Development of a de novo cerebral arteriovenous malformation in a child with sickle cell disease and moyamoya arteriopathy

Abstract The erythrocyte redox environment may contribute to increased hemolysis and decreased nitric oxide (NO) bioavailability in pulmonary hypertension (PH) of sickle cell disease (SCD). Glutathione (GSH) is the principal thiol redox buffer in erythocytes and its depletion has been linked to hemolysis. Glutamine plays an additional anti-oxidant role through preservation of the intracellular nicotinamide adenine dinucleotide (NAD) levels, required for reducing GSSG back to GSH. We hypothesized that altered GSH and glutamine metabolism promotes hemolysis and contributes to PH in SCD. Glutamine, total glutathione (GSH+GSSG) and its precursors (glutamate, cysteine, glycine) were assayed in plasma and erythrocytes of 40 SCD patients and 9 controls. PH is defined by echocardiogram as a tricuspid regurgitant jet velocity (TRV) ≥ 2.5m/s. Total plasma glutathione was lower in SCD vs control patients (2.7 ± 0.3 μM vs. 4.1± 0.8 μM, p<0.05). Similarly, total erythrocyte glutathione levels were decreased in SCD vs. control patients (310 ± 26 μM vs. 683 ± 110 μM, p<0.0001). A trend towards higher GSH precursor levels identified in plasma and erythrocyte compartments suggests that the total glutathione (GSH+GSSG) deficit is due to heightened rate of GSH utilization rather than decreased synthesis capacity. While severity of erythrocyte GSH depletion was similar in SCD patients with and without PH, erythrocyte glutamine levels differed significantly (482±92μM, n=17 vs 934±134μM, n=23, p<0.02) and values inversely correlated to TRV (r = −0.51, p<0.0001). As glutamine is required for de novo synthesis of NAD(P)+ essential for GSH recycling, lower steady-state glutamine levels may reflect enhanced GSH utilization rates in the SCD erythrocytes. A significant reduction in the erythrocyte glutamine:glutamate ratio occurred in SCD patients compared with normal volunteers, with the lowest ratios observed in SCD patients with PH. The glutamine:glutamate ratio, potentially a gauge of NADPH biosynthesis and oxidative stress, was inversely correlated with TRV (r = −0.62, p<0.001), implicating glutamine bioavailability as a novel factor in the pathophysiology of PH. Changes in the glutamine:glutamate ratio were predominantly caused by decreased erythrocyte glutamine levels rather than increased glutamate levels, ruling out an effect on the ratio from increased cellular glutamine uptake. Erythrocyte glutamine:glutamate ratio correlated with age in patients with SCD (r = −0.33, p=0.04), and inversely correlated with plasma arginase concentratoin (r= −0.45, p=0.012), and plasma-Hb (r= −0.41, p=0.01), linking lower glutamine bioavailability to increased red cell derived plasma arginase, hemolysis and potentially with increased mortality in PH of SCD as previously reported (Morris et al, JAMA 2005). Decreased erythrocyte total glutathione and glutamine levels contribute to alterations in the erythrocyte redox environment, which compromise erythrocyte integrity and NO bioavailability and may play a role in hemolysis and the pathogenesis of PH of SCD.

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Extracellular Hemin Auto-Amplification Intensifies Tissue Injury In Sickle Cell Disease

Blood ◽

10.1182/blood.v122.21.972.972 ◽

2013 ◽

Vol 122 (21) ◽

pp. 972-972

Author(s):

Samit Ghosh ◽

Solomon F. Ofori-Acquah

Keyword(s):

Sickle Cell Disease ◽

Oxygen Saturation ◽

Organ Failure ◽

Sickle Cell ◽

De Novo ◽

Tissue Injury ◽

Time Interval ◽

Cell Disease ◽

Intravascular Hemolysis ◽

Breath Rate

Abstract Acute organ failure is a major clinical concern in sickle cell disease (SCD). However, the mechanism responsible for this potentially lethal complication is poorly understood. We tested the hypothesis that extracellular hemin liberates an intracellular danger molecule that promotes acute organ failure in SCD. Transgenic homozygous SCD (SS), sickle-trait (AS) and normal human hemoglobin (Hb) AA mice were infused with purified hemin (35 µmoles/kg), which raised total plasma hemin by ∼0.45 mM (equivalent to 0.72 g/dl Hb) within 5 min in all three groups of mice. In agreement with our previous results, SS but not AA and AS mice (n= 6 for each genotype) developed cardiopulmonary depression at 30 min evident by reductions in oxygen saturation (99.88±0.23% to 92.1±1.3%, p<0.001), breath rate (175.4±20.6 to 77.36±2.25, p<0.001, breath per min), heart rate (574.5±22.7 to 361.9±23.25 beats per min, p<0.001) and pulse distension (512.8±18.7 to 238.8±17.6 µm, p<0.001), and ∼70% of these animals died within 2 hours. Markedly raised lung wet/dry weight ratio in SS mice that succumbed to hemin suggests that the cardiopulmonary depression was secondary to a severe pulmonary edema. To identify biological correlates for the acute adverse effects in the SS mice, cohorts of both sickle and control mice were challenged with the same dose of hemin, blood samples were drawn at baseline (i.e. time=0 min), and 5 and 30 min after the hemin infusion and analyzed for markers of oxidative stress, tissue damage, plasma scavengers and high mobility group box-1 (HMGB-1), a prototypical danger molecule. Plasma hemopexin decreased by ∼80% at 5 min compared to baseline values in all three groups of mice regardless of the Hb genotype. The catabolism of hemopexin was associated with clearance of ∼50% of the hemin infusion from the circulation of AS and AA mice at 30 min. Paradoxically, the plasma concentration of hemin in the SS mice during this same time interval increased by ∼0.2 mM (p<0.001, n=6). The magnitude of this increase was dependent on the dose of hemin administered exogenously. We discovered that the de novo hemin release in the SS mice was preceded by acute intravascular hemolysis (mean decrease in total Hb: ∼1.4 g/dl, p<0.001, n=9, mean increase in cell-free Hb: 1.0 g/dl, p=0.001, n=9), oxidation of oxyHbS to metHbS (mean increase: 12%, p<0.001, n=6) and persistence of metHbS. It is noteworthy that de novo hemin release did not occur in AS mice suggesting that this phenomenon is dominantly influenced by sickle erythrocytes and not by the presence of intracellular HbS per se. Auto-amplification of hemin may help to explain an observation made nearly fifty years ago that SCD patient plasma contains more hemin than the plasma of patients with more severe intravascular hemolysis involving normal adult Hb (e.g. paroxysmal nocturnal hemoglobinuria), who have higher plasma Hb. To determine whether this phenomenon is critical to the cardiopulmonary depression in the SS mice, recombinant human hemopexin was administered 5 min after the infusion to sequester the endogenous hemin release. In hemin challenged SS mice with respiratory distress, intravenous recombinant human hemopexin rapidly halted the decline in oxygen saturation and breath rate and averted inevitable respiratory failure. In conclusion, we have identified a phenomenon of extracellular hemin auto-amplification that appears to be unique to SCD, and may play a critical role in propagating tissue injury in this disorder. Factors that inhibit erythrocyte lysis and accelerate metHb reduction may help to limit extracellular hemin amplification and preserve organ function during episodes of acute exacerbations in SCD. Disclosures: No relevant conflicts of interest to declare.

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Treatment of a cerebral pial arteriovenous fistula in a patient with sickle cell disease-related moyamoya syndrome: case report

Journal of Neurosurgery Pediatrics ◽

10.3171/2014.12.peds14486 ◽

2015 ◽

Vol 16 (2) ◽

pp. 207-211 ◽

Cited By ~ 3

Author(s):

Anna Lo Presti ◽

Alexander G. Weil ◽

Aria Fallah ◽

Eric C. Peterson ◽

Toba N. Niazi ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Moyamoya Disease ◽

De Novo ◽

Transarterial Embolization ◽

Management Strategies ◽

Superficial Temporal Artery ◽

Temporal Artery ◽

Venous Aneurysm ◽

Cell Disease

Sickle cell disease (SCD) is an autosomal recessive hematological disorder, characterized by sickling of the red blood cells in response to a hypoxic stress and vaso-occlusive crises. It is associated with moyamoya-like changes on cerebral angiographic imaging in 43% of patients. Cerebral aneurysms, arteriovenous malformations, and dural arteriovenous fistulas (AVFs) have been described in association with SCD and moyamoya disease. However, the description of a pial AVF (pAVF) in a patient with SCD and/or moyamoya formation has not yet been reported. The authors present the case of a 15-year-old boy with SCD-associated moyamoya disease harboring a pAVF who developed a de novo venous aneurysm 8 months after undergoing indirect superficial temporal artery-middle cerebral artery (MCA) bypass that was complicated by bilateral ischemia of the MCA territory. The pAVF was successfully treated with transarterial embolization using Onyx. The authors describe the possible pathophysiological mechanisms and management strategies for this rare occurrence.

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Base Editing-Mediated Dissection of the -200 Region of the γ-Globin Promoters to Induce Fetal Hemoglobin and Rescue Sickle Cell Disease and β-Thalassemia

Blood ◽

10.1182/blood-2021-149710 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 562-562

Author(s):

Panagiotis Antoniou ◽

Giulia Hardouin ◽

Pierre Martinucci ◽

Tristan Felix ◽

Letizia Fontana ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

De Novo ◽

Clinical Severity ◽

Erythroid Cell ◽

Cell Phenotype ◽

Cell Disease ◽

Ineffective Erythropoiesis ◽

Base Editing ◽

Target Activity

Abstract β-hemoglobinopathies are caused by mutations affecting the adult hemoglobin production. In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. In β-thalassemia, reduced β-globin production leads to precipitation of uncoupled α-chains causing ineffective erythropoiesis and the production of poorly hemoglobinized RBCs. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. The clinical severity of β-hemoglobinopathies is alleviated by the co-inheritance of mutations causing hereditary persistence of fetal Hb (HPFH). HPFH mutations clustering 200 nucleotides upstream of the TSS of the fetal γ-globin (HBG) genes either disrupt the binding site (BS) of the fetal Hb (HbF) repressor LRF or generate a de novo BS for the KLF1 activator. To reactivate γ-globin expression, nuclease-based approaches have been explored. However, nucleases generate double-strand breaks (DSBs), raising safety concerns for clinical applications. Base editing (BE) allows the introduction of point mutations without generating DSBs. In this study, we designed BE systems to introduce a variety of HPFH or HPFH-like mutations in the -200 region of the HBG promoters. First, we screened in erythroid cell lines known and novel BEs, and we selected combinations of BEs and guide RNAs that edit alternative bases of the -200 region. We then developed a clinically-relevant protocol based on RNA-transfection to deliver the BE system to HSPCs. The expression profile of genes activated by RNA stimuli revealed no immune response in HSPCs. A progenitor assay indicated no alteration in the growth and multilineage differentiation of edited HSPCs. We applied this protocol to SCD and β-thalassemia HSPCs, achieving editing efficiencies up to ~70% of the HBG promoters. In RBCs differentiated from edited SCD HSPCs, RT-qPCR, HPLC and flow cytometry showed a potent γ-globin reactivation with a high frequency of HbF + cells and a concomitant decrease in the HbS content/cell. Importantly, the pathological RBC sickling phenotype was corrected in the samples derived from edited HSPCs. Similarly, in β-thalassemia samples, RT-qPCR and HPLC analyses showed strong γ-globin induction and decrease of the α-globin precipitates. HbF expression rescued the delay in erythroid differentiation and ineffective erythropoiesis characterizing β-thalassemia, as demonstrated by the increased RBC enucleation rate and the reduced apoptosis and oxidative stress. We then compared BE strategies that either disrupt the LRF BS or create a de novo KLF1 BS in single colonies derived from erythroid progenitors. Generation of the KLF1 BS was associated with higher levels of HbF compared to the LRF BS disruption. These results suggest that eviction of the LRF repressor is sufficient to reactivate HBG genes, but recruitment of an activator is more effective to achieve high levels of gene expression. HbF expression induced by both LRF BS disruption and KLF1 BS generation was sufficient to rescue the SCD cell phenotype, but higher HbF levels - achieved only through KLF1 BS generation - were necessary to fully correct the β-thalassemia phenotype. In the majority of cases, we detected no DSB-induced insertions, deletions, or large genomic rearrangements in base-edited samples. Accordingly, DSB-induced DNA damage response (DDR) was absent in base-edited HSPCs, as measured by evaluating the expression of p21, a readout of p53-induced DDR. DNA off-target activity was assessed by GUIDE-seq and targeted sequencing of the potential off-target sites in edited HSPCs, while RNA off-target activity was evaluated by RNA-seq in HSPCs. Finally, BE-treated HSPCs were transplanted in immunodeficient mice to evaluate the engraftment and differentiation capability of edited HSCs. We detected good frequencies of human cells with up to ~60% of edited promoters in the peripheral blood of transplanted mice. In conclusion, we developed a clinically-relevant strategy to perform efficient BE in the HBG promoters that led to therapeutically-relevant HbF levels and rescued both the SCD and β-thalassemia phenotypes, thus providing sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies. Disclosures El Nemer: Hemanext: Consultancy.

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