scholarly journals Effects of Targeting the Interleukin-1 Receptor in a Murine Model of Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2259-2259
Author(s):  
Jessica Venugopal ◽  
Jintao Wang ◽  
Chiao Guo ◽  
Daniel Eitzman
Keyword(s):  
Sickle Cell Disease ◽  
Stroke Volume ◽  
Sickle Cell ◽  
Whole Blood ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Cell Disease ◽  
Post Stroke ◽  
Formalin Fixed

Background: Sickle cell disease (SCD) is associated with chronic hemolytic anemia and a heightened inflammatory state. Multiple inflammatory pathways have been shown to be activated in SCD, however the causal role of these pathways in mediating organ damage and contributing to stroke is unclear. Methods: Bone marrow transplantation (bmt) from SCD (Townes) to wild-type (WT) recipient mice was used to generate SCD mice (WTSCD). To generate mice with non-hematopoietic deficiency of the interleukin-1 receptor (IL1R), SCD marrow was transplanted into IL1R deficient recipients (IL1R-/- SCD). Other WTSCD mice were given the IL1R inhibitor, anakinra, or PBS for 2 or 4 weeks before sacrifice. Iron and collagen staining were performed in multiple formalin-fixed tissues with Prussian blue and Masson's Trichrome, respectively. Complete blood counts and reticulocyte percentages were analyzed 15 weeks following BMT. Plasma levels of soluble E-selectin (sE-sel) and soluble P-selectin (sP-sel) were measured 14 weeks following bmt with ELISA. Ischemic stroke was induced by middle cerebral artery (MCA) photothrombosis at 16 weeks of age. WTSCD mice were given anakinra following stroke induction. Seventy-two hours after MCA occlusion, stroke volume was assessed by staining brain sections with 2, 3, 5-triphenyltetrazolium chloride. Formalin-fixed brain sections were also stained for macrophages. Results: All SCD mice were anemic and the severity of anemia was not different between WTSCD and IL1R-/- SCD mice. Increased circulating erythrocytes (9.01 ± 0.78 vs 6.77 ± 0.18 M/uL; p<0.05) and decreased reticulocytes (25.31 ± 1.79 vs 32.45 ± 2.74%; p<0.05) were observed in WTSCD mice treated with anakinra for 4 weeks. Two week anakinra treatment was sufficient to decrease organ iron deposition and lung collagen deposition. In vivo treatment with anakinra also decreased erythrocyte sickling in a whole blood ex vivo sickling assay (15.14 ± 0.94 vs 24.18 ± 1.95 % sickled; p<0.01). Further, in vitro treatment of WTSCD whole blood with IL-1β increased sickling (26.70 ± 0.39 vs 22.34 ± 0.95; p<0.05), and this was ameliorated with in vitro pretreatment with anakinra. Alterations to ex vivo sickling were not observed when conditions were repeated on erythrocytes alone. Stroke volume was significantly reduced in IL1R-/- SCD mice compared to WTSCD mice 3 days following MCA occlusion (8.08 ± 0.91 vs 11.10 ± 1.16 % hemisphere; p<0.05). Post-stroke treatment of WTSCD mice with anakinra also decreased stroke size compared to vehicle-treated mice (14.45 ± 1.44 vs 9.7 ± 3.23 % hemisphere; p=0.05). Non-hematopoietic deficiency of IL1R decreased sE-sel (vs 50.10 ± 2.31 vs 32.12 ± 2.08 pg/mL; p<0.05) and sP-sel (155.98 ± 24.44 vs 327.23 ± 18.31 ng/mL; p<0.05) levels in post-stroke animals and decreased the number of macrophages observed in the peri-stroke area (0.26 ± 0.05 vs 0.722 ± 0.15 % hemisphere; p<0.05). Conclusions: Deficiency of non-hematopoietic IL1R or treatment with an IL1R antagonist was appears beneficial towards several phenotypes in a mouse model of SCD. Given the safety of IL1R inhibitors in various patient populations, this study reveals a potential therapeutic intervention for treatment of sickle cell patients. Disclosures No relevant conflicts of interest to declare.

Regulation of Na+/Mg2+ Exchange in Sickle Erythrocytes By Endothelin-1

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4064-4064
Author(s):  
Pablo A. Rivera ◽  
Yaritza Inostroza ◽  
Jose R. Romero ◽  
Alicia Rivera
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ex Vivo ◽  
Red Cells ◽  
Cell Disease ◽  
Magnesium Homeostasis ◽  
Increased Activity ◽  
Exchange Activity

Abstract Excess levels of endothelin-1 (ET-1), erythrocyte sickling and chronic inflammation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have shown that ET-1 receptor antagonists improve hematological parameters by reducing Gardos channel activity in two transgenic mouse models of SCD while reducing oxidant stress by decreasing circulating levels of protein disulfide isomerase. Magnesium (Mg2+) deficiency, mediated in part via increased erythrocyte Na+/Mg2+ exchanger activity, has been demonstrated to contribute to erythrocyte dehydration, K+ loss and sickling in SCD. However, the relationship between ET-1 and the Na+/Mg2+ exchanger in SCD remains unclear. We measured Na+/Mg2+ exchange activity in ex vivo red cells and observed increased activity following in vitro incubation of human (2.2 ± 0.2 to 3.2 ± 0.1 mmol/1013 cell x h, P<0.03, n=5) and mouse red blood cells with ET-1 (P<0.001, n=5); events that were significantly blocked by pre-incubation of cells with 1 μM BQ788, a selective inhibitor of ET-1 type B receptors. In addition, in vitro deoxygenation of sickle red cells led to increased exchanger activity that was inhibited by impramine, a Na+/Mg2+ exchange inhibitor, and associated with reduced deoxygenation-stimulated sickle cell dehydration. These results suggest an important role for ET-1 and cellular magnesium homeostasis in sickle cell disease. To this end, we studied Na+/Mg2+ exchange activity in ex vivo erythrocytes from three transgenic sickle mouse models and observed increased activity in these cells when compared to red cells from either Hb A transgenic or C57BL/J6 wild-type mice (P<0.03, n=4). We then tested the in vivo effects of ET-1 receptor antagonists on erythrocyte Na+/Mg2+ exchange activity in the BERK mouse, a transgenic model of SCD. We blocked ET-1 receptors type A and B by in vivo treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed lower erythrocyte exchanger activity when compared to cells from vehicle treated BERK mice (P<0.02, n=6). Thus our results suggest that ET-1 receptor blockade represents an important therapeutic approach to control erythrocyte volume and magnesium homeostasis that may lead to improved inflammatory and vascular complications observed in SCD. Supported by NIH R01HL090632 to AR. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evaluation of persistence and fate of ex vivo edited-HSC modified with donor template and Its role in correcting Sickle Cell Disease

2021 ◽  
Author(s):  
Sowmya Pattabhi ◽  
Samantha N Lotti ◽  
Mason P Berger ◽  
David J Rawlings
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ex Vivo ◽  
Cell Disease ◽  
Gene Correction ◽  
Healthy Human ◽  
Peripheral Blood Stem Cells ◽  
Donor Template

Sickle cell disease (SCD) is caused by a single nucleotide transversion in exon 1 of the HBB gene that changes the hydrophobicity of adult globin (βA), leading to substantial morbidity and reduced lifespan. Ex vivo autologous gene editing utilizing co-delivery of a designer nuclease along with a DNA donor template allows for precise homology-directed repair (HDR). These gene corrected cells when engrafted into the bone marrow (BM) can prove to be therapeutic and serves as an alternative to HLA-matched BM transplantation. In the current study, we extensively explored the role of single stranded oligonucleotide (ssODN) and recombinant adeno-associated 6 (rAAV6) donor template delivery to introduce a codon-optimized change (E6optE) or a sickle mutation (E6V) change following Crispr/Cas9-mediated cleavage of HBB in healthy human mobilized peripheral blood stem cells (mPBSCs). We achieved efficient HDR in vitro in edited cells and observed robust human CD45+ engraftment in the BM of NBSGW mice at 16-17 weeks. Notably, recipients of ssODN-modified HSC exhibited a significantly higher proportion of HDR-modified cells within individual BM, CD34+ and CD235+ compartments of both E6optE and E6V cohorts. We further assessed key functional outcomes including RNA transcripts analysis and globin sub-type expression. Our combined findings demonstrate the capacity to achieve clinically relevant HDR in vitro and in vivo using both donor template delivery method. The use of ssODN donor template-delivery is consistently associated with higher levels of gene correction in vivo as demonstrated by sustained engraftment of HDR-modified HSC and erythroid progeny. Finally, the HDR-based globin protein expression was significantly higher in the E6V ssODN-modified animals compared to the rAAV6-modified animals confirming that the ssODN donor template delivery outperforms rAAV6-donor template delivery.

scholarly journals Rheological Effects of L-Glutamine in Patients with Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3567-3567
Author(s):  
Celeste K. Kanne ◽  
Varun Reddy ◽  
Vivien A. Sheehan
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Blood Viscosity ◽  
Whole Blood ◽  
Blood Cells ◽  
Cell Disease ◽  
Phase 3 ◽  
Whole Blood Viscosity

Background: ENDARITM (oral pharmaceutical L-glutamine powder) received FDA approval in 2017 as a treatment for sickle cell disease (SCD). A pivotal phase 3 clinical study conducted by Emmaus Medical, Inc. showed that L-glutamine resulted in a lower incidence of vaso-occlusive crises (VOC) as well as a lower rate of hospitalizations and shorter hospital stays. No changes in standard clinical laboratory values were noted. The clinical improvements associated with sickle cell complications are believed to be due to an increase in the proportion of the reduced form of nicotinamide adenine dinucleotides in the red blood cells (RBC) of patients with SCD, reducing the oxidative stress. While the endpoints in the phase 3 study are clinically important, it is essential that we identify biomarkers or measurable laboratory changes that can serve as endpoints for future clinical trials assessing dose optimization and the efficacy and safety of L-glutamine in SCD individuals, including those with hepatic and renal dysfunction. RBC rheology is markedly abnormal in SCD; blood is more viscous for a given hematocrit than normal individuals, dense red blood cells (DRBC) are packed with HbS, potentiating sickling, and RBCs are less deformable than those of HbAA or HbAS individuals. High whole blood viscosity, high DRBCs, and poor RBC deformability are associated with higher rates of VOC. Given the demonstrated reduction in pain events, we hypothesized that L-glutamine might improve RBC rheology and sought to test this in vitro and in vivo using a battery of rheological tests. Methods: For the in vitro study, 6 mL of whole blood was drawn into an EDTA vacutainer from ten pediatric patients with sickle cell anemia (HbSS or HbSβ0) during routine clinical checkups under an IRB approved protocol. The cohort included 3 female and 7 male patients, ages 2-19 years old. All patients were on a steady dose of hydroxyurea and did not receive a transfusion within the 3 months prior to sample collection. A 200 mM stock solution of L-glutamine and water was mixed and filtered under light-protected conditions. Aliquots were stored at -20°C to avoid multiple freeze/thaw cycles. L-glutamine was added to 3 mL of whole blood for a final concentration of 1 mM (average in vivo L-glutamine plasma concentration in patients with SCD treated with L-glutamine); 3 mL of the same patient sample with water added served as a control. After a 24-hour incubation period at 4°C, whole blood viscosity was measured using a cone and plate viscometer at 37°C (DV3T Rheometer, AMETEK Brookfield, USA), %DRBCs were measured on an ADVIA 120 Hematology System (Siemens Healthcare Diagnostics, Inc., USA), and deformability measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca®) (RR Mechatronics, the Netherlands) with the Oxygenscan module. The Oxygenscan measures RBC deformability at normoxia (Elmax), deformability upon deoxygenation (EImin), and point of sickling (PoS), the oxygen tension at which deformability begins to decline, reflecting the patient-specific pO2 at which sickling begins. Paired samples (with and without added L-glutamine) were analyzed using Student's t-test. For the in vivo study, rheological tests were performed on peripheral blood from one patient (18-year-old male on hydroxyurea) at baseline and treated with L-glutamine as part of his routine clinical care. Results and conclusions: Addition of L-glutamine in vitro significantly reduced the PoS, meaning RBCs incubated with L-glutamine could tolerate a lower pO2 before sickling compared to the control. RBCs incubated with L-glutamine also had significantly higher EImin, meaning deoxygenated RBCs were more flexible and deformable. Whole blood viscosity at 45s-1 and 225s-1 did not change significantly following incubation with L-glutamine; %DRBCs also did not change significantly (Table 1). The in vivo patient sample tested exhibited a similar improvement in PoS and EImin (Figure 1). We therefore propose to further test the performance of the PoS and EImin as possible biomarkers of response to L-glutamine in vivo. If validated, these biomarkers may also help further elucidate the mechanisms of action of L-glutamine in SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals GTx011, a Potent Allosteric Modifier of Hemoglobin Oxygen Affinity, Prevents RBC Sickling in Whole Blood and Prolongs RBC Half-Life in Vivo in a Murine Model of Sickle Cell Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 217-217 ◽  
Author(s):  
Kobina Dufu ◽  
Donna Oksenberg ◽  
Chengjing Zhou ◽  
Athiwat Hutchaleelaha ◽  
David R. Archer
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Half Life ◽  
Ex Vivo ◽  
Cell Disease ◽  
Employment Equity ◽  
Hypoxic Conditions ◽  
Equity Ownership

Abstract Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene leading to production of hemoglobin S (HbS) that polymerizes under hypoxic conditions with subsequent formation of sickled red blood cells (RBCs). We have developed a novel small molecule, GTx011, which attains effective concentrations in blood upon oral dosing in multiple species. GTx011 increases the affinity of oxygen (O2) for HbS, delays in vitro HbS polymerization and prevents sickling of isolated RBCs under hypoxic conditions. We report here that GTx011 prevents in vitro sickling of RBCs in blood from sickle cell patients. Moreover, in a murine model of sickle cell disease (Townes SS mice), GTx011 prevents ex vivo sickling of RBCs and prolongs RBC half-life. We previously reported that GTx011 prevents sickling of isolated sickle cell RBCs (SSRBCs) subjected to a fixed hypoxic condition (pO2 of ~30 mm Hg) for 30 min. For a more physiologically relevant evaluation, we determined the anti-sickling activity of GTx011 in blood under variable hypoxic conditions over a shorter duration of time. Sickling of SSRBCs in blood was evaluated using a combination of hemoximetry and morphometric measurements. Whole blood from sickle cell patients was modified in vitro with GTx011 prior to hemoximetry. Conversely, blood from SS mice with GTx011 orally dosed acutely or chronically for 10-12 days was used for hemoximetry. SSRBCs were harvested during hemoximetry at various O2 tensions and immediately fixed in a deoxygenated solution of 2% glutaraldehyde/PBS prior to morphological quantitative analysis with CellVigene software or imaging flow cytometry (AMNIS ImageStreamX MkII). To evaluate the effect of GTx011 on RBC half-life in SS mice, N-hydroxysuccinimide biotin was injected into SS mice on day 5 of chronic dosing, producing a pulse-label. Flow cytometry was performed using fluorescently labeled streptavidin to determine the decay of biotinylation and RBC half-life. Reticulocyte counts were measured at different intervals during the dosing regimen by determining the percentage of blood cells that were Ter-119+, Thiazole-Orange+ and CD45- by flow cytometry. In a dose-dependent manner, GTx011 decreased the p50 value of human blood indicating an increase in Hb-O2 affinity. In parallel, GTx011 dose-dependently reduced the number of sickled SSRBCs under all hypoxic conditions (pO2 of <40 mm Hg) evaluated. Moreover, at an O2 tension mimicking typical hypoxic conditions in tissue capillaries (40 mm Hg), 300 µM of GTx011 was sufficient to prevent sickling of human SSRBCs in whole blood (20% Hct). Similarly, ex vivo sickling analysis indicated that, relative to blood from vehicle-treated SS mice, blood from GTx011-treated SS mice showed a pronounced reduction in the number of sickled RBCs under hypoxic conditions with a concurrent reduction in p50. For example, at a pO2 of 10 mm Hg, 19% of SSRBCs in blood from GTx011-treated mice sickled ex vivo compared with 56% in blood from vehicle-treated SS mice. In SS mice chronically dosed with GTx011, a prolongation of the RBC half-life from 2.4 days to 3.8 days was achieved together with a marked decrease in reticulocyte count. This increase in RBC half-life and accompanying reduction in reticulocyte count was observed in mice with GTx011 concentrations in blood that corresponded to >30% calculated Hb target occupancy. Taken together, these data suggest that GTx011 has the potential to be a beneficial therapeutic agent for the chronic treatment of SCD. Table SS mice RBC half life Reticulocytes Sickled RBCs Hemoximetry Chronic treatment, PO, BID, 10-12 days (Days) (%) (% at 10 mm Hg) p20 (mm Hg) p50 (mm Hg) Vehicle-treated 2.4 53 56 18 32 GBT440-treated (100mg/kg) 3.8 32 19 4.5 21 Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Zhou:Global Blood Therapeutics: Research Funding. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Archer:Global Blood Therapeutics: Consultancy, Research Funding.

scholarly journals Novel Structurally-Modified Allosteric Effectors of Hemoglobin Exhibit Superior Antisickling Properties

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 218-218
Author(s):  
Osheiza Abdulmalik ◽  
Tanvi Deshpande ◽  
Mohini Ghatge ◽  
Yan Zhang ◽  
Jurgen Venitz ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Phase I ◽  
Sickle Cell ◽  
Clinical Studies ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Cell Disease ◽  
Hb S ◽  
Dose Dependent

Abstract Sickle cell disease (SCD) continues to cause significant morbidity, mortality and healthcare disparities. Despite considerable progress in understanding the underlying pathophysiology and investigating various therapeutic strategies, novel pharmacologic approaches to ameliorate SCD continue to hold immense potential and promise, especially for patients in developing countries. Our group and others have recently renewed and refocused attention to candidate drugs that directly bind to hemoglobin (Hb) and increase oxygen (O2) affinity, preventing the fundamental pathophysiology of the disease, i.e., sickle Hb (Hb S) polymerization and red blood cell (RBC) sickling. While several candidate drugs have shown biological activity in-vitro, ex-vivo and in animal studies, their ultimate success in clinical studies was hampered by toxicity concerns and/or low oral bioavailability. Recent promising reports from a phase I/II study on 5-HMF renews optimism for this therapeutic approach. We reasoned that modifications of vanillin--a previously reported antisickling agent and food constituent without known toxicities--to enhance its efficacy, would represent a feasible approach in rationally developing clinically useful candidate drugs. Consequently, we designed and synthesized two classes of compounds: INN and TD series. The former are pyridyl derivatives of vanillin, rationalized to stereospecifically inhibit deoxy-Hb S polymer formation while increasing the fraction of the soluble oxy-Hb S in regions of low O2 tension. The TD compounds represent further modification of corresponding INN compounds (with a methoxyl group on the pyridine ring), rationalized to exhibit similar dual antisickling effects, but with enhanced direct polymer destabilization properties. We subjected a prototypical compound from each class (INN-270 and TD-7) to our battery of exploratory in-vitro assays, specifically: 1) rates of Hb S binding/modification, 2) corresponding change in O2 affinity, 3) direct inhibition of Hb S polymerization, and 4) inhibition of RBC sickling under hypoxia. We incubated 0.5, 1, or 2 mM of either INN-270 or TD-7 with RBCs from patients with homozygous SCD, under hypoxia (4% O2/96% N2 gas mixture) in a shaker-incubator at 37 ˚C for 3 h. Assays were conducted in at least three replicates utilizing different samples on different days. At the conclusion of each assay, aliquot samples (~ 10 μl each) were drawn into a fixing solution under hypoxia to preserve RBC morphology for analyses. Residual RBC suspensions were washed, hemolyzed, and subjected to: cation-exchange HPLC (to determine Hb modification); P50 analyses to establish change in O2 affinity; and temperature-dependent delay time studies to establish a delay in Hb S polymerization. Our results show that both compounds permeated RBC membranes without causing hemolysis, bound to and modified intracellular Hb at high levels in a dose dependent manner, increased O2 affinity significantly, and inhibited sickling of RBCs under hypoxia. TD-7 modified Hb S in a dose-dependent manner (to 92.3 ± 5.2 %, n=4 at 2 mM), shifted O2 equilibrium to the left (Δp50 = 45.6 ± 8.2 %, n=3 at 2 mM), and inhibited RBC sickling (by 95 -100 %, n=4). Preliminary delay time analyses also showed that at 2 mM, TD-7 increased the Hb S polymerization times from 18.1 ± 1.0 min to 24.5 ± 0.5 min. INN-270 showed a similar profile, however with a lower efficacy (at 2 mM) for Hb S modification (to ~ 75 %), Δp50 of 40.3 %, sickling inhibition by ~ 70 %, and increased delay times from 15.6 ± 0.5 min to 19.7 ± 1.0 min. We have elucidated the dual antisickling mechanism of action of INN-270 and TD-7 by X-ray crystallography. Two molecules of each compound bind to Hb via Schiff-base, and a series of hydrogen-bond/hydrophobic interactions that favor a high-O2-affinity Hb state. Importantly, the methoxyl group on the pyridine ring of TD-7 forms hydrogen-bond interactions with the surface-located αF-helix, resulting in a conformational change, possibly explaining the improved potency. Based on our results, both TD7 and INN 270 exhibited greater than a 40- and 3-fold superiority in efficacy compared to vanillin and 5-HMF, respectively. We conclude that our findings justify a prospective, structure-based approach to designing novel antisickling agents with enhanced potency. In-vitro/ex-vivo murine and human PK/PD studies are currently ongoing to help guide planned in-vivo PK/PD studies in mice. Disclosures Venitz: Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: Consultancy. Safo:Baxter and AesRx companies have licensed our patented antisickling compounds. Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: #7160910; #7119208 Patents & Royalties, Consultancy, Research Funding.

Cocaine-Induced Hemolysis Activates Platelets in Whole Blood

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3836-3836
Author(s):  
William Hobbs ◽  
Adam Munday ◽  
Jose A. Lopez
Keyword(s):  
Sickle Cell Disease ◽  
Platelet Activation ◽  
Sickle Cell ◽  
Whole Blood ◽  
Cocaine Exposure ◽  
Cell Disease ◽  
Cocaine Use ◽  
Risk Of Death ◽  
Chronic Hemolysis

Abstract The clinical phenotype of sickle cell disease (SCD) is primarily a consequence of two processes: chronic hemolysis and vaso-occlusive phenomena, the latter a result of the enhanced adhesiveness of erythrocytes, leukocytes, and platelets. These processes produce the myriad clinical manifestations of the disease, which include endothelial dysfunction, chronic inflammation, episodic ischemia/reperfusion injury, tissue infarction, and oxidative injury. The proximate cause of SCD is a mutation in the gene encoding the beta-globin chain of hemoglobin, converting glutamate at the sixth position of the polypeptide chain to valine. This seemingly innocuous change results in a hemoglobin molecule prone to polymerize under low oxygen tension, altering the shape of the erythrocyte, its mechanical stability, and its adhesiveness. Very little is known about the mechanisms that render some patients more susceptible to the consequences of chronic hemolysis or vaso-occlusion. Cocaine abuse is known to trigger pathways consistent with both increased adhesivity (vaso-occlusive pain crises) and hemolysis (priapism) in SCD. Cocaine use has also been associated with a 29% increased risk of death/year following even a single documented cocaine exposure in SCD patients. It is also noteworthy that cocaine is associated with thrombotic processes that suggest both endothelial and platelet activation, both of which have been implicated in SCD pathophysiology. We hypothesized that cocaine activates platelets, which would be augmented in sickle cell disease. We assessed the activation of platelets exposed in vitro to cocaine by assaying two markers of platelet activation: surface P-selectin exposure reflecting activation-dependent release of platelet a granules, and exposure of an activation-dependent epitope in the integrin aIIbb3 recognized by the antibody PAC-1. Surprisingly, neither washed platelets nor platelets in plasma exposed to cocaine in vitro showed evidence of activation above background. In fact, treatment of platelets with cocaine under these conditions rendered them hyporesponsive to subsequent activation by ADP, epinephrine, or thrombin with similar dose–response curves. In contrast, when whole blood was exposed to cocaine, platelet activation was observed by both increased platelet P-selectin exposure and PAC-1 binding. When individual blood components were then mixed with washed platelets, only addition of erythrocytes resulted in platelet activation to cocaine. We explored the mechanism of this response using isolated erythrocytes. By flow cytometry, we observed accumulation of erythrocyte-derived microparticles within 10–20 min of cocaine exposure, which also occurred in cocaine treated whole blood. Erythrocytes from patients with SCD were more sensitive to cocaine, producing microparticles at lower concentrations of the drug than non-SCD erythrocytes. Using DIC microscopy and video recording, we found that erythrocytes (both SCD and non-SCD) underwent rapid morphologic changes upon cocaine exposure, changing from discocytes to echinocytes then rapidly to stomatocytes. The echinocytes appeared within 15–30 sec of cocaine exposure, then transformed into stomatocytes within the next 45 sec to 2 min. We did not observe reversal of this morphologic transition, with 90–100% of erythrocytes undergoing the full transformation into stomatocytes and retaining this morphology through 60 minutes of observation. We also observed release of small membrane vesicles from the erythrocytes. A fraction of the cells undergoing this transformation lysed after reaching the stomatocyte phase, with increased hemolysis observed with sickle erythrocytes compared to erythrocytes from ethnically matched non-sickle controls. These results suggest that cocaine activates platelets indirectly by a mechanism that involves erythrocyte lysis. Other aspects of cocaine’s effects on erythrocytes would be expected to potentiate a prothrombotic phenotype, including microparticle release and nitric oxide scavenging resulting from hemolysis. Because of the increased fragility of their erythrocytes, individuals with sickle cell anemia would be expected to be especially susceptible to the cardiovascular complications of cocaine use.

3057 – MATURATION OF EX VIVO-CULTURED HUMAN ERYTHROCYTES AND SICKLE CELL DISEASE MODELLING USING CRISPR-CAS9

2020 ◽  
Vol 88 ◽  
pp. S55
Author(s):  
Yelena Boccacci ◽  
Guillaume Margaillan ◽  
Nellie Dumont ◽  
Mathieu Drouin ◽  
Yannick Doyon ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ex Vivo ◽  
Human Erythrocytes ◽  
Disease Modelling ◽  
Cell Disease

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.

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

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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