Sickle Cell Pathophysiology Research Articles

The role of nucleation in sickle cell pathophysiology: opportunities for innovative treatments

The role of nucleation in sickle cell pathophysiology: opportunities for innovative treatments

Stroke in sickle cell patients, epidemiology, pathophysiology, systemic and surgical treatment options and prevention strategies

AbstractBackgroundA hereditary haemoglobinopathy known as sickle cell disease (SCD) affects over 100 000 people in the United States severely. Cerebrovascular disease is a prominent consequence of SCD. By the age of 30, 53% of patients have silent cerebral infarcts (SCIs) (a stroke that occurs without any obvious symptoms because it damages a small part of the brain that isn't responsible for any essential functions), and by the age of 40, 3.8% have overt strokes.Main bodyThe multidimensional burden of cerebrovascular illness in SCD is reviewed in detail in this article, which includes both clinical strokes and the frequently asymptomatic SCIs. The intricate pathophysiology of SCD and stroke is explored. With SCD, there are currently very few methods for preventing primary and secondary stroke; the most common ones are hydroxyurea and blood transfusion. Nevertheless, not enough research has been done on the possible contributions of anticoagulation and aspirin to strokes linked to SCD. Promising evidence is also highlighted in the study, suggesting that new drugs intended to treat SCD may be able to alleviate leg ulcers and renal impairment in addition to reducing unusually high transcranial Doppler flow velocity – a crucial component of cerebrovascular events. Given that these novel medications specifically target haemolysis and vaso‐occlusion, the two main causes of strokes in this population, more research is desperately needed to determine whether they are effective in avoiding strokes in people with SCD. The literature review also emphasizes how common healthcare inequities are and how they hinder advancements in SCD research and management in the United States.ConclusionTo successfully address these inequities, the evaluation recommends more funding for both SCD management and research, as well as for patient and clinician education. This multimodal viewpoint highlights the intricate terrain of cerebrovascular problems associated with SCD and the urgent need for all‐encompassing and fair strategies to improve patient outcomes and advance research.

Improvements in Health-Related Quality of Life for Patients Treated with LentiGlobin for Sickle Cell Disease (bb1111) Gene Therapy

Improvements in Health-Related Quality of Life for Patients Treated with LentiGlobin for Sickle Cell Disease (bb1111) Gene Therapy

Correlation of lipid peroxidation and nitric oxide metabolites, trace elements, and antioxidant enzymes in patients with sickle cell disease.

BackgroundLipid peroxidation plays a very important role in sickle cell pathophysiology. The formation of malondialdehyde (MDA) in patients with sickle cell disease (SCD) may lead to endothelial dysfunction. Nitric oxide (NO) is a known vasodilator which plays a role in endothelial function. The current study determined the association between MDA and NO metabolites (NOx), trace elements, and antioxidant enzymes (SOD and CAT) in patients with SCD. The ratio of MDA/NOx was also determined as an index of oxidative stress in the study groups.MethodsThis was a cross‐sectional study involving 90 patients with SCD and 50 “healthy” controls. Blood samples (n = 140) were collected from the study groups. The plasma, sera, and red cells were kept at −20°C for biochemical analyses. Hemoglobin (Hb) and NOx levels were determined in the plasma using Labsystem Multiskan MS and Griess reagent system, respectively. Super oxide dismutase (SOD) and catalase (CAT) levels were determined in the red cells using assay kits from Cayman chemicals. Lipid peroxidation biomarker MDA was determined in the sera using the TBARS assay. Levels of iron (Fe), copper (Cu), and zinc (Zn) were also determined in the sera using Variant 240FS. MDA and NOx ratio was computed for the study groups and compared.ResultsLevels of Hb, NOx, SOD, CAT, and Zn were significantly lower in the patients with SCD (P < .001). MDA, Fe, and MDA/ NOx ratio were, however, significantly higher in the patients with SCD (P < .001). There was no significant correlation between MDA and NOx, SOD, CAT, Fe, and Zn in the study groups. MDA, however, correlated positively and significantly with Cu in the HbSS patients with vaso‐occlusive crises (VOC). Gender did not affect the levels of oxidative stress markers.ConclusionsFindings from this study suggest a link between lipid peroxidation and Cu in HbSS patients with VOC. Increased MDA/NOx ratio may contribute to sickle cell pathophysiology by promoting oxidative stress.

Sickle Cell Disease-Genetics, Pathophysiology, Clinical Presentation and Treatment.

Sickle cell disease (SCD) is a monogenetic disorder due to a single base-pair point mutation in the β-globin gene resulting in the substitution of the amino acid valine for glutamic acid in the β-globin chain. Phenotypic variation in the clinical presentation and disease outcome is a characteristic feature of the disorder. Understanding the pathogenesis and pathophysiology of the disorder is central to the choice of therapeutic development and intervention. In this special edition for newborn screening for haemoglobin disorders, it is pertinent to describe the genetic, pathologic and clinical presentation of sickle cell disease as a prelude to the justification for screening. Through a systematic review of the literature using search terms relating to SCD up till 2019, we identified relevant descriptive publications for inclusion. The scope of this review is mainly an overview of the clinical features of pain, the cardinal symptom in SCD, which present following the drop in foetal haemoglobin as young as five to six months after birth. The relative impact of haemolysis and small-vessel occlusive pathology remains controversial, a combination of features probably contribute to the different pathologies. We also provide an overview of emerging therapies in SCD.

Sickle cell disease: an inherited thrombophilia.

Activation of the hemostatic system occurs in patients with sickle cell disease. The extent to which this activation contributes to sickle cell pathophysiology is uncertain. Clinical trials of anticoagulants or platelet inhibitors have demonstrated the ability to decrease biomarkers of hemostatic activation, but this has generally not resulted in improvement in clinically relevant outcomes. Venous thromboembolism (VTE: deep venous thrombosis and pulmonary embolism) has been until recently an underappreciated complication of sickle cell disease, with incident event and recurrence rates consistent with a strong thrombophilia. There is no strong evidence that management should differ than for other patients with VTE, with the possible exception that secondary prophylaxis be extended regardless of provocation, given the persistent strong thrombophilic state.

Commonly Used Clinical Intravenous Fluid Formulations Differentially Affect Sickle Red Blood Cell Stiffness and Transit Time

Background:Disruption of red blood cell (RBC) volume regulation and water homeostasis is a major component of sickle cell pathophysiology. As such, hydration is a mainstay of prevention and treatment of vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). However, evidence for guiding clinicians' choice for intravenous (IV) fluid hydration in the acute setting is lacking (Okomo, Coch Data Syst Rev, 2012). Pediatric hematologists typically discourage rapid infusion of hypotonic fluid given the risk of hyponatremia, although such fluids as 5% dextrose with 34meq/L or 77meq/L sodium are often used after stabilizing various acute clinical situations relevant to SCD. Although altered sodium concentration and osmolarity have been shown to affect erythrocyte swelling and rheology, dextrose-containing fluids used clinically such as D5 ¼ and D5 ½ normal saline (NS), have not been studied (Reinhart et al., Mic Res, 2015; Hijiya et al., J Lab Clin Med, 1991). To those ends, we sought to investigate the effect of different clinically relevant IV fluid formulations on normal and sickle RBC stiffness and deformability.Methods:Fresh blood was obtained from healthy volunteers and patients with sickle cell anemia (SS) on hydroxyurea and not transfused for at least 100 days. Sterile, clinical grade fluids stored at room temperature were used for the experiments (Baxter, Figure 1A). Our laboratory has previously published a description of a microfluidic device comprised of multiple parallel microchannels 5μm wide recapitulating the in vivo geometry of capillaries (Rosenbluth et al., Lab Chip, 2008). This microvasculature-on-a-chip enables measurements of single-RBC transit times, which correlate with cell stiffness (Figure 1B,C). For the transit time experiments, a master silicon wafer was used to mold the microfluidic channels out of polydimethylsiloxane (PDMS) silicone. Centrifuged RBCs were washed with phosphate-buffered saline (PBS) and then diluted to 0.5% hematocrit (HCT) in the various fluids prior to flow. Cell suspensions were perfused into a microfluidic device pre-coated with 2% bovine serum albumin (BSA) at an average linear flow rate of 0.50 mm s-1 in the smallest channels with a syringe pump (Harvard Apparatus) and then imaged at 20x at 20 frames per second. Images were recorded for future analysis. For the experiments assessing RBC shape, washed RBCs were diluted with PBS to 0.5% HCT and imaged at 40x in plastic wells at time 0. PBS was removed and 100 μL of each clinical fluid formulation was added to the wells and images were then obtained over time. RBC circularity was calculated using custom-written scripts in Matlab (Figure 1D).Results:RBC transit times in both healthy and sickle blood were affected by osmolarity and the various solute concentrations (Figures 2A,B). Transit times of sickle RBCs in all IV fluid formulations were significantly higher, over 10 times, than that of RBCs from healthy donors. Transit times for both healthy and sickle donors were least in the D5 ¼ NS solution. Of note, sickle RBC transit time was greatest in the NS solution. Sickle RBC circularity also changed with solute concentration and osmolarity with statistical significance (Figure 2C). Cells with the highest change in circularity from baseline were also those exposed to the D5 ¼ NS solution.Conclusion:Our results suggest the stiffness of sickle RBCs is affected by different formulations of clinical IV fluids. Increased transit time of sickle RBCs in NS through our device may in part be explained by the decreased circularity, indicating that RBCs adopt more irregular shapes in this fluid. This, in turn, could lead to increased propensity of microchannel obstruction. Although the exact mechanisms are unclear, this begs the question of whether NS is an appropriate choice for initial fluid resuscitation for VOC and other SCD-related complications as it could exacerbate the already high stiffness and shape irregularity of sickle RBCs, further increasing microvascular occlusion. As these in vitro results have significant clinical implications, ongoing experiments involve investigating how these fluid-dependent effects may alter sickle RBC adhesion in 'endothelialized' microfluidic devices, how different oxygen tensions affect these fluid-mediated effects, potential differences on and off of hydroxyurea, and the underlying mechanisms of this IV fluid formulation-dependent effect. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Extensive Ethnogenomic Diversity of Endothelial Nitric Oxide Synthase (eNOS) polymorphisms

Nitric oxide (NO) is highly reactive, produced in endothelial cells by endothelial NO synthase (eNOS) and has been implicated in sickle cell pathophysiology. We evaluated the distribution of functionally significant eNOS variants (the T786C variant in the promoter region, the Glu298Asp variant in exon 7, and the variable number of tandem repeats (VNTR) in intron 4) in Africans, African Americans and Caucasians. The C-786 variant was more common in Caucasians than in Africans and African Americans. Consistent with other findings, the Asp-298 variant had the highest frequency in Caucasians followed by African Americans, but was completely absent in Africans. The very rare intron 4 allele, eNOS 4c, was found in some Africans and African Americans, but not in Caucasians. eNOS 4d allele was present in 2 Africans. These findings suggest a consistent and widespread genomic diversity in the distribution of eNOS variants in Africans, comparative to African Americans and Caucasians.

Turnover of Heme-Bound Iron Is Associated with Activation of TLR4 and Chemokine Receptor Pathways in the Peripheral Blood Mononuclear Cell Transcriptome in Sickle Cell Anemia

AbstractAbstract 819 IntroductionIt is widely accepted that inflammation plays an important role in the pathophysiology of Sickle Cell Anemia (SCA). Recently a number of studies indicated that peripheral blood mononuclear cell (PBMC) iron may contribute to inflammation in some diseases In SCA, PBMCs are continuously exposed to high turnover of iron in haptoglobin/hemoglobin complexes and free heme due to severe intravascular hemolysis. Therefore, we hypothesized that PBMC iron flux is marked by altered expression of iron-regulated genes, and in turn associated with the up regulation of genes in inflammatory pathways. To explore this hypothesis we correlated the expression of a predefined set of iron regulated genes to the SCA PBMC transcriptome as a whole. MethodsSteady state patients with SCA were selected as published earlier. (Bereal-Williams et al. Haematologica 2012) Most noteworthy exclusion criteria were recent blood transfusion, liver or kidney dysfunction or a recent acute complication. Affymetrix Human Genome U133 Plus 2.0 arrays were used to measure PBMC mRNA profile. The primary source of free iron in PBMC in SCA is iron released from heme by heme oxygenase-1 (HMOX1), the first committed biochemical step in production of bilirubin. Therefore, we planned to validate the clustering of the study participants by correlating the cluster rank with bilirubin plasma levels. Genes which are published to be differentially expressed upon iron loading or chelation were used to prospectively create an ‘iron-regulated' gene set (figure). Differentially expressed genes with the filter of a change greater than 40% between the clustering groups and 10% false discovery rate (FDR) were further analyzed with Ingenuity Pathway Analysis (IPA) System. ResultsTwenty-five subjects with SCA and 9 healthy subjects were analyzed. Hierarchical clustering using the predefined iron regulated gene list identified 3 groups of subjects with high, low and intermediate expression of iron activated genes (figure 1). As expected, none of the healthy controls was found in the high iron cluster. The cluster grouping was validated by correlation of serum bilirubin levels to the cluster rank (Spearman rho=0.358 p=0.044) and the expression of HMOX1 (Spearmen rho=0.387 p=0.034). In analysis of these three iron regulated gene clusters, we found 98 genes which were consistently and significantly differentially expressed, notably many genes important to crucial inflammatory pathways, especially Toll-like receptor 4 and 7 (TLR-4,7), chemokine receptor CCR1, and interleukin 15. The 98 markers identified ten canonical pathways in Ingenuity Pathway Analysis, most involving inflammation (each p<0.004, Table ).Expression profiling using a defined set of iron regulated genes identifies co-regulation of genes and pathways related to inflammatory cytokines, signaling cascades and innate immunity pathways. Our human PBMC findings confirm and extend recently presented data from several other groups linking TLR4-mediated heme-iron toxicity to pathological responses in sickle cell mice and in cultured cells. The role of heme-associated iron in sickle cell pathophysiology merits additional investigation.Tabletop ten canonical pathways associated with increased expression of the pre-defined iron regulated gene set. (p<0.004)NameDifferential regulated genesRole of Pattern Recognition Receptors in Recognition of Bacteria and VirusesTLR4, NLRP3, TLR7, PRKCH, PRKCAIL-10 SignalingCCR1, HMOX1, BLVRA, CD14LPS-stimulated MAPK SignalingTLR4, CD14, PRKCH, PRKCAAltered T Cell and B Cell Signaling in Rheumatoid ArthritisTLR4, TNFSF13, IL15(includes EG:16168), TLR7Communication between Innate and Adaptive Immune CellsTLR4, TNFSF13, IL15(includes EG:16168), TLR7Beta-Adrenergic SignalingGNB4, PRKCH, SLC8A1, PRKCAPhospholipase C SignalingHMOX1, GNB4, ZAP70, PRKCH, MARCKS, PRKCAToll-like Receptor SignalingTLR4, TLR7, CD14fMLP Signaling in NeutrophilsGNB4, CYBB, PRKCH, PRKCAIL-8 Signaling.HMOX1, GNB4, CYBB, PRKCH, PRKCA [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Sickle cell disease: looking back but towards the future

Sickle cell disease (SCD) is a multisystem disease associated with episodes of acute illness and progressive organ damage, and one of the most common monogenic disorders worldwide, affecting an estimated 30 million people. SCD represents a major public health problem because of its associated morbidity and mortality(1,2). Last year marked the 100th year since SCD was first described by James B. Herrick. The myriad clinical features of this hemolytic anemia result from a deceptively simple amino acid substitution of valine for glutamic acid in the sixth position of the s-subunits of hemoglobin (Hb). The result is intracellular polymerization under conditions of hypoxia and dehydration, which triggers the characteristic sickle-shaped erythrocytes(3). Modern advances in molecular and cellular biology have resulted in an accumulation of data on the sickle cell pathophysiology related to erythrocyte and extra-erythrocyte events(1). Since the seminal discovery by Dr. Herrick, we have learnt that SCD is as much a disease of endothelial dysfunction as it is a hemoglobinopathy. Oxidative stress, chronic endothelial damage and hemolysis initiate a cascade of events that result in episodic vaso-occlusion, subsequent ischemia-reperfusion injury, inflammation and organ dysfunction(3).

Measuring the Direct Effects of Sickle Cell Vaso-Occlusion on Endothelial Cells Using Microfluidic Technology

Abstract 897Sickle cell disease is a complex process involving biophysical and biological phenomenon such as microvascular occlusion due to rigid sickle erythrocytes, hemolysis, and aberrant cellular interactions involving endothelial cells and sickle erythrocytes and leukocytes. Indeed, a key aspect of sickle cell pathophysiology is endothelial cell dysfunction. Cardiovascular research in recent years has shown that endothelial cells biologically respond to the local mechanical environment, particularly to the changes in the applied shear stresses (Chiu and Chien, Physiological Reviews, 2011). Interestingly, no studies investigating how the biophysical alterations in sickle cell disease may directly affect endothelial function have been published. The classic view has been that vaso-occlusion is simply due to sickled erythrocytes becoming stuck in microvasculature at low oxygen tensions leading to decreased blood flow and tissue ischemia. However, the mechanical aspects of sickle cell vaso-occlusion themselves, that is, the physical phenomenon of sickling erythrocytes tightly packed in an occluded blood vessel, may directly affect endothelial biology and lead to dysfunction. We hypothesize that these pathologic forces induced by sickling erythrocytes directly lead to dysfunction of endothelial cells, which are mechanosensitive, and contribute to sickle cell pathophysiology. However, these sickling-induced forces and their effects on endothelial cells have been difficult to measure, in part due to a lack of available tools.To that end, we have developed two microfluidic tools to assess the role of sickle-cell vaso-occlusion on endothelial cells. The first device is an in vitro microfluidic platform featuring microchannels the size of post-capillary venules (30 μm) with human endothelial cells cultured within and completely lining the entire inner surface of those microchannels (Figure 1A). This “microvasculature-on-a-chip” enables the visualization of blood cell-endothelial cell interactions during vaso-occlusion under a controlled hemodynamic environment and provides a platform to study the effect of vaso-occlusion on endothelial cells. To date we have characterized this “endothelialized” microfluidic device, showing that endothelial cells are confluent using anti-VE-cadherin immunostaining and adequately generate nitric oxide. Furthermore, we have flowed blood samples from patients with sickle cell disease and found that hydroxyurea treatment both reduces the number of occlusions and increases the mean velocity of the blood traveling through the device, as expected (Figure 1B–E).To decouple whether it is a biochemical or biophysical phenomenon that causes endothelial cell dysfunction during vaso-occlusion, a second micromechanical device was created to quantitatively measure the forces generated by sickling events. The device captures whole blood and will deform outward when forces are applied by the sickle erythrocytes as shown in Figure 2. The membrane above the sickle cells has been coated with 2 μm fluorescent beads which will change focus during deflection. Deflections of one or two beads indicates that a single sickle cell is locally applying force, whereas deflections of large numbers of beads indicates that the cells are collectively applying a pressure to the membrane. The device has been fully fabricated and loaded with blood cells. An accompanying experimental setup enabling the deoxygenation of the device coupled with microscopy has also been created and preliminary tests show successful deoxygenation of sickle erythrocytes from patients with hemoglobin SS disease and the Berkeley sickle cell mouse model.By combining insights gained from each device, future work will determine how the mechanical process of sickling and vaso-occlusion directly affect endothelial function and will lead to a new understanding of sickle cell pathophysiology. Sickle cell vaso-occlusion will be induced in the “endothelialized” microfluidic device while monitoring nitric oxide production and the upregulation of inflammatory markers, such as adhesion molecules and free radicals. The second device will provide quantitative numbers of forces produced by sickling erythrocytes, leading to experiments in which these forces are applied to endothelial cells while monitoring the same metrics. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Sickle Cell Disease Is Associated with Reduced Adenosine Deaminase Catalytic Activity, Resulting in Altered Adenosine Metabolism

Abstract 1078Sickle cell disease (SCD), a chronic hemolytic anemia due to a mutant beta-globin, is associated with prominent inflammatory features with elevated levels of inflammation mediating molecules and an oxidative state, with elevated levels of reactive oxygen species (ROS). Additionally, elevated levels of adenosine (Ado) have recently been shown to play a role in exacerbating sickle cell pathophysiology. However, the etiology of increased Ado is unclear; and presumed to occur secondary to increased ATP release from cellular damage (Xu et al, Nature Medicine, 2010). Herein, we show that increased levels of Ado in the plasma of patients with SCD and the mouse model of SCD [Berkeley sickle (SS) mice] were accompanied by significantly reduced catalytic activity of the Ado degrading enzyme, adenosine deaminase (ADA) in sickle red blood cell (RBCs). ADA activity in Berkeley sickle (SS) RBCs was 246 ± 50 U/L as compared to 570 ± 36 U/L in normal mouse RBCs (derived from Berkeley AA/WT mice), as measured by a color-based enzymatic assay. Notably, despite lower enzyme activity of ADA, immunoblotting assays showed that the levels of ADA protein in sickle RBC were similar, or even slightly higher compared to that in wild type (WT) RBC, suggesting that ADA catalytic activity was impaired in sickle RBCs. In vitro plasma swap experiments, where WT RBC are incubated with sickle plasma and vice versa for an hour, revealed that sickle plasma induced a significant decrease in ADA activity in WT RBC, suggesting that plasma from sickle mice contains a soluble factor that is capable of impairing ADA enzyme activity. Interestingly enough, the decrease in ADA activity correlated inversely with the levels of intracellular RBC ROS in sickle mice. Next we directly assess in vivo the effect of induced oxidative stress on RBC ADA activity in sickle mice by injecting sickle mice with 35umoles/Kg body weight of hemin and saline respectively. Preliminary data suggests a decrease in RBC ADA activity in hemin–injected mice (294 ± 197 U/L) compared to the saline-injected group (346 ± 185.8 U/L), and this data was consistent with a concomitant increase in intracellular RBC and white blood cells ROS levels. We conclude that increase in oxidative stress levels mediates a post translational alteration of ADA catalytic activity and alters Ado metabolism in SCD. Disclosures:No relevant conflicts of interest to declare.

Regulation of Endothelin-1 Stimulated-Ca2+-Activated K+ Channel Activity by Protein Disulfide Isomerase Gene Knockdown in Endothelial Cells

Abstract 1080Endothelial cell activation is as an important contributor to vaso-occlusion in the pathophysiology of Sickle Cell Disease. We have reported a role for dual endothelin-1 (ET-1) receptor antagonists in improving sickle cell pathophysiology in vivo via modulation of the Ca2+-activated K+ channel, KCNN4 (Rivera A., 2008, Amer J Physiol). Protein disulfide isomerase (PDI) catalyzes disulfide interchange reactions in the plasma membrane, mediates redox modifications and is up-regulated under hypoxic conditions. Thus KCNN4 maybe regulated by PDI in endothelial cells. We now report on the detection of KCNN4, PDI and ET-1 receptor B by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We studied the effects ET-1 on cytosolic Ca2+ levels by spectrofluorimetry of FURA-2AM loaded endothelial cells and observed that 10−9−10−7 M ET-1 led to dose-dependent increases in cytosolic Ca2+ levels that were blocked by pre-incubation with the ET-1 receptor B antagonist, BQ-788. We then studied the effects of 100 nM ET-1 on Ca2+-activated K+ channels and observed increased activity (153.7 vs. 187.0 mmol/L × h × mg protein) that was sensitive to clotrimazole (95.8 mmol/L × h × mg protein), a well-known inhibitor of KCNN4. We then studied the effects of two well-described PDI inhibitors, monoclonal antibodies to PDI and bacitracin, on channel activity and showed that these inhibitors significantly decreased channel activity in EA cells. Consistent with these observations, we show that siRNA against PDI likewise led to a reduction in channel activity (153.7 vs 29.5 mmol/L × h × mg protein, P<0.01, n=6) that was associated with significantly reduced PDI mRNA levels but not with scrambled siRNA as determined by quantitative RT-PCR with TaqMan detection probes and GAPDH and beta-2 microglobulin as endogenous controls. We then studied the effects of ET-1 on EA cells as well as in early cultures of mouse aortic endothelial cells (MAEC) using quantitative RT-PCR with TaqMan probes and GAPDH and beta-actin as endogenous controls and observed that stimulation of EA cells with 100 nM 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 MAEC incubated with 100 nM ET-1 for 4 hr. Furthermore, incubation of EA cells for 12 hr with 10 nM ET-1 led to increases in cell-associated PDI levels by western blot analyses. We then tested the in vivo effects of ET receptor antagonist in sickle transgenic BERK mice and observed a reduction of plasma PDI activity when compared to vehicle treatment (67.7±3 to 34.3±6, Relative Fluorescence Units, P<0.03, n=3). In these mice, we also observed a positive correlation in erythrocyte Gardos channel activity and PDI activity. Thus, our results implicate PDI as a novel regulator of KCNN4. We posit that regulation of PDI activity represents a novel target aimed at ameliorating complications associated with the pathophysiology of Sickle Cell Disease. Supported by NIH R01HL090632 to AR. Disclosures:No relevant conflicts of interest to declare.

Alloimmunization to transfused HOD red blood cells is not increased in mice with sickle cell disease

Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease. Transgenic sickle mice, which express human α and β(S) globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffy(b) ) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array. Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals. In sum, homozygous β(S) expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.

Sickle cell anemia pathophysiology: Back to the data

Sickle cell anemia pathophysiology: Back to the data

Current Understanding in the Management of Sickle Cell Disease

Sickle cell disease (SCD), the commonest monogenetic disorder worldwide, represents a major public health burden because of its significant morbidity and mortality. Advances in molecular and cellular biology have resulted in an accumulation of knowledge on sickle cell pathophysiology and broadened our understanding of the complexity of this molecular disease with heterogeneous manifestations. Natural history studies and clinical trials have provided incremental data on clinical features, complications, and predictors of severity in SCD and, above all, have laid important recommendations for prevention and treatment of complications. Disease modifying therapies that have significantly improved survival of SCD patients have been identified over recent years. Despite increasingly successful therapies and better overall survival, patients continue to die especially with increasing age and health providers caring for SCD patients face major challenges. This article will highlight modern management of SCD and its impact on the lives of affected patients.

Identification of genetic polymorphisms associated with risk for pulmonary hypertension in sickle cell disease

AbstractUp to 30% of adult patients with sickle cell disease (SCD) will develop pulmonary hypertension (pHTN), a complication associated with significant morbidity and mortality. To identify genetic factors that contribute to risk for pHTN in SCD, we performed association analysis with 297 single nucleotide polymorphisms (SNPs) in 49 candidate genes in patients with sickle cell anemia (Hb SS) who had been screened for pHTN by echocardiography (n = 111). Evidence of association was primarily identified for genes in the TGFβ superfamily, including activin A receptor, type II–like 1 (ACVRL1), bone morphogenetic protein receptor 2 (BMPR2), and bone morphogenetic protein 6 (BMP6). The association of pHTN with ACVRL1 and BMPR2 corroborates the previous association of these genes with primary pHTN. Moreover, genes in the TGFβ pathway have been independently implicated in risk for several sickle cell complications, suggesting that this gene pathway is important in overall sickle cell pathophysiology. Genetic variation in the β-1 adrenergic receptor (ADRB1) was also associated with pHTN in our dataset. A multiple regression model, which included age and baseline hemoglobin as covariates, retained SNPs in ACVRL1, BMP6, and ADRB1 as independently contributing to pHTN risk. These findings may offer new promise for identifying patients at risk for pHTN, developing new therapeutic targets, and reducing the occurrence of this life-threatening SCD complication.

The proverbial chicken or the egg? Dissection of the role of cell-free hemoglobin versus reactive oxygen species in sickle cell pathophysiology

SICKLE CELL PATHOPHYSIOLOGY comprises a complex interplay of episodic vasoocclusive events, ischemia-reperfusion injury, overproduction of reactive oxygen species (ROS), inflammation, endothelial activation, and hemolysis, all somehow driven by a single amino acid substitution in the -globin chain of hemoglobin. Hemolysis and oxidative stress act synergistically to promote vascular dysfunction in sickle cell disease (SCD). As a result of chronic hemolysis, levels of free plasma hemoglobin are increased at baseline and nitric oxide (NO) bioavailability is diminished, producing endothelial dysfunction that has been linked to chronic vasculopathic complications of SCD such as pulmonary hypertension, cutaneous leg ulceration, priapism, and sudden death (14, 18, 19, 22, 23, 25). NO regulates vasorelaxation and also possesses antioxidant, antiadhesive, and antithrombotic properties (33). NO is produced from the substrate L-arginine by endothelial nitric oxide synthase (eNOS) and mediates vasorelaxation through a paracrine action on vascular smooth muscle cells underlying the endothelium. Endothelial dysfunction, characterized by impaired vascular responsiveness resulting from decreased NO bioavailability, is associated with atherosclerosis, diabetes mellitus, hypertension, hypercholesterolemia, smoking, and obesity, illustrating the central importance of NO in the physiological regulation of vasomotor activity (3, 6).

Sickle Cell Disease: New Insights into Pathophysiology and Treatment

&lt;P&gt;Sickle cell disease, the most common monogenetic disorder worldwide affecting an estimated 30 million people, represents a major public health concern because of its associated significant morbidity and mortality. Modern advances in molecular and cellular biology have resulted in an accumulation of data on sickle cell pathophysiology related to erythrocyte and extra-erythrocyte events. These emerging data have broadened our understanding of the complexity of this molecular disease with heterogeneous manifestations and have paved the route to the development of novel and targeted therapeutic interventions. With the increasing scope of knowledge about this disease, it is imperative that pediatricians acquaint themselves with the many clinical presentations and the new insights into pathophysiology and treatment of this disorder.&lt;/P&gt;&lt;H4&gt;ABOUT THE AUTHORS&lt;/H4&gt;&lt;P&gt;Adlette Inati, MD, is with Rafik Hariri University Hospital, Division of Pediatric Hematology and Oncology, Beirut, Lebanon, the Chronic Care Center, Hazmieh, Lebanon, and the University of Balamand, Koura, Lebanon. Suzan Koussa, MD, is with the Chronic Care Center, Hazmieh. Ali Taher, MD, is with the American University of Beirut Medical Center, Department of Internal Medicine, and Chronic Care Center, Hazmieh. Susan Perrine, MD, is with the Boston University School of Medicine, Boston, Massachusetts.&lt;/P&gt;&lt;P&gt;Address correspondence to: Adlette Inati, MD, Division of Pediatric Hematology Oncology, Children’s Center for Cancer and Blood Diseases, Rafik Hariri University Hospital, Beirut, Lebanon; or e-mail &lt;a href="mailto:khorina@dm.net.lb"&gt;khorina@dm.net.lb&lt;/a&gt;.&lt;/P&gt;&lt;P&gt;Dr. Inati, Dr. Koussa, and Dr. Taher have disclosed no relevant financial relationships. Dr. Perrine has disclosed the following relevant financial relationships: HemaQuest Pharmaceuticals: Employee/stockholder. &lt;/P&gt;&lt;H4&gt;EDUCATIONAL OBJECTIVES&lt;/H4&gt;&lt;OL&gt;&lt;LI&gt;Discuss the current pathophysiologic model of sickle cell disease and genotype-phenotype correlation.&lt;/LI&gt;&lt;LI&gt;Describe the predictors of disease severity and identify reasons for improved survival.&lt;/LI&gt;&lt;LI&gt;Review disease-modifying therapies and understand the mechanism of action of novel targeted treatments.&lt;/LI&gt;&lt;/OL&gt;

Oxidative Modifications to Flippase and Scramblase Affect Phosphatidylserine Exposure in Mouse Sickle Cells.

A subset of sickle red blood cells shows phosphatidylserine (PS) exposure on the red blood cell (RBC) surface. PS exposure has consequences for sickle cell pathophysiology, including an imbalance in hemostasis, enhanced cell recognition and adhesion to the endothelium. PS-exposing RBC are found among young reticulocytes as well as old dense sickle cells, but it is poorly understood how the PS-exposing cells are formed. PS exposure occurs when phospholipids are randomized across the membrane by the calcium-activated phospholipid scramblase. In addition, the flippase, which is responsible for transporting PS from the outer to the inner monolayer, needs to be inactivated. Indeed, we have previously found that all highly PS-exposing sickle cells have lost their flippase activity. We hypothesized then that transient calcium influx in RBC without flippase activity would result in PS exposure in sickle cells. However, brief calcium influx in normal red cells is not sufficient to induce PS exposure. Here we show that the scramblase activity is also altered by oxidative damage in sickle cells, which contributes to a higher susceptibility to calcium-induced PS exposure. We modified the sulfhydryl moieties of the flippase and the scramblase to see their respective effect on PS scrambling in sickle mouse (Berkeley type) and normal mouse RBC. We evaluated the scrambling rate by monitoring the appearance of PS-exposing cells over time after loading the cells with 0.1 mM calcium using ionophore. Sickle cells scrambled at twice the rate of normal cells when loaded with the same level of calcium. This could be accounted for by reduced flippase activity, since flippase inhibition by vanadate increased the formation of PS-exposing cells by 2.5-fold in normal mouse cells. However, a similar stimulation was also seen in sickle cells, indicating that not only flippase inhibition, but also enhanced scrambling contributes to PS exposure in sickle cells. The sulfhydryl alkylating reagent n-ethylmaleimide (NEM) inhibits the flippase and enhances scrambling in human RBC, probably by changing the scramblase into an active conformation. This effect was similar in mouse RBC, leading to a markedly enhanced scrambling rate with more than 70% of the cells exposing PS after 10 minutes incubation. While most sickle cells also scrambled rapidly after NEM treatment, about 20% of mouse sickle cells had lost their ability to scramble. Another sulfhydryl modifying reagent, pyridyldithioethylamine (PDA), suppresses the scrambling process in human RBC even though it inhibits the flippase, indicating a different type of sulfhydryl modification of the scramblase. Mouse RBC were slightly less susceptible to PDA than human cells, but the observed scrambling resulted in a lower degree of PS exposure per cell than normally found. Sickle cells, on the other hand, doubled their scrambling rate in presence of PDA, showing that the inhibiting effect on the scramblase was inactivated in these cells. In conclusion, mouse sickle cells show a higher rate of scrambling than normal RBC at the same level of intracellular calcium, although it is suppressed in a subpopulation. Sulfhydryl modification of the scramblase at the site that can be modified by PDA could prevent its downregulation and increase its susceptibility to PS exposure in response to transient calcium influx. Our data show that both flippase and scramblase sustain damage by oxidative stress in sickle cells.