The excessive release of heme during hemolysis contributes to the severity of sickle cell anemia (SCA) by exacerbating hemoglobin S (HbS) autoxidation, inflammation and systemic tissue damage. The present study investigated the effect of hydroxyurea (HU) on free radical neutralization and its stimulation of antioxidant genes in human peripheral blood mononuclear cells (PBMC) and human umbilical vein endothelial cells (HUVEC) in the presence or absence of hemin. HU (100 and 200 µM) significantly reduced the production of intracellular reactive oxygen species (ROS) induced by hemin at 70 µM in HUVEC. HUVECs treated with HU+hemin presented significant increases in nitric oxide (NO) production in culture supernatants. HU alone or in combination with hemin promoted the induction of superoxide dismutase-1 (SOD1) and glutathione disulfide-reductase (GSR) in HUVECs and PBMCs, and glutathione peroxidase (GPX1) in PBMCs. Microarray analysis performed in HUVECs indicated that HU induces increased expression of genes involved in the antioxidant response system: SOD2, GSR, microsomal glutathione S-transferase (MGST1), glutathione S-transferase mu 2 (GSTM2), carbonyl reductase 1 (CBR1) and klotho B (KLB). Significant increases in expression were observed in genes with kinase activity: protein kinase C beta (PRKCB), zeta (PRKCZ) and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta (PIK3C2B). HU also induced a significant increase in expression of the gene p62/sequestosome (p62/SQSTM1) and a significant decrease in the expression of the transcriptional factor BACH1 in HUVECs. Upstream analysis predicted the activation of Jun, miR-155-5p and mir-141-3p. These results suggest that HU directly scavenges free radicals and induces the expression of antioxidant genes via induction of the Nrf2 signaling pathway.

Introduction: The pathophysiology of sickle cell anemia (HF) is characterized by hemolytic and intermittent vasoconstrictive events with increased redox status in the vascular microenvironment that favors the chronic inflammation. Objectives: To investigate whether hydroxyurea (HU) acts in the inhibition/minimization of reactive oxygen/nitrogen species (ROS/RNS) and in the modulation of in vitro the antioxidant genes expression. Methods: DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay was performed to determine the antioxidant activity of HU using L-ascorbate (L-Asc) and butylated hydroxytoluene (BHT) as antioxidant controls. Human umbilical vein endothelial cell (HUVEC) and peripheral blood mononuclear cells (PBMC) were challenged with HU in the presence or absence of hemin for evaluation the cytotoxicity, inhibition of superoxide anions, nitrate/nitrite production and expression of antioxidant enzymes SOD1 (superoxide dismutase-1); GPx (glutathione peroxidase); GSR (glutathione-disulfide reductase); and HMOX1 (heme oxygenase-1 by RT-qPCR. Microarray analyses were performed on HUVEC stimulated with HU. Results: HU showed scavenging activity (similar to BHT) at concentrations equivalent to that found in the plasma of patients taking the drug (~200 μM). Treatments with HU alone or in combination with hemin did not induce toxicity in PBMC and HUVEC. HU decreases the accumulation of superoxide anions in PBMC in the presence or absence of hemin and the combined treatment of HU with hemin stimulated nitrate/nitrite production in PBMC. HU increases expression of SOD1 and GPx in PBMC and HUVEC. The increase in GSR expression was observed in PBMC and HUVEC submitted to the combined treatment of HU with hemin. HU did not induce HMOX1 expression and did not decrease its expression in combination with hemin in both, PBMC and HUVEC (Figure 1). The microarray assay showed that HU induces the expression of cellular antioxidant components, such as SOD2, GSR, GST1 (glutathione S-transferase-1, GSTM2 (glutathione S-transferase mu 2), MGST1 (microsomal glutathione S-transferase 1), CBR1 (carbonyl reductase 1), protein kinases phosphatidylinositol 3-phosphate C (PRKCB, PRKCZ, PIK3C2B) and p62/SQSTM1 (sequestosome 1) (Table 1). In contrast, a decrease in BACH1 (BTB domain and CNC homolog 1) transcriptional factor expression was observed. Upstream analysis demonstrated prediction of activation for the transcriptional factor JUN and miR-155-5p. Conclusion: The findings indicate that HU can act (i) directly inhibiting on the direct inhibition of ROS/RNS; (ii) inducing nitrite/nitrate production in the presence of hemin; (iii) besides stimulating the antioxidant cellular response by inducing the antioxidant/electrophile response element (ARE/EpRE) mediated by Nrf2 under p62/SQSTM1 regulation. Disclosures No relevant conflicts of interest to declare.

BackgroundStroke is one of the highest complications of sickle-cell anemia (SCA). The Transcranial Doppler (TCD) has been adopted worldwide as a gold standard method for detecting alterations in the blood velocity in cerebral arteries. In this study, we investigated the association between laboratory parameters and increased cerebral blood flow velocity in Brazilian SCA pediatric patients.MethodsThe study included 159 pediatric patients with SCA, submitted to TCD velocity screening, and the time-averaged maximum mean velocity (TAMMV) was determined in the middle cerebral artery (MCA), anterior cerebral artery (ACA), and distal intracranial internal carotid artery (ICA). We compared cerebral blood flow in patients stratified by the following: TCD1—defined as normal, with TAMMV inferior to 170 cm/s; TCD2—conditional, with TAMMV above 170 cm/s, but less than 199 cm/s; TCD3—altered, with TAMMV greater than or equal to 200 cm/s.ResultsTAMMV was negatively correlated with age and weight (p < 0.05). Moreover, TAMMV was associated or correlated with reductions in HbF, RBC, hemoglobin, hematocrit, HDL, and haptoglobin and, increases in MCV, MCH, RDW, reticulocytes, WBC, lymphocytes, monocytes, eosinophils, total and indirect bilirubin, LDH, AST, ALT, glucose, ferritin, and AAT (p < 0.05).ConclusionThe current study highlights the importance of the investigation of hemolytic and inflammatory biomarkers for monitoring the clinical outcome of SCA pediatric patients, to avoid acute or chronic stroke. Moreover, glucose and HDL-C appear useful for predicting higher TAMMV.