Oxidative Stress and Vaso-Occlusion in Sickle Cell Disease: Role of Activated Leukocytes and Redox Active Iron.
Authors: Belcher John D. , Mahaseth Hemachandra , Welch Thomas E. , Boakye-Agyeman Felix , Hebbel Robert P. , Vercellotti Gregory M. 
Published: April 2020

Abstract
Sickle cell disease (SCD) is a disease of oxidative stress. We and others have demonstrated increased oxidative stress, inflammation, endothelial cell activation and white blood cell counts in human patients and transgenic murine models of SCD. Leukocytosis in SCD is associated with increases in the incidence of pain crisis, acute chest syndrome, stroke and mortality. We hypothesize that reactive oxygen species (ROS) derived from leukocytes and excess redox active iron promote vascular inflammation and vaso-occlusion. Leukocytes were activated in S+S-Antilles sickle mice compared to normal C57BL/6 control mice as measured by the percentage of leukocytes expressing CD11b on their surface in ambient air (25.4% vs. 19.3%, p&lt;0.05) and after exposure of mice to hypoxia-reoxygenation (31.7% vs. 23.0%, p&lt;0.05). In addition, resting leukocytes from S+S-Antilles mice produce 1.8-fold more H2O2 than normal mice (p&lt;0.05) as measured by Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) fluorescence. These leukocyte oxidants are especially toxic in the presence of excess redox active iron. Histopathology of the lungs and livers of 10 week old S+S-Antilles and BERK sickle mice showed red blood cell (RBC) congestion compared to normal. In addition, the sickle livers had multiple areas of infarction and inflammatory leukocyte infiltration. The heme contents of S+S-Antilles sickle lungs and livers were increased by 37- and 4.9-fold, respectively, compared to normals (p&lt;0.05 for both organs). Furthermore, there was significantly more chelatable iron that is potentially redox active as measured by Ferene-S in sickle lungs (21.0-fold, p&lt;0.05) and livers (2.4-fold, p&lt;0.05) compared to normals. Thus, these data demonstrate there is an explosive pro-oxidative environment in sickle mice. These excess oxidants lead to NF-kB activation, VCAM-1 and ICAM-1 expression, and increased oxidative injury, as seen histopathologically by nitro-tyrosine and dihydroethidium staining in organs. Hypoxia-reoxygenation, which induces RBC sickling and enhances ROS production in sickle mice, causes an increase in leukocyte rolling (4.4-fold, S+S-Antilles vs. normal, p&lt;0.05) and adhesion (6.5-fold, p&lt;0.05). Hypoxia-reoxygenation induces transient vaso-occlusion in 12% and 24% of the subcutaneous venules of S+S-Antilles and BERK mice respectively. No vessels become static in normal mice (p&lt;0.05 sickle vs. normal). Hypoxia-reoxygenation-induced vaso-occlusion can be inhibited by antibodies to P-selectin, VCAM-1 or ICAM-1. Furthermore, scavenging ROS with the SOD and catalase mimetic, polynitroxyl albumin or the iron chelator Trimidox, inhibited hypoxia-reoxygenation-induced vaso-occlusion (p&lt;0.05). We conclude that oxidative stress derived from activated leukocytes and excess redox active iron plays a critical role in promoting vaso-occlusion and organ injury in SCD. We speculate that iron chelators, leukocyte adhesion molecule blockade and anti-oxidants will modulate vaso-occlusion in patients with SCD.