scholarly journals Neutrophil-Platelet Micro-Emboli Enable Vaso-Occlusion in Sickle Cell Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2695-2695
Author(s):  
Prithu Sundd ◽  
Maritza Jimenez ◽  
Enrico M Novelli ◽  
Mark T Gladwin
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Cellular Adhesion ◽  
Epifluorescence Microscopy ◽  
Single Point Mutation ◽  
Cellular Interactions ◽  
Cell Disease ◽  
Cellular Adhesion Molecule

Abstract Introduction: Sickle Cell Disease (SCD) is an autosomal-recessive-hemolytic disorder caused by a single point mutation in the β-globin gene that leads to sickling of RBCs under deoxygenated condition. Sickle RBCs (sRBCs) are not only rigid but also express adhesion molecules, which are not normally expressed on RBCs. The sticky and rigid sRBCs are believed to get trapped in blood vessels along with leukocytes to cause vaso-occlusion, which is the predominant pathophysiology underlying acute pain crisis in SCD patients. The process of sickling and vaso-occlusion leads to sRBC hemolysis, which releases hemoglobin, ADP and other RBC contents into the blood giving rise to a pro-inflammatory and pro-coagulant state, characterized by activated leukocytes, platelets, endothelial cells (ECs), tissue factor (TF) and enhanced adhesion of these cells to each other. Leukocyte–endothelium adhesion starts with leukocyte rolling mediated by P-selectin-glycoprotein-ligand (PSGL)-1 on leukocytes binding to P-selectin on endothelium. Rolling is followed by firm arrest, which is mediated by activated β­2-integrins (LFA-1 and Mac-1) on the leukocytes binding to inter-cellular-adhesion-molecule (ICAM)-1 on endothelium. Although neutrophils have been shown to play a role in the onset of vaso-occlusion by interacting with sRBCs and platelets in cremaster venules of SCD mice; the cellular, molecular and biophysical mechanisms that enable vaso-occlusion in SCD patients are not known. Materials and Methods: Freshly collected heparinized blood from SCD patients and race matched control subjects was perfused through a polydimethylsiloxane (PDMS) based microfluidic flow chamber with a glass bottom coated with either human micro-vascular endothelial cells or a cocktail of recombinant human P-selection, ICAM-1 and IL-8 at a venular/arteriolar wall shear stress. Fluorochrome conjugated Abs against CD16, CD235a and CD49b were added to the blood to stain neutrophils, sRBCs and platelets, respectively, and cellular interactions were recorded using multi-color Quantitative Dynamic Footprinting (qDF; Sundd et al Nature Methods 2010) or epifluorescence microscopy. Specificity of cellular interactions was tested using function blocking Abs against human Mac-1, LFA-1, P-selectin and PSGL-1. Results: SCD patients had much higher number of circulating neutrophils than control patients. Neutrophils rolled, arrested and then captured free flowing platelets in both SCD and control blood. However, significantly larger number of neutrophils rolled and arrested in SCD blood than control blood. As a result, much higher number of platelets was captured by arrested neutrophils in SCD blood than control blood, which led to the formation of neutrophil-platelet micro-emboli. The micro-emboli formation was mediated by a unique biophysical mechanism, which involved PSGL-1 and Mac-1 on neutrophils binding to P-selectin and GPIbα on platelets, respectively. Conclusion: Vaso-occlusion involves a cascade of adhesive events. First, neutrophils roll and arrest at the site of vaso-occlusion. Second, arrested neutrophils capture free flowing platelets and RBCs to form micro-emboli. Third, eventually these micro-emboli give rise to micro-thrombi, which cause stasis of blood flow. Acknowledgments: This study is supported by 11SDG7340005 from the American Heart Association (P.S.), VMI start-up funds (P.S.) and CBTP-T32 fellowship HL076124 (M.J). Disclosures No relevant conflicts of interest to declare.

Platelet Nucleation on Arrested Neutrophils Drives Vaso-Occlusion in Sickle Cell Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 414-414
Author(s):  
Maritza A. Jimenez ◽  
Prithu Sundd ◽  
Enrico M Novelli ◽  
Gregory J Kato
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Fluorescence Microscopy ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Genetic Disorder ◽  
Epifluorescence Microscopy ◽  
Cellular Interactions ◽  
Cell Disease ◽  
Neutrophil Aggregation

Abstract Introduction: Sickle Cell Disease (SCD) is an autosomal recessive genetic disorder that leads to sickling and hemolysis of RBCs under hypoxic conditions. As a result of chronic hemolysis, SCD is associated with a hyper-inflammatory and hyper-coagulation state, which accounts for enhanced adhesion of leukocytes, platelets, RBCs and vascular endothelial cells leading to vaso-occlusion. Acute vaso-occlusive pain crisis (VOC) is the primary reason for emergency medical care by SCD patients. Although neutrophils have been shown to play a role in the on-set of vaso-occlusion by interacting with sickle RBCs and platelets in cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SCD patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SCD (SS) patients and race matched control subjects was perfused through polydimethylsiloxane (PDMS) based microfluidic flow channels (30 µm x 500 µm) with a glass bottom coated with either human microvascular endothelial cells or a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological shear stress (6 dyn cm-2). Fluorescent Abs against CD16 and CD49b were added to the blood for in-situ staining of neutrophils and platelets, respectively. Cellular interactions were recorded using quantitative microfluidic fluorescence microscopy (qMFM)1, which is a combination of quantitative dynamic footprinting1 and epifluorescence microscopy. Results and Discussion: Neutrophils in SS blood were observed to roll, arrest and then capture freely flowing platelets leading to the formation of vaso-occlusive aggregates. RBCs were observed getting trapped within the platelet-neutrophil aggregates. The number of platelet-neutrophil interactions, lifetime of these interactions and the extent of platelet-neutrophil aggregation were several folds higher in SS than control subject blood. Bacterial lipopolysaccharide (LPS; 500 ng/ml) pretreatment led to enhanced platelet-neutrophil aggregations in SS but not control blood. The enhanced platelet-neutrophil aggregations in SS blood (+/-LPS) was attenuated to the level observed in control blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils with functional blocking Abs. Conclusion: Our data demonstrates that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of platelet-neutrophil aggregates and trapping of RBCs in these aggregates. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced platelet-neutrophil aggregation than control blood. Vaso-occlusion can be ameliorated in SS blood by simultaneous inhibition of platelet P-selectin and neutrophil Mac-1. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapies that can prevent VOC in SS patients. References: 1. Jimenez MA, Tutuncuoglu E, Barge S, Novelli EM, Sundd P. Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in Sickle Cell Disease. Haematologica, 2015. 2 Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods 7, 821-824, doi:10.1038/nmeth.1508 (2010). Disclosures No relevant conflicts of interest to declare.

scholarly journals Beyond the Definitions of the Phenotypic Complications of Sickle Cell Disease: An Update on Management

2012 ◽  
Vol 2012 ◽  
pp. 1-55 ◽  
Author(s):  
Samir K. Ballas ◽  
Muge R. Kesen ◽  
Morton F. Goldberg ◽  
Gerard A. Lutty ◽  
Carlton Dampier ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Point Mutation ◽  
Sickle Cell ◽  
Globin Gene ◽  
Single Point ◽  
Acute Chest Syndrome ◽  
Single Point Mutation ◽  
Cell Disease ◽  
Exon 1 ◽  
Abnormal Hemoglobin

The sickle hemoglobin is an abnormal hemoglobin due to point mutation (GAG → GTG) in exon 1 of theβglobin gene resulting in the substitution of glutamic acid by valine at position 6 of theβglobin polypeptide chain. Although the molecular lesion is a single-point mutation, the sickle gene is pleiotropic in nature causing multiple phenotypic expressions that constitute the various complications of sickle cell disease in general and sickle cell anemia in particular. The disease itself is chronic in nature but many of its complications are acute such as the recurrent acute painful crises (its hallmark), acute chest syndrome, and priapism. These complications vary considerably among patients, in the same patient with time, among countries and with age and sex. To date, there is no well-established consensus among providers on the management of the complications of sickle cell disease due in part to lack of evidence and in part to differences in the experience of providers. It is the aim of this paper to review available current approaches to manage the major complications of sickle cell disease. We hope that this will establish another preliminary forum among providers that may eventually lead the way to better outcomes.

scholarly journals Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease

2021 ◽  
Vol 13 (598) ◽  
pp. eabf2444
Author(s):  
Annalisa Lattanzi ◽  
Joab Camarena ◽  
Premanjali Lahiri ◽  
Helen Segal ◽  
Waracharee Srifa ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Drug Product ◽  
Globin Gene ◽  
Monogenic Disease ◽  
Single Point Mutation ◽  
Cell Disease ◽  
Gene Correction ◽  
Hematopoietic Stem ◽  
Virus Serotype

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the β-globin gene (HBB). Ex vivo β-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)–mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.

scholarly journals Nrf2 activation in myeloid cells and endothelial cells differentially mitigates sickle cell disease pathology in mice

2019 ◽  
Vol 3 (8) ◽  
pp. 1285-1297 ◽  
Author(s):  
Nadine Keleku-Lukwete ◽  
Mikiko Suzuki ◽  
Harit Panda ◽  
Akihito Otsuki ◽  
Fumiki Katsuoka ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Vascular Function ◽  
Cell Activation ◽  
Ischemia Reperfusion ◽  
Globin Gene ◽  
Cell Disease ◽  
Cell Counts ◽  
Nrf2 Activation

Abstract Sickle cell disease (SCD) is caused by a monogenic mutation of the β-globin gene and affects millions of people worldwide. SCD is associated with sustained hemolytic anemia, vasoocclusion, ischemia-reperfusion injury, oxidative tissue damage, inflammatory cell activation, and systemic endothelial dysfunction. The transcription factor Nrf2 coordinates the expression of a wide variety of genes encoding antioxidant, detoxification, and metabolic enzymes. Nrf2 participates in suppressing proinflammatory cytokines and organ protection in SCD. However, little is known regarding the mechanisms by which Nrf2 ameliorates SCD pathology or how some cells respond to Nrf2 stimuli to alleviate SCD pathology. Here, we asked whether monocytes/granulocytes and/or endothelial cells are particularly critical in alleviating the pathology of SCD. By targeting these cells with a Cre recombinase system, we generated SCD::Keap1F/F::LysM-Cre and Tie1-Cre mice with constitutive Nrf2 activation in monocytes/granulocytes and endothelial cells, respectively. Analyses of SCD::Keap1F/F::LysM-Cre and SCD::Keap1F/F::Tie1-Cre mice revealed significantly reduced inflammation, along with decreased white blood cell counts and lower Tnfα and Il1β expression in the lungs. Notably, SCD::Keap1F/F::LysM-Cre mice exhibited reduced heme distribution in the liver, consistent with a decrease in the damaged areas. Vascular function in SCD::Keap1F/F::Tie1-Cre mice was significantly improved, with a 50% decrease in vascular leakage and low expression of the adhesion molecules Vcam1 and P-selectin. Thus, Nrf2 activation in monocytes/granulocytes and endothelial cells contributes differentially and cooperatively to the improvement of SCD pathology.

scholarly journals Selenium Deficiency in a Mouse Model of Sickle Cell Disease Resulted in Increased Oxygen Consumption and Aberrant Mitochondrial Retention (OR11-05-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Lenny Hong ◽  
Ramasamy Jagadeeswaran ◽  
Robert Molokie ◽  
Donald Lavelle ◽  
Angela Rivers ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Sickle Cell Disease ◽  
Mouse Model ◽  
Sickle Cell ◽  
Globin Gene ◽  
Single Point ◽  
Selenium Deficiency ◽  
Single Point Mutation ◽  
Cell Disease ◽  
Deficient Diet

Abstract Objectives Sickle Cell Disease (SCD) is caused by a single point mutation in the β-globin gene, resulting in the polymerization of the altered hemoglobin βS in hypoxic conditions, affecting millions of people worldwide. Previous studies have shown that there are lower selenium levels and reduced activity of the antioxidant selenoprotein GPX1 in SCD patients. The objective of this study was to investigate the consequences of selenium deficiency in a SCD mouse model. Methods Humanized SCD (HbSS) mice (Townes model) and wild type (HbAA) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were fed either a selenium-deficient (<0.01 mg/kg) or a selenium adequate (0.1 mg/kg) diet for 4 weeks. Hematological testing was performed using the ADVIATM 120 analyzer (Bayer Corporation, NY). Mitochondrial retention and reactive oxygen species (ROS) were measured by flow cytometry with a BD LSRFortessaTM analyzer using Kaluza analysis software (Beckman Coulter, CA). The oxygen consumption rate (OCR) was measured from isolated red blood cells (RBCs) in real time using the Seahorse Extracellular Analyzer (Agilent, CA). Results RBCs normally eject their mitochondria before reaching maturity. However, a previous study demonstrated that there was increased RBC mitochondrial retention in SCD mice and patients when compared to controls. Feeding SCD mice a selenium deficient diet resulted in increased retention of mitochondria in RBCs (26% + 6.9%, 5% + 3.5%, n = 3, P < 0.01), decreased hemoglobin levels (5.7 + 0.17 g/dl, 7.0 + 0.83 g/dl, n = 3, P < 0.05), and an increased OCR of the RBCs (P < 0.01) in these animals when compared to SCD mice fed a selenium adequate diet. Conclusions Providing humanized SCD mice a selenium deficient diet resulted in increased mitochondrial retention in mature RBCs, decrease hemoglobin levels, and increased RBC oxygen consumption. RBC retention of mitochondria is associated with increased ROS and hemolysis, potentially contributing to the pain and vaso-occlusive crises that occur in the disease. These studies indicate that selenium deficiency may contribute to the severity of symptoms experienced by patients with SCD. Funding Sources This work is supported by a grant from the NIH.

scholarly journals Sickle Cell Anemia: A review on the most severe form of Sickle Cell Disease

Bionatura ◽  
2019 ◽  
Vol 02 (Bionatura Conference Serie) ◽  
Author(s):  
María Belén Paredes ◽  
María Eugenia Sulen
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Single Point ◽  
Clinical Manifestations ◽  
Severe Form ◽  
Single Point Mutation ◽  
Cell Disease ◽  
Hemoglobin S

Sickle cell disease (SCD) is a group of hereditary disorders caused by a single point mutation in the β-globin gene. This mutation results in the formation of a mutated hemoglobin S (HbS) and the consequent sickle phenotype of erythrocytes. SCD is common in regions of malaria endemicity. However, changes in population dynamics enabled the movement of the mutated gene to other areas such as North America and Europe. Sickle cell anemia (SCA) is the most severe form of SCD and affects millions of people around the globe. The clinical manifestations of SCA arise primarily from the polymerization of deoxygenated hemoglobin S (deoxyHbS) leading to vascular occlusion and hemolytic anemia. Clinical complications of the disease are derived from deoxyHbS polymerization, but there are several therapeutic strategies to reduce the severity of the symptoms. Gene therapy has arisen as a new therapeutic approach aimed to cure rather than to treat the symptomatology of SCA by targeting the altered β-globin gene for gene correction.

Rapid and reliable β-globin gene cluster haplotyping of sickle cell disease patients by FRET Light Cycler and HRM assays

2011 ◽  
Vol 412 (13-14) ◽  
pp. 1257-1261 ◽  
Author(s):  
Philippe Joly ◽  
Philippe Lacan ◽  
Caroline Garcia ◽  
Angelique Delasaux ◽  
Alain Francina
Keyword(s):  
Sickle Cell Disease ◽  
Gene Cluster ◽  
Sickle Cell ◽  
Globin Gene ◽  
Cell Disease ◽  
Globin Gene Cluster ◽  
Light Cycler

scholarly journals 3,3′,5-Triiodothyroacetic acid (TRIAC) induces embryonic ζ-globin expression via thyroid hormone receptor α

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Huiqiao Chen ◽  
Zixuan Wang ◽  
Shanhe Yu ◽  
Xiao Han ◽  
Yun Deng ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Globin Gene ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Potent Inducer ◽  
Thyroid Hormone Receptor Α

AbstractThe human ζ-globin gene (HBZ) is transcribed in primitive erythroid cells only during the embryonic stages of development. Reactivation of this embryonic globin synthesis would likely alleviate symptoms both in α-thalassemia and sickle-cell disease. However, the molecular mechanisms controlling ζ-globin expression have remained largely undefined. Moreover, the pharmacologic agent capable of inducing ζ-globin production is currently unavailable. Here, we show that TRIAC, a bioactive thyroid hormone metabolite, significantly induced ζ-globin gene expression during zebrafish embryogenesis. The induction of ζ-globin expression by TRIAC was also observed in human K562 erythroleukemia cell line and primary erythroid cells. Thyroid hormone receptor α (THRA) deficiency abolished the ζ-globin-inducing effect of TRIAC. Furthermore, THRA could directly bind to the distal enhancer regulatory element to regulate ζ-globin expression. Our study provides the first evidence that TRIAC acts as a potent inducer of ζ-globin expression, which might serve as a new potential therapeutic option for patients with severe α-thalassemia or sickle-cell disease.

scholarly journals Sickle Cell Disease: Role of Oxidative Stress and Antioxidant Therapy

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 296
Author(s):  
Rosa Vona ◽  
Nadia Maria Sposi ◽  
Lorenza Mattia ◽  
Lucrezia Gambardella ◽  
Elisabetta Straface ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Organ Damage ◽  
Cell Disease ◽  
Vessel Occlusion ◽  
Antioxidant Agents ◽  
Oxidant Status

Sickle cell disease (SCD) is the most common hereditary disorder of hemoglobin (Hb), which affects approximately a million people worldwide. It is characterized by a single nucleotide substitution in the β-globin gene, leading to the production of abnormal sickle hemoglobin (HbS) with multi-system consequences. HbS polymerization is the primary event in SCD. Repeated polymerization and depolymerization of Hb causes oxidative stress that plays a key role in the pathophysiology of hemolysis, vessel occlusion and the following organ damage in sickle cell patients. For this reason, reactive oxidizing species and the (end)-products of their oxidative reactions have been proposed as markers of both tissue pro-oxidant status and disease severity. Although more studies are needed to clarify their role, antioxidant agents have been shown to be effective in reducing pathological consequences of the disease by preventing oxidative damage in SCD, i.e., by decreasing the oxidant formation or repairing the induced damage. An improved understanding of oxidative stress will lead to targeted antioxidant therapies that should prevent or delay the development of organ complications in this patient population.

Hypoxia-induced acute lung injury in murine models of sickle cell disease

2004 ◽  
Vol 286 (4) ◽  
pp. L705-L714 ◽  
Author(s):  
Kirkwood A. Pritchard ◽  
Jingsong Ou ◽  
Zhijun Ou ◽  
Yang Shi ◽  
James P. Franciosi ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Lung Injury ◽  
Sickle Cell ◽  
Globin Gene ◽  
Heat Shock Protein 90 ◽  
Cell Disease ◽  
Vascular Congestion ◽  
Nitrite Nitrate ◽  
Endothelial Nitric Oxide

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human α/sickle β-globin (hαβS) transgene (SCD mice) or are heterozygous for the normal murine β-globin gene and express the hαβStransgene (mβ+/-, hαβS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing ·NO generation and predisposing the lung to vaso-occlusion.

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