Antisickling Effects of Quercetin may be Associated with Modulation of Deoxyhaemoglobin, 2, 3-bisphosphoglycerate mutase, Redox Homeostasis and Alteration of Functional Chemistry in Human Sickle Erythrocytes.

It is now glaring that sickle cell anaemia is still one of the highest leading inbred hemoglobinopathy amongst Africans. This study examined the antisickling effects of quercetin via modulation of deoxy-haemoglobin, redox homeostasis and alteration of functional chemistry in human sickle erythrocyte using in silico and in vitro models while espousing preventive and curative approaches. Quercetin was docked against deoxy-haemoglobin and 2, 3-bisphosphoglycerate mutase, with binding energies (−30.427 and −21.106 kcal/mol) and Ki of 0.988μM and 0.992μM at their catalytic sites via strong hydrophobic and hydrogen bond interactions. Induction of sickling was done using 2% metabisulphite at 3h. Treatment with quercetin prevented sickling outstandingly at 5.0μg/mL and reversed same at 7.5μg/mL, 83.6% and 75.9%, respectively. Quercetin also significantly (P<0.05) maintained the integrity of erythrocyte membrane apparently from the observed % haemolysis relative to untreated. Quercetin significantly (P<0.05) prevented and counteracted lipid peroxidation while stimulating GSH and CAT levels which were detected to considerably (P<0.05) increase with simultaneous significant (P<0.05) reduction in SOD level based on curative approach. Umpiring from our FTIR results, a favorable alteration in the part of functional chemistry in terms of shifts (bend and stretches) and functional groups were observed relative to the induced erythrocyte/untreated. Thus, antisickling effects of quercetin may be associated with modulation of deoxy-haemoglobin, redox homeostasis and alteration of functional chemistry in human sickle erythrocytes.

Decreased Erythrocyte Binding Capability for Neutrophil Siglec-9 Is a Source of Oxidative Stress in Sickle Cell Disease

Introduction Oxidative stress and inflammation promote hemolysis and vaso-occlusion in sickle cell disease (SCD). Erythrocytes can play a pro-oxidative and anti-oxidative role in disease-associated inflammation through iron-driven free radical production and endogenous anti-oxidants. In SCD, HbS accelerated auto-oxidation, iron de-compartmentalization, and inflammatory cell-derived oxidants drive this oxidative stress. CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids in "Self-Associated Molecular Patterns" (SAMPs) and typically inhibit innate immune cell functions via cytosolic signaling. Recent studies have shown that Siglec-9 on human neutrophils in circulating blood interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactivity, including reactive oxygen species (ROS) production. Modification of erythrocyte membrane sialic acids interferes with their ability to inhibit neutrophil activation and oxidative burst. As erythrocytes age and undergo cellular damage there is a loss of membrane bound sialoglycoproteins. Several studies have indicated that this reduction in the sialome is accelerated on the sickle erythrocyte. We hypothesize that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and that decreased binding of neutrophil Siglec-9 to sickle erythrocyte sialic acid enhances neutrophil activation and oxidative burst. Methods & Results Binding of recombinant Siglec-9-Fc protein to AA (n=9) and SS erythrocytes (n=7) was measured using flow cytometry. SS erythrocytes displayed significantly less Siglec-9-Fc binding 45% ± 11.9 (mean ± SEM) compared to AA erythrocytes 82% ± 5.4 (p=.03). Treatment of AA erythrocytes with neuraminidase to remove sialic acid decreased binding to 4% ± 7.9. Neutrophil ROS production was measured by flow cytometry using dihydrorhodamine 123 (DHR123). Neutrophils were purified from AA donors (n=5) and SS (n=11) or AA erythrocytes (n=5) were added to the neutrophils at a ratio of 50 erythrocytes to 1 neutrophil. Oxidative burst was stimulated using phorbol 12-myristate 13-acetate (PMA). AA erythrocytes decreased PMA-stimulated neutrophil ROS production by 84% ± 6.7. In contrast, SS erythrocytes decreased PMA-stimulated neutrophil ROS production by 53% ± 6.8 (p=0.03,). Recent studies have shown that neutrophil extracellular trap (NET) formation is pathogenic in SCD. We added AA and SS erythrocytes (50:1) to neutrophils stimulated with PMA and NET formation was assessed using Syto Orange, Syto Green and confocal microscopy. PMA-stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation. In contrast, AA erythrocytes treated with neuraminidase to remove sialic acid had increased NET formation. PMA-stimulated neutrophils incubated with SS erythrocytes showed increased NET formation. Conclusions A constant disease state of oxidative stress and inflammation underlies SCD pathophysiology. These data demonstrate that SS erythrocytes with decreased membrane sialic acid are deficient in binding to neutrophil Siglec-9. The decreased binding of SS erythrocytes to neutrophil Siglec-9 diminishes the ability of SS erythrocytes to properly modulate neutrophil activation, which may contribute to the oxidative stress and increased state of basal inflammation inherent to SCD. The overall reduction in number of RBCs in SS may also be a factor? Figure. Figure. Disclosures Belcher: CSL Behring: Research Funding. Vercellotti:CSL Behring: Research Funding.

Metabolomic Profiling Identifies Hypoxia-Induced Imbalanced Lands' Cycle Promotes Sickling and Disease Progression

Sickle cell disease (SCD) is a prevalent hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although it is well accepted that deoxygenation and polymerization of sickle hemoglobin (HbS) are initial triggers for sickling, it has been known for more than three decades that abnormal membrane lipid organization and composition are found in sickle erythrocyte. Early studies showed some lipids are altered in sickle erythrocytes, however, no studies have identified overall membrane lipid alteration and functional role of those altered specific lipids in SCD. Using unbiased metabolomic profiling, we found that lysophospholipids (LPLs), particularly lysophosphocholines (LysoPCs), were significantly elevated inside erythrocytes of mice with SCD due to imbalanced Lands' cycle. Lands' cycle containing two concerted enzymes: phospholipases A2 (PLA2s) and lysophospholipid acyltransferases (LPLATs) was initially discovered in 1958. However, its function and cellular regulation in membrane homeostasis in SCD remain unrecognized prior to our metabolomics screening. Here, we demonstrated that enhancing imbalanced Lands' cycle promotes a process of sickling and disease progression in mice by inducing LysoPC content inside erythrocytes. Significantly, correcting impaired Lands' cycle reduced LysoPC levels within erythrocytes and attenuated sickling and disease progression in mice. Mechanistically, we revealed that hypoxia-mediated MEK/ERK activation underlies imbalanced Lands' cycle by preferentially inducing activity of PLA2 but not LPCAT1 in mouse sickle erythrocytes. Additionally, the detrimental role of impaired Lands' cycle-induced LysoPC production in sickling via MEK/ERK-dependent activation of PLA2 in SCD patients mirrors our mouse finding. Overall, our studies have identified a pathological role of imbalanced Lands' cycle in SCD, revealed molecular basis regulating Lands' cycle and immediately provided novel therapeutic possibilities for the disease. Disclosures No relevant conflicts of interest to declare.

Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity

Key Points Adenosine signaling via ADORA2B induces SphK1 activity in sickle and normal erythrocytes via PKA-mediated ERK1/2 activation. Lowering adenosine by PEG-ADA or interfering ADORA2B activation by specific antagonist decreases SphK1 activity in normal and sickle RBCs.

The Anti-Sickling Agent Aes-103 Decreases Sickle Erythrocyte Fragility, Hypoxia-Induced Sickling and Hemolysis In Vitro

Background The polymerization of hemoglobin S results in red cell morphological changes and fragility, which promotes hemolysis. Aes-103 (5-hydroxymethylfurfural, 5-HMF) is a clinical-stage candidate anti-sickling agent that binds to alpha subunits of hemoglobin, increases its oxygen affinity and stabilizes the R-state. In vitro at, millimolar levels, it inhibits hypoxia-induced sickling and in vivo protects sickle cell mice against hypoxia-induced death. We have found in vitro that Aes-103 increases oxygen affinity of red cells from healthy control subjects and from patients with sickle cell disease either on or off hydroxyurea. We further investigated the ability of Aes-103 to protect red cells from patients with sickle cell anemia from a variety of effects of sickling. First, we used transmission electron microscopy (TEM) to investigate fiber formation in human sickle blood incubated with 5 mM Aes-103 for one hour at 37° prior to deoxygenation (2% oxygen for two hours) and fixed with glutaraldehyde, and embedded by standard techniques. Semi-quantitative analysis of the images showed that the addition of Aes-103 was able to significantly reduce the percentage of cells displaying elongated fibers (p=0.048). Furthermore, we confirmed that Aes-103 incubation prior to hypoxia also reduces morphological sickling as quantified by sickle imaging flow cytometry (P=0.0027). Fiber formation was associated with morphological sickling (R=0.522, P=0.027). Second, we investigated the effects of Aes-103 on sickle erythrocyte fragility. In vitro shear stress induced by rotation on a vertical rotator at 21 revolutions per minute for 3 hours promoted hemolysis in blood samples from patients with SCA (free hemoglobin 29.4 ± 3.4 vs. 8.4 ± 0.9 mM, p<0.001, n=10). Addition of Aes-103 at increasing concentrations for one hour prior to testing reduced the extent of shear-stress induced hemolysis, ranging from decreases of 15% at 1 mM to 28% at 2mM to 37% at 5 mM (p<0.001). Interestingly, although shear stress promoted less hemolysis in blood samples from healthy controls, Aes-103 reduced hemolysis in healthy control blood to a comparable extent, suggesting a red cell stabilizing mechanism distinct from any anti-sickling effect. Third, we evaluated the degree of hemolysis associated with erythrocyte sickling induced by deoxygenation. Hypoxia (2% oxygen for 2 hours) induced hemolysis, and the severity of hemolysis was reduced by the addition of Aes-103 for one hour (prior to hypoxia) at increasing concentrations up to 5 mM (free hemoglobin 31.3 ± 2.9 vs. 21.3 ± 2.8 mM, p = .002, n=11). In summary, treatment of sickle blood with Aes-103 in vitro, decreases fiber formation, increases oxygen affinity of sickle red cells, reduces sickle cell mechanical fragility, and reduces hemolysis caused by hypoxia-induced sickling. Aes-103 merits investigation as a potential alternative or adjunct to hydroxyurea as a treatment for sickle cell disease. We have completed a phase I clinical trial of oral Aes-103 at the NIH Clinical Center, and a separate abstract reports its apparent safety and tolerability. A phase 2 twenty-eight day study is being initiated in the U.K. in adults with sickle cell anemia. Disclosures: No relevant conflicts of interest to declare.

Protein Disulfide Isomerase Regulates Sickle Erythrocyte Volume Via ET-1 Dependent Casein Kinase II Mechanism

Abstract 2114 Disordered K+ efflux and osmotically induced water loss leads to red blood cell (RBC) dehydration and plays a role in the pathophysiology of Sickle Cell Disease. We previously reported that activation of endothelin-1 (ET-1) receptors in sickle erythrocyte was partially responsible for dense sickle cell formation. However, the mechanism by which ET-1 regulates RBC volume remains unclear. Serine/threonine kinases have been shown to regulate K+ transport in RBC. Casein Kinase II (CK2), a serine/threonine kinase, phosphorylates acidic proteins, regulates calmodulin activity and cytoskeletal proteins and is present in RBC. CK2 activity is blocked by apigenin, emodin, heparin, and ornithine decarboxylase. Previous reports have shown a role for flavonoids such as apigenin as substrates for erythrocyte plasma membrane oxidoreductases. We recently observed a role for Protein Disulfide Isomerase (PDI) in regulating cellular hydration and K+ efflux in human RBC. PDI catalyzes disulfide interchange reactions in the plasma membrane, mediates redox modifications and is up-regulated under hypoxic conditions. However the relationship between CK2 and PDI in the setting of cellular hydration status is un-explored. Our results indicate that erythrocyte membrane CK2 activity increases when sickle cells are incubated with 500 nM ET-1 for 30 min (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 106 cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 106 cell, n=3, p<0.04) and 20 μM apigenin (2.7 ± 0.4 nmol/min/mL * 106 cell, n=3, p<0.04). We examined the role of CK2 activation on cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), a specific CK2 inhibitor, and measured the changes in erythrocyte density by phthalate oil density analysis. We observed that inhibition of CK2 led to reduced deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). We then studied the role of CK2 inhibitors on PDI activity by Insulin Turbidity Assay and observed that apigenin and TBB led to significant reductions in PDI activity in vitro (64% and 42% respectively). We also studied the effects of the flavonoids: naringenin, naringin, apigenin and rutin on PDI activity and observed reductions in PDI activity that were greater with apigenin>rutin>TBB>naringin>naringenin (n=2, P<0.05). Furthermore, we observed that K+ flux via Gardos channel activation is correlated with PDI activity in vitro in sickle erythrocytes. Taken together our results implicate CK2 and PDI as intermediate regulators of ET-1 stimulated cellular volume systems in red blood cells. Supported by NIH R01-HL09632 to AR. Disclosures: No relevant conflicts of interest to declare.

Endothelin-1 Receptor Antagonists Regulate Cell Surface-Associated Protein Disulfide Isomerase In Sickle Cell Disease

Abstract 265 Erythrocyte hydration status and endothelial cell activation have been proposed as important contributors to vaso-occlusion and impaired blood flow in the pathophysiology of sickle cell disease (SCD). However, the physiological mechanism(s) that mediate the interplay between erythrocytes hydration status and the endothelium in SCD are unclear. We have recently reported a role for dual endothelin-1 receptor antagonists in improving sickle erythrocyte hydration status and K+ transport in vivo via modulation of Gardos channel activity (Rivera A., 2008, Amer J Physiol). The Gardos channel is an important contributor to sickle erythrocyte dehydration that maybe modulated by protein disulfide isomerase (PDI). PDI in leukocytes has been reported to catalyze disulfide interchange reactions, mediate redox modifications and has been observed to be up-regulated under hypoxic conditions. We report the detection of PDI by western blot analyses in membranes from both human and mouse sickle erythrocytes. We observed greater levels of cell surface associated PDI in sickle vs Hb A-containing erythrocytes. We also quantified PDI activity and observed a significant correlation between Gardos channel activity and cell surface associated PDI activity in human sickle erythrocytes and Hb A-containing cells (n=40, r2=0.3046, p=0.0002). In fact, closer examination revealed that sickle erythrocyte membranes had higher PDI activity than Hb A-containing erythrocyte membranes (5.07±0.4 vs 1.30±0.1%, n=22 and 18, respectively p<0.0001). Similar results were observed in membrane preparations of erythrocytes isolated from the BERK sickle transgenic mouse model when compared with wild-type controls. Consistent with a functional role for PDI in Gardos channel activation, we also observed that sickle erythrocytes incubated in cycles of oxygenation/de-oxygenation for 3 hr in the presence of PDI antibodies were associated with reduced sickle dense cell formation. Similar results were observed with bacitracin, another PDI inhibitor. We then treated BERK mice with dual ET-1 receptor antagonists (BQ123/BQ788) for 14 days and measured erythrocyte surface associated PDI activity. We observed that as with Gardos channel activity, cell surface associated PDI activity was significantly reduced following treatment with BQ123/BQ788 (8.80±0.5 to 6.4±0.6%, n=3 P<0.02). These changes were associated with an increase in erythrocyte MCV (31.3±1.63 to 40.4±0.35 fL, n=3, p<0.002) and a decrease in MCHC (40.4±0.8 to 27.4±3 g/dL, n=3, p<0.003). We then studied the direct effects of ET-1 on the human endothelial cell line, EA.hy926 (EA), as well as in primary cultures of BERK mouse aortic endothelial cells (BMAEC). Using quantitative RT-PCR with Taqman chemistries and GAPDH and beta-actin as endogenous controls, we observed that stimulation of EA cells with 100nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Similar results were observed on PDI mRNA expression in BMAEC isolated and cultured for 10 days then incubated with 100 nM ET-1 for 4 hr. Thus, our results strongly implicate cell surface associated PDI in cellular hydration status and its regulation by ET-1. We posit that aberrant regulation of PDI activity and/or its expression and secretion from either erythrocytes or endothelial cells represent a novel target aimed at ameliorating the complications associated with the pathophysiology of Sickle Cell Disease. Supported by NIH R01HL090632 to AR. Disclosures: No relevant conflicts of interest to declare.