Mitochondrial reactive oxygen species scavenging attenuates thrombus formation in a murine model of sickle cell disease

2021 ◽  
Author(s):  
Gowtham Annarapu ◽  
Deirdre Nolfi‐Donegan ◽  
Michael Reynolds ◽  
Yinna Wang ◽  
Sruti Shiva
Keyword(s):  
Reactive Oxygen Species ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Murine Model ◽  
Thrombus Formation ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Mitochondrial Reactive Oxygen Species ◽  
Cell Disease

scholarly journals Potential role of LSD1 inhibitors in the treatment of sickle cell disease: a review of preclinical animal model data

2018 ◽  
Vol 315 (4) ◽  
pp. R840-R847 ◽  
Author(s):  
Angela Rivers ◽  
Ramasamy Jagadeeswaran ◽  
Donald Lavelle
Keyword(s):  
Reactive Oxygen Species ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Reactive Oxygen ◽  
Organ Damage ◽  
The United States ◽  
Oxygen Species ◽  
Cell Disease ◽  
Hematopoietic Stem

Sickle cell disease (SCD) is caused by a mutation of the β-globin gene (Ingram VM. Nature 180: 326–328, 1957), which triggers the polymerization of deoxygenated sickle hemoglobin (HbS). Approximately 100,000 SCD patients in the United States and millions worldwide (Piel FB, et al. PLoS Med 10: e1001484, 2013) suffer from chronic hemolytic anemia, painful crises, multisystem organ damage, and reduced life expectancy (Rees DC, et al. Lancet 376: 2018–2031, 2010; Serjeant GR. Cold Spring Harb Perspect Med 3: a011783, 2013). Hematopoietic stem cell transplantation can be curative, but the majority of patients do not have a suitable donor (Talano JA, Cairo MS. Eur J Haematol 94: 391–399, 2015). Advanced gene-editing technologies also offer the possibility of a cure (Goodman MA, Malik P. Ther Adv Hematol 7: 302–315, 2016; Lettre G, Bauer DE. Lancet 387: 2554–2564, 2016), but the likelihood that these strategies can be mobilized to treat the large numbers of patients residing in developing countries is remote. A pharmacological treatment to increase fetal hemoglobin (HbF) as a therapy for SCD has been a long-sought goal, because increased levels of HbF (α2γ2) inhibit the polymerization of HbS (Poillin WN, et al. Proc Natl Acad Sci USA 90: 5039–5043, 1993; Sunshine HR, et al. J Mol Biol 133: 435–467, 1979) and are associated with reduced symptoms and increased lifespan of SCD patients (Platt OS, et al. N Engl J Med 330: 1639–1644, 1994; Platt OS, et al. N Engl J Med 325: 11–16, 1991). Only two drugs, hydroxyurea and l-glutamine, are approved by the US Food and Drug Administration for treatment of SCD. Hydroxyurea is ineffective at HbF induction in ~50% of patients (Charache S, et al. N Engl J Med 332: 1317–1322, 1995). While polymerization of HbS has been traditionally considered the driving force in the hemolysis of SCD, the excessive reactive oxygen species generated from red blood cells, with further amplification by intravascular hemolysis, also are a major contributor to SCD pathology. This review highlights a new class of drugs, lysine-specific demethylase (LSD1) inhibitors, that induce HbF and reduce reactive oxygen species.

Mitochondrial Uncoupling Protein 2 Is Present in Human Platelets and Regulates Platelet Activation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4147-4147 ◽  
Author(s):  
Sruti Shiva ◽  
Enrico M Novelli ◽  
Grant C Bullock ◽  
Elizabeth Kenny ◽  
Gabrielle Hill ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Sickle Cell Disease ◽  
Platelet Activation ◽  
Sickle Cell ◽  
Mitochondrial Membrane ◽  
Reactive Oxygen ◽  
Human Platelets ◽  
Oxygen Species ◽  
Cell Disease ◽  
Superoxide Generation

Abstract It has long been recognized that platelets contain functional mitochondria and accumulating data suggest that several aspects of mitochondrial function, including reactive oxygen species production, modulate platelet thrombotic function. We have recently shown that platelet mitochondria isolated from patients with Sickle Cell Disease generate significantly greater concentrations of reactive oxygen species than healthy African American subjects and that this mitochondrial oxidant generation contributes to higher levels of basal platelet activation in these patients (Blood. 2014 May 1;123(18):2864-72). Based on these data, we have now investigated the factors that regulate platelet mitochondrial superoxide generation in healthy and Sickle Cell Disease platelets. Here we demonstrate that human platelets express mitochondrial uncoupling protein-2 (UCP2), a protein that is known to decrease the efficiency of oxidative phosphorylation and oxidant generation in other cell types but has previously not been identified in platelets. In this study we show that UCP2 protein is expressed in healthy human platelets and is fully functional as it facilitates proton leak across the inner mitochondrial membrane, leading to decreased mitochondrial membrane potential. Further, we demonstrate that the expression of this protein attenuates platelet mitochondrial superoxide generation, as treatment of platelets with Genipin (2-10µM), a pharmacological inhibitor of UCPs, concentration-dependently increases mitochondrial membrane potential and reactive oxygen species production. Further, an approximately 70% inhibition of UCP activity results in platelet activation demonstrated by increased membrane p-selectin expression (65±7% versus 6±3% in untreated controls) and augmented glycoprotein IIb/IIIa activation (57±9% versus 9±4% in untreated controls). The use of the mitochondrial-targeted antioxidant mitoTEMPO (10µM) decreases genipin-induced superoxide generation and significantly attenuates platelet activation. Notably, preliminary data presented here also suggest that UCP expression is decreased in platelets isolated from Sickle Cell Disease patients (3-fold) compared to healthy African American subjects. Additionally, ongoing studies are investigating platelet function in mice deficient in UCP2. Taken together, these data demonstrate a novel mechanism of regulation of platelet thrombotic function whose physiological relevance is apparent in the context of Sickle Cell Disease. More broadly this study advances the understanding of the role of the mitochondrion in platelet biology and thrombotic disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Nicotinamide Improves Anemia and Decreases Reactive Oxygen Species in Sickle Cell Disease Mice

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2019-2019
Author(s):  
Jagadeesh Ramasamy ◽  
Vinzon Ibanez ◽  
Vijaya Lakshmi Yalagala ◽  
Yogenthiran Saunthararajah ◽  
Robert E. Molokie ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Sickle Cell Disease ◽  
Redox Potential ◽  
Sickle Cell ◽  
Therapeutic Option ◽  
Statistical Significance ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Cell Disease ◽  
Nad Synthesis

Abstract The polymerization of deoxygenated HbS molecules in the red blood cells (RBCs) of patients with sickle cell disease (SCD) causes RBC destruction resulting in chronic pain, debilitating acute pain crises, strokes, multi-organ damage and a reduced life span. Therapeutic options remain limited. Bone marrow transplantation can be curative but is not an option for the majority of patients. Gene therapy interventions that also offer the promise of a cure are under investigation but are not likely to be available to the vast majority of patients in the near future. Hydroxyurea, the first drug approved for treatment of SCD, increases levels of Fetal Hemoglobin (HbF) that inhibit polymerization of HbS molecules but is not effective in all patients while a more powerful HbF-inducing drug, the DNA methyltransferase inhibitor decitabine has yet to be approved. L-glutamine, another approved therapeutic option, increases NAD redox potential and decreases reactive oxygen species (ROS) in the sickle RBCs to reduce symptoms. In our laboratory we have observed that increased ROS is associated with the retention of mitochondria in the SCD RBCs and have hypothesized that the abnormal presence of mitochondria in these cells is a major source of ROS (Jagadeeswaran et al Exp Hematol 50:46-52, 2017). In this investigation we have tested the hypothesis that chronic oral supplementation with nicotinamide, a direct precursor of NAD synthesis, would improve NAD redox potential, decrease mitochondrial retention and ROS in SCD RBCs, and reduce anemia in the SCD mouse model. The effect of nicotinamide was tested in SCD mice whose drinking water was supplemented for three months with 1% nicotinamide. The percentage of RBCs retaining mitochondria and the levels of ROS were determined by flow cytometric assays using the mitochondrial-specific dye TMRM and the ROS probe CM-H2DCFDA, respectively. In SCD mice receiving nicotinamide the fraction of RBCs retaining mitochondria was reduced 22.1% (p<0.05) and the level of ROS in RBCs was reduced 41% (p<0.01) compared to control SCD mice. The reticulocyte percentage was reduced 28% in nicotinamide-treated SCD mice compared to control SCD mice (p<0.01). The total RBC count was 30% higher (p<0.05) in nicotinamide-treated mice (6.61±0.76 X 10 6/μl) compared to control SCD mice (5.08±0.70 X 10 6/μl). Similar differences in hematocrit and total hemoglobin were also observed but failed to reach statistical significance. Total NAD levels were not significantly different in SCD mice receiving nicotinamide compared to control SCD mice (p<0.05), but the NADH/NAD total ratio was increased 2 fold (p<0.05). These results show that oral administration of high doses of nicotinamide decreases mitochondrial retention and ROS in SCD RBCs and improves NAD redox potential and anemia in SCD mice. These effects strongly suggest that additional studies be performed to investigate nicotinamide as a therapeutic option in SCD. Figure 1 Figure 1. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

scholarly journals Endothelin-1 contributes to the progression of renal injury in sickle cell disease via reactive oxygen species

2015 ◽  
Vol 173 (2) ◽  
pp. 386-395 ◽  
Author(s):  
J Brett Heimlich ◽  
Joshua S Speed ◽  
Paul M O'Connor ◽  
Jennifer S Pollock ◽  
Tim M Townes ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Renal Injury ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Cell Disease ◽  
Endothelin 1

scholarly journals Ginkgo biloba extract (EGb 761) attenuates oxidative stress induction in erythrocytes of sickle cell disease patients

2012 ◽  
Vol 48 (4) ◽  
pp. 659-665 ◽  
Author(s):  
Aline Emmer Ferreira Furman ◽  
Railson Henneberg ◽  
Priscila Bacarin Hermann ◽  
Maria Suely Soares Leonart ◽  
Aguinaldo José do Nascimento
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Lipid Peroxidation ◽  
Sickle Cell ◽  
Reduced Glutathione ◽  
Reactive Oxygen ◽  
Intracellular Reactive Oxygen Species ◽  
Oxygen Species ◽  
Egb 761 ◽  
Cell Disease

Sickle cell disease promotes hemolytic anemia and occlusion of small blood vessels due to the presence of high concentrations of hemoglobin S, resulting in increased production of reactive oxygen species and decreased antioxidant defense capacity. The aim of this study was to evaluate the protective action of a standardized extract of Ginkgo biloba (EGb 761), selected due to its high content of flavonoids and terpenoids, in erythrocytes of patients with sickle cell anemia (HbSS, SS erythrocytes) subjected to oxidative stress using tert-butylhydroperoxide or 2,2-azobis-(amidinepropane)-dihydrochloride, in vitro. Hemolysis indexes, reduced glutathione, methemoglobin concentrations, lipid peroxidation, and intracellular reactive oxygen species were determined. SS erythrocytes displayed increased rates of oxidation of hemoglobin and membrane lipid peroxidation compared to normal erythrocytes (HbAA, AA erythrocytes), and the concentration of EGb 761 necessary to achieve the same antioxidant effect in SS erythrocytes was at least two times higher than in normal ones, inhibiting the formation of intracellular reactive oxygen species (IC50 of 13.6 µg/mL), partially preventing lipid peroxidation (IC50 of 242.5 µg/mL) and preventing hemolysis (IC50 of 10.5 µg/mL). Thus, EGb 761 has a beneficial effect on the oxidative status of SS erythrocytes. Moreover, EGb 761 failed to prevent oxidation of hemoglobin and reduced glutathione at the concentrations examined.

Modified Actriib-Fc Fusion Protein (ACE-536) Mitigates Sickling and Red Cell Pathology in a Murine Model of Sickle Cell Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4055-4055
Author(s):  
Rajasekhar NVS Suragani ◽  
Robert Li ◽  
Sharon M Cawley ◽  
R. Scott Pearsall ◽  
Ravindra Kumar
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Murine Model ◽  
Half Life ◽  
Annexin V ◽  
Red Cell ◽  
Oxygen Species ◽  
Cell Disease ◽  
Employment Equity ◽  
Equity Ownership

Abstract Sickle cell disease (SCD) is a debilitating hereditary disorder caused by a single point mutation in the β-globin gene resulting in the production of sickle hemoglobin variant (HbS). In the deoxygenated state, HbS is labile and undergoes auto-oxidation and polymerizes to generate rigid and irreversibly sickled erythrocytes. The pathophysiology of SCD include increased red cell hemolysis, reactive oxygen species and phosphatidylserine (PS) exposure on RBC membranes which leads to a very short red cell half-life, increased reticulocytosis and splenomegaly. Sickled RBCs show enhanced adherence to activated endothelium causing chronic inflammation leading to frequent and acute painful vaso-occlusive crises in SCD patients. Hydroxyurea (HU) augments fetal hemoglobin production, decreases irreversible sickle cells and painful events, and is the only approved therapy for SCD patients. However, recent studies have shown dose limiting myelosupression with HU treatment. Approximately one-third of patients do not respond to HU therapy thereby highlighting the need for alternative treatment strategies. ACE-536 is a modified type IIB activin receptor-Fc fusion protein (ACE-536)1 which functions as a ligand trap for certain members of the TGFβ superfamily. In a murine model of β-thalassemia, RAP-536 (murine ortholog of ACE-536) treatment reduced hemichromes on RBC membranes, decreased reactive oxygen species, reduced hemolysis, improved red cell half-life and thus corrected anemia and mitigated disease complications of β-thalassemia syndrome2. In the present study, we evaluated RAP-536 as a monotherapy and combination therapy with HU in the murine model of sickle cell disease (βS/βS)3. SCD mice were dosed with RAP-536 (1 mg/kg, twice weekly, s.c.) or TBS vehicle (VEH) control (N=5/group) for 3 months. A combination treatment with HU (100mg/kg, i.p.) and RAP-536 (10mg/kg, s.c) twice weekly for 2 months was performed and compared with vehicle or HU monotherapy treated SCD mice. Non-symptomatic compound heterozygote (β/βS) littermates were treated similarly (N=5/group) and used as controls to confirm disease in SCD (βS/βS) mice. At study baseline, SCD mice had reduced RBC number (-28%, P<0.01) and hemoglobin (-14.5%, P<0.05) and increased reticulocytes (+50%, P<0.001) compared to compound heterozygote mice. Following one month of treatment, RAP-536 (1mg/kg) significantly reduced spleen weight (-20.5%, P<0.05), decreased serum bilirubin content (-17%, P<0.01) and cell free hemoglobin (-30.7%, P=0.06) compared to vehicle treated mice indicating decreased hemolysis. Most remarkably, blood smears from RAP-536 treated SCD mice displayed a decrease in number of irreversibly sickled erythrocytes (-66.5%, P<0.001) as well as reduced annexin V/PS exposure (-18.75%, N.S), suggesting improved membrane phospholipid asymmetry. RAP-536 treatment showed increased RBC number (+15.2%, P<0.01) and hemoglobin (+9.28%, P<0.05) compared to VEH treatment with concomitant decrease in reticulocytes (-13.5%, P< 0.05), suggestive of an increase in red cell half-life. Furthermore, histopathological analysis of spleen, kidneys and heart revealed a trend toward reduced intravascular congestion in RAP-536 treated SCD mice. Preliminary data from the combination treatment of HU and RAP-536 in SCD mice displayed additive beneficial effects as compared to HU alone. The combination of RAP-536 and HU produced a greater reduction in annexin V/PS exposure on peripheral blood cells than did HU alone compared to vehicle treatment (-35.6%, P<0.001 vs. -22.2%, N.S, respectively). Similarly, HU+RAP-536 showed a greater reduction in spleen size than HU alone (-50.7%, P<0.05 vs. -20.2%, N.S) respectively, compared to vehicle treated SCD mice. Additional analyses are in progress. Taken together, these data demonstrates that RAP-536 reduces the RBC sickling and red blood cell pathology in SCD and also shows its utility as monotherapy and in combination with HU to further mitigate the disease severity. ACE-536 is currently being tested in Phase 2 clinical trials in MDS and β-thalassemia patients, and merits evaluation as a therapy for SCD patients. References: Suragani RN et.al; Nature Medicine 2014; 20: 408-14Suragani RN et.al; Blood 2014; 123: 3864-72Wu LC etal; Blood. 2006; 108:1183-8. Disclosures Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Cawley:Acceleron Pharma Inc: Employment. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

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