Circulating Extracellular Vesicles from Patients with Sickle Cell Disease Progressively Disrupt Different Types of Endothelial Intercellular Junctions

Introduction: Aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of crises in sickle cell disease (SCD), including acute chest syndrome (ACS). We previously demonstrated that the plasma of SCD patients contains circulating small extracellular vesicles (EVs) and that those vesicles can disrupt endothelial integrity in vitro, including a decrease in VE-cadherin. The current study was designed to examine the effects of those EVs on additional components of the endothelial junctions including tight (zonula occludens 1, ZO-1) and gap junctions (connexin43, Cx43) and to test the hypothesis that the junctions would be more severely affected by EVs isolated from patients during an episode of ACS than by those isolated from the same patient at baseline. Methods: We identified subjects with SCD in our biobank who had plasma isolated at baseline and at the beginning of an admission for ACS (prior to transfusion). Samples were considered baseline if the patient was more than 4 weeks since transfusion and had no new health-related complaints. ACS was defined by the presence of an infiltrate on chest x-ray combined with fever, pain, hypoxia or cough. EVs were isolated from plasma using established methodologies. To determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with EVs for 48 h. Cells were fixed and studied by fluorescence microscopy (after immunolocalization of Cx43, ZO-1 and/or VE-cadherin and staining of nuclei with DAPI). Proteins were detected and quantified by immunoblotting. mRNA expression was determined by RT-qPCR. Gap junction mediated intercellular communication was assessed following microinjection of Lucifer yellow and neurobiotin. Results: Microscopy confirmed our previous observation that EVs isolated from subjects with SCD caused in vitro disruption of endothelial monolayers and that damage is significantly worse when EVs are isolated during an episode of ACS. The distribution and abundance of VE-cadherin and ZO-1 at the plasma membrane of undisturbed cells were minimally affected by SCD EVs. While baseline EVs did not detectably affect the distribution of Cx43, EVs isolated during ACS caused a loss of Cx43 from the plasma membrane. The integrated intensity of Cx43 membrane staining was decreased by ~20% following treatment with ACS EVs. Cx43 protein decreased on average by 32 % and Cx43 mRNA levels by 21% in cells treated with ACS EVs compared to baseline from the same patient. EVs isolated during ACS caused significant disruption in intercellular transfer compared to EVs isolated at baseline (67-94% reduction) (Figure 1). Conclusions: Our results show that subjects with SCD produce small EVs that cause disruption of the endothelial monolayer in vitro. Gap junctions composed of Cx43 are the most sensitive of the cell-cell junctions in this setting, since their abundance and function are reduced by ACS EVs even when the endothelial monolayer appears intact. Disruption of endothelial intercellular communication mediated by Cx43 appears to be an early and sensitive event in the endothelial disturbance caused by EVs in SCD patients. Disclosures No relevant conflicts of interest to declare.

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Gap Junctions between Endothelial Cells Are Disrupted by Circulating Extracellular Vesicles from Sickle Cell Patients with Acute Chest Syndrome

International Journal of Molecular Sciences ◽

10.3390/ijms21238884 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8884

Author(s):

Joanna Gemel ◽

Yifan Mao ◽

Gabrielle Lapping-Carr ◽

Eric C. Beyer

Keyword(s):

Endothelial Cells ◽

Sickle Cell Disease ◽

Gap Junctions ◽

Extracellular Vesicles ◽

Sickle Cell ◽

Intercellular Junctions ◽

The Other ◽

Acute Chest Syndrome ◽

Cell Disease ◽

Intercellular Connections

Intercellular junctions maintain the integrity of the endothelium. We previously found that the adherens and tight junctions between endothelial cells are disrupted by plasma extracellular vesicles from patients with sickle cell disease (especially those with Acute Chest Syndrome). In the current study, we evaluated the effects of these vesicles on endothelial gap junctions. The vesicles from sickle cell patients (isolated during episodes of Acute Chest Syndrome) disrupted gap junction structures earlier and more severely than the other classes of intercellular junctions (as detected by immunofluorescence). These vesicles were much more potent than those isolated at baseline from the same subject. The treatment of endothelial cells with these vesicles led to reduced levels of connexin43 mRNA and protein. These vesicles severely reduced intercellular communication (transfer of microinjected Neurobiotin). Our data suggest a hierarchy of progressive disruption of different intercellular connections between endothelial cells by circulating extracellular vesicles that may contribute to the pathophysiology of the endothelial disturbances in sickle cell disease.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00288.2002 ◽

2004 ◽

Vol 286 (4) ◽

pp. L705-L714 ◽

Cited By ~ 57

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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Endothelial Barrier Integrity Is Disrupted In Vitro by Heme and by Serum From Sickle Cell Disease Patients

Frontiers in Immunology ◽

10.3389/fimmu.2020.535147 ◽

2020 ◽

Vol 11 ◽

Author(s):

Vanessa Araujo Gomes Santaterra ◽

Maiara Marx Luz Fiusa ◽

Bidossessi Wilfried Hounkpe ◽

Francine Chenou ◽

Wouitchekpo Vincent Tonasse ◽

...

Keyword(s):

Sickle Cell Disease ◽

Healthy Volunteers ◽

Sickle Cell ◽

Endothelial Barrier ◽

Acute Chest Syndrome ◽

Cell Disease ◽

Experimental Conditions ◽

Cell Monolayers ◽

Junction Protein

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.

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Mineral Content and Antisickling Activity of Annona senegalensis, Alchornea cordifolia and Vigna unguiculata Used in the Management of Sickle Cell Disease in the Kwilu Province (Congo, DR)

International Blood Research & Reviews ◽

10.9734/ibrr/2020/v11i330131 ◽

2020 ◽

pp. 18-27

Author(s):

Jules M. Kitadi ◽

Clément L. Inkoto ◽

Emmanuel M. Lengbiye ◽

Damien S. T. Tshibangu ◽

Dorothée D. Tshilanda ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mineral Composition ◽

Sickle Cell ◽

Mineral Elements ◽

Spectrometric Method ◽

Spectrometric Analysis ◽

Composition Analysis ◽

Cell Disease ◽

Republic Of The Congo

Aims: To determine the mineral composition of some plants (Annona senegalensis Pers., Alchornea cordifolia (Schumach. & Thonn.) Müll. Arg. and Vigna unguiculate (L.) Walp.) used in the management of sickle cell disease by traditional practitioners in Kwilu province and to evaluate their antisickling activity in vitro. Study Design: Plant collection in the Kwilu province, sample preparation, antisickling tests and fluorescence spectrometric analysis. Place and Duration of Study: This work was performed at the Faculty of Science, University of Kinshasa, Congo DR, from October 2016 to January 2018. Methodology: These three plants were harvested in the province of Kwilu in Democratic Republic of the Congo. The mineral composition analysis was carried out using the fluorescence spectrometric method while the in vitro antisickling activity was evaluate using Emmel and hemolysis tests. Results: Twenty three mineral elements were identified in each of these three plants: Potassium (K), Phosphorus (P), Calcium (Ca), Sodium (Na), Magnesium (Mg), Sulphur (S), Chlorine (Cl) and trace elements as: Aluminum (Al), Silicon (Si), Vanadium (V), Chromium (Cr), Manganese (Mn), Iron (Fe), Nickel (Ni), Copper (Cu), Zinc (Zn), Selenium (Se), Brome (Br), Molybdenum (Mo), Tin (Sn), Iodine (I), Barium (Ba) and Lead (Pb). Annona senegalensis Pers., Alchornea cordifolia (Schumach. & Thonn.) Müll.Arg. and Vigna unguiculate (L.) Walp. aqueous extracts showed the capacity to prevent the sickling and the hemolysis of red blood cells. Conclusion: The obtained results confirm the antisickling activity thus justifying the use of these plants in Traditional Medicine for the management of sickle cell disease. The presence of some mineral elements like Fe, Zn, Mg and Se are useful for sickle cell disease patients.

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In vivo survival studies of 51Cr-labeled methyl acetimidate treated erythrocytes in patients with sickle cell disease

Blood ◽

10.1182/blood.v56.6.1041.1041 ◽

1980 ◽

Vol 56 (6) ◽

pp. 1041-1047 ◽

Cited By ~ 5

Author(s):

TG Gabuzda ◽

TL Chao ◽

MR Berenfeld ◽

T Gelbart

Keyword(s):

Sickle Cell Disease ◽

Survival Time ◽

Sickle Cell ◽

Clinical Testing ◽

Cell Disease ◽

Show Evidence ◽

Survival Studies ◽

Circulating Antibody

Abstract Studies of the survival time of 51Cr labeled erythrocytes treated in vitro with methyl acetimidate (MAI) were conducted in 13 patients with sickle cell disease in order to assess the suitability of this antisickling agent for more extensive clinical testing. In comparison with previously measured control values (average t1/2 8.4 +/- 1.1 days a), the survival time of the treated erythrocytes in 10 of the patients who were not transfused was initially prolonged (average t1/2 24.4 +/- 4.6 days). However, 5 of the 13 patients studied developed circulating antibody against the MAI treated erythrocytes, markedly reducing the survival time of MAI treated erythrocytes in subsequent studies. Two patients, each challenged 3 times with infused MAI treated erythrocytes, failed to show evidence of antibody production, suggesting that not all subjects become immunized even after repeated exposure. In spite of many other promising properties of MAI as an antisickling agent of potential value, consideration of its use in further clinical testing must depend on successful avoidance of immunization in patients receiving infusions of treated erythrocytes.

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Placenta growth factor in sickle cell disease: association with hemolysis and inflammation

Blood ◽

10.1182/blood-2009-04-217950 ◽

2010 ◽

Vol 115 (10) ◽

pp. 2014-2020 ◽

Cited By ~ 35

Author(s):

Julia E. Brittain ◽

Ben Hulkower ◽

Susan K. Jones ◽

Dell Strayhorn ◽

Laura De Castro ◽

...

Keyword(s):

Growth Factor ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Predictive Accuracy ◽

Cross Sectional Study ◽

Acute Chest Syndrome ◽

Cell Disease ◽

Placenta Growth Factor ◽

Cross Sectional

Abstract Placenta growth factor (PlGF) is released by immature erythrocytes and is elevated in sickle cell disease (SCD). Previous data generated in vitro suggest that PlGF may play a role in the pathophysiology of SCD-associated pulmonary hypertension (PHT) by inducing the release of the vasoconstrictor, endothelin-1. In this cross-sectional study of 74 patients with SCD, we confirm that PlGF is significantly elevated in SCD compared with healthy control subjects. We found significantly higher levels of PlGF in SCD patients with PHT but observed no association of PlGF with the frequency of acute pain episodes or history of acute chest syndrome. The observed correlation between PlGF and various measures of red cell destruction suggests that hemolysis, and the resultant erythropoietic response, results in the up-regulation of PlGF. Although relatively specific, PlGF, as well as N-terminal pro-brain natriuretic peptide and soluble vascular cell adhesion molecule, has low predictive accuracy for the presence of PHT. Prospective studies are required to conclusively define the contribution of PlGF to the pathogenesis of PHT and other hemolytic complications in SCD.

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Voxelotor Treatment Interferes With Quantitative and Qualitative Hemoglobin Variant Analysis in Multiple Sickle Cell Disease Genotypes

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqaa067 ◽

2020 ◽

Vol 154 (5) ◽

pp. 627-634

Author(s):

Nicola J Rutherford-Parker ◽

Sean T Campbell ◽

Jennifer M Colby ◽

Zahra Shajani-Yi

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

High Performance ◽

Clinical Laboratory ◽

The United States ◽

Cell Disease ◽

Hemoglobin Variant ◽

Hbd Punjab ◽

The Impact

Abstract Objectives Voxelotor was recently approved for use in the United States as a treatment for sickle cell disease (SCD) and has been shown to interfere with the quantitation of hemoglobin (Hb) S percentage. This study aimed to determine the effect of voxelotor on the quantitation of hemoglobin variant levels in patients with multiple SCD genotypes. Methods In vitro experiments were performed to assess the impact of voxelotor treatment on hemoglobin variant testing. Whole blood samples were incubated with voxelotor and then analyzed by routinely used quantitative and qualitative clinical laboratory methods (high-performance liquid chromatography [HPLC], capillary zone electrophoresis [CZE], and acid and alkaline electrophoresis). Results Voxelotor modified the α-globin chain of multiple hemoglobins, including HbA, HbS, HbC, HbD-Punjab, HbE, HbA2, and HbF. These voxelotor-hemoglobin complexes prevented accurate quantitation of multiple hemoglobin species, including HbS, by HPLC and CZE. Conclusions Technical limitations in quantifying HbS percentage may preclude the use of HPLC or CZE for monitoring patients treated with voxelotor. Furthermore, it is unclear whether HbS-voxelotor complexes are clinically equivalent to HbS. Consensus guidelines for reporting hemoglobin variant percentages for patients taking voxelotor are needed, as these values are necessary for determining the number of RBC units to exchange in acute situations.

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