A Novel Hemoglobin Binding Peptide Increases Intracellular Heme and Potentiates Hemoglobin-Induced HO-1 Levels in Endothelial Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1065-1065
Author(s):  
Madelyn S. Hanson ◽  
Timothy C. Flewelen ◽  
Hao Xu ◽  
Kirkwood A Pritchard ◽  
Nancy J Wandersee ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Vascular Dysfunction ◽  
Murine Models ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Free Hemoglobin ◽  
Hemoglobin Binding

Abstract Abstract 1065 The hemolysis that occurs in many forms of hereditary and acquired hemolytic anemia, including sickle cell disease, saturates the hemoglobin/heme scavenging system resulting in increased levels of cell-free hemoglobin circulating in the plasma. Several recent studies have suggested a central role for intravascular hemolysis and cell-free hemoglobin in the development of vascular dysfunction, including pulmonary hypertension, in affected humans potentially by imposing oxidative and inflammatory stress. In agreement, mouse models of sickle cell disease and severe hereditary spherocytosis also develop vascular dysfunction and pulmonary hypertension. However, the role of intravascular hemolysis and cell-free hemoglobin in vascular dysfunction has proved controversial and a resolution of this important issue requires new experimental tools. This controversy highlights the importance of understanding if cell-free hemoglobin does indeed contribute to vascular complications associated with sickle cell disease. To address the role of cell-free hemoglobin in vascular pathology, we have synthesized a novel hemoglobin-binding peptide, hE-Hb-B10. This peptide is linked to a small fragment of apolipoprotein-E (apoE) to facilitate the endocytic clearance of cell-free hemoglobin through the ubiquitous heparin sulfate proteoglycan (HSPG)-associated lipoprotein pathway versus hemoglobin/heme scavenging system. We have shown previously that hE-Hb-B10 reduces cell-free hemoglobin levels and restores NO-dependent vascular function in murine models of hemolytic anemia. In the current studies, we investigate the cellular response of endothelial cells to hemoglobin uptake facilitated by hE-Hb-B10. We show that treatment of bovine aortic endothelial cells (BAECs) with oxyhemoglobin in the presence of hE-Hb-B10 augments intracellular heme concentration compared to oxyhemoglobin alone. Additionally, incubation of BAECs with methemoglobin increases heme oxygenase-1 (HO-1) protein levels and this induction is potentiated by hE-Hb-B10. hE-Hb-B10 also augments HO-1 induction by oxyhemoglobin, suggesting that hemoglobin uptake facilitated by hE-Hb-B10 is not dependent on the oxidation state of hemoglobin. In contrast, both Hb-B10, a peptide lacking the apoE fragment, and the scrambled hE-Hb-sB10 peptide in which the hemoglobin-binding sequence is scrambled, inhibit HO-1 induction caused by hemoglobin. Taken together, these data suggest that hE-Hb-B10 facilitates hemoglobin uptake into endothelial cells, augmenting both intracellular heme concentration and the induction of HO-1 by hemoglobin. While HO-1 expression is indicative of oxidative stress, enzymatic products of HO-1 can provide important protective functions against oxidative stress and iron overload. Therefore, altering HO-1 expression in SCD could potentially improve or worsen the severity of this disease. Indeed, potentiating HO-1 levels in models of SCD has been shown to be protective in murine models of SCD. Overall, our findings demonstrate that hE-Hb-B10 is a useful tool in determining the role of Cell-free hemoglobin in SCD pathology and suggests a mechanism by which this novel peptide could impact disease outcome. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Influence of Haptoglobin Polymorphism on Stroke in Sickle Cell Disease Patients

Genes ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 144
Author(s):  
Olivia Edwards ◽  
Alicia Burris ◽  
Josh Lua ◽  
Diana J. Wilkie ◽  
Miriam O. Ezenwa ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Blood Cells ◽  
Cell Disease ◽  
Free Hemoglobin ◽  
Cerebral Tissue ◽  
Sickle Hemoglobin ◽  
Haptoglobin Polymorphism

This review outlines the current clinical research investigating how the haptoglobin (Hp) genetic polymorphism and stroke occurrence are implicated in sickle cell disease (SCD) pathophysiology. Hp is a blood serum glycoprotein responsible for binding and removing toxic free hemoglobin from the vasculature. The role of Hp in patients with SCD is critical in combating blood toxicity, inflammation, oxidative stress, and even stroke. Ischemic stroke occurs when a blocked vessel decreases oxygen delivery in the blood to cerebral tissue and is commonly associated with SCD. Due to the malformed red blood cells of sickle hemoglobin S, blockage of blood flow is much more prevalent in patients with SCD. This review is the first to evaluate the role of the Hp polymorphism in the incidence of stroke in patients with SCD. Overall, the data compiled in this review suggest that further studies should be conducted to reveal and evaluate potential clinical advancements for gene therapy and Hp infusions.

Heme Induces Endothelial Tissue Factor Expression: Potential Role in Hemostatic Activation in Patients with Intravascular Hemolysis Including Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1902-1902
Author(s):  
Yamaja Setty ◽  
Suhita Gayen Betal ◽  
Jie Zhang ◽  
Nigel S Key ◽  
Marie Stuart
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Dependent Manner ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Tnf Α ◽  
Endothelial Tissue

Abstract Plasma levels of heme in the 20 to 600 μM range are found in clinical conditions associated with intravascular hemolysis including paroxysmal nocturnal hemoglobinuria and sickle cell disease, conditions also associated with a thrombotic tendency. Objectives: To investigate whether heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease (SCD), could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Additionally, in SCD patient-related studies, we assessed whether any association existed between whole blood TF activity (WBTF) and levels of surrogate markers of intra-vascular hemolysis including lactate dehydrogenase (LDH) and reticulocyte counts. Methods: Following incubation of human endothelial cells (from umbilical vein and/or lung microvasculature) with heme (1 to 100 μM) for various times (30 minutes to 8 hours), levels of TF protein were assessed using ELISA, flow cytometry and/or Western blotting; and TF mRNA by a semi-quantitative RT-PCR. An assay for TF functional activity was performed using a chromogenic tenase activity kit where specificity of TF activity was tested in antibody-blocking experiments. Three TF-specific antibodies including a rabbit polyclonal and two mouse monoclonal (clones hTF-1 and TF9-10H10) antibodies were used in assays involving TF protein analysis. All experiments were performed in media containing polymyxin B to neutralize any potential endotoxin contamination. In patient-related studies, 81 subjects with SCD (1 to 21 years) were evaluated for levels of WBTF, LDH, and reticulocyte counts and data analyzed for potential relationships. Results: Heme induced TF protein expression on the surface of both macro- and micro-vascular endothelial cells in a concentration-dependent manner with 12- to 50-fold induction noted (ELISA assays) between 1 and 100 μM heme (P<0.05, n=3 to 6). Complementary flow cytometry studies showed that the heme-mediated endothelial TF expression was quantitatively similar to that induced by the cytokine TNF-α. Heme also up-regulated endothelial expression of TF mRNA (8- to 26-fold, peak expression at 2 hours postagonist treatment), protein (20- to 39-fold, peak expression at 4 hours) and procoagulant activity (5- to 13-fold, peak activity at 4 hours post-agonist treatment) in a time-dependent manner. Time-course of heme-mediated TF antigen expression paralleled induction of procoagulant activity with antibody blocking studies demonstrating specificity for TF protein. Potential involvement of endogenously released cytokines including IL-1α and TNF-α in mediating the heme effect was next explored. We found that the latter cytokines are not involved, since antibodies against IL-1α and TNF-α, and an IL-1- receptor antagonist failed to block heme-induced endothelial TF expression. Inhibition of heme-induced TF mRNA expression by sulfasalazine and curcumin suggested that the transcription factor NFκB was involved in mediating heme-induced effect. In patient-related studies, whole blood TF levels in SCD correlated positively with both LDH (r=0.72, p<0.000001), and reticulocyte count (r=0.60, p<0.000001). Conclusions: Our findings demonstrate that heme induces TF expression in endothelial cells, and that the observed effects occurred at patho-physiologically relevant heme concentrations. Our results suggest that heme-induced endothelial TF expression may provide a pathophysiologic link between the intravascular hemolytic milieu and the hemostatic perturbations previously noted in patients with hemolytic anemia including sickle cell disease.

scholarly journals Persistence of Chronic Inflammation Despite Years of Transfusion Program in SCD Patients: Changing Red Blood Cells Is Not Sufficient to Treat Sickle Cell Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 764-764
Author(s):  
Abdoul Karim Dembele ◽  
Patricia Hermand-Tournamille ◽  
Florence Missud ◽  
Emmanuelle Lesprit ◽  
Malika Benkerrou ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Chronic Inflammation ◽  
Sickle Cell ◽  
Blood Cells ◽  
Healthy Controls ◽  
Cell Disease ◽  
The Impact

Abstract Sickle cell disease (SCD) is a severe hemoglobinopathy due to abnormal hemoglobin S (HbS). Although red blood cell dysfunction is at the core of the SCD pathophysiology, several studies have highlighted the important role of inflammatory cells like neutrophils. One of the most serious complications of SCD is cerebral vasculopathy (CV), due to the occlusion of one or more intracranial or cervical arteries. In 1998, the STOP study demonstrated that monthly blood transfusions could reduce the risk of stroke by 90% in children with CV. However, there is large heterogeneity in the evolution of CV under chronic transfusion, sometimes requiring exchange transfusion (ET) program for years without succeeding in healing the CV. The aim of the study is to investigate the impact of long-term transfusion program on neutrophil dysfunction, in order to understand if persistent inflammation could contribute to the non-healing of CV despite HbS permanently below 40%. In SCD children undergoing ET program for at least 1 year, we analysed i)the phenotype of neutrophils with 8 markers of activation/adhesion/ageing, ii)the plasmatic levels of elastase, witnessing the NETose activity of neutrophils, and iii)the ex-vivo adhesion of neutrophils on activated endothelial cells. One hundred and two SCD children with an ET transfusion program for at least 6 months because of CV were included in the study. ET session, carried out every 5 weeks and most of the time by erythrapheresis, reached their biological objectives with a mean HbS rate after ET session of 14.1%, and 35.4% before the next ET session, which means that these patients globally live at an average HbS level of 24% for at least 1 year. We managed to limit iron overload with a mean ferritinemia of 207 µg/L in the whole cohort. Despite these satisfactory results in terms of HbS reduction, the efficiency in curing the CV was modest in accordance with the previously described efficiency of ET program in SCD children: after a mean ET program duration of 4.4 years only 22% of them had an improvement of their CV since the beginning of the ET program, while 60% of them had a stagnation of their CV, and 18% of them worsened their vascular lesions. Considering inflammatory parameters, the patients had persistence of high leukocytosis and high neutrophils count (respective mean of 9810 G/L and 5742 G/L), significantly not different of neutrophils count before inclusion in the ET program. In a random subgroup of 20 patients, we analysed neutrophils phenotype, NETose and endothelial adhesion and compared them to healthy controls and SCD children without ET, treated or not with Hydroxyurea (HU). Overall, we observed as expected an activated, aged and adherent profile of neutrophils from untreated SCD children compared to healthy controls, characterized by an overexpression of CD18/CD11b (p=0,03), CD18/CD11a (p=0,02), CD162 (p=0,01), CD66a (p=0,01) and the ageing markers CD184 high/CD62Llow (p=0,04) as well as a higher plasmatic level of elastase (p=0. 01) and higher adhesion of neutrophils to endothelial cells. All these parameters were alleviated in SCD patients treated with HU. In SCD patient undergoing ET program, we found a similar profile of activated neutrophils to that of untreated SCD patients with a similar expression of activation molecules, high level of elastase and the same increase of neutrophils adhesion to endothelial cells compared to controls, witnessing a persistence of chronic inflammation despites years of ET. Overall, our study highlights that the replacement of sickle red blood cells, even for years, is not sufficient to reverse the deleterious inflammatory phenotype of neutrophils. Given the major role of inflammation in endothelial dysfunction, these could contribute to the persistence of CV in a majority of patients despite efficient ET programs. This raises the question of systematically combining ET program with anti-inflammatory treatment such as HU or P-selectin inhibitors in children with CV. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Role of Macrophage Stimulating Protein 1 (MSP1) in the Development of Glomerulopathy in Sickle Cell Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4583-4583
Author(s):  
Marina Jerebtsova ◽  
Elena Afia Adjei ◽  
Alfia Khaibullina ◽  
Zena Quezado ◽  
Sergei A. Nekhai
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ischemia Reperfusion ◽  
Cell Disease ◽  
Macrophage Stimulating Protein ◽  
Capillary Formation ◽  
Glomerular Lesions

Abstract Background More than 50% patients with sickle cell disease (SCD) develop renal glomerular disease. Its pathophysiology is likely to be multifactorial, being affected by hyperfiltration, glomerular hypertension, ischemia-reperfusion injury, oxidative stress, and endothelial dysfunction. Ischemia-reperfusion is associated with significant recruitment of glomerular and interstitial macrophages. Recently, Macrophage Stimulating Protein 1 (MSP1) was shown to be involved in the development of anti-Thy1 glomerulonephritis in rat model that also associated with renal macrophages infiltration. MSP1 is produced by the liver and secreted into the circulation as a non-active protein. It is cleaved and activated locally by a macrophage membrane-associated proteinase. We hypothesesize that infiltrating macrophages in SCD activate MSP1 protein that accumulates in the renal glomeruli and induces endothelial cell and podocytes injury. Objective We analyzed MSP1 expression in vivo in SCD mouse model and the effect of recombinant MSP1 on cultured human podocytes and endothelial cells. Methods Transgenic SCD mice were obtained from Jackson Laboratory (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow/J Townes strain). Townes mice produce approximately 94% human sickle (HbS), 6% human fetal hemoglobin (HbF), and no murine hemoglobin. Control animals are carrying two copies of the transgene encoding human α1-globin gene and two copies of the Hbbtm3(HBG1,HBB)Tow mutation (human hemoglobin gamma (Aγ) gene and the human wildtype hemoglobin beta (βA) gene). Townes mice spontaneously develop renal glomerular lesions. Kidneys were collected from 5 months old mice and immunohistochemistry (IHC) was carried to detect macrophages, endothelial cells (CD34), podocytes (WT-1) and MSP1. Human glomerular endothelial cell line (HGEC) and two human podocyte lines (PD1 and PD2) were treated with recombinant MCP1 and the cellular motility, permeability, growth and capillary formation were analyzed. Results SCD mice developed focal segmental glomerulosclerosis. It was associated with glomerular capillary aneurisms, loss of podocytes and increased macrophages infiltration. IHC demonstrated accumulation of MSP1 in the capillaries of affected glomeruli. In vitro, MSP1 treatment increased motility of both endothelial cells and podocytes. In addition, MSP1 significantly reduced podocytes growth and viability. MSP1 also inhibited capillary formation by endothelial cells on Matrigel. Conclusion Activated MSP1 is likely to be involved in the development of glomerular lesions in SCD which could be due to the modulation of endothelial cell and podocytes function. Further analysis is needed to elucidate the role of MSP1 in the development of glomerular disease in humans. Acknowledgments This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005 and 5G12MD007597). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential Acetone Extraction of Total and Hemoprotein-Unbound Heme to Quantify Heme Binding Capacity of Plasma in Patients with Sickle Cell Disease: The Role of Heme Scavengers

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-15
Author(s):  
Madhav Vissa ◽  
Eric Soupene ◽  
Sandra K. Larkin ◽  
Lynne D. Neumayr ◽  
Elliott P. Vichinsky ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Research Funding ◽  
Binding Capacity ◽  
Principal Investigator ◽  
Heme Binding ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Free Hemoglobin ◽  
Neutral Ph

Introduction: Heme, an iron-containing protoporphyrin, is an essential component of hemoglobin that binds oxygen for delivery to tissues. In sickle cell disease, intravascular hemolysis leads to the presence of cell-free hemoglobin and heme, which may contribute to oxidative damage and activation of inflammatory pathways. Hemoproteins such as haptoglobin and hemopexin provide pathways to remove hemoglobin and heme, respectively, from circulation. Due to its hydrophobic nature, heme also intercalates in cell membranes and binds to plasma components such as albumin and lipoproteins, though with varying affinity. Hemopexin has high affinity for heme and removes heme from other heme pools in blood to counter the highly toxic properties of heme unbound to hemoproteins. Due to chronic hemolysis, hemopexin is depleted in individuals with sickle cell disease. We hypothesize that the reduction in heme binding capacity leads to increased unbound heme in blood and contributes to the pathogenesis of sickle cell disease. To define the different heme binding pools in patients with sickle cell disease, we developed a method requiring small amounts of plasma which allows measurement of total and hemoprotein-unbound heme. With this method, we can quantify the binding capacity of plasma for heme and correlate that with measurement of heme scavenging proteins. Methods: Blood from healthy individuals and sickle cell patients was collected in EDTA as anticoagulant under IRB approval. Plasma was separated by centrifugation from whole blood, and either processed fresh or after freezing at -80°C. Plasma protein was precipitated with a 4-fold volume of acetone at neutral pH (NA) or acidic pH (AA). Under acidic condition, heme is released from all heme binding pools, including hemoglobin, and provides detection of the total heme present in plasma. Under neutral pH condition, only heme unbound to plasma proteins is extracted. Once extracted, samples were dried and resuspended in DMSO. Heme concentration was spectrophotometrically determined at 400nm using standard curves prepared from hemin added in AA or NA. To determine heme binding capacity, hemin was added to serial dilutions of plasma and extracted in NA and AA as above. The appearance of heme in NA relative to AA represents the point at which heme binding capacity of plasma was saturated. This was compared to measurement of hemopexin and haptoglobin using commercially available ELISA measurements. Hemopexin and albumin were added to samples to modulate heme binding capacity. Results: Heme concentration closely correlates with spectroscopic measurement of heme in DMSO confirming reliable quantification of total and unbound heme in acidic and neutral acetone extractions as low as 2.5µM. We next show that heme binding capacity can be determined. Heme added to plasma was effectively recovered in AA extracts and begins to appear in the NA extract when binding sites start to become saturated. We note that not all sites appear to be fully saturated before heme is detected in NA extract. Addition of hemopexin to plasma increased the binding capacity on an equimolar basis, indicating that hemopexin effectively binds heme present in plasma. In samples from patients with sickle cell disease, concentration of total and unbound heme varied widely, and did not necessarily correlate with degree of intravascular hemolysis, estimated based on the measurement of cell free hemoglobin. Both the capacity of plasma to bind heme and levels of hemopexin indicated that, in a number of patients, the amount of heme present was greater than the ability of hemopexin to bind cell free heme. Discussion: We present a novel method to quantitatively differentiate hemoprotein-bound and unbound heme in plasma, the latter of which is pathologically relevant in sickle cell disease. Our data show significant variation in the concentration of total and unbound heme in sickle cell patient samples, and that the binding capacity in sickle cell plasma only partially correlates to the degree of hemolysis measured based on cell free hemoglobin. Patients are currently enrolled in a clinical study to measure intra-patient differences in heme and heme-binding capacity during steady state and during acute sickle cell-related illness. Understanding the clinical implications of heme and heme scavengers may provide insights into diagnostic and therapeutic targets for patients with sickle cell disease. Disclosures Neumayr: Emmaus: Consultancy; Bayer: Consultancy; CTD Holdings: Consultancy; Pfizer: Consultancy; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Micelle: Other: Site principal investigator; GBT: Other: Site principal investigator; PCORI: Other: site principal investigator; Novartis: Other: co-investigator; Bluebird Bio: Other: co-investigator; Sangamo Therapeutics: Other; Silarus: Other; Celgene: Other; La Jolla Pharmaceuticals: Other; Forma: Other; Centers for Disease Control and Prevention: Other; Seattle Children's Research: Other; Imara: Other; National Heart, Lung, and Blood Institute: Other; Health Resources and Services Administration: Other. Vichinsky:Bluebird Bio: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; GBT: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Kuypers:Forma Therapeutics, Inc.: Research Funding.

scholarly journals Xanthine Oxidase Drives Hemolysis and Vascular Malfunction in Sickle Cell Disease

2020 ◽  
Author(s):  
Heidi M. Schmidt ◽  
Katherine C. Wood ◽  
Sara E. Lewis ◽  
Scott A. Hahn ◽  
Xena M. Williams ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Xanthine Oxidase ◽  
Sickle Cell ◽  
Vascular Function ◽  
Cell Disease ◽  
Free Hemoglobin ◽  
Chronic Hemolysis ◽  
Oxidant Production

Objective: Chronic hemolysis is a hallmark of sickle cell disease (SCD) and a driver of vasculopathy; however, the mechanisms contributing to hemolysis remain incompletely understood. Although XO (xanthine oxidase) activity has been shown to be elevated in SCD, its role remains unknown. XO binds endothelium and generates oxidants as a byproduct of hypoxanthine and xanthine catabolism. We hypothesized that XO inhibition decreases oxidant production leading to less hemolysis. Approach and Results: Wild-type mice were bone marrow transplanted with control (AA) or sickle (SS) Townes bone marrow. After 12 weeks, mice were treated with 10 mg/kg per day of febuxostat (Uloric), Food and Drug Administration–approved XO inhibitor, for 10 weeks. Hematologic analysis demonstrated increased hematocrit, cellular hemoglobin, and red blood cells, with no change in reticulocyte percentage. Significant decreases in cell-free hemoglobin and increases in haptoglobin suggest XO inhibition decreased hemolysis. Myographic studies demonstrated improved pulmonary vascular dilation and blunted constriction, indicating improved pulmonary vasoreactivity, whereas pulmonary pressure and cardiac function were unaffected. The role of hepatic XO in SCD was evaluated by bone marrow transplanting hepatocyte-specific XO knockout mice with SS Townes bone marrow. However, hepatocyte-specific XO knockout, which results in >50% diminution in circulating XO, did not affect hemolysis levels or vascular function, suggesting hepatocyte-derived elevation of circulating XO is not the driver of hemolysis in SCD. Conclusions: Ten weeks of febuxostat treatment significantly decreased hemolysis and improved pulmonary vasoreactivity in a mouse model of SCD. Although hepatic XO accounts for >50% of circulating XO, it is not the source of XO driving hemolysis in SCD.

Haptoglobin Genotypes among Pediatric and Adult Sickle Cell Patients: An Emerging Genetic Modifier?.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3805-3805
Author(s):  
Heather Y. Hughes ◽  
Ferdane Kutlar ◽  
Betsy Clair ◽  
Dedrey Elam ◽  
Kathleen M. McKie ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Allele Frequency ◽  
Sickle Cell ◽  
Pediatric Patients ◽  
Susceptibility Gene ◽  
Diabetic Patients ◽  
Cell Disease ◽  
Free Hemoglobin ◽  
Α Chain

Abstract The role of hemolysis and its end product, cell free hemoglobin, as a Nitric Oxide (NO) scavenger in the pathogenesis of some complications of sickle cell disease (SCD) such as pulmonary hypertension has recently been recognized. Hemoglobin (Hb) released from red blood cells as a result of hemolysis complexes with haptoglobin (Hp). Hb-Hp complexes are removed from the circulation by tissue macrophages in the liver and spleen through CD163, the newly identified hemoglobin scavenger receptor expressed on the monocytes and macrophages. Hp is a polymorphic protein with two well-known alleles, Hp-1 and Hp-2 with worldwide distribution. Although the worldwide frequencies of these two allelles are approximately equal, there is significant ethnic and geographic variation in the distribution of Hp-1 and Hp-2 among different populations. Hp locus is on chromosome 16q22. each Hp monomer consists of one α-chain and one β-chain. An intragenic duplication event during evolution resulted in an elongated α-chain thus generating the Hp-2 allele. Thus individuals homozygous for the long α-2 chain express large multimeric molecules (Hp 2-2). Over the past 5 years, Hp-2 allele has emerged as a major susceptibility gene for development of vascular complications such as coronary artery restenosis and risk of cardiovascular disease, particularly in diabetic patients. It has been reported that the Hb-Hp-2 complexes have a 10-fold greater affinity for the CD163 receptor compared to Hb-Hp-1, and the interaction of Hb-Hp-2 complexes with CD163 on macrophages generates a more powerful inflammatory response with the release of pro-inflammatory cytokines such as IL-1β, IL-6, and GM-CSF. These observations led us to examine the role of Hp polymorphisms in sickle cell disease. We performed genotyping of the Hp-alleles with PCR in 21 pediatric patients (age 4 mo-10 years) and 41 adults patients (age 16–60). The results of this genotyping are shown in the table below. The Hp-1 allele frequency was 0.24 among pediatric patients and 0.52 among adults; while that of Hp-2 was 0.76 in the pediatric age group and 0.48 in adults. This difference was significant (p=0.0023). The significantly higher allele frequency of Hp-2 in pediatric patients compared to adults may indicate a selection against this genotype and suggests that Hp-1 allele may be associated with a survival advantage. This is in keeping with the deleterious effects of Hp-2 allele reported in the literature and suggests yet another mechanism whereby hemolysis may lead to adverse outcomes associated with Hp polymorphisms, in addition to the NO scavenging effects of cell free hemoglobin. Detailed studies are currently being undertaken to elucidate the mechanism whereby Hp polymorphisms may impact on the pathophysiology of SCD. PERCENTAGE OF HAPTOGLOBIN GENOTYPES BETWEEN ADULT AND PEDIATRIC SICKLE CELL PATIENTS ADULT PEDS sHp 1–1 41.4% 9.5% Hp 1–2 22.5% 28.6% Hp 2–2 36.6% 62.0%

Hypocholesterolemia in a Large Sickle Cell Cohort: Correlations of Serum Lipids to Markers of Intravascular Hemolysis and Vascular Dysfunction

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 123-123
Author(s):  
Suzana M. Zorca ◽  
Lita A. Freeman ◽  
Patricia L. Littel ◽  
Gregory J. Kato
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Total Bilirubin ◽  
Reticulocyte Count ◽  
Vascular Dysfunction ◽  
Healthy Controls ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Cholesterol Levels ◽  
The Mean

Abstract Background: Hypocholesterolemia in sickle cell disease (SCD) has been documented in small cohorts from Nigeria, Cameron, Iran and the United States. Although not uniformly consistent, these studies found decreased total plasma cholesterol levels, HDL- cholesterol (HDL-C) and LDL-cholesterol (LDL-C), when compared to healthy controls. Consistent with the low levels of LDL-C, atherosclerosis is largely absent in SCD patients. However, vascular dysfunction is prevalent, and higher-than-average triglyceride (TG) levels, a known risk factor for vascular diseases, have also been reported in SCD populations. Methods: In a large SCD cohort, consisting of 405 SCD patients with normal and mildto- significantly elevated pulmonary artery pressures (tricuspid regurgitant jet velocities (TRVs) ranging from 1 to 4.9) as well as 32 healthy African American controls, we determined mean serum lipid levels for total cholesterol, HDL-C, LDL-C and TG. We also determined apolipoprotein levels, ApoA-I, ApoB and the ratio ApoA-I/ApoB. We then investigated the correlations between plasma serum lipids and apolipoproteins against a battery of markers of intravascular hemolysis and vascular dysfunction. Results: In our large cohort, we observe significantly lower mean total cholesterol levels in SCD patients versus healthy controls (135.2 and 187.5 mg/dL, respectively, p<0.001). Total serum cholesterol was negatively correlated to markers of intravascular hemolysis (absolute reticulocyte count, p<0.0001; total bilirubin, p<0.0001, indirect bilirubin, p<0.0001), and positively associated with hemoglobin and hematocrit (p=0.0036 and p=0.0028, respectively). Mean HDL-C levels were statistically significantly lower in the SCD cohort versus healthy controls (41.27 and 58.91, respectively, p<0.0001), and were also negatively correlated to absolute reticulocyte count (p< 0.0001), total bilirubin (p<0.0001) as well as to LDH, another well-known marker of intravascular hemolysis (p<0.0001). Mean LDL-C levels were significantly lower in SCD patients than in control subjects (78.22 and 117.1 mg/dL, respectively, p<0.0001), and were negatively correlated to reticulocyte count (p=0.0002), and total bilirubin (p= 0.0025). In contrast, mean triglyceride levels were significantly higher in the SCD cohort than in healthy subjects (123.2 vs. 76.42 mg/dL respectively, p<0.0001), and were inversely correlated to hemoglobin (p= 0.0005) and hematocrit (p= 0.0003). TGs were also significantly positively correlated to markers of hemolysis: plasma hemoglobin (p= 0.0004), LDH (p= 0.0005), and with arginase (p=0.003), ornithine (p< 0.0001), and SE selectin (p= 0.0386). Moreover, when the SCD cohort was divided among low-TRV (<2.5), moderate-TRV (2.5–2.9) or high-TRV (>2.9) cohorts, the mean plasma TG levels were significantly different between the low and high TRV groups. Specifically, for low-TRV patients (TRV<2.5, n=197), the mean plasma TG level was 110.5 mg/dL (95% confidence interval 102.5–118.6, median 102.0); for moderate-TRV patients (TRV=2.5–2.9, n=103) the mean TG level did not differ significantly from the low-TRV group (TG=115.3 mg/dL, 95% CI 104.6–126.1; median 105.0); however, for high TRV patients (TRV>2.9, n = 58), the TG mean was significantly higher (153.4, 95% CI 131.4–175.5, median=136.0). Interestingly, ApoA-I levels were also low in SCD patients, and there was a trend towards lower ApoA-I levels in the higher TRV patient groups, but this trend did not achieve statistical significance. Lastly, ApoA-I levels were significantly positively correlated with NTProBNP, a marker of pulmonary hypertension (p= 0.0026). Conclusions: To our knowledge, this constitutes the first evidence that hypocholesterolemia in sickle cell disease is correlated with markers of intravascular hemolysis and vascular dysfunction. Further study is needed to explore the mechanistic basis for these correlations; however, one possibility is that the fatty acids carried by triglycerides may become oxidized in SCD patients with hemolytic oxidative stress and serve as signaling molecules, or alternatively, hemolytic oxidative stress may coordinately regulate these pathways. Our findings provide a link between plasma lipids, particularly triglyceride levels, and vascular dysfunction in sickle cell disease.

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