The Effect of a Selective Inhibitor of Phosphodiesterase‐9 on Oxidative Stress, Inflammation and Cytotoxicity in Neutrophils from Patients with Sickle Cell Anaemia

Abstract

The aim of the study was to investigate the possible anti-inflammatory and antioxidant effects of BAY 73-6691 on neutrophils from SCA patients. This study included 35 patients with a molecular diagnosis of SCA, whose neutrophils were isolated and treated with BAY 73-6691 at the concentrations 100, 10, 1.0 and 0.1 lg/mL. LDH release and MTT assays were performed to verify cell viability. To evaluate oxidative stress, the following parameters were determined by spectrophotometric assays: NO and malondialdehyde (MDA) levels and activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx). As inflammatory markers, myeloperoxidase (MPO) levels were evaluated by colorimetric assay and TNF-a by enzyme immunoassay. The results showed that neutrophils from SCA patients not treated with hydroxyurea (HU) had significantly lower NO levels and catalase and SOD activity, as well as significantly higher MDA, MPO and TNF-a levels when compared with neutrophils from SCA patients treated with HU and neutrophils from control group. Treatment of SCA neutrophils with BAY 73-6691 resulted in 94%, 200% and 168% increase in NOx levels, SOD and catalase activity, respectively. In addition, there was a reduction of approximately 46% and 45% in TNF-a and MPO levels, respectively. In SCAHU neutrophils, there was a 30% and 44% increase in NOx levels and SOD activity, respectively, and a 28% and 37% decrease in TNF-a and MPO levels, respectively. However, these effects were observed at cytotoxic doses only. The results of this study are original and demonstrate that inhibition of phosphodiesterase-9 in neutrophils from SCA patients with BAY 73-6691 was able to increase the NO bioavailability and attenuate oxidative stress and inflammation in neutrophils from patients not treated with HU.Sickle cell anaemia (SCA) is an inherited disease that results from a single mutation in the b-globin gene, leading to the production of unstable haemoglobin S (HbS) [1]. The haemoglobin polymerizes when deoxygenated and alters the normal biconcave-shaped erythrocyte to a sickle-shaped, unstable and rigid cell with a high expression of adhesion molecules [2]. Thus, SCA is characterized by intravascular haemolysis, chronic inflammation, vaso-occlusive events, endothelial damage and oxidative stress, which culminate in progressive microvascular damage in all organs [3][4][5].Leucocytes are now thought to play a fundamental role in the vaso-occlusive process, adhering to the endothelium and participating in reactive oxygen species (ROS) formation and inflammatory processes in the vasculature [6]. In addition, leucocytosis has been described as a risk factor for sickle crisis, stroke, silent cerebral infarction, acute chest syndrome and disease-related mortality [6][7][8]. Previous studies reported that in SCA individuals, leucocytes appear to exist in a chronic activated state in circulation, even in the basal state, and this activation causes the production of ROS in a NADPH oxidase-mediated respiratory burst [9,10].The reduced bi...

Increased oxidative stress alters nucleosides metabolite levels in sickle cell anemia

“…It is therefore useful as a defense agent against oxidative stress [67]. However, there are some discrepancies between studies on catalase levels in this disease, with some studies observing increased activity [68] and others reporting decreased catalase activity [69,70]. Our results showed an increase in catalase activity in SCA patients.…”

Neutrophil extracellular trap regulators in sickle cell disease: Modulation of gene expression of PADI4, neutrophil elastase, and myeloperoxidase during vaso‐occlusive crisis

“…Interestingly, levels of these complexes were positively correlated with nucleosome levels. 16 In addition, both the expression 27,28 and the activity 29 of MPO, whose upregulation is crucial to the oxidative burst of neutrophils during inflammation, have been described in SCD. Accordingly, our results provide additional evidence of neutrophil activation in acute VOC in SCD.…”

Influence of Oxidative Stress Biomarkers and Genetic Polymorphisms on the Clinical Severity of Hydroxyurea-Free Senegalese Children with Sickle Cell Anemia

“…As a consequence, the decreased amount of free circulating hemoglobin and heme would reduce oxidative stress reactions and the production of ROS. It might also explain the decreased MPO activity in SCA children with alpha-thalassemia since free heme has been reported to be a strong stimulator of neutrophils activation [ 48 ]. Overall, alpha-thalassemia would act by lowering the oxidative burst input and not by stimulating antioxidant defenses.…”