Rheological Impact of GBT1118 Cessation in a Sickle Mouse Model.

In sickle cell disease (SCD), higher whole blood viscosity is a risk factor for vaso-occlusive crisis, avascular necrosis, and proliferative retinopathy. Blood viscosity is strongly impacted by hemoglobin (Hb) levels and red blood cell (RBC) deformability. Voxelotor is a hemoglobin S (HbS) polymerization inhibitor with anti-sickling properties that increases the Hb affinity for oxygen, thereby reducing HbS polymerization. In clinical trials, voxelotor increased Hb by an average of 1g/dl, creating concern that this rise in Hb could increase viscosity, particularly when the drug was cleared. To investigate this potential rebound hyperviscosity effect, we treated SCD mice with GBT1118, a voxelotor analog, and stopped the treatment to determine the effect on blood viscosity and RBC deformability under a range of oxygen concentrations. GBT1118 treatment increased Hb, improved RBC deformability by increasing the elongation index under normoxic (EImax) and hypoxic conditions (EImin), and decreased the point of sickling (PoS) without increasing blood viscosity. The anti-sickling effects and improvement of RBC deformability balanced the effect of increased Hb such that there was no increase in blood viscosity. Forty-eight hours after ceasing GBT1118, Hb declined from the rise induced by treatment, viscosity did not increase, and EImin remained elevated compared to control animals. Hb and PoS were not different from control animals, suggesting a return to native oxygen affinity and clearance of the drug. RBC deformability did not return to baseline, suggesting some residual rheological improvement. These data suggest that concerns regarding viscosity rise above pre-treatment levels upon sudden cessation of voxelotor are not warranted.

Mutant KLF1 in Adult Anemic Nan Mice Leads to Profound Transcriptome Changes and Disordered Erythropoiesis.

Anemic Nan mice carry a mutation (E339D) in the second zinc finger of erythroid transcription factor KLF1. Nan-KLF1 fails to bind a subset of normal KLF1 targets and ectopically binds a large set of genes not normally engaged by KLF1, resulting in a corrupted fetal liver transcriptome. Here, we performed RNAseq using flow cytometric-sorted spleen erythroid precursors from adult Nan and WT littermates rendered anemic by phlebotomy to identify global transcriptome changes specific to the Nan Klf1 mutation as opposed to anemia generally. Mutant Nan-KLF1 leads to extensive and progressive transcriptome corruption in adult spleen erythroid precursors such that stress erythropoiesis is severely compromised. Terminal erythroid differentiation is defective in the bone marrow as well. Principle component analysis reveals two major patterns of differential gene expression predicting that defects in basic cellular processes including translation, cell cycle, and DNA repair could contribute to disordered erythropoiesis and anemia in Nan. Significant erythroid precursor stage specific changes were identified in some of these processes in Nan. Remarkably, however, despite expression changes in large numbers of associated genes, most basic cellular processes were intact in Nan indicating that developing red cells display significant physiological resiliency and establish new homeostatic set points in vivo.

Neomorphic effects of the neonatal anemia (Nan-Eklf) mutation contribute to deficits throughout development.

Transcription factor control of cell-specific downstream targets can be significantly altered when the controlling factor is mutated. We show that the semi-dominant neonatal anemia (Nan) mutation in the EKLF/KLF1 transcription factor leads to ectopic expression of proteins that are not normally expressed in the red blood cell, leading to systemic effects that exacerbate the intrinsic anemia in the adult and alter correct development in the early embryo. Even when expressed as a heterozygote, the Nan-EKLF protein accomplishes this by direct binding and aberrant activation of genes encoding secreted factors that exert a negative effect on erythropoiesis and iron use. Our data form the basis for a novel mechanism of physiological deficiency that is relevant to human dyserythropoietic anemia and likely other disease states.

Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability.

The rules of engagement between zinc finger transcription factors and DNA have been partly defined by in vitro DNA-binding and structural studies, but less is known about how these rules apply in vivo. Here, we demonstrate how a missense mutation in the second zinc finger of Krüppel-like factor-1 (KLF1) leads to degenerate DNA-binding specificity in vivo, resulting in ectopic transcription and anemia in the Nan mouse model. We employed ChIP-seq and 4sU-RNA-seq to identify aberrant DNA-binding events genome wide and ectopic transcriptional consequences of this binding. We confirmed novel sequence specificity of the mutant recombinant zinc finger domain by performing biophysical measurements of in vitro DNA-binding affinity. Together, these results shed new light on the mechanisms by which missense mutations in DNA-binding domains of transcription factors can lead to autosomal dominant diseases.

Plasma concentration of platelet-derived microparticles is related to painful vaso-occlusive phenotype severity in sickle cell anemia.

High plasma level of microparticles (MPs) deriving mainly from erythrocytes and platelets has been detected in sickle cell anemia (SCA) patients. Flow cytometry was used to determine the concentration of MPs in two groups of SCA patients exhibiting marked differences in painful vaso-occlusive crisis rates [a non-severe group (n = 17) and a severe group (n = 12)], and in a control group composed of healthy subjects (n = 20). A 3- to 4-fold increase of total MP plasma concentration was detected in SCA patients. Higher platelet-derived MPs concentration was detected in the severe SCA group while erythrocyte-derived MPs concentration was increased in the non-severe SCA patient group only. Our results suggest that plasma concentration of MPs shed by platelets is a biomarker of the vaso-occlusive phenotype-related severity.

Fetal hemoglobin and hydroxycarbamide moduate both plasma concentration and cellular origin of circulating microparticles in sickle cell anemia children.

Microparticles are cell membrane-derived microvesicles released during cell apoptosis and activation processes. They have been described as bio-markers in various vascular diseases, including sickle cell anemia, and associated with an increased risk of thrombosis. We investigated the effects of fetal hemoglobin level, a factor known to modulate the clinical expression of sickle cell anemia, and that of hydroxycarbamide treatment which reduces the frequency of vasoocclusive crises, the canonical clinical manifestation of the disease, on both the plasma concentration and the cellular origin of circulating microparticles. Flow cytometry was used to characterize microparticles in 62 sickle cell anemia children at steady state aged 2 months-16 years; 13 of them were treated with hydroxycarbamide. In untreated children, we observed negative correlations between fetal hemoglobin levels and the absolute plasma concentration of microparticles as well as that of microparticles specifically derived from platelets, erythrocytes, and monocytes. Compared to untreated children, those treated with hydroxyurea showed lower concentrations of total microparticles as a consequence of decreased microparticles shed by platelets and erythrocytes. In conclusion, in our sickle cell patients, neonatal decline of fetal hemoglobin coincided with an increase in circulating microparticles derived from erythrocytes, platelets, and monocytes. Hydroxyurea treatment was associated with a decrease in microparticles derived from erythrocytes and platelets.

Relationship between acute chest syndrome and the sympatho-vagal balance in adults with hemoglobin SS disease; a case control study.

The present study evaluated the relationship between acute chest syndrome (ACS) and autonomic nervous system (ANS) activity in patients with hemoglobin SS disease (Hb SS). Nine patients had suffered ACS were matched by age and gender to patients who had not suffered ACS and ANS activity, pulmonary function and history of painful crisis were compared. Correlations between number of episodes of ACS suffered and these variables were determined. The results demonstrated that 1) patients with a history of ACS ever had lower parasympathetic nervous system (PNS) activity and lower global ANS activity than patients with no ACS ever (p < 0.05), 2) the number of ACS episodes ever negatively correlated (p < 0.05) with PNS activity and global ANS activity and 3) There were no significant associations between lung function or a history of painful crisis in these patients. In conclusion, a history of ACS was associated with ANS dysfunction in adults with Hb SS disease.

Frequency of pain crises in sickle cell anemia and its relationship with the sympatho-vagal balance, blood viscosity and inflammation.

BACKGROUND: Recent evidence suggests that autonomic nervous system activity could be involved in the pathophysiology of sickle cell disease, but it is unclear whether differences in autonomic nervous system activity are detectable during steady state in patients with mild and severe disease. The aim of the present study was to compare the autonomic nervous system activity, blood rheology, and inflammation in patients with sickle cell anemia according to the frequency of acute pain crisis. DESIGN AND METHODS: Twenty-four healthy volunteers, 20 patients with sickle cell anemia with milder disease, and 15 patients with sickle cell anemia with more severe disease were recruited. Milder disease was defined as having no pain crisis within the previous year. More severe disease was defined as having had within the previous year three or more pain crises which were documented by a physician and required treatment with narcotics. The autonomic nervous system activity was determined by spectral analysis of nocturnal heart rate variability. Blood viscosity determination and measurements of several inflammatory markers (interleukin-6, soluble vascular cell adhesion molecule-1, soluble CD40 ligand and sL-selectin) were made on blood samples collected in steady-state conditions. RESULTS: Results showed that: 1) patients who had suffered more frequent pain crises had lower parasympathetic activity and greater sympatho-vagal imbalance than both controls and patients with milder disease. However, when adjusted for age, no significant difference was detected between the two sickle cell anemia patient groups; 2) patients who had suffered more frequent pain crises had higher blood viscosity than patients with milder disease, and this was not dependent on age. CONCLUSIONS: Results from the present study indicate that both the autonomic nervous system activity and blood viscosity are impaired in patients with sickle cell anemia exhibiting high frequency of pain crisis in comparison with those who did not experience a crisis within the previous year.

Endurance running trial in tropical environment: a blood rheological study.

PURPOSE: The aim of this study was to clarify whether exercising in a tropical climate induces blood rheology alterations despite ad libitum hydration. METHODS: Hematological, biochemical and hemorheological changes were investigated in young healthy adults (N = 9 men, 20.7 ± 0.8 yrs) after a 10-km race in hot and humid conditions. Subjects' maximal aerobic abilities were tested using a maximal ramp exercise. Blood was sampled at rest (TR), at the end of the race (TEx), and after 24 hours of recovery (T24). Ad libitum hydration was allowed during the race. Blood viscosity (ηb), red blood cell deformability (EI), aggregation (AI) and disaggregation shear rate (γ) were measured. RESULTS: Hematocrit, hemoglobin and plasma concentration of chlorine, sodium and potassium did not change in response to exercise. No functional consequence was observed on RBC deformability since EI remained unchanged. Percentages of echinocytes, schizocytes and stomatocytes remained in the subclinical range at all times. AI, γ and ηb did not present change. CONCLUSION: Running exercise in tropical climate with ad libitum hydration does not alter the main rheological properties of blood.

Alpha-thalassemia is associated with a decreased occurrence and a delayed age-at-onset of albuminuria in sickle cell anemia patients.

The aim of this study was to identify possible risk factors for albuminuria, an early marker of sickle cell anemia (SCA) glomerulopathy, in a cohort of 189 SCA adult patients followed at the Sickle Cell Center of Guadeloupe, a French Caribbean island. Biological parameters obtained at baseline, alpha-globin gene status, and beta(S) haplotypes were compared in patients stratified accordingly to graded albuminuria. Abnormal albumin excretion rate was detected in half of the studied adult patients and macroalbuminuria occurred in 21.6%. Graded albuminuria was associated with advanced age (p=0.006), systolic blood pressure (p=0.031), and worsened anemia, i.e. low hemoglobin rate (p<0.0001) and red blood cell count (p<0.0001). Alpha-thalassemia frequency was lower in microalbuminuric and macroalbuminuric patients than in normoalbuminuric patients, 12.5%, 13.75% and 26%, respectively (p=0.0057). Comparison of albuminuria-free survival curves in SCA patients without and with alpha-thalassemia showed that the median time of albuminuria onset was delayed in the later ones (p=0.021). In contrast, no association of albuminuria was detected with the Bantou beta(S) haplotype. Our results strongly suggest a protective effect of alpha-thalassemia against glomerulopathy in SCA adult patients which could be related to a decreased hemolytic rate.

Effect of interleukin-8 and RANTES on the Gardos channel activity in sickle human red blood cells: role of the Duffy antigen receptor for chemokines.

We investigated the effects of the chemokines IL-8 and RANTES on the activity of the Gardos channel (GC) of sickle red blood cells (SSRBCs). SSRBCs expressing the Duffy antigen receptor for chemokines (DARC) incubated under oxygenated conditions exhibit GC activation. The deoxygenation-stimulated K(+) loss via the GC is activated by the chemokines in the Duffy-positive SSRBCs. The percentage of cells with high density is 17 times higher in the Duffy-positive group. These findings are consistent with a greater susceptibility of Duffy-positive SSRBCs to inflammatory chemokines leading to GC activation and cellular dehydration and suggest a coupling, promoted by the sickling process, between DARC and the GC.

Association between Duffy antigen receptor for chemokines expression and levels of inflammation markers in sickle cell anemia patients.

Since inflammation plays a prominent role in the pathogenesis of sickle cell anemia (SCA) and Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes, we analyzed the relationship between the erythrocyte DARC phenotype and clinical expression of SCA. DARC locus was genotyped in 212 SS adult patients followed by the sickle cell center of Guadeloupe (French West Indies). After patients' stratification according to RBC DARC expression, the prevalence of renal disease, leg ulcers, priapism and osteonecrosis was compared between patient groups as well as hematological variables and plasma levels of chemokines. Duffy-positive patients exhibited higher counts of white blood cells (9.95+/-2.36 vs 8.88+/-2.32 10(9)/L, p=0.0066), polynuclear neutrophils (5.1+/-1.73 vs 4.51+/-1.71 10(9)/L, p=0.0227), higher plasma levels of IL-8 (4.46+/-1.22 vs 1.47+/-0.5 pg/mL, p=0.0202) and RANTES (27.8+/-4.3 vs 18.1+/-2.3 ng/mL, p=0.04) than Duffy-negative patients. No association was detected between RBC expression of DARC and the studied complications.