Pathophysiology Of Sickle Cell Disease Research Articles

Phase-I Study of ETA Receptor Antagonist Ambrisentan in Sickle Cell Disease

Endothelin-1 (ET1) is a potent vasoconstrictor, mitogen, proinflammatory mediator and a mediator of nociception. Its synthesis is increased by hypoxia, ischemia, shear stress, oxidative stress and reduced nitric oxide (NO) bioavailability, all of which are well documented mechanisms in the pathophysiology of sickle cell disease. Two distinct receptors, ETA and ETB mediate opposing actions of ET1; while ETA mediates vasoconstriction, inflammation, cellular adhesion and vascular remodeling, ETB stimulation leads to increased NO production, vasodilatation, anticoagulation and anti-adhesion. We studied the role of ET1 in SCD in transgenic mouse models, and showed that selective ETA receptor blockade with ambrisentan provides renal protection by preventing the development of glomerular hyperfiltration, and preventing proteinuria (Kasztan et al, 2017; Taylor et al, 2019; Kasztan & Pollock, 2019).Additionaly, ETA receptor blockade was shown to decrease pulmonary inflammation in response to different stimuli, including hypoxia/reoxygenation and LPS (Meiler et al). Lutz et al (2018) showed that pharmacologic inhibition, or neuron specific knockdown of ETA receptor in primary sensory neurons of dorsal root ganglia in Berk mice alleviated basal and post-hypoxia evoked pain hypersensitivity. We then conducted a Phase 1, double blind, placebo controlled study to elucidate the role of ET-1 in 26 patients with SCD (SS or Sb0thal); patients were randomized to either ambrisentan 5 mg/day (N=13) or placebo (N=13) for 12 weeks. The primary end point was safety and tolerability. We also measured microalbuminuria, pressure pain threshold, transcranial doppler (TCD), echocardiograms (TR jet velocity), vascular endothelial function by flow mediated dilation (FMD), and inflammatory cytokines. Urine specimens were collected for measurement of markers of glomerular and tubular injury (NGAL, KIM-1, netrin and nephrin) at baseline and at the end of study (Day 85). Ambrisentan was well tolerated without any adverse events or fluid retention. No change in systolic and diastolic blood pressures were observed in either group (p=0.88, and p=049, respectively). No significant change in weight or serum creatinine was observed in the ambrisentan group (69.1→68.6 Kg, p=0.99, and 0.74→0.69 mg/dl, p=0.43). In the ambrisentan group, there was a tendency toward reduced microalbuminuria, (-36.6 mg/g Cr, vs +91.6), especially in the subgroup of patients who had been on a stable dose of ACE or ARB for 6 months (n=6, 286.1 mg/g Cr at baseline to 197.7 mg/g on day 85, p=0.06). FMD measurements showed increased arterial diameter, and improved microvascular function. Data collected for secondary end points including, TCD, TRV, inflammatory cytokines, markers of glomerular and tubular injury, quantitative sensory testing (QST) with pressure pain measurements, and von Frey monofilament readings, as well as quality of life measures (ASCQMe) are in the process of being analyzed. These data suggest that ETA receptor blockade is safe, well tolerated and has the potential to impact various aspects of disease pathophysiology in SCD. Disclosures Kutlar: Novartis: Consultancy; Global Blood Therapeutics, Inc. (GBT): Research Funding; Bluebird Bio: Other: DSMB Member; Novo Nordisk: Research Funding; Micelle Biopharma: Other: DSMB Chair. OffLabel Disclosure: Ambrisentan is an approved FDA drug used for treatment of another condition but it is expected that it will also help improve and/or prevent kidney damage that can be caused by sickle cell disease.

Polymorphisms in Inflammatory Genes Modulate the Occurrence of Clinical Complications in Patients with Sickle Cell Disease

Background: Patients (pts) with sickle cell disease (SCD) show a high phenotype variability that is not fully understood. Because inflammation plays a major role in SCD pathophysiology, we hypothesized that single nucleotide polymorphisms (SNPs) in genes of innate and adaptative inflammatory response modulate the occurrence of SCD complications. Objective: to establish associations between SNPs and clinical complications in pts with SCD. Methods: Case-control retrospective study; 500 pts were included, followed at Senegal (n=56), Brazil (n=230) and France (n=214). We analyzed the effect of 20 SNPs in 6 clinical complications: acute chest syndrome (ACS), stroke, leg ulcers, cholelithiasis, osteonecrosis and retinopathy. Using TaqMan 5'-nuclease assay, we genotyped SNPs in genes encoding Toll-like receptor (TLR) 1 (rs4833095), 2 (rs4308099, rs4308100, rs4696480), 6 (rs5743810), 10 (rs11466653, rs11096957), natural killer (NK) group 2 member D (NKG2D) receptor (rs1982536, rs2617160, rs2617169, rs2617170, rs2617171, rs1049174, rs2246809, rs2255336), human leukocyte antigen (HLA)-G (rs9380142), HLA-E (rs2517523), major histocompatibility complex class I polypeptide-related sequence A (MICA, rs1051792) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) (rs5742909, rs231775). All SNPs had a call rate >90% and minimum allele frequency >1%. We performed analyses of correspondence for indicating associations between SNPs and number of clinical complications. Logistic regressions were used to identify associations between SNPs and each complication, using geographical origin, SCD genotype, rate of transfusions and gender for modeling adjustment; the significance was adjusted for multiple testing using false discovery rate. Comparisons of genotype frequencies with the population of African descent from 1000 genomes database were done by chi-square. Results: Pts were originally from Brazil (n=228), Sub-Saharan Africa (n=200), French West Indies (n=53), North Africa (n=7) and 12 unknown. SCD genotype (available n=498) was SS (n=402), SC (n=46), SB (n=42), SD or SE (n=4). 280 pts were female; median age was 32 years (range 0-69); 184 pts received at least 20 transfusions. 71 pts presented stroke, 200 had at least 1 episode of ACS, 69 had leg ulcers, 271 had cholelithiasis, 150 had retinopathy and 90 osteonecrosis. 97 pts did not present complications, 135 had 1 type of clinical complication, 134 had 2, 94 had 3, 34 had 4 and 6 had 5. 21 pts underwent hematopoietic stem cell transplantation. 11 pts died during follow-up, mostly from ACS and hemorrhagic stroke. Indication of association with number of complications: TLR2 rs4696480 TA, TLR2rs3804099 CC and HLA-Grs9380142 AA were associated with occurrence of 0-1 clinical complications, MICA rs1051792 AA/AG with up to 2 complications and NKG2D rs2617169 AA with 5 complications. Association between genotypes/haplotypes and each complication: no association was found between SNPs and stroke, ACS, leg ulcers and osteonecrosis. Rs9380142 was the only SNP significantly associated with cholelithiasis in the logistic regression additive model. The G allele increased the risk of cholelithiasis (AG x AA, OR 1.57, 95%CI 1.16-2.15; GGxAA, OR 2.47, 95%CI 1.34-4.64; P=0.02). For retinopathy, in the logistic regression additive model, the presence of the A allele decreased the risk of retinopathy for rs2246809 in pts of same origin (AAxGG: OR 0.22, 95%CI 0.09-0.50; AGxGG: OR 0.47, 95%CI 0.31-0.71; P=0.004), rs2617160 (ATxTT: OR 0.67, 95%CI 0.48-0.92; AAxTT: OR 0.45, 95%CI 0.23-0.84; P=0.04) and rs2617169 in pts of same SCD genotype (AAxTT: OR 0.33,95%CI 0.13-0.82; ATxTT: OR 0.58, 95%CI 0.36-0.91, P=0.049). No haplotype was associated with complications. The genotype distribution of SNPs rs4696480, rs3804099, rs1051792 and rs2617169 differed significantly from the population of African descent from the 1000 genomes database. Discussion: We have previously shown that TLR2 rs4696480 TA decreases occurrence of bacterial infections in SCD; in this study, TA is also associated with less complications. Also, HLA-G rs9380142 AA had less complications and cholelithiasis, and 3 SNPs in NKG2D modulated occurrence of retinopathy. TLR and NKG2D may be activated by heat shock proteins, released in ischemia-reperfusion injury that occurs in SCD. Our findings help to better understand the role of inflammation in phenotype heterogeneity in SCD. Disclosures No relevant conflicts of interest to declare.

Iron Catalyzed Hydrogen Sulfide and Its Prevalence during Oxidative Events in Sickle Cell Disease

In sickle cell disease (SCD), red blood cell (RBC) oxidation-reduction (redox) status is a critical determinant of hemolysis and RBC adhesion/vaso-occlusive crises (VOC). Iron, while essential for hemoglobin and RBC production, also is an important catalyst in processes that produce reactive oxygen species (ROS) which damage RBCs and ultimately other tissues. This has necessitated mechanisms for iron sequestration to control its activity, primarily achieved through binding of iron to heme, hemoglobin, and ferritin. In hemolytic disorders such as SCD, however, there is overload of protein-free, unbound iron, which by increasing ROS, significantly contributes to disease pathology. There is hence a need for novel druggable targets and treatments to attenuate this pathway of ROS production and pathophysiology. Previously, we determined a novel catalytic role of iron in which hydrogen sulfide (H2S) gas is produced both in vitro and in blood ex vivo by iron- and Vitamin B6- mediated catalysis of cysteine. We additionally determined that the bound versus unbound status of iron as well as the integrity of RBCs dictates this non-enzymatic capacity for H2S production. H2S serves as a potent regulatory redox factor throughout the body, and here we hypothesize that excess H2S in blood produced via iron catalysis protects against oxidative stress generated during SCD-related hemolytic crisis and VOC. In the current study, we further show that H2S produced in vitro from iron and Vitamin B6 catalysis of cysteine not only exists as a gas in the headspace, but itself reacts with iron in the solution. This was determined by the formation of acid labile iron sulfide, which formed a light yellow precipitate. The addition of a strong acid removed the yellow colored precipitate and resulted in further release of H2S in to the headspace. This indicates that H2S was interacting with free iron (Fe3+) and formed the iron sulfide product, which may have decreased capabilities for the formation of tissue- and cellular-damaging ROS. On-going work will examine the inhibition of H2S on iron-induced ROS generation in vitro and test the therapeutic potential of applying D-/L-cysteine and/or Vitamin B6- supplemented dietary interventions to enhance iron/B6 catalyzed H2S production in blood to lessen oxidative stress and VOC events in SCD mice in vivo. Free-iron in SCD is conventionally considered only to be a liability. However, here we exploit iron as a catalyst for beneficial H2S production in blood that can protect against oxidative stress and SCD-related pathologies. This recently discovered pathway has the potential to create novel opportunities and approaches to modulate the devastating pathophysiology of SCD. Figure Disclosures Saunthararajah: Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties.

Impaired Bile Secretion Promotes Chronic Liver Injury in Sickle Cell Disease

Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary crisis remains poorly understood. We examined the liver pathophysiology of SCD using a humanized mouse model (townes SCD mice; homozygous for Hbatm1(HBA)Tow, homozygous for Hbbtm2(HBG1,HBB*)Tow). These mice have the major features (irreversibly sickled red cells, splenomegaly, anemia, multiorgan pathology) found in humans with SCD and, as such, represent a useful in vivo system to study hepatobiliary changes in SCD disease. SCD mice manifested progressive hepatomegaly, liver injury and hyperbilirubinemia. RNA-sequence analysis of total RNA from SCD mouse liver compared to control (AS) identified dysregulation of genes encoding proteins responsible for fibrosis, bile acid synthesis, bile transport and cholesterol metabolism. Immunohistochemical analysis confirmed inflammation, fibrosis and increased ductular reaction in SCD mice. To mechanistically address the cholestatic phenotype in SCD mice, we used our recently developed multi-photon-excitation (MPE) enabled in vivo real-time fluorescence microscopy of intact liver in live mice. We used Texas-Red (TXR) dextran to visualize the blood flow through liver sinusoids and carboxyfluorescein (used as a surrogate of bile flow) to visualize bile flow through biliary canaliculi. Real time imaging show that sinusoidal ischemia occurs in the liver of transgenic-humanized SCD mice under basal condition. Intravital imaging also revealed impaired bile secretion into the bile canaliculi, which was associated with loss of apical bile acid transporters and bile acid biosynthetic enzymes, hepatic bile accumulation, and activation of Farnesoid X receptor (FXR) and small heterodimer partner (Shp) in the liver of SCD mice. These findings are the first to identify that impaired bile acid synthesis and misexpression of bile transporters promote intrahepatic bile accumulation and impaired bile secretion, leading to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of new therapies to treat sickle cell hepatic crisis. Disclosures Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases . Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding.

The Relationships between Target Gene Transduction, Engraftment of HSCs and RBC Physiology in Sickle Cell Disease Gene Therapy

The Relationships between Target Gene Transduction, Engraftment of HSCs and RBC Physiology in Sickle Cell Disease Gene Therapy

Stiff Erythrocyte Subpopulations Biomechanically Induce Endothelial Inflammation in Sickle Cell Disease

Background: Originally described as a monogenic hemoglobin disorder resulting in increased red blood cell (RBC) stiffness leading to vaso-occlusion, sickle cell disease (SCD) is now known to be a vasculopathic disease with some semblance to cardiovascular disease in which the endothelium is inflamed. While adhesive RBC-endothelial interactions, inflammatory cytokines, and hemolysis all contribute to SCD vasculopathy, whether the increased stiffness of sickle RBCs directly contributes to endothelial inflammation is unknown. Endothelial cells are now known to mechanotransduce shear forces into biological signals. Pathological alteration of such forces leads to proinflammatory endothelial cell signaling including upregulation of VCAM-1 and E-selectin, which contribute to atherosclerotic plaques leading to myocardial infarction and stroke (Abe, ATVB, 2014). In addition, under normal homeostatic conditions, RBCs do not come into contact with the endothelium due to a cell-free layer created by the Fåhræus-Lindqvist effect. Studies including our own have shown in silico that increasing RBC stiffness diminishes or eliminates the cell-free layer, allowing stiff RBCs to contact the vessel wall (Kumar, Phys Rev E, 2011). This is particularly pertinent in SCD, as all patients have a small population (1-10%) of sickle RBCs that are permanently stiff and misshapen. We therefore hypothesize that purely physical interactions - akin to "scratches" or collisions - between endothelial cells and stiff SCD RBCs breaking through the cell-free layer are sufficient to cause endothelial inflammation in the absence of adhesion or vaso-occlusion (Fig. 1A). Methods: We performed computational direct numerical simulations using the boundary integral method for a binary suspension of flexible biconcave discs and stiff curved prolate spheres modeling healthy RBCs and ISCs, respectively. Experimentally, we leveraged our microfluidic microvasculature models of human umbilical vein endothelial cells cultured throughout each microchannel (Fig. 2). RBCs from SCD patients were "spiked" into normal RBC suspensions to comprise 5 and 10% of the overall population (a representation of ISCs in vivo), suspended in media to 25% hematocrit mimicking conditions seen in SCD patients, and perfused into the microfluidics for 4 hours. Samples of 100% normal RBCs or SCD RBCs were run in parallel. To isolate the stiffness effects of sickle RBCs without confounding hemolytic and adhesive effects, parallel experiments were conducted using nystatin-treated normal RBCs to create artificially stiffened RBC subpopulations, defined by elevated mean corpuscular hemoglobin concentrations (MCHCs), at the same proportion of the overall RBC population (0, 5, 10 and 100%). The endothelialized models were then fixed, permeabilized, and immunostained with antibodies against VCAM-1 and E-selectin. Mean fluorescence intensity was measured to quantify endothelial inflammation. Results: In silico, we observed that ISCs strongly marginate towards the vessel walls due to their stiffness and "pointy" shape, and heterogeneous suspensions with small fractions of stiff, pointy cells (5 and 10%) caused the highest degree of margination (Fig. 1B). Experimentally, endothelium exposed to 5, 10, and 100% SCD RBCs exhibited increased VCAM-1 and E-selectin expression over normal RBCs, and the degree of expression increased with higher percentages of SCD RBCs. While endothelial cells exposed to nystatin-stiffened RBCs also showed increased VCAM-1 and E-selectin expression, those exposed to a lower percentage of stiff cells (5 and 10%) exhibited higher expression than the homogenously stiff (100%) condition (Fig. 3), which is consistent with our computer simulations. Conclusions: Here we demonstrate that purely non-adhesive, physical interactions between endothelial cells and SCD RBCs are sufficient to cause endothelial inflammation. Furthermore, heterogeneous RBC populations, comprised of a small minority of stiff cells, cause more inflammation than uniformly stiff RBCs. Studies elucidating the underlying mechanisms, using different endothelial cell types, and analyzing the effect of vessel curvature are ongoing. Our results introduce a new paradigm for understanding SCD pathophysiology and may help explain how chronic diffuse vasculopathy develops, which could lead to more biophysically-based therapeutic strategies. Disclosures Carden: GBT: Honoraria; NIH: Research Funding. Mannino:Sanguina, LLC: Employment, Equity Ownership. Lam:Sanguina, LLC: Equity Ownership.

Biomarkers of Cardiopulmonary, Renal, and Liver Dysfunction in an Adult Sickle Cell Disease Cohort

Introduction In sickle cell disease (SCD), chronic anemia, hypoxia, and NO scavenging related to intravascular hemolysis contribute to diastolic dysfunction and pulmonary hypertension. Free hemoglobin and heme released during hemolysis cause oxidative stress and exacerbate vascular inflammation. Iron overload from frequent blood transfusions provokes reactive oxygen species and consequently cellular damage. We examined correlation of biomarkers reflecting major aspects of SCD patho-physiology (hemolysis, anemia, hypoxia, inflammation, and iron overload) with cardiopulmonary, renal, and liver dysfunction in an adult SCD cohort. The study provides observational evidence for etiologies of sickle cell organ damage by assessing the relative strength of outcome association for these biomarkers. Methods The study included 442 SCD patients with median age of 31 years, 77% with severe β hemoglobin mutation genotypes (HbSS, HbSβ0-thalassemia, HbSOarab) and 58% female. Biomarkers were identified for the SCD etiological conditions listed in Table 1. Primary cardiopulmonary outcomes were left heart enlargement (left atrial and ventricular diameter) and elevated systolic pulmonary artery pressure measured by tricuspid regurgitation peak velocity (TRV). The primary renal outcome was elevated urine albumin to creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2. Primary hepatic outcomes were hepatocyte integrity estimated as the first principal component (PC) of alanine transaminase (ALT) and aspartate aminotransferase (AST), cholestasis estimated as the first PC of alkaline phosphatase (ALP) and direct bilirubin, and functioning liver mass measured by serum albumin1. Clinical associations between biomarkers and primary outcomes first applied single marker regression with covariates of age, gender, and severity of β hemoglobin genotype. Significant biomarkers representative of the major SCD etiological conditions were then analyzed by multi-marker regression and model selection. Results Left atrial diameter showed significant associations with biomarkers of hemolysis (LDH, percent reticulocytes, AST, total bilirubin), anemia (HCT, HGB), hypoxia (HCT, HGB, O2 saturation), and inflammation (WBC) (Table 1). Left ventricular diameter had a profile of biomarker associations similar to left atrial diameter. TRV showed significant associations with biomarkers of hemolysis (LDH, percent reticulocytes, AST), anemia (HCT, HGB), hypoxia (HCT, HGB, O2 saturation), inflammation (WBC), and iron overload (ferritin) (Table 1). In multi-marker analysis, anemia and hypoxia (low HCT, low O2 saturation) associated with greater left atrial diameter and with high TRV (Figure 1). Urine ACR significantly associated with biomarkers of hemolysis (LDH, AST, percent reticulocytes), anemia (HCT, HGB), hypoxia (HCT, HGB, O2 saturation), inflammation (WBC, serum albumin), and iron overload (ferritin) (Table 1). Impaired kidney function based on eGFR <60 mL/min/1.73m2 had a profile of biomarker associations similar to ACR. In multi-marker analysis, hemolysis (high LDH), anemia (low HCT), and iron overload (high ferritin) associated with increased ACR that indicates renal damage (Figure 2). Hepatocyte integrity significantly associated with biomarkers of hemolysis (percent reticulocytes), anemia (HCT, HGB), hypoxia (HCT, HGB, O2 saturation), inflammation (WBC, serum albumin), and iron overload (ferritin) (Table 2). Cholestasis had a profile of biomarker associations similar to decreased hepatocyte integrity (Table 2). Functioning liver mass showed significant association with hemolysis (percent reticulocyte), anemia (HGB, HCT), and iron overload (ferritin) (Table 2). In multi-marker analysis, hemolysis (high percent reticulocyte) and iron overload (high ferritin) associated with decreased hepatocyte integrity (Figure 3A, 3B), whereas anemia (low HCT) and iron overload (high ferritin) associated with cholestasis (Figure 3C, 3D) and with low functioning liver mass (Figure 3E, 3F). Conclusion Our study indicated significant correlations of anemia and hypoxia with both left heart enlargement and elevated pulmonary arterial systolic pressure. Our study also underscores a significant impact of iron overload on renal and liver damages. Acknowledgement We thank CSL Behring Biotherapies for Life™ for their support of this study. Disclosures Saraf: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding. Gordeuk:Pfizer: Research Funding; Inctye: Research Funding; Imara: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Modus Therapeutics: Consultancy, Honoraria.

Sickle Cell Anaemia: Midwifery and Team Management

Sickle cell anaemia (SCA) affects more than 12,000 women/birthing people in the UK (Public Health England (PHE) 2018), so midwives need to be familiar with its impact on pregnancy and childbearing. Physiological adaptation to pregnancy combined with pathophysiological mechanisms of SCA can trigger fetal and maternal complications, including venous thromboembolism (VTE), intra-uterine growth restriction (IUGR), neural tube defects, bacterial infections and urinary tract infections (UTIs). Midwives need to understand the pathophysiology of SCA in order to promote positive pregnancy outcomes.

Nitric oxide loading reduces sickle red cell adhesion and vaso-occlusion in vivo.

Sickle red blood cells (SSRBCs) are adherent to the endothelium, activate leukocyte adhesion, and are deficient in bioactive nitric oxide (NO) adducts such as S-nitrosothiols (SNOs), with reduced ability to induce vasodilation in response to hypoxia. All these pathophysiologic characteristics promote vascular occlusion, the hallmark of sickle cell disease (SCD). Loading hypoxic SSRBCs in vitro with NO followed by reoxygenation significantly decreased epinephrine-activated SSRBC adhesion to the endothelium, the ability of activated SSRBCs to mediate leukocyte adhesion in vitro, and vessel obstruction in vivo. Because transfusion is frequently used in SCD, we also determined the effects of banked (SNO-depleted) red blood cells (RBCs) on vaso-occlusion in vivo. Fresh or 14-day-old normal RBCs (AARBCs) reduced epinephrine-activated SSRBC adhesion to the vascular endothelium and prevented vaso-occlusion. In contrast, AARBCs stored for 30 days failed to decrease activated SSRBC adhesivity or vaso-occlusion, unless these RBCs were loaded with NO. Furthermore, NO loading of SSRBCs increased S-nitrosohemoglobin and modulated epinephrine's effect by upregulating phosphorylation of membrane proteins, including pyruvate kinase, E3 ubiquitin ligase, and the cytoskeletal protein 4.1. Thus, abnormal SSRBC NO/SNO content both contributes to the vaso-occlusive pathophysiology of SCD, potentially by affecting at least protein phosphorylation, and is potentially amenable to correction by (S)NO repletion or by RBC transfusion.

Emerging disease-modifying therapies for sickle cell disease.

Sickle cell disease afflicts millions of people worldwide and approximately 100,000 Americans. Complications are myriad and arise as a result of complex pathological pathways ‘downstream’ to a point mutation in DNA, and include red blood cell membrane damage, inflammation, chronic hemolytic anemia with episodic vaso-occlusion, ischemia and pain, and ultimately risk of cumulative organ damage with reduced lifespan of affected individuals. The National Heart, Lung, and Blood Institute’s 2014 evidence-based guideline for sickle cell disease management states that additional research is needed before investigational curative therapies will be widely available to most patients with sickle cell disease. To date, sickle cell disease has been cured by hematopoietic stem cell transplantation in approximately 1,000 people, most of whom were children, and significantly ameliorated by gene therapy in a handful of subjects who have only limited follow-up thus far. During a timespan in which over 20 agents were approved for the treatment of cystic fibrosis by the Food and Drug Administration, similar approval was granted for only two drugs for sickle cell disease (hydroxyurea and L-glutamine) despite the higher prevalence of sickle cell disease. This trajectory appears to be changing, as the lack of multimodal agent therapy in sickle cell disease has spurred engagement among many in academia and industry who, in the last decade, have developed new drugs poised to prevent complications and alleviate suffering. Identified therapeutic strategies include fetal hemoglobin induction, inhibition of intracellular HbS polymerization, inhibition of oxidant stress and inflammation, and perturbation of the activation of the endothelium and other blood components (e.g. platelets, white blood cells, coagulation proteins) involved in the pathophysiology of sickle cell disease. In this article, we present a crash-course review of disease-modifying approaches (minus hematopoietic stem cell transplant and gene therapy) for patients with sickle cell disease currently, or recently, tested in clinical trials in the era following approval of hydroxyurea.

A Phase 3 Randomized Trial of Voxelotor in Sickle Cell Disease

BackgroundDeoxygenated sickle hemoglobin (HbS) polymerization drives the pathophysiology of sickle cell disease. Therefore, direct inhibition of HbS polymerization has potential to favorably modify disease outcomes. Voxelotor is an HbS polymerization inhibitor.MethodsIn a multicenter, phase 3, double-blind, randomized, placebo-controlled trial, we compared the efficacy and safety of two dose levels of voxelotor (1500 mg and 900 mg, administered orally once daily) with placebo in persons with sickle cell disease. The primary end point was the percentage of participants who had a hemoglobin response, which was defined as an increase of more than 1.0 g per deciliter from baseline at week 24 in the intention-to-treat analysis.ResultsA total of 274 participants were randomly assigned in a 1:1:1 ratio to receive a once-daily oral dose of 1500 mg of voxelotor, 900 mg of voxelotor, or placebo. Most participants had sickle cell anemia (homozygous hemoglobin S or hemoglobin Sβ0-thalassemia), and approximately two thirds were receiving hydroxyurea at baseline. In the intention-to-treat analysis, a significantly higher percentage of participants had a hemoglobin response in the 1500-mg voxelotor group (51%; 95% confidence interval [CI], 41 to 61) than in the placebo group (7%; 95% CI, 1 to 12). Anemia worsened between baseline and week 24 in fewer participants in each voxelotor dose group than in those receiving placebo. At week 24, the 1500-mg voxelotor group had significantly greater reductions from baseline in the indirect bilirubin level and percentage of reticulocytes than the placebo group. The percentage of participants with an adverse event that occurred or worsened during the treatment period was similar across the trial groups. Adverse events of at least grade 3 occurred in 26% of the participants in the 1500-mg voxelotor group, 23% in the 900-mg voxelotor group, and 26% in the placebo group. Most adverse events were not related to the trial drug or placebo, as determined by the investigators.ConclusionsIn this phase 3 randomized, placebo-controlled trial involving participants with sickle cell disease, voxelotor significantly increased hemoglobin levels and reduced markers of hemolysis. These findings are consistent with inhibition of HbS polymerization and indicate a disease-modifying potential. (Funded by Global Blood Therapeutics; HOPE ClinicalTrials.gov number, NCT03036813.)

Targeting sickle cell disease root-cause pathophysiology with small molecules

The complex, frequently devastating, multi-organ pathophysiology of sickle cell disease has a single root cause: polymerization of deoxygenated sickle hemoglobin. A logical approach to disease modification is, therefore, to interdict this root cause. Ideally, such interdiction would utilize small molecules that are practical and accessible for worldwide application. Two types of such small molecule strategies are actively being evaluated in the clinic. The first strategy intends to shift red blood cell precursor hemoglobin manufacturing away from sickle hemoglobin and towards fetal hemoglobin, which inhibits sickle hemoglobin polymerization by a number of mechanisms. The second strategy intends to chemically modify sickle hemoglobin directly in order to inhibit its polymerization. Important lessons have been learnt from the pre-clinical and clinical evaluations to date. Open questions remain, but this review summarizes the valuable experience and knowledge already gained, which can guide ongoing and future efforts for molecular mechanism-based, practical and accessible disease modification of sickle cell disease.

Sickle cell disease: a review for the internist.

Sickle cell disease (SCD) is the most important hemoglobinopathy worldwide in terms of frequency and social impact, recently recognized as a global public health problem by the World Health Organization. It is a monogenic but multisystem disorder with high morbidity and mortality. Vaso-occlusion, hemolytic anemia and vasculopathy are the hallmarks of SCD pathophysiology. This review focuses both on "time-dependent" acute clinical manifestations of SCD and chronic complications commonly described in adults with SCD. The review covers a broad spectrum of topics concerning current management of SCD targeted at the internists and emergency specialists who are increasingly involved in the care of acute and chronic complications of SCD patients.

Effects of Individualized Treadmill Endurance Training on Oxidative Stress in Skeletal Muscles of Transgenic Sickle Mice.

Oxidative stress is a key feature in the pathophysiology of sickle cell disease. Endurance training has been shown to reduce oxidative stress in the heart and the liver of sickle mice. However, the effects of endurance training on skeletal muscles, which are major producers of reactive oxygen species during exercise, are currently unknown. The aim of this study was to evaluate the effect of sickle genotype on prooxidant/antioxidant response to individualized endurance training in skeletal muscles of sickle mice. Healthy and homozygous Townes sickle mice were divided into trained or sedentary groups. Maximal aerobic speed and V̇O2 peak were determined using an incremental test on a treadmill. Trained mice ran at 40% to 60% of maximal aerobic speed, 1 h/day, 5 days/week for 8 weeks. Oxidative stress markers, prooxidant/antioxidant response, and citrate synthase enzyme activities were assessed in the gastrocnemius, in the plantaris, and in the soleus muscles. Maximal aerobic speed and V̇O2 peak were significantly reduced in sickle compared to healthy mice (-57% and -17%; p < 0.001). NADPH oxidase, superoxide dismutase, and catalase activities significantly increased after training in the gastrocnemius of sickle mice only. A similar trend was observed for citrate synthase activity in sickle mice (p = 0.06). In this study, we showed an adaptive response to individualized endurance training on the prooxidant/antioxidant balance in the gastrocnemius, but neither in the plantaris nor in the soleus of trained sickle mice, suggesting an effect of sickle genotype on skeletal muscle response to endurance treadmill training.

Combined genetic disruption of K-Cl cotransporters and Gardos channel KCNN4 rescues erythrocyte dehydration in the SAD mouse model of sickle cell disease

Excessive red cell dehydration contributes to the pathophysiology of sickle cell disease (SCD). The densest fraction of sickle red cells (with the highest corpuscular hemoglobin concentration) undergoes the most rapid polymerization of deoxy-hemoglobin S, leading to accelerated cell sickling and increased susceptibility to endothelial activation, red cell adhesion, and vaso-occlusion. Increasing red cell volume in order to decrease red cell density can thus serve as an adjunct therapeutic goal in SCD. Regulation of circulating mouse red cell volume and density is mediated largely by the Gardos channel, KCNN4, and the K-Cl cotransporters, KCC3 and KCC1. Whereas inhibition of the Gardos channel in subjects with sickle cell disease increased red cell volume, decreased red cell density, and improved other hematological indices in subjects with SCD, specific KCC inhibitors have not been available for testing. We therefore investigated the effect of genetic inactivation of KCC3 and KCC1 in the SAD mouse model of sickle red cell dehydration, finding decreased red cell density and improved hematological indices. We describe here generation of mice genetically deficient in the three major red cell volume regulatory gene products, KCNN4, KCC3, and KCC1 in C57BL6 non-sickle and SAD sickle backgrounds. We show that combined loss-of-function of all three gene products in SAD mice leads to incrementally increased MCV, decreased CHCM and % hyperchromic cells, decreased red cell density (phthalate method), increased resistance to hypo-osmotic lysis, and increased cell K content. The data show that combined genetic deletion of the Gardos channel and K-Cl cotransporters in a mouse SCD model decreases red cell density and improves several hematological parameters, supporting the strategy of combined pharmacological inhibition of these ion transport pathways in the adjunct treatment of human SCD.

Angiopoietin-2 as a Marker of Retinopathy in Children and Adolescents With Sickle Cell Disease: Relation to Subclinical Atherosclerosis.

Angiopoietin-2 (Ang-2) is a multifaceted cytokine that functions in both angiogenesis and inflammation. A proangiogenic state has been found in adults with sickle cell disease (SCD), mainly because of elevated Ang-2 levels. We determined Ang-2 level in 40 children and adolescents with SCD compared with 40 healthy controls and assessed its relation to retinopathy as well as carotid intimamedia thickness (CIMT). Hematologic profile, serum ferritin, and serum Ang-2 were measured. CIMT was assessed using high-resolution ultrasound. Fundus examination was performed followed by fundus fluorescein angiography. Optical coherence tomography angiography (OCTA) was used to find small vascular changes not clinically manifested. Ang-2 levels and CIMT were significantly higher in SCD patients compared with controls. The incidence of nonproliferative retinopathy was 45%. SCD patients with retinopathy were older in age with a history of sickling crisis of >3 attacks per year and had a higher incidence of sickle cell anemia than sickle β-thalassemia. Ang-2 cutoff value 9000 pg/mL could significantly detect the presence of retinopathy among SCD patients with 100% sensitivity and specificity. Serum Ang-2 levels were positively correlated with HbS and CIMT. Logistic regression analysis revealed that Ang-2 and HbS significantly contribute to retinopathy among patients with SCD. Elevated Ang-2 highlights the role of angiogenesis in the pathophysiology of SCD and may be considered a promising marker for screening of patients at risk of sickle retinopathy and vascular dysfunction.

Innate-like T cells in children with sickle cell disease.

BackgroundThe implication of lymphocytes in sickle cell disease pathogenesis is supported by a number of recent reports. These studies provided evidence for the activation of invariant natural killer T (iNKT) cells in adult patients, but did not investigate the involvement of other innate-like T cell subsets so far.MethodsHere we present a monocentric prospective observational study evaluating the number and functional properties of both circulating conventional and innate-like T cells, namely iNKT, Mucosal-Associated Invariant T (MAIT) and gammadelta (γδ) T cells in a cohort of 39 children with sickle cell disease.ResultsRelative to age-matched healthy controls, we found that patients had a higher frequency of IL-13- and IL-17-producing CD4+ T cells, as well as higher MAIT cell counts with an increased frequency of IL-17-producing MAIT cells. Patients also presented increased Vδ2 γδ T cell counts, especially during vaso-occlusive crisis, and a lower frequency of IFNγ-producing Vδ2 γδ T cells, except during crisis. iNKT cell counts and the frequency of IFNγ-producing iNKT cells were unchanged compared to controls. Our study revealed positive correlations between 1) the frequency of IFNγ-producing CD4+, CD8+ and Vδ2 γδ T cells and the number of hospitalizations for vaso-occlusive crisis in the previous year; 2) the frequency of IFNγ-producing iNKT cells and patients’ age and 3) the frequency of IL-17-producing Vδ2 γδ T cells and hemoglobin S level.ConclusionThese results strongly suggest a role of innate-like T cells in sickle cell disease pathophysiology, especially that of IL-17-producing MAIT and γδ T cells.

Oxysterol concentrations are associated with cholesterol concentrations and anemia in pediatric patients with sickle cell disease

Sickle cell disease (SCD) causes anemia, oxidative stress, chronic inflammation, and lipid abnormalities. Oxysterols are oxidized derivatives of cholesterol and affect cholesterol metabolism and eryptosis. Our aim was to determine whether the plasma concentrations of 7-ketocholesterol (7-KC) and cholestane-3β,5α,6β-triol (C-triol) were associated with hemolysis and lipid profile in patients with SCD. A total of 32 steady-state pediatric patients with SCD (22 HbSS and 10 HbSß+) and 25 healthy controls were included in the study. Hemolysis parameters, ferritin, serum iron, lipids, 7-KC and C-triol concentrations of all subjects were measured. Oxysterols were quantified with N,N-dimethylglycine derivatization via LC-MS/MS. 7-KC and C-triol concentrations were found to be increased in SCD patients, while there was no difference between the HbSS and HbSß+ subgroups. 7-KC concentrations s were correlated negatively with hemoglobin and positively with lactate dehydrogenase concentrations, while C-triol concentrations were negatively correlated with HDL cholesterol. Furthermore, while 7-KC and C-triol concentrations were highly correlated among controls, there was no correlation in patients. The findings of our study suggest that 7-KC and C-triol may have a role in SCD pathophysiology. The lack of correlation in patients’ 7-KC and C-triol concentrations suggest alterations in oxysterol production in patients with SCD.

PS1519 DECREASED ACTIVITY AND STABILITY OF PYRUVATE KINASE IN SICKLE CELL DISEASE AND THALASSEMIA: A POTENTIAL TARGET FOR THERAPY

Background:Reactive oxygen species (ROS) play an important role in the complex and multifactorial pathophysiology of sickle cell disease (SCD) and thalassemia. In SCD, auto‐oxidation of red blood cell (RBC) hemoglobin produces high levels of ROS that exceed anti‐oxidant capacity. In β–thalassemia, auto‐oxidation of unpaired α‐globins produces high levels of ROS. Increased intracellular levels of oxidative stress disrupt normal cell functioning and may contribute to premature clearance from the circulation. Pyruvate kinase (PK) is a key regulatory enzyme of glycolysis, the cell's only source of energy. Because PK is very sensitive to redox balance we hypothesized that increased levels of oxidative stress in hemoglobinopathies impairs proper enzyme function and thereby compromizes RBC energy metabolism. This may contribute to disease pathophysiology.Aims:To investigate if secondary deficiency of PK is common in SCD and thalassemia, and to investigate if PK in these disorders is able to respond to treatment with the allosteric PK activator AG‐348.Methods:Enzymatic activities of red cell PK and hexokinase (HK) were measured together with PK thermal stability (residual activity of PK) in order to assess relative PK activity and enzyme stability. RBC lysates were incubated with AG‐348 (3.33 uM) for 2 hours after which PK thermal stability, PK activity and ATP response was measured. Red blood cells of SCD patients were also analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior (Rab et al, Am J Hematol, 2019).Results:Twenty‐eight patients and 18 healthy controls (HC) were included. The patient cohort consisted of patients with HbSS (n = 16), HbS/β‐thalassemia (n = 1), β‐thalassemia major (n = 3, regularly transfused), and HbSC (n = 3). Patients showed reticulocytosis and a concomitant increase in HK activity. However, PK activity was relatively decreased significantly compared to HK in SCD and β‐thalassemia patients, but not in HbSC patients (Figure 1A). PK thermal stability also was significantly decreased in SCD patients compared to healthy controls. In HbSC and β‐thalassemia patients PK‐thermal stability was comparable to HC (Figure 1B). When purified RBCs were incubated with 3.33uM of the allosteric PK‐activator AG‐348 an increase in PK activity was seen in all patients and HCs, with a median of 122% in SCD (range 120–138%, n = 3), 127% in β‐thalassemia and 142% in HC (range 130–166%, n = 5, Figure 1D). Accordingly, ATP‐levels increased in all patients and HCs, with a median of 126% in SCD (range 121–129%, n = 3), 146% in 1 patient with β‐thalassemia, and 122% in HCs (range 100–148%). In SCD patients PK‐thermal stability inversely correlated significantly with oxygenscan parameters, in particular the point‐of‐sickling (PoS). This indicates that RBCs tend to sickle at a higher pO2 when PK is more unstable. Interestingly, the 3 patients with most stable PK are HbSC patients (2) and a HbS/dβ‐thalassemia patient with HbF levels of 35% (Figure 1C).Summary/Conclusion:PK enzyme activity and stability is compromised in patients with SCD and β‐thalassemia. In SCD, reduced PK thermal stability is associated with higher PoS, which is associated with more severe disease. Current studies are in progress to further substantiate the underlying mechanism(s), and to investigate whether AG‐348 may ameliorate pathophysiological features such as hemolysis and sickling tendency.image

Interleukin-6 in pregnancy with sickle cell disease

BackgroundDespite advances in health care for sickle cell disease patients, as well as in the improvement in reproductive issues mainly in women with the disease, pregnancy is still a challenge, both for the mother and the child, with high rates of maternal and fetal morbidity and mortality. Besides their chronic hemolytic status and vaso-occlusive events that confer systemic complications, pregnant women also have higher rates of pain episodes, infections, abortion, intrauterine growth retardation, pre-term births, eclampsia, stillbirth and the hemolysis, elevated liver enzymes and low platelets syndrome. The physiologic mechanisms of the disease in pregnancy are still unknown and chronic inflammatory responses may interfere in the adverse outcomes. The cytokine and chemokine profiles in pregnancy with sickle cell disease remain unknown. The aim of this study was to evaluate the cytokine profile of the inflammatory response of pregnant women with sickle cell disease. MethodBlood samples from 20 pregnant women with sickle cell disease, 24 women with sickle cell disease in steady state, 16 healthy pregnant women and a control group with 9 women at childbearing age were assayed for interleukin-6. Main resultsPregnant women with sickle cell disease presented high serum levels of interleukin-6, compared to healthy pregnant women (p=0.0115). ConclusionThese data suggest that the increased production of interleukin-6 may occur during pregnancy with sickle cell disease and that the role of this cytokine in the sickle cell disease pathophysiology and pregnancy complications should be further studied.