Protective Effect of Fetal Hemoglobin On Inflammation-Induced Expression of Hypoxia-Inducible Factor-1α (HIF-1α) in Sickle Mice: Microvascular Implications

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3250-3250
Author(s):  
Dhananjay K. Kaul ◽  
Mary E. Fabry ◽  
Sandra M Suzuka ◽  
Janki Shah
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Hypoxia Inducible Factor ◽  
Heme Oxygenase 1 ◽  
No Production ◽  
Cell Disease ◽  
Activation Markers ◽  
No Bioavailability

Abstract Abstract 3250 Chronic inflammation is a salient feature of human sickle cell disease (SCD) and transgenic-knockout sickle (BERK) mouse model. Although tissue ischemia is the primary instigator of hypoxia-inducible factor (HIF) activation, a number of inflammatory factors/pathways and oxidative stress can potentially induce expression of HIF-1α. Increased oxidative stress and inflammation are implicated in the activation of HIF-1α under normoxic conditions. HIF can trigger transcription of genes for vasoactive molecules such as vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1) and endothelin, which are implicated in the pathophysiology of SCD. We hypothesize that, in SCD, inflammation coupled with nitric oxide (NO) depletion will induce expression of HIF-1α. To this end, we have examined the expression of HIF-1α in normoxic BERK mice expressing exclusively human α- and βS- globins, and evaluated the effect of HbF in BERK mice (i.e., <1.0%, 20% and 40% HbF). We have previously shown that HbF exerts anti-sickling and anti-inflammatory effects (Kaul et al. J Clin Invest, 2004; Dasgupta et al. Am J Physiol, 2010). Here, we show that HIF-1α is expressed in BERK mice under normoxic conditions (i.e., normal hemoglobin oxygen saturation levels). In BERK mice expressing HbF, HIF-1α expression decreased concomitantly with increasing HbF, commensurately with increased NO bioavailability, and showed a strong inverse correlation with plasma NO metabolites (NOx) levels. Reduced HIF-1α expression in BERK mice expressing HbF was associated with decreased HO-1 and VEGF expression, and reduced serum endothelin-1 (ET-1) levels, which are among the target vasoactive molecules of HIF-1α. Furthermore, the commensurate decrease in HIF-1α expression with increase in HbF levels in BERK mice was accompanied by a distinct decrease in soluble (s) forms of endothelial activation markers such as sP-selectin and vascular cell adhesion molecule-1 (sVCAM-1). Notably, arteriolar dilation, enhanced volumetric blood flow and low blood pressure in normoxic BERK mice all showed a trend toward normalization with the introduction of HbF. Also, arginine treatment reduced HIF-1α expression as well as ET-1 levels in normoxic BERK mice, supporting a role of decreased NO bioavailability in HIF-1α activation. The present in vivo studies show that reduced inflammation and increased NO production in normoxic BERK mice (expressing HbF or treated with arginine) are distinctly associated with suppression of HIF-1α activation and inhibition of vasodilators, resulting in improved microvascular and hemodynamic parameters in the BERK model of sickle cell disease. The unique feature of inflammation in SCD is that it can be ameliorated by increased HbF, thereby coupling HbS polymerization/sickling to NO depletion, HIF-1α expression and inflammation in this disease. Disclosures: No relevant conflicts of interest to declare.

Reduced Oxidative Stress and Enhanced Nitric Oxide (NO) Bioavailability in Transgenic-Knockout Sickle Mice Expressing Fetal Hemoglobin (HbF) Is Mediated by Decreased Sickling and Hemolysis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 842-842
Author(s):  
Trisha Dasgupta ◽  
Mary E. Fabry ◽  
Dhananjay K. Kaul
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Genetic Manipulation ◽  
Fetal Hemoglobin ◽  
Organ Damage ◽  
Multiple Organ ◽  
Protective Effects ◽  
Cell Disease ◽  
No Bioavailability

Abstract The primary event in the vaso-occlusive pathophysiology of sickle cell disease (SCD) is polymerization of hemoglobin S under deoxygenated conditions. In SCD, sub-clinical transient vaso-occlusive events caused by red cell sickling are likely to be more frequent resulting in “reperfusion injury” that generates reactive oxygen species and results in chronic oxidative stress that will contribute to multiple organ damage. In fact, previous studies have suggested that sickling is etiologic to repefusion injury and oxidative stress (Kaul and Hebbel, JCI, 2000), although the effect of antisickling therapy on oxidative stress has not been evaluated. Increasing the levels of antisickling fetal hemoglobin (HbF) by hydroxyurea therapy markedly reduces polymer formation. HbF exerts an ameliorating effect in sickle cell disease patients both on red cells and in the prevention of multiple organ damage. Here, we hypothesize that induction of HbF by genetic manipulation (in the absence of pharmacological manipulation) will reduce organ oxidative stress by reducing sickling and hemolysis, and thereby increase NO bioavailability. To test our hypothesis, we measured activity of selected antioxidants and lipid peroxidation (LPO) in BERK mice expressing exclusively human α- and βS-globins and varying levels of HbF, i.e., BERK (<1% HbF), BERKγM (20% HbF) and BERKγH (40% HbF). Percent sickled cells in venous samples (drawn in 2.5% glutaraldehyde solution in 0.1M cacodylate buffer) showed a distinct decrease with increased %HbF (P<0.05, multiple comparisons). Consistent with maximal sickling, BERK mice showed 5.4–6.9-fold increase in LPO in various tissues (muscle, kidney and liver) compared with C57BL controls (P<0.001). In contrast, BERKγM and BERKγH mice showed a marked decrease (73% and 80%, respectively) in LPO compared with BERK mice (P<0.001). Also, activity/levels of antioxidants (superoxide dismutase [SOD], catalase, glutathione peroxidase [GPx] and reduced glutathione [GSH]) showed significant decreases in BERK mice (P<0.001–0.00001). On the other hand, BERKγM and BERKγH mice showed significant increases in antioxidant activity (P<0.05–0.0001). Induction of HbF was associated with increased levels of NO metabolites (NOx) and reduced hemolysis; the latter is in agreement with our previous observations in BERKγM mice (Kaul et al. JCI, 2004). These results strongly suggest that reduced sickling and hemolysis in the presence of HbF cause increased NO bioavailability. NO is well known to exert antioxidative effects. Thus, we show for the first time that the induction of antisickling HbF leads to an increase in NO bioavailability and a decrease in oxidative stress, and that these protective effects are mediated primarily by reduced intravascular sickling.

scholarly journals Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing non-nitric oxide vasodilators, hemolysis, and oxidative stress

2008 ◽  
Vol 295 (1) ◽  
pp. H39-H47 ◽  
Author(s):  
Dhananjay K. Kaul ◽  
Xiaoqin Zhang ◽  
Trisha Dasgupta ◽  
Mary E. Fabry
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Vascular Reactivity ◽  
Microvascular Function ◽  
Cell Disease ◽  
No Bioavailability ◽  
Cox 2 ◽  
And Oxidative Stress

In sickle cell disease, nitric oxide (NO) depletion by cell-free plasma hemoglobin and/or oxygen radicals is associated with arginine deficiency, impaired NO bioavailability, and chronic oxidative stress. In transgenic-knockout sickle (BERK) mice that express exclusively human α- and βS-globins, reduced NO bioavailability is associated with induction of non-NO vasodilator enzyme, cyclooxygenase (COX)-2, and impaired NO-mediated vascular reactivity. We hypothesized that enhanced NO bioavailability in sickle mice will abate activity of non-NO vasodilators, improve vascular reactivity, decrease hemolysis, and reduce oxidative stress. Arginine treatment of BERK mice (5% arginine in mouse chow for 15 days) significantly reduced expression of non-NO vasodilators COX-2 and heme oxygenase-1. The decreased COX-2 expression resulted in reduced prostaglandin E2(PGE2) levels. The reduced expression of non-NO vasodilators was associated with significantly decreased arteriolar dilation and markedly improved NO-mediated vascular reactivity. Arginine markedly decreased hemolysis and oxidative stress and enhanced NO bioavailability. Importantly, arteriolar diameter response to a NO donor (sodium nitroprusside) was strongly correlated with hemolytic rate (and nitrotyrosine formation), suggesting that the improved microvascular function was a response to reduced hemolysis. These results provide a strong rationale for therapeutic use of arginine in sickle cell disease and other hemolytic diseases.

scholarly journals Hydroxyurea Reverses Dysfunctional Ubiquitin-Proteasomal System in Sickle CELL Disease and Suppresses Posttranslational Alterations in Hemoglobin and CELL Membranes

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4822-4822
Author(s):  
Swee Lay Thein ◽  
Arun S Shet ◽  
Michael Brad Strader ◽  
Fanato Meng ◽  
Michael Heaven ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Longitudinal Study ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Membranes ◽  
Conflicts Of Interest ◽  
Side Reactions ◽  
Cell Disease ◽  
Post Translational Modification ◽  
Oxidative Modifications

HYDROXYUREA REVERSES DYSFUNCTIONAL UBIQUITIN-PROTEASOMAL SYSTEM IN SICKLE CELL DISEASE AND SUPPRESSES POSTTRANSLATIONAL ALTERATIONS IN HEMOGLOBIN AND CELL MEMBRANES Sirsendu Jana, PhD1, Michael Brad Strader, PhD1, Fantao Meng, PhD1,Michael Heaven, PhD2, Arun Shet, MD3, Swee Lay Thein, MD3, and Abdu I. Alayash, PhD1. 1Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), 2Vulcan Analytical, Birmingham Al, 3Sickle Cell Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH). Introduction: Intracellular oxidative stress and oxidative modifications of sickle cell hemoglobin (HbS) play an important role in the pathogenesis of sickle cell disease (SCD). We recently reported transgenic mice studies revealing microparticles (MP) proteome differences between SCD and control mice expressing human HbS and HbA, respectively. Hb-dependent oxidative reactions and consequent posttranslational modifications of Hb βCys93 were central to red cell membrane changes that included modification of band3, and ubiquitination of proteins (Jana S et al., JCI Insights, 2018 3:120451). Ubiquitination is an important post-translational modification required for several biological functions including degradation by the ubiquitin-proteasomal system (UPS) proteolytic pathway. Proteins susceptible to oxidative damage are therefore likely degraded by UPS machinery. When these animals were treated with hydroxyurea (HU) they were able to reduce oxidative stress by controlling Hb oxidation side reactions. As a follow-up study, we have characterized human RBC derived MP proteomes of control, untreated and HU treated SCD patient samples to identify the mechanistic basis of how HU treatment reduces oxidative stress. Methods: We used a variety of biochemical and hematological methods to investigate a group of sickle cell disease (SCD) patients (n= 22) who are either on HU treatment (n=10) or without HU treatment (n=12) and a group of ethnically matched controls (n=4). We also performed an additional proteomic analysis on a subset of these patients, including a separate longitudinal study in which SCD patients (n=2) were followed before and after treatment with HU. Results: Immunoprecipitation experiments on RBCs obtained from untreated SCD patients revealed the presence of extensive ubiquitination contrary to those samples obtained from HU treated patients and controls. High proteasomal activity was found in SCD RBCs suggesting accumulated polyubiquitinated proteins found in these samples were not a byproduct of proteasomal inhibition but rather due to imbalance in the redox state of SS RBCs. In addition to Hb oxidation and oxidative modifications (including βCys93), our results revealed differences in the SCD proteome (from both control and untreated groups) including upregulation of phosphorylation and ubiquitination of proteins that are known to interact directly with band3 and are functionally involved in MP formation. Ubiquitination of Hb βLys145 and βLys96 were more abundant in SS patient's samples as well as phosphorylation of band3 (a prerequisite process for band3 clustering and MPs release). As revealed by the separate longitudinal study, HU treatment uniformly reversed ubiquination and phosphorylation of proteins involved in SCD induced MP formation. Conclusion: These mechanistic analyses of SCD RBC derived MPs suggest a potential involvement of ubiquitination and phosphorylation in SCD pathogenesis and provide additional insight into the therapeutic mechanisms of HU treatment. Disclosures No relevant conflicts of interest to declare.

Quantitative Analysis of Small Molecular Weight Thiols and Disulfides in Blood from a Sickle Cell Disease Patient Infused with N-Acetyl-L-Cysteine

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2662-2662
Author(s):  
Shelby A Cate ◽  
Tahsin Ozpolat ◽  
Junmei Chen ◽  
Colette Norby ◽  
Barbara A. Konkle ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Small Molecule ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Antioxidant Properties ◽  
Disease Patient ◽  
Cell Disease ◽  
Mixed Disulfides

Abstract N-acetyl-L-cysteine (NAC) is an FDA approved drug used to treat acetaminophen overdose or as a mucolytic agent in respiratory disorders. The commonly accepted mechanism of action is that NAC undergoes deacetylation to cysteine, which is then used to synthesize glutathione (GSH), a major intracellular antioxidant. Like other thiol-containing compounds, NAC can also act as a reducing agent to break protein disulfide bonds or as a scavenger of reactive oxygen species. Due to its antioxidant properties, NAC has been proposed as a potential treatment for many diseases associated with oxidative stress, including sickle cell disease (SCD), neurological disorders, infectious diseases, and cancers. Though NAC has been widely studied, a full understanding of the mechanism by which NAC is effective in vivo has been limited by challenges in accurately quantifying NAC and its metabolites. As part of a clinical trial of NAC therapy in SCD, we have developed a liquid chromatography-mass spectrometry (LC-MS) based assay to quantify small molecule free thiols and disulfides using isotopically labeled internal standards. We applied this method to quantify small molecular thiols and disulfides in whole blood, red blood cells, and plasma from a SCD patient before (pre) and at 1, 8, 24 and 72 hr time points of intravenous administration of NAC at a dose of 300 mg/kg (a bolus infusion of 150 mg/kg for 1 hour followed by 150 mg/kg given over the next 7 hr). The cysteine concentration in whole blood increased to 286 μM at 1 hr from 97 μM at baseline, indicating that NAC is indeed rapidly metabolized (deacetylated) to cysteine. Interestingly, although cysteine concentration in RBCs increased over 4 fold at 1 hr and remained high compared to baseline, the highest concentration of total GSH in blood was observed at 24 hr (743 μM compared to 494 μM at baseline). Intracellular availability of cysteine is known as a rate-limiting step for GSH synthesis, and the delayed accumulation of GSH may suggest that NAC is involved in the extracellular deficit of reducing equivalents before it serves as a substrate in GSH synthesis. To explore this possibility, we quantitated NAC and its oxidation products, homo- and mixed disulfides. We found that total NAC concentration reached 1.58 mM in whole blood at 1 hr, but 44% of NAC was oxidized to N-acetyl-cystine (NAC-ss) or formed mixed disulfides with GSH (GS-ss-NAC) and Cys (Cys-ss-NAC), whereas the NAC used for infusion contained less than 0.5% in the oxidized form (NAC-ss). Concurrent with the formation of NAC disulfides, the levels of oxidized GSH (GSSG, GS-ss-Cys) and cysteine (cystine) were significantly decreased. These observations suggest that NAC administration of SCD not only increases GSH levels by raising the cysteine concentration, but also directly functions as an antioxidant to reduce oxidative stress. SCD patients are known to have low levels of GSH and frequently experience oxidative stress. NAC treatment is likely to address both issues. We plan to analyze the effects of NAC on blood small molecule thiol concentrations in several more SCD patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Impact of arginine therapy on mitochondrial function in children with sickle cell disease during vaso-occlusive pain

Blood ◽  
2020 ◽  
Vol 136 (12) ◽  
pp. 1402-1406 ◽  
Author(s):  
Claudia R. Morris ◽  
Lou Ann S. Brown ◽  
Michael Reynolds ◽  
Carlton D. Dampier ◽  
Peter A. Lane ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mitochondrial Function ◽  
Protein Carbonyls ◽  
Mitochondrial Activity ◽  
No Production ◽  
Loading Dose ◽  
Cell Disease

Abstract Altered mitochondrial function occurs in sickle cell disease (SCD), due in part to low nitric oxide (NO) bioavailability. Arginine, the substrate for NO production, becomes acutely deficient in SCD patients with vaso-occlusive pain episodes (VOE). To determine if arginine improves mitochondrial function, 12 children with SCD-VOE (13.6 ± 3 years; 67% male; 75% hemoglobin-SS) were randomized to 1 of 3 arginine doses: (1) 100 mg/kg IV 3 times/day (TID); (2) loading dose (200 mg/kg) then 100 mg/kg TID; or (3) loading dose (200 mg/kg) followed by continuous infusion (300 mg/kg per day) until discharge. Platelet-rich plasma mitochondrial activity, protein expression, and protein-carbonyls were measured from emergency department (ED) presentation vs discharge. All VOE subjects at ED presentation had significantly decreased complex-V activity compared to a steady-state cohort. Notably, complex-V activity was increased at discharge in subjects from all 3 arginine-dosing schemes; greatest increase occurred with a loading dose (P &lt; .001). Although complex-IV and citrate synthase activities were similar in VOE platelets vs steady state, enzyme activities were significantly increased in VOE subjects after arginine-loading dose treatment. Arginine also decreased protein-carbonyl levels across all treatment doses (P &lt; .01), suggesting a decrease in oxidative stress. Arginine therapy increases mitochondrial activity and reduces oxidative stress in children with SCD/VOE. This trial was registered at www.clinicaltrials.gov as #NCT02536170.

scholarly journals Oxidative Stress and Induction of Heme Oxygenase-1 in the Kidney in Sickle Cell Disease

2001 ◽  
Vol 158 (3) ◽  
pp. 893-903 ◽  
Author(s):  
Karl A. Nath ◽  
Joseph P. Grande ◽  
Jill J. Haggard ◽  
Anthony J. Croatt ◽  
Zvonimir S. Katusic ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Heme Oxygenase ◽  
Sickle Cell ◽  
Heme Oxygenase 1 ◽  
Cell Disease

Determinants Of Heme-Oxygenase-1 Upregulation In Patients With Sickle Cell Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2235-2235 ◽  
Author(s):  
Olufolake Adisa ◽  
Benjamin Yaw Owusu ◽  
Yijuan Hu ◽  
Samit Ghosh ◽  
Fang Tan ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Heme Oxygenase ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Dinucleotide Repeat ◽  
Acute Chest Syndrome ◽  
Heme Oxygenase 1 ◽  
Cell Disease ◽  
The Mean

Abstract Inflammation is a cardinal component of the pathogenesis of sickle cell disease (SCD). Increased plasma concentration of the inflammatory agonist hemin increases the odds of acute chest syndrome (ACS) in children with SCD (Adisa et al., Br. J Haematol, 2013). In addition, free hemin promotes the development of a lethal ACS-like disease in transgenic sickle mice (Ghosh et al., J Clin Invest, 2013). Hemin degradation is controlled by the rate-limiting enzyme heme oxygenase-1 (HO-1). Polymorphism of a (GT)n dinucleotide repeat in the HO-1 promoter, which enhances expression of the gene, is associated with lower rates of hospitalization for ACS in children. Over-expression of HO-1 reduces stasis in a mouse model of SCD vaso-occlusion. However, the role of plasma HO-1 in SCD patients is entirely unknown. In this study, we measured steady-state plasma HO-1 in two cohorts of patients. Cohort 1 in Atlanta (n=98) consisted of children with a mean age of 10.07±0.42 years (range 2-19 years) and cohort 2 from Accra (n=80) consisted of older patients (mean age 25.30±1.0 years, range 13-58 years). The mean plasma HO-1 of both cohorts was significantly higher compared to the mean value of age- and ethnic-matched individuals with normal adult Hb; Atlanta: 10.19±5.80 vs. 2.08± 1.16, p<0.0001 and Accra: 13.7±8.14 vs. 2.57± 0.82, p<0.0001. Plasma HO-1 varied by 25-fold in both cohorts and it correlated with the white blood cell count (Atlanta: r=0.3361, p<0.0001, Accra: r=0.25, p=0.02). Fifty-four percent (n=53) of subjects in the Atlanta cohort were on hydroxyurea. The mean plasma HO-1 of this subgroup was lower (8.1 ± 4.5) compared to the hydroxyurea naïve Accra cohort (p=<0.0001). Further studies of the Accra cohort revealed significant correlations between HO-1 and multiple markers of vascular inflammation; sICAM-1(r=0.2794, p=0.03, n=60), sE-selectin (r= 0.4209, p=0.0017, n=58) and sP-selectin (r=0.3855, p=0.0028, n=58). The number of the (GT)n dinucleotide in the HO-1 promoter ranged 17 to 45; the distribution was trimodal with peaks at 23, 30 and 41 repeats. The overwhelming majority of patients had medium and large size alleles that are generally hypo-response to induction. Plasma HO-1 level correlated with the length of the (GT)n dinucleotide repeat (p=0.003, n=80). In a multivariable regression model, WBC, sICAM-1, sE-selectin and sP-selectin accounted for 13.4% of the total variance of plasma HO-1 level, and the (GT)n polymorphism accounted for 9.8%. In conclusion, the concentration of plasma HO-1 is generally raised among SCD patients at steady-state. However, a large proportion of patients have a relatively modest level that is probably inadequate to counter the severity of inflammation typical of SCD, due in part to a hypo-responsive HO-1 promoter. Therapeutic strategies that complement induction of the endogenous HO-1 gene may be critical to ameliorate inflammation in SCD. Disclosures: No relevant conflicts of interest to declare.

Type-2 Phosphatidylserine (PS)-Positive Erythrocytes and Their Association with Markers of Hemolysis and Hemostatic Activation in Children with Sickle Cell Disease (SCD)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 943-943
Author(s):  
Yamaja B. Setty ◽  
Suhita Gayennebetal ◽  
Nigel S. Key ◽  
Marie Stuart
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Red Cells ◽  
Red Cell ◽  
Cell Disease ◽  
Activation Markers ◽  
D Dimer

Abstract Introduction: Type-2 phosphatidylserine (PS)-positive red cells are a subpopulation of erythrocytes that are highly positive for PS, contain low levels of fetal hemoglobin, are specific for sickle cell disease (SCD) and have been identified in the dense red cell fraction. Studies have implicated PS-positive red cells in enhancing anemia due to phagocytosis and hemolysis. Shielding of red cell PS by diannexin, a synthetic homodimer of human annexin-V, has been demonstrated to provide protection against hemolysis and prevent activation of prothrombinase. Methods: Using flow cytometry, we measured the levels of type-1 (red cells with low PS positivity) and type-2 PS-positive red cells in 50 children with SCD (31 with HbSS and 19 with HbSC), and assessed their association with various markers of hemolysis and hemostatic activation. Markers of hemolysis evaluated included plasma lactate dehydrogenase (LDH), reticulocyte count, and hemoglobin. Whole blood tissue factor (WBTF), pro-thrombin fragment F1+2, and D-dimer were evaluated as markers of hemostatic activation. Results: We demonstrate that the levels of type-2 PS-positive red cells are significantly increased in HbSS patients (1.37 ± 0.97%, p<0.01) compared to children with HbSC disease (0.32 ± 0.21%) and age- and race-matched controls (0.15 ± 0.15%, n=19). WBTF and D-dimer showed significant associations with both type-1 and -2 red cells with no significant differences in the strength of their association. However, significantly greater correlations were noted between type-2 PS red cells and hemolytic markers compared to those noted with type-1 (Steiger's Z=3.05 to 4.59, p<0.01). In addition our in vitro studies demonstrate increased osmotic fragility of these red cells. Table 1. Association of PS-positive RBCs with markers of hemolysis and hemostatic activation Biomarker Type-1 PS-positive RBCs Type-2 PS-positive RBCs Markers of Hemolysis LDH r = 0.44, p<0.002 r = 0.63, p<0.00001 % Reticulocyte r = 0.43, p=0.002 r = 0.66, p<0.00001 Hemoglobin r =-0.35, p=0.014 r =-0.63, p<0.00001 Markers of Hemostatic Activation WBTF r = 0.41, p=0.008 r = 0.56, p<0.0002 F1+2 r = 0.26, p=0.07 r = 0.31, p<0.03 D-dimer r = 0.46, p<0.001 r = 0.56, p<0.0005 Conclusions: Type-2 PS-positive red cells are elevated in SCD and the number of these cells correlates significantly with both markers of hemolysis and hemostasis. These findings provide a patho-physiologic link between the intravascular hemolytic milieu of SCD and the hemostatic perturbations previously noted in this disease. Disclosures No relevant conflicts of interest to declare.

Oral Arginine Increases Erythrocyte Glutathione Levels in Sickle Cell Disease: Implications for Pulmonary Hypertension.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1208-1208 ◽  
Author(s):  
Claudia R. Morris ◽  
Jung Suh ◽  
Elliott P. Vichinsky ◽  
Elizabeth S. Klings ◽  
Martin H. Steinberg ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pulmonary Hypertension ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Strong Predictor ◽  
No Production ◽  
Cell Disease ◽  
Plasma Hemoglobin ◽  
Before And After ◽  
Tandem Mass Spectrometric

Abstract Background: Pulmonary hypertension (PHT) is a strong predictor of mortality in sickle cell disease (SCD), and is associated with dysregulation of the arginine-nitric oxide (NO) pathway. This is in part the consequence of hemolysis, as erythrocyte release of plasma hemoglobin consumes NO, while simultaneous release of arginase consumes arginine, the substrate for NO production. The arginase-induced shift towards ornithine metabolism may then contribute to the proliferative changes in the lungs and vasculature associated with PHT through excess production of proline and polyamines. Glutathione (GSH) depletion may contribute to oxidative stress and pre-dispose sickle erythrocytes to hemolysis. We have found that erythrocyte GSH depletion is associated with severity of anemia and PHT measured by Doppler echocardiography (Morris C, Klings E, unpublished data). Kaul et al recently demonstrated that arginine supplementation in BERK mice markedly reduced hemolysis (&gt; 60% reduction in plasma hemoglobin), increased NO generation, and decreased COX-2 expression and PGE2 levels. Since short-term arginine therapy improves PHT in SCD, we hypothesize that oral arginine therapy may impact GSH, a key erythrocyte anti-oxidant. Methods: Total erythrocyte GSH was analyzed using a sensitive liquid chromatography coupled to tandem mass spectrometric technique before and after arginine therapy at 0.1 mg/kg three times daily for 1 month, followed by 0.2 mg/kg three times daily for two more months, in 8 SCD patients already on stable hydroxyurea therapy. Results: Erythrocyte GSH levels increased significantly in SCD patients after arginine therapy (Mean±SEM: 1222±150 vs. 1593± 144mM, p=0.03; Figure 1). Conclusion: Arginine therapy increases erythrocyte GSH levels, which may decrease oxidative stress and hemolysis. Therapies that attenuate hemolysis and oxidative stress will likely benefit PHT in SCD. Figure 1: Arginine Therapy Figure 1:. Arginine Therapy

Heme Oxygenase-1 Gene Therapy in a Murine Model of Sickle Cell Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1527-1527
Author(s):  
Belcher D. Belcher ◽  
Julie V. Vineyard ◽  
Carol M Bruzzone ◽  
Chunsheng Chen ◽  
Joan D Beckman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Gene Therapy ◽  
Sickle Cell Disease ◽  
Transposable Element ◽  
Heme Oxygenase ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Sleeping Beauty ◽  
Heme Oxygenase 1 ◽  
Cell Disease

Abstract Abstract 1527 Poster Board I-550 We previously demonstrated that increases in heme oxygenase-1 (HO-1) and its products, carbon monoxide and biliverdin, inhibit inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin promoter driven Sleeping Beauty transposase plasmid with a wild type rat hmox-1 gene (wt-HO-1) transposable element (289 amino acids) was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had 3-fold increases in HO-1 activity in their livers, similar to hemin-treated mice. mRNA transcription of the transposable element was confirmed by qRT-PCR RFLP analysis. HO-1 overexpressing mice had marked activation of the phospho-p38 MAPK and phospho-Akt proteins and decreased NF-kB p65 in liver nuclear extracts. Hypoxia-induced stasis, a characteristic of SCD but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice. None of these effects were seen in SCD mice injected with Sleeping Beauty containing a nonsense rat hmox-1 gene (ns-HO-1) that encodes for amino acids 1-241 with little or no enzymatic activity. Immunohistochemistry of HO-1 in livers demonstrated perinuclear HO-1 staining in both hepatocytes and sinusoidal Kupffer cells in wt- and ns-HO-1-treated mice. We conclude HO-1 gene therapy targeted to the liver is beneficial in SCD by activating cytoprotective signaling pathways and inhibiting vascular stasis at sites distal to transgene expression. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document