allopurinol and Anemia--Sickle-Cell

Sickle cell disease is a class of hemoglobinopathy in humans, which is the most common inherited disease in the world. Although complications of sickle cell disease start from polymerization of red blood cells during its deoxygenating phase, the oxidative stress resulting from the biological processes associated with this disease (ischaemic and hypoxic injuries, hemolysis and inflammation) has been shown to contribute to its pathophysiology. It is widely known that chronic exercise reduces oxidative stress in healthy people, mainly via improvement of antioxidant enzyme efficiency. In addition, recent studies in other diseases, as well as in sickle cell trait carriers and in a mouse model of sickle cell disease, have shown that regular physical activity could decrease oxidative stress. The purpose of this review is to summarize the role of oxidative stress in sickle cell disease and the effects of acute and chronic exercise on the pro-oxidant/antioxidant balance in sickle cell trait and sickle cell disease.

Topics: Anemia, Sickle Cell; Animals; Disease Models, Animal; Exercise; Heme; Hemoglobin, Sickle; Hemolysis; Humans; Hypoxia; Iron; Mice; NADPH Oxidases; Nitric Oxide; Oxidative Stress; Reperfusion Injury; Sickle Cell Trait; Xanthine Oxidase

Endothelial dysfunction and impaired nitric oxide bioavailability have been implicated in the pathogenesis of sickle cell anemia. Nitric oxide is a diatomic gas with a role in vascular homeostasis. Hemoglobin polymerization resulting from the HbS mutation produces erythrocyte deformation and hemolysis. Free hemoglobin, released into plasma by hemolysis scavenges on nitric oxide, and leads to reduced nitric oxide bioavailability. Pulmonary hypertension is a known consequence of sickle cell anemia. It occurs in 30-40% of patients with sickle cell anemia, and is associated with increased mortality. Several studies have implicated intravascular hemolysis, and impaired nitric oxide bioavailability in the pathogenesis of pulmonary hypertension. In this review, we summarize the mechanisms of altered nitric oxide bioavailability in sickle cell anemia and its possible role in the pathogenesis of pulmonary hypertension.

Topics: Anemia, Sickle Cell; Animals; Endothelium, Vascular; Genetic Predisposition to Disease; Hemoglobins; Hemolysis; Humans; Hypertension, Pulmonary; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase; Oxidants; Oxidative Stress; Xanthine Oxidase

1. Sickle cell disease (SCD) is an inherited disorder of haemoglobin synthesis that is associated with significant morbidity and mortality due to sequelae of episodic vaso-occlusive events: pain crises and multiorgan damage. The microvascular responses to the initiation, progression and resolution of vaso-occlusive events are consistent with an inflammatory phenotype as suggested by activation of multiple cell types, an oxidatively stressed environment and endothelial cell dysfunction. 2. Decreased anti-oxidant defences in SCD patients and mice are accompanied by activation of enzymatic (NADPH oxidase, xanthine oxidase) and non-enzymatic (sickle haemoglobin auto-oxidation) sources of reactive oxygen species. The resultant oxidative stress leads to dysfunction/activation of arteriolar and venular endothelial cells, resulting in impaired vasomotor function and blood cell-endothelial cell adhesion. 3. Changes in substrate and cofactor availability for endothelial cell nitric oxide synthase may underlie reactive oxygen- and nitrogen-induced events that contribute to SCD-induced vasculopathy. 4. The emerging role of reactive oxygen and nitrogen species in the pathogenesis of SCD provides a platform for the development of novel agents to treat this painful and lethal disease.

Topics: Anemia, Sickle Cell; Animals; Antioxidants; Antisickling Agents; Endothelium, Vascular; Enzyme Inhibitors; Erythrocyte Aggregation; Hemoglobin, Sickle; Hemolysis; Humans; Hydroxyurea; Iron Chelating Agents; Leukocyte Rolling; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Platelet Adhesiveness; Reactive Nitrogen Species; Reactive Oxygen Species; Vasodilation; Xanthine Oxidase

Chronic hemolysis is a hallmark of sickle cell disease (SCD) and a driver of vasculopathy; however, the mechanisms contributing to hemolysis remain incompletely understood. Although XO (xanthine oxidase) activity has been shown to be elevated in SCD, its role remains unknown. XO binds endothelium and generates oxidants as a byproduct of hypoxanthine and xanthine catabolism. We hypothesized that XO inhibition decreases oxidant production leading to less hemolysis. Approach and Results: Wild-type mice were bone marrow transplanted with control (AA) or sickle (SS) Townes bone marrow. After 12 weeks, mice were treated with 10 mg/kg per day of febuxostat (Uloric), Food and Drug Administration-approved XO inhibitor, for 10 weeks. Hematologic analysis demonstrated increased hematocrit, cellular hemoglobin, and red blood cells, with no change in reticulocyte percentage. Significant decreases in cell-free hemoglobin and increases in haptoglobin suggest XO inhibition decreased hemolysis. Myographic studies demonstrated improved pulmonary vascular dilation and blunted constriction, indicating improved pulmonary vasoreactivity, whereas pulmonary pressure and cardiac function were unaffected. The role of hepatic XO in SCD was evaluated by bone marrow transplanting hepatocyte-specific XO knockout mice with SS Townes bone marrow. However, hepatocyte-specific XO knockout, which results in >50% diminution in circulating XO, did not affect hemolysis levels or vascular function, suggesting hepatocyte-derived elevation of circulating XO is not the driver of hemolysis in SCD.. Ten weeks of febuxostat treatment significantly decreased hemolysis and improved pulmonary vasoreactivity in a mouse model of SCD. Although hepatic XO accounts for >50% of circulating XO, it is not the source of XO driving hemolysis in SCD.

Topics: Anemia, Sickle Cell; Animals; Disease Models, Animal; Enzyme Inhibitors; Erythrocytes; Febuxostat; Hemodynamics; Hemolysis; Liver; Male; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Artery; Ventricular Function; Xanthine Oxidase

Proinflammatory high-density lipoprotein (p-HDL) is a biomarker of cardiovascular disease. Sickle cell disease (SCD) is characterized by chronic states of oxidative stress that many consider to play a role in forming p-HDL. To measure p-HDL, apolipoprotein (apo) B containing lipoproteins are precipitated. Supernatant HDL is incubated with an oxidant/LDL or an oxidant alone and rates of HDL oxidation monitored with dichlorofluorescein (DCFH). Although apoB precipitation is convenient for isolating HDL, the resulting supernatant matrix likely influences HDL oxidation. To determine effects of supernatants on p-HDL measurements we purified HDL from plasma from SCD subjects by anion exchange (AE) chromatography, determined its rate of oxidation relative to supernatant HDL. SCD decreased total cholesterol but not triglycerides or HDL and increased cell-free (cf) hemoglobin (Hb) and xanthine oxidase (XO). HDL isolated by AE-HPLC had lower p-HDL levels than HDL in supernatants after apoB precipitation. XO+xanthine (X) and cf Hb accelerated purified HDL oxidation. Although the plate and AE-HPLC assays both showed p-HDL directly correlated with cf-Hb in SCD plasma, the plate assay yielded p-HDL data that was influenced more by cf-Hb than AE-HPLC generated p-HDL data. The AE-HPLC p-HDL assay reduces the influence of the supernatants and shows that SCD increases p-HDL.

Topics: Anemia, Sickle Cell; Animals; Anion Exchange Resins; Case-Control Studies; Cell-Free System; Cholesterol; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Hemoglobins; Humans; Inflammation Mediators; Lipoproteins, HDL; Mice, Inbred C57BL; Online Systems; Oxidation-Reduction; Reference Standards; Triglycerides; Xanthine; Xanthine Oxidase

Although blood cell-endothelial cell adhesion and oxidative stress have been implicated in the pathogenesis of sickle cell disease (SCD), the nature of the linkage between these vascular responses in SCD remains unclear. The objective of this study was to determine whether superoxide derived from endothelial cell-associated NADPH oxidase mediates the leukocyte-endothelial (L/E) and platelet-endothelial cell (P/E) adhesion that is observed in the cerebral microvasculature of sickle cell transgenic (betaS) mice. Intravital fluorescence microscopy was used to monitor L/E and P/E adhesion in brain postcapillary venules of wild-type (WT), SOD1 transgenic (SOD1-TgN), and gp91phox (NADPH oxidase)-deficient mice that were transplanted with bone marrow from betaS mice. Hypoxia/reoxygenation (H/R) yielded intense P/E and L/E adhesion responses in cerebral venules of betaS/WT chimeras that were significantly attenuated in both betaS/SOD1-TgN, and betaS/gp91phox-/- chimeras. Pretreatment of betaS/WT chimeras with the iron-chelator desferroxamine blunted the blood cell-endothelial cell adhesion responses to H/R, whereas pretreatment with the xanthine oxidase inhibitor allopurinol had no effect. These findings suggest that superoxide derived from endothelial cell NADPH-oxidase and catalytically active iron contribute to the proinflammatory and prothrombogenic responses associated with sickle cell disease.

Topics: Allopurinol; Anemia, Sickle Cell; Animals; Bone Marrow Transplantation; Brain; Deferoxamine; Endothelial Cells; Gene Expression; Hemorheology; Humans; Inflammation; Iron; Iron Chelating Agents; Leukocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microcirculation; Microscopy, Fluorescence; NADPH Oxidases; Platelet Adhesiveness; Superoxide Dismutase; Thrombosis; Transplantation Chimera; Venules; Xanthine Oxidase

Topics: Anemia, Sickle Cell; Animals; Disease Models, Animal; Humans; Mice; Nitric Oxide; Reactive Oxygen Species; Xanthine Oxidase

Plasma xanthine oxidase (XO) activity was defined as a source of enhanced vascular superoxide (O(2)( *-)) and hydrogen peroxide (H(2)O(2)) production in both sickle cell disease (SCD) patients and knockout-transgenic SCD mice. There was a significant increase in the plasma XO activity of SCD patients that was similarly reflected in the SCD mouse model. Western blot and enzymatic analysis of liver tissue from SCD mice revealed decreased XO content. Hematoxylin and eosin staining of liver tissue of knockout-transgenic SCD mice indicated extensive hepatocellular injury that was accompanied by increased plasma content of the liver enzyme alanine aminotransferase. Immunocytochemical and enzymatic analysis of XO in thoracic aorta and liver tissue of SCD mice showed increased vessel wall and decreased liver XO, with XO concentrated on and in vascular luminal cells. Steady-state rates of vascular O(2)( *-) production, as indicated by coelenterazine chemiluminescence, were significantly increased, and nitric oxide (( *)NO)-dependent vasorelaxation of aortic ring segments was severely impaired in SCD mice, implying oxidative inactivation of ( *)NO. Pretreatment of aortic vessels with the superoxide dismutase mimetic manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin markedly decreased O(2)( small middle dot-) levels and significantly restored acetylcholine-dependent relaxation, whereas catalase had no effect. These data reveal that episodes of intrahepatic hypoxia-reoxygenation associated with SCD can induce the release of XO into the circulation from the liver. This circulating XO can then bind avidly to vessel luminal cells and impair vascular function by creating an oxidative milieu and catalytically consuming (*)NO via O(2)( small middle dot-)-dependent mechanisms.

Topics: Alanine Transaminase; Anemia, Sickle Cell; Animals; Endothelium, Vascular; Erythrocytes; Humans; In Vitro Techniques; Mice; Mice, Knockout; Muscle Relaxation; Nitric Oxide; Superoxides; Xanthine Oxidase

Reperfusion of tissues after interruption of their vascular supply causes free-radical generation that leads to tissue damage, a scenario referred to as "reperfusion injury." Because sickle disease involves repeated transient ischemic episodes, we sought evidence for excessive free-radical generation in sickle transgenic mice. Compared with normal mice, sickle mice at ambient air had a higher ethane excretion (marker of lipid peroxidation) and greater conversion of salicylic acid to 2,3-dihydroxybenzoic acid (marker of hydroxyl radical generation). During hypoxia (11% O(2)), only sickle mice converted tissue xanthine dehydrogenase to oxidase. Only the sickle mice exhibited a further increase in ethane excretion during restitution of normal oxygen tension after 2 hours of hypoxia. Only the sickle mice showed abnormal activation of nuclear factor-kappaB after exposure to hypoxia-reoxygenation. Allopurinol, a potential therapeutic agent, decreased ethane excretion in the sickle mice. Thus, sickle transgenic mice exhibit biochemical footprints consistent with excessive free-radical generation even at ambient air and following a transient induction of enhanced sickling. We suggest that reperfusion injury physiology may contribute to the evolution of the chronic organ damage characteristic of sickle cell disease. If so, novel therapeutic approaches might be of value.

Topics: Allopurinol; Anemia, Sickle Cell; Animals; Biomarkers; Enzyme Inhibitors; Ethane; Hemoglobin, Sickle; Humans; Hydroxybenzoates; Hydroxyl Radical; Kidney; Lipid Peroxidation; Liver; Mice; Mice, Inbred C57BL; Mice, Transgenic; NF-kappa B; Reference Values; Reperfusion Injury; Salicylic Acid; Xanthine Dehydrogenase; Xanthine Oxidase

1. Malondialdehyde (MDA) levels of erythrocyte membranes of sickle-cell disease patients were measured as an indicator of peroxidative damage and allopurinol was used as a lipid peroxide scavenger. 2. Incubating sickle-cell erythrocytes with allopurinol significantly reduced MDA levels of erythrocyte membranes compared with before-treatment values. 3. Allopurinol seems to reduce the peroxidative damage in sickle-cell erythrocytes, but the mechanism is still unknown.

Topics: Adult; Allopurinol; Anemia, Sickle Cell; Antioxidants; Blood Cell Count; Erythrocyte Membrane; Female; Humans; Lipid Peroxidation; Male; Malondialdehyde

Topics: Adolescent; Allopurinol; Anemia, Sickle Cell; Bone Marrow Examination; Carbon Radioisotopes; Electrophoresis, Disc; Electrophoresis, Starch Gel; Erythrocytes; Female; FIGLU Test; Folic Acid Deficiency; Hemoglobins; Hemoglobins, Abnormal; Humans; Leucine; Leukemia, Lymphoid; Methotrexate; Prednisone; Vincristine