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Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects ∼100,000 Americans and millions of people worldwide. Erythrocyte sickling, vaso-occlusion, sterile inflammation, and hemolysis are the major pathophysiological pathways leading to liver injury in SCD. Although hepatic dysfunction affects up to 10%–40% of patients with SCD, therapeutic approaches to prevent liver injury in SCD are not known, and the molecular mechanisms promoting progressive liver injury in SCD remain poorly understood. Animal models have been beneficial in bridging the gap between preclinical and translational research in SCD. Recent advances in methodology have allowed the development of several humanized animal models to address various aspects of SCD-related liver diseases. This review provides an overview of current knowledge of the molecular mechanisms and potential therapeutic options of SCD-associated liver dysfunction using the Townes mouse model.

The up-regulation of kynurenine metabolism induces immunomodulatory responses via incompletely understood mechanisms. We report that increases in cellular and systemic kynurenine levels yield the electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA), as evidenced by the accumulation of thiol conjugates and saturated metabolites. Kyn-CKA induces NFE2 like bZIP transcription factor 2– and aryl hydrocarbon receptor–regulated genes and inhibits nuclear factor κB– and NLR family pyrin domain containing 3-dependent proinflammatory signaling. Sickle cell disease (SCD) is a hereditary hemolytic condition characterized by basal inflammation and recurrent vaso-occlusive crises. Both transgenic SCD mice and patients with SCD exhibit increased kynurenine and Kyn-CKA metabolite levels. Plasma hemin and kynurenine concentrations are positively correlated, indicating that Kyn-CKA synthesis in SCD is up-regulated during pathogenic vascular stress. Administration of Kyn-CKA abrogated pulmonary microvasculature occlusion in SCD mice, an important factor in lung injury development. These findings demonstrate that the up-regulation of kynurenine synthesis and its metabolism to Kyn-CKA is an adaptive response that attenuates inflammation and protects tissues.

In the US, mortality in sickle cell disease (SCD) increases after age 18–20 years. Biomarkers of mortality risk can identify patients who need intensive follow-up and early or novel interventions. We prospectively enrolled 510 SCD patients aged 3–20 years into an observational study in 2006–2010 and followed 497 patients for a median of 88 months (range 1–105). We hypothesized that elevated pulmonary artery systolic pressure as reflected in tricuspid regurgitation velocity (TRV) would be associated with mortality. Estimated survival to 18 years was 99% and to 25 years, 94%. Causes of death were known in seven of 10 patients: stroke in four (hemorrhagic two, infarctive one, unspecified one), multiorgan failure one, parvovirus B19 infection one, sudden death one. Baseline TRV ≥2.7 m/second (>2 SD above the mean in age-matched and gender-matched non-SCD controls) was observed in 20.0% of patients who died vs 4.6% of those who survived (P = .012 by the log rank test for equality of survival). The baseline variable most strongly associated with an elevated TRV was a high hemolytic rate. Additional biomarkers associated with mortality were ferritin ≥2000 μg/L (observed in 60% of patients who died vs 7.8% of survivors, P < .001), forced expiratory volume in 1 minute to forced vital capacity ratio (FEV1/FVC)

Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although platelet–neutrophil aggr...

To address the global burden of sickle cell disease and the need for novel therapies, the American Society of Hematology partnered with the US Food and Drug Administration to engage the work of 7 panels of clinicians, investigators, and patients to develop consensus recommendations for clinical trial end points. The panels conducted their work through literature reviews, assessment of available evidence, and expert judgment focusing on end points related to patient-reported outcome, pain (non–patient-reported outcomes), the brain, end-organ considerations, biomarkers, measurement of cure, and low-resource settings. This article presents the findings and recommendations of the end-organ considerations, measurement of cure, and low-resource settings panels as well as relevant findings and recommendations from the biomarkers panel.

Free heme activates erythroblasts to express and secrete Placenta Growth Factor (PlGF), an angiogenic peptide of the VEGF family. High circulating levels of PlGF have been associated in experimental animals and in patients with sickle cell disease with echocardiographic markers of pulmonary hypertension, a life-limiting complication associated with more intense hemolysis. We now show that the mechanism of heme regulation of PlGF requires the contribution of the key antioxidant response regulator NRF2. Mimicking the effect of heme, the NRF2 agonist sulforaphane stimulates the PlGF transcript level nearly 30-fold in cultured human erythroblastoid cells. Heme and sulforaphane also induce transcripts for NRF2 itself, its partners MAFF and MAFG, and its competitor BACH1. Furthermore, heme induction of the PlGF transcript is significantly diminished by the NRF2 inhibitor brusatol and by siRNA knockdown of the NRF2 and/or MAFG transcription factors. Chromatin immunoprecipitation experiments show that heme induces NRF2 to bind directly to the PlGF promoter region. In complementary in vivo experiments, mice injected with heme show a significant increase in their plasma PlGF protein as early as 3 h after treatment. Our results reveal an important mechanism of PlGF regulation, adding to the growing literature that supports the pivotal importance of the NRF2 axis in the pathobiology of sickle cell disease.

People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80–102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

The six-minute walk test (6MWT) has been used in patients with sickle cell disease (SCD), in conjunction with tricuspid regurgitant velocity (TRV) and plasma N-terminal pro-brain natriuretic peptide (NT-pro BNP), to assess risk of having pulmonary hypertension. Exercise-induced vital sign changes (VSCs) are predictors of clinical outcomes in other diseases. In this study, we assess the predictors and prognostic value of 6MWT VSC in adult SCD patients. Data from a multinational study of SCD patients (Treatment of Pulmonary Hypertension with Sildenafil: walk-PHaSST) were used to calculate the 6MWT VSC. Predictors of VSC were identified by a multivariable analysis, and a survival analysis was conducted by the Cox proportional hazard method. An increase in heart rate was observed in 90% of the 630 SCD adults, 77% of patients had an increase in systolic blood pressure (SBP), and 50% of patients had a decrease in oxygen saturation. TRV (odds ratio [OR] = 1.82, p = .020), absolute reticulocyte count (OR = 1.03, p

Background: Pulmonary complications of sickle cell disease (SCD) are diverse and encompass acute and chronic disease. The understanding of the natural history of pulmonary complications of SCD is limited, no specific therapies exist, and these complications are a primary cause of morbidity and mortality. Methods: We gathered a multidisciplinary group of pediatric and adult hematologists, pulmonologists, and emergency medicine physicians with expertise in SCD-related lung disease along with an SCD patient advocate for an American Thoracic Society–sponsored workshop to review the literature and identify key unanswered clinical and research questions. Participants were divided into four subcommittees on the basis of expertise: 1) acute chest syndrome, 2) lower airways disease and pulmonary function, 3) sleep-disordered breathing and hypoxia, and 4) pulmonary vascular complications of SCD. Before the workshop, a comprehensive literature review of each subtopic was conducted. Clinically important questions were developed after literature review and were finalized by group discussion and consensus. Results: Current knowledge is based on small, predominantly observational studies, few multicenter longitudinal studies, and even fewer high-quality interventional trials specifically targeting the pulmonary complications of SCD. Each subcommittee identified the three or four most important unanswered questions in their topic area for researchers to direct the next steps of clinical investigation. Conclusions: Important and clinically relevant questions regarding sickle cell lung disease remain unanswered. High-quality, multicenter, longitudinal studies and randomized clinical trials designed and implemented by teams of multidisciplinary clinician-investigators are needed to improve the care of individuals with SCD.

Sickle Cell Disease (SCD) is one of the most common monogenic disorders caused by a point mutation in the β-globin gene. This mutation results in polymerization of hemoglobin (Hb) under reduced oxygenation conditions, causing rigid sickle-shaped RBCs and hemolytic anemia. This clearly defined fundamental molecular mechanism makes SCD a prototypical target for precision therapy. Both the mutant β-globin protein and its down-stream pathophysiology are pharmacological targets of intensive research. SCD also is a disease well-suited for biological interventions like gene therapy. Recent advances in hem-atopoietic stem cell (HSC) transplantation and gene therapy platforms, like Lentiviral vec-tors and gene editing strategies, expand the potentially curative options for patients with SCD. This review discusses the recent advances in precision therapy for SCD and the pre-clinical and clinical advances in autologous HSC gene therapy for SCD.

Genetic modifiers of anemia in Plasmodium falciparum infection and sickle cell disease (SCD) are not fully known. Both conditions are associated with oxidative stress, hemolysis and anemia. The CYB5R3 gene encodes cytochrome b5 reductase 3, which converts methemoglobin to hemoglobin through oxidation of NADH. CYB5R3(c.350C > G) encoding CYB5R3(T117S), the most frequent recognized African-specific polymorphism, does not have known functional significance, but its high allele frequency (23% in African Americans) suggests a selection advantage. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection from oxidants; its African-polymorphic X-linked A+ and A− alleles, and other variants with reduced activity, coincide with endemic malaria distribution, suggesting protection from lethal infection. We examined the association of CYB5R3(c.350C > G) with severe anemia (hemoglobin G) offered protection against severe malarial anemia in children without G6PD deficiency (G6PD wild type or A+/A− heterozygotes) (odds ratio 0.29, P = .022) but not in G6PD A+ or A− hemizygotes/homozygotes. We also examined the relationship of CYB5R3(c.350C > G) with hemoglobin concentration among 267 children and 321 adults and adolescents with SCD in the US and UK and found higher hemoglobin in SCD patients without G6PD deficiency (β = 0.29, P = .022 children; β = 0.33, P = .004 adults). Functional studies in SCD erythrocytes revealed mildly lower activity of native CYB5R3(T117S) compared to wildtype CYB5R3 and higher NADH/NAD+ ratios. In conclusion, CYB5R3(c.350C > G) appears to ameliorate anemia severity in malaria and SCD patients without G6PD deficiency, possibly accounting for CYB5R3(c.350C > G) selection and its high prevalence.

Purpose Patients with sickle cell disease (SCD) regularly experience abnormal sleep, characterized by frequent arousals and reduced total sleep time. However, obstructive sleep apnea syndrome (OSAS) is a common comorbidity of SCD, making it unclear whether the disease per se is impacting sleep, or sleep disruption is secondary to the presence of OSAS. Thus, we assessed sleep, independent of OSAS, using a mouse model of SCD. Methods Sleep was compared between 10-to-12-week-old Townes knockout-transgenic mice with the sickle cell phenotype SS (n = 6) and Townes mice with sickle cell trait AS (n = 6; control). The mice underwent chronic polysomnographic electrode implantation (4EEG/2EMG) to assess sleep architecture. Results The SS mice had significantly lower hemoglobin concentration compared to control AS mice (7.3 ± 1.3 vs. 12.9 ± 1.7 g/dL; p

BACKGROUND AND AIMS Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.

The six-minute walk test (6MWT) is a well-established assessment of the cardiopulmonary function of sickle cell disease (SCD) patients. The test is used for people with SCD who are suspected of having hypoxia or an elevated estimated pulmonary arterial systolic pressure, which increases the risk for early death. Low 6MWT distances are associated with cardiopulmonary compromise. Six-minute walk distance examination has been proposed as a screening test, used in conjunction with plasma levels of brain natriuretic peptide and Doppler-echocardiography, through which to identify high-risk patients. Post-exercise tachycardia or lack of heart rate recovery following a 6MWT has been shown to be a predictor of pulmonary hypertension and mortality in people with pulmonary fibrosis. A rise in blood pressure after exercise is associated with impaired capacity for vasodilation in ischemic heart disease. The prognostic significance of patterns of vital sign change in adult SCD patients completing the 6MWT is currently unknown. In this study, we aimed to assess the distribution and predictors of vital sign change during 6MWT in adult SCD patients and test the association of these changes with patient survival. Data from a multinational observational study of SCD patients (Walk-PhassT), was used to calculate the change in vital signs (heart rate, O2 saturation, systolic blood pressure, diastolic blood pressure, pulse pressure) normalized for walk distance, after a 6MWT. Bivariate and LASSO regression analyses were performed to ascertain the significant predictors of change in each vital sign, in addition to Cox proportional hazard analysis to assess the impact of vital sign change and time to death. The median age of the 630 adult SCD patients was 37 years. 47% were male, 77% had the HbSS phenotype, and 22 (3.7% of 592 with follow-up data) died during a median time of follow up of 29 months. The most frequent changes in vital signs identified were increases in the heart rate (90%) followed by increases in systolic blood pressure (77%, Table 1b). Bivariate analysis revealed significant but weak positive correlations between tricuspid regurgitation velocity (TRV) and increases in both heart rate (r= 0.08; p These findings support links between changes of vital signs during the 6MWT and established markers of hemolysis and cardiovascular dysfunction in SCD patients. Evidence of a protective effect of increased systolic pressure is a novel finding. This might indicate that the ability to increase systolic pressure during submaximal exercise relates to cardiac output and conveys a physiological advantage for SCD patients. These findings could be used as the basis for future mechanistic studies of exercise effects on cardiovascular function in SCD patients. Disclosures Gordeuk: Imara: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; CSL Behring: Consultancy, Research Funding; Ironwood: Research Funding. Gibbs:Pfizer: Consultancy; United Therapeutics: Consultancy; MSD: Consultancy; GSK: Consultancy; Complexa: Consultancy; Bayer: Consultancy; Actelion: Consultancy; Acceleron: Consultancy. Little:BioChip Labs: Patents & Royalties: SCD Biochip (patent, no royalties); GBT: Membership on an entity's Board of Directors or advisory committees; NHLBI: Research Funding; Hemex Health, Inc.: Patents & Royalties: Microfluidic electropheresis (patent, no royalties); Bluebird Bio: Research Funding; GBT: Research Funding.

Key PointsQuestionCan poloxamer 188, an agent that is reported to reduce blood viscosity and cell-cell interactions, effectively reduce the duration of vaso-occlusive episodes (painful crises) in hospitalized patients with sickle cell disease? FindingsIn this randomized clinical trial that included 388 children and adults with sickle cell disease, treatment with poloxamer 188 vs placebo resulted in mean time to last dose of parenteral opioids during vaso-occlusive episodes of 81.8 vs 77.8 hours, a difference that was not statistically significant. MeaningAmong patients with sickle cell disease, poloxamer 188 did not significantly shorten the duration of painful vaso-occlusive episodes. ImportanceAlthough effective agents are available to prevent painful vaso-occlusive episodes of sickle cell disease (SCD), there are no disease-modifying therapies for ongoing painful vaso-occlusive episodes; treatment remains supportive. A previous phase 3 trial of poloxamer 188 reported shortened duration of painful vaso-occlusive episodes in SCD, particularly in children and participants treated with hydroxyurea. ObjectiveTo reassess the efficacy of poloxamer 188 for vaso-occlusive episodes. Design, Setting, and ParticipantsPhase 3, randomized, double-blind, placebo-controlled, multicenter, international trial conducted from May 2013 to February 2016 that included 66 hospitals in 12 countries and 60 cities; 388 individuals with SCD (hemoglobin SS, SC, S-beta(0) thalassemia, or S-beta(+) thalassemia disease) aged 4 to 65 years with acute moderate to severe pain typical of painful vaso-occlusive episodes requiring hospitalization were included. InterventionsA 1-hour 100-mg/kg loading dose of poloxamer 188 intravenously followed by a 12-hour to 48-hour 30-mg/kg/h continuous infusion (n=194) or placebo (n=194). Main Outcomes and MeasuresTime in hours from randomization to the last dose of parenteral opioids among all participants and among those younger than 16 years as a separate subgroup. ResultsOf 437 participants assessed for eligibility, 388 were randomized (mean age, 15.2 years; 176 [45.4%] female), the primary outcome was available for 384 (99.0%), 15-day follow-up contacts were available for 357 (92.0%), and 30-day follow-up contacts were available for 368 (94.8%). There was no significant difference between the groups for the mean time to last dose of parenteral opioids (81.8 h for the poloxamer 188 group vs 77.8 h for the placebo group; difference, 4.0 h [95% CI, -7.8 to 15.7]; geometric mean ratio, 1.2 [95% CI, 1.0-1.5]; P=.09). Based on a significant interaction of age and treatment (P=.01), there was a treatment difference in time from randomization to last administration of parenteral opioids for participants younger than 16 years (88.7 h in the poloxamer 188 group vs 71.9 h in the placebo group; difference, 16.8 h [95% CI, 1.7-32.0]; geometric mean ratio, 1.4 [95% CI, 1.1-1.8]; P=.008). Adverse events that were more common in the poloxamer 188 group than the placebo group included hyperbilirubinemia (12.7% vs 5.2%); those more common in the placebo group included hypoxia (12.0% vs 5.3%). Conclusions and RelevanceAmong children and adults with SCD, poloxamer 188 did not significantly shorten time to last dose of parenteral opioids during vaso-occlusive episodes. These findings do not support the use of poloxamer 188 for vaso-occlusive episodes. Trial RegistrationClinicalTrials.gov Identifier: NCT01737814 This phase 3 trial examines the effectiveness of poloxamer 188 in reducing the duration of painful vaso-occlusive episodes in children and adults with sickle cell disease compared with placebo.

Summary In sickle cell disease (SCD), abnormal microvascular function combined with chronic anaemia predisposes patients to perfusion-demand mismatch. We hypothesized that skeletal muscle and myocardial perfusion, normalized to the degree of anaemia, is reduced at basal-state compared to controls, and that this defect is ameliorated by hydroxycarbamide (HC; also termed hydroxyurea) therapy. Twenty-one SCD patients, of whom 15 were treated with HC, and 27 controls underwent contrast-enhanced ultrasound (CEU) perfusion imaging of the forearm as well as the myocardium. HC treatment was associated with lower white cell and reticulocyte counts, and higher fetal haemoglobin and total haemoglobin levels. When corrected for the degree of anaemia in SCD patients, skeletal flow in HC-treated patients was significantly higher than in untreated SCD patients (217·7 ± 125·4 vs. 85·9 ± 40·2, P = 0·018). Similarly, when normalized for both anaemia and increased myocardial work, resting myocardial perfusion was also significantly higher in HC-treated patients compared with untreated SCD patients (0·53 ± 0·47 vs. 0·13 ± 0·07, P = 0·028). Haemoglobin F (HbF) levels correlated with skeletal muscle microvascular flow (r = 0·55, P = 0·01). In conclusion, patients with SCD not on HC therapy have resting flow deficits in both skeletal muscle and myocardial flow. HC therapy normalizes flow and there is a direct correlation with HbF levels. Clinical trial registration ClinicalTrials.gov Identifier: NCT01602809; https://clinicaltrials.gov/ct2/show/NCT01602809?term=sACHDEV&rank=9.

Hemolysis is a fundamental feature of sickle cell anemia that contributes to its pathophysiology and phenotypic variability. Decompartmentalized hemoglobin, arginase 1, asymmetric dimethylarginine, and adenine nucleotides are all products of hemolysis that promote vasomotor dysfunction, proliferative vasculopathy, and a multitude of clinical complications of pulmonary and systemic vasculopathy, including pulmonary hypertension, leg ulcers, priapism, chronic kidney disease, and large-artery ischemic stroke. Nitric oxide (NO) is inactivated by cell-free hemoglobin in a dioxygenation reaction that also oxidizes hemoglobin to methemoglobin, a non–oxygen-binding form of hemoglobin that readily loses heme. Circulating hemoglobin and heme represent erythrocytic danger-associated molecular pattern (eDAMP) molecules, which activate the innate immune system and endothelium to an inflammatory, proadhesive state that promotes sickle vaso-occlusion and acute lung injury in murine models of sickle cell disease. Intravascular hemolysis can impair NO bioavailability and cause oxidative stress, altering redox balance and amplifying physiological processes that govern blood flow, hemostasis, inflammation, and angiogenesis. These pathological responses promote regional vasoconstriction and subsequent blood vessel remodeling. Thus, intravascular hemolysis represents an intrinsic mechanism for human vascular disease that manifests clinical complications in sickle cell disease and other chronic hereditary or acquired hemolytic anemias.