production directly promotes sickling in cultured mouse and human SCD erythrocytes. Mechanistically, we revealed that hypoxia-mediated ERK activation underlies imbalanced Lands’ cycle by preferentially inducing the activity of PLA2 but not LPCAT in human and mouse SCD erythrocytes. Overall, our studies have identified a pathological role of imbalanced Lands’ cycle in SCD erythrocytes, novel molecular basis regulating Lands’ cycle and therapeutic opportunities for the disease.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications.

Clot Contraction-Mediated Erythrocyte Packing Is Significantly Altered in Sickle Cell Disease

Background: The genetic mutation of sickle cell disease (SCD) leads to polymerized hemoglobin, causing altered erythrocyte shape, increased stiffness, and dehydration, all of which are associated with vascular occlusion and negative clinical outcomes (Bartolucci, Blood, 2012). Stroke in SCD is a major cause of mortality and morbidity (Bernaudin, Blood, 2011), but little is known about the underlying mechanisms. Recently, in vivo arterial thrombi were noted to comprise a core of tightly packed, polyhedral shaped erythrocytes formed during platelet-mediated clot contraction (Cines 2014). Such a core would likely be altered in SCD due to the altered shape and increased stiffness of SCD erythrocytes causing altered clot mechanics, with important implications in clot dissolution, thromboembolism, and cerebral infarct. Here we present initial data indicating that the unique shape and stiffness of SCD erythrocytes greatly affects the packing and fibrin density in the erythrocyte core of SCD clots.Results: We examined clots in oxygenated conditions from healthy individuals (AA hemoglobin genotype) and SCD patients not on hydroxyurea under oxygenated and deoxygenated conditions (SS hemoglobin genotype). Within 12 hours of collection for each sample, thirty microliters of citrated blood were recalcified and clotted with 1U/mL thrombin. After one hour, clots are extensively washed in a sodium cacodyl buffer, fixed in 2% glutaraldehyde, dehydrated, cleaved (Fig 1A), and imaged using scanning electron microscopy (Fig 1B-D). Fibrin density was quantified by dividing SEM images into 399 individual regions of equal size and recording either a presence or absence of fibrin in each region (Fig 1E). Red cell type percentage was quantified by distinguishing three cell types (amorphous, polyhedral, and sickled) and counting the number of each cell type present in a given image (Fig 1F). Red cells from AA individuals had the same polyhedral shape as previously observed (Fig 1B). In SCD (Fig 1C), fibrin is much more abundant, and present in nearly 50% of the image vs 10% for AA cells (Fig 1E). In SCD, the planar and smooth polyhedral sides of packed erythrocytes is less pronounced, and many of the cells have a more rounded or amorphous appearance, with very few cells displaying the classic polyhedral shape (4%) (Fig 1F). In deoxygenated conditions the erythrocytes appear more elongated, presumably due to increased polymerization of sickle hemoglobin. We also observe situations where a sickled erythrocyte is completely enveloped by a neighboring erythrocyte, indicating a high degree of heterogeneity in deoxygenated conditions. Finally, unlike AA erythrocytes, SCD erythrocytes do not appear to share common geometrical boundaries indicating a lower packing densityConclusions and Ongoing Efforts: Taken together, the altered packing density, amorphous shape, and higher fibrin content could play a pivotal role in thromboembolism. As SCD erythrocytes have a higher variance in mechanical stiffness as compared to normal erythrocytes, the heterogeneous packing density and increased fibrin content in SCD clots under physiologically flow conditions could potentially cause weaker parts of the clot to break apart in large, stiff cellular aggregates, contributing to thromboemboli that lead to microvascular occlusion or stroke. Whereas the extra fibrin stabilizes some portions of the clot, the heterogeneous packing could lead to microfissures which destabilize other portions of the clot. Other complementary work such as microfluidic experiments to measure stability and embolic tendency of SCD clots under flow as well as mechanical measurements of SCD clots are currently ongoing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Photophysical characterization of sickle cell disease hemoglobin by multi-photon microscopy.

The photophysical properties of human sickle cell disease (SCD) Hemoglobin (Hb) is characterized by multi-photon microscopy (MPM). The intrinsic two-photon excited fluorescence (TPEF) signal associated with extracted hemoglobin was investigated and the solidified SCD variant (HbS) was found to demonstrate broad emission peaking around 510 nm when excited at 800 nm. MPM is used to dynamically induce and image HbS gelling by photolysis of deoxygenated HbS. For comparison, photolysis conditions were applied to a healthy variant of human hemoglobin (HbA) and found to remain in solution not forming fibers. The use of this signal to study the mechanism of HbS polymerization associated with the sickling of SCD erythrocytes is discussed.

Deformability analysis of sickle blood using ektacytometry.

Sickle cell disease (SCD) is characterized by decreased erythrocyte deformability, microvessel occlusion and severe painful infarctions of different organs. Ektacytometry of SCD red blood cells (RBC) is made difficult by the presence of rigid, poorly-deformable irreversibly sickled cells (ISC) that do not align with the fluid shear field and distort the elliptical diffraction pattern seen with normal RBC. In operation, the computer software fits an outline to the diffraction pattern, then reports an elongation index (EI) at each shear stress based on the length and width of the fitted ellipse: EI=(length-width)/(length+width). Using a commercial ektacytometer (LORCA, Mechatronics Instruments, The Netherlands) we have approached the problem of ellipse fitting in two ways: (1) altering the height of the diffraction image on a computer monitor using an aperture within the camera lens; (2) altering the light intensity level (gray level) used by the software to fit the image to an elliptical shape. Neither of these methods affected deformability results (elongation index-shear stress relations) for normal RBC but did markedly affect results for SCD erythrocytes: (1) decreasing image height by 15% and 30% increased EI at moderate to high stresses; (2) progressively increasing the light level increased EI over a wide range of stresses. Fitting data obtained at different image heights using the Lineweaver-Burke routine yielded percentage ISC results in good agreement with microscopic cell counting. We suggest that these two relatively simple approaches allow minimizing artifacts due to the presence of rigid discs or ISC and also suggest the need for additional studies to evaluate the physiological relevance of deformability data obtained via these methods.