Laurel Mendelsohn

Chronic Hyper-Hemolysis in Sickle Cell Anemia: Association of Vascular Complications and Mortality with Less Frequent Vasoocclusive Pain

Background Intravascular hemolysis in sickle cell anemia could contribute to complications associated with nitric oxide deficiency, advancing age, and increased mortality. We have previously reported that intense hemolysis is associated with increased risk of vascular complications in a small cohort of adults with sickle cell disease. These observations have not been validated in other populations. Methods The distribution of serum lactic dehydrogenase (LDH) values was used as a surrogate measure of intravascular hemolysis in a contemporaneous patient group and an historical adult population from the Cooperative Study of Sickle Cell Disease (CSSCD), all with sickle cell anemia. Chronic hyper-hemolysis was defined by the top LDH quartile and was compared to the lowest LDH quartile. Results Hyper-hemolysis subjects had higher systolic blood pressure, higher prevalence of leg ulcers (OR 3.27, 95% CI 1.92-5.53, P<0.0001), priapism (OR 2.62, 95% CI 1.13-6.90, P = 0.03) and pulmonary hypertension (OR 4.32, 95% CI 2.12-8.60, P<0.0001), while osteonecrosis (OR 0.32, 95% CI 0.19-0.54, P<0.0001) and pain (OR 0.23, 95% CI 0.09-0.55, P = 0.0004) were less prevalent. Hyper-hemolysis was influenced by fetal hemoglobin and α thalassemia, and was a risk factor for early death in the CSSCD population (Hazard Ratio = 1.97, P = 0.02). Conclusions Steady state LDH measurements can identify a chronic hyper-hemolysis phenotype which includes less frequent vasooclusive pain and earlier mortality. Clinicians should consider sickle cell specific therapies for these patients, as is done for those with more frequent acute pain. The findings also suggest that an important class of disease modifiers in sickle cell anemia affect the rate of hemolysis.

Production of gene-corrected adult beta globin protein in human erythrocytes differentiated from patient iPSCs after genome editing of the sickle point mutation

Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene‐corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β‐globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild‐type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno‐free and feeder‐free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%–10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16‐kDa β‐globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome‐edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient‐derived iPSCs to generate disease‐free cells for cell and gene therapies. Stem Cells 2015;33:1470–1479

Prevalence and risk factors for pulmonary artery systolic hypertension among sickle cell disease patients in Nigeria

Pulmonary artery systolic hypertension is common and associated with increased mortality among adult sickle cell disease (SCD) patients in the United States. Although the prevalence of SCD is highest in sub‐Saharan Africa, the frequency of pulmonary artery systolic hypertension and the risk factors for the development of pulmonary hypertension have not been reported from Africa. We studied 208 hydroxyurea naïve Nigerian SCD patients at steady state and 94 healthy controls. Pulmonary artery systolic hypertension was defined prospectively as tricuspid regurgitant jet velocity ≥2.5 m/sec. Results were compared with a previously published US prospective SCD cohort. Only 7% of Nigerians compared with 46% of US adults with SCD were >35 years. Tricuspid regurgitant jet velocity was ≥2.5 m/sec in 25% of Nigerian SCD patients. Higher jet velocity was associated with greater serum globulin (P = 0.002), blood urea nitrogen (P = 0.019) and lactate dehydrogenase concentrations (P = 0.026) and with inability to walk >300 m in 6 min (P = 0.042). Compared with the US cohort, Nigerian patients had more hemolysis as indicated by lower hemoglobin and higher lactate dehydrogenase concentrations (P ≤ 0.003). Pulmonary hypertension is common among Nigerian SCD patients. The public health implication of this finding is significant considering the potential number of individuals at risk for this complication. Better understanding of the long term outcome of pulmonary hypertension and causes of death in SCD and the institution of preventive measures are major public health challenges for Africa. The inclusion of African sites in sickle cell pulmonary hypertension clinical trials should be encouraged. Am. J. Hematol., 2008.

High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.

Pulmonary hypertension is associated with reduced nitric oxide bioavailability and early mortality in sickle cell disease (SCD). We previously demonstrated that placenta growth factor (PlGF), an angiogenic factor produced by erythroid cells, induces hypoxia-independent expression of the pulmonary vasoconstrictor endothelin-1 in pulmonary endothelial cells. Using a lentivirus vector, we simulated erythroid expression of PlGF in normal mice up to the levels seen in sickle mice. Consequently, endothelin-1 production increased, right ventricle pressures increased, and right ventricle hypertrophy and pulmonary changes occurred in the mice within 8 weeks. These findings were corroborated in 123 patients with SCD, in whom plasma PlGF levels were significantly associated with anemia, endothelin-1, and tricuspid regurgitant velocity; the latter is reflective of peak pulmonary artery pressure. These results illuminate a novel mechanistic pathway linking hemolysis and erythroid hyperplasia to increased PlGF, endothelin-1, and pulmonary hypertension in SCD, and suggest that strategies that block PlGF signaling may be therapeutically beneficial.

NT-pro Brain Natriuretic Peptide Levels and the Risk of Death in the Cooperative Study of Sickle Cell Disease

Epidemiological studies support a hypothesis that pulmonary hypertension (PH) is a common complication of sickle cell disease (SCD) that is associated with a high risk of death and evolves as a complication of haemolytic anaemia. This fundamental hypothesis has been recently challenged and remains controversial. In order to further test this hypothesis in a large and independent cohort of SCD patients we obtained plasma samples from the Cooperative Study of Sickle Cell Disease (CSSCD) for analysis of a biomarker, N‐terminal‐pro brain natriuretic peptide (NT‐proBNP), which is elevated in the setting of pulmonary arterial and venous hypertension. A NT‐pro‐BNP value previously identified to predict PH in adults with SCD was used to determine the association between the risk of mortality in 758 CSSCD participants (428 children and 330 adults). An abnormally high NT‐proBNP level ≥160 ng/l was present in 27·6% of adult SCD patients. High levels were associated with markers of haemolytic anaemia, such as low haemoglobin level (P < 0·001), high lactate dehydrogenase (P < 0·001), and high total bilirubin levels (P < 0·007). A NT‐proBNP level ≥160 ng/l was an independent predictor of mortality (RR 6·24, 95% CI 2·9–13·3, P < 0·0001). These findings provide further support for an association between haemolytic anaemia and cardiovascular complications in this patient population.

INCREASED PULMONARY PRESSURES AND MYOCARDIAL WALL STRESS IN CHILDREN WITH SEVERE MALARIA

BACKGROUND Chronic intravascular hemolysis leads to nitric oxide (NO) depletion and pulmonary hypertension in sickle cell disease. To test whether this pathophysiology occurs in malaria, we examined in Mali 53 children who were admitted to the hospital with severe malaria (excluding cerebral malaria) and 31 age-matched controls. METHODS Severity of hemolysis was assessed from plasma levels of free hemoglobin and arginase-1. NO metabolism was assessed by whole-blood nitrite levels and plasma NO consumption. Effects on the cardiovascular system and endothelial function were assessed by using echocardiography to measure peak tricuspid regurgitant jet velocity and by evaluating plasma levels of N-terminal prohormone brain natriuretic peptide (NT-proBNP) and soluble vascular cell adhesion molecule-1. RESULTS Children with severe malaria had higher plasma levels of hemoglobin and arginase-1, reduced whole-blood levels of nitrite, and increased NO consumption relative to controls. They also had increased pulmonary arterial pressures (P< .05) with elevated levels of NT-proBNP and soluble vascular cell adhesion molecule-1 (P< .001). CONCLUSION Children with severe malaria have increased pulmonary pressures and myocardial wall stress. These complications are consistent with NO depletion from intravascular hemolysis, and they indicate that the pathophysiologic cascade from intravascular hemolysis to NO depletion and its cardiopulmonary effects is activated in children with severe malaria.

Iron, inflammation, and early death in adults with sickle cell disease

Rationale: Patients with sickle cell disease (SCD) have markers of chronic inflammation, but the mechanism of inflammation and its relevance to patient survival are unknown. Objective: To assess the relationship between iron, inflammation, and early death in SCD. Methods and Results: Using peripheral blood mononuclear cell transcriptome profile hierarchical clustering, we classified 24 patients and 10 controls in clusters with significantly different expression of genes known to be regulated by iron. Subsequent gene set enrichment analysis showed that many genes associated with the high iron cluster were involved in the toll-like receptor system (TLR4, TLR7, and TLR8) and inflammasome complex pathway (NLRP3, NLRC4, and CASP1). Quantitative PCR confirmed this classification and showed that ferritin light chain, TLR4, and interleukin-6 expression were >100-fold higher in patients than in controls (P<0.001). Further linking intracellular iron and inflammation, 14 SCD patients with a ferroportin Q248H variant that causes intracellular iron accumulation had significantly higher levels of interleukin-6 and C-reactive protein compared with 14 matched SCD patients with the wild-type allele (P<0.05). Finally, in a cohort of 412 patients followed for a median period of 47 months (interquartile range, 24–82), C-reactive protein was strongly and independently associated with early death (hazard ratio, 3.0; 95% confidence interval, 1.7–5.2; P<0.001). Conclusions: Gene expression markers of high intracellular iron in patients with SCD are associated with markers of inflammation and mortality. The results support a model in which intracellular iron promotes inflammatory pathways, such as the TLR system and the inflammasome, identifying important new pathways for additional investigation.

Heme-bound iron activates placenta growth factor in erythroid cells via erythroid Krüppel-like factor

In adults with sickle cell disease (SCD), markers of iron burden are associated with excessive production of the angiogenic protein placenta growth factor (PlGF) and high estimated pulmonary artery pressure. Enforced PlGF expression in mice stimulates production of the potent vasoconstrictor endothelin-1, producing pulmonary hypertension. We now demonstrate heme-bound iron (hemin) induces PlGF mRNA >200-fold in a dose- and time-dependent fashion. In murine and human erythroid cells, expression of erythroid Krüppel-like factor (EKLF) precedes PlGF, and its enforced expression in human erythroid progenitor cells induces PlGF mRNA. Hemin-induced expression of PlGF is abolished in EKLF-deficient murine erythroid cells but rescued by conditional expression of EKLF. Chromatin immunoprecipitation reveals that EKLF binds to the PlGF promoter region. SCD patients show higher level expression of both EKLF and PlGF mRNA in circulating blood cells, and markers of iron overload are associated with high PlGF and early mortality. Finally, PlGF association with iron burden generalizes to other human diseases of iron overload. Our results demonstrate a specific mechanistic pathway induced by excess iron that is linked in humans with SCD and in mice to markers of vasculopathy and pulmonary hypertension. These trials were registered at www.clinicaltrials.gov as #NCT00007150, #NCT00023296, #NCT00081523, and #NCT00352430.

Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease.

In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patients disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged “sickled” and “normal” erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = −0.558, P = 0.027), negatively with pH (R = −0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes‐103 (5‐hydroxymethyl‐2‐furfural) (R = −0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator‐independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes‐103. SIFCA is a new method that may be useful in sickle cell drug development. Am. J. Hematol. 89:598–603, 2014.

Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors

There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10(6)-10(7)-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~10(68)-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine.