Antioxidants Inhibit Sickle Cell Adhesion to Activated Endothelium in an Ex Vivo Preparation.
Authors: Kaul Dhananjay K. , Liu Xiao-du , Zhang Xiaoqin , Ma Li , Nagel Ronald L. 
Published: October 2019

Abstract
In sickle cell anemia (SS), intravascular sickling and attendant transient occlusive events are implicated in the generation of reactive oxygen species (ROS) and vascular endothelial activation. Oxidants (as well as cytokines) result in up-regulation of endothelial adhesion molecules that may lead to increased adhesion of sickle red blood cells (SS RBC) and contribute to vaso-occlusion. We hypothesize that endothelial activation will augment the expression of adhesion molecules involved in SS RBC-endothelium interaction, and that antioxidants will have an inhibitory effect on this interaction. To this end, we have tested selected antioxidants for their efficacy to inhibit SS RBC adhesion in the ex vivo mesocecum vasculature of the rat stimulated by platelet-activating factor (PAF). PAF, a potent inflammatory agent, results in endothelial ROS generation (Suematsu et al. Am. J. Physiol. 1993), as well as in increased adhesion of SS RBCs (Kaul et al. Blood, 2000). Here, we have tested the efficacy of superoxide dismutase (SOD), catalase and polynitroxylated albumin (PNA). While SOD and catalase remove superoxide (O2 ·−) and H2O2, respectively, nitroxides covalently attached to albumin (i.e., PNA) act as intra-vascular SOD mimetic and also facilitate heme-mediated catalytic removal of H2O2. To directly demonstrate the involvement of ROS (O2 •- and H2O2), the ex vivo vasculature was pre-treated with PAF (200 pg/ml in Ringer-albumin solution) followed by infusion with SOD (1500 U in 5 ml Ringer-albumin) or catalase (10,000 U in 5 ml) (each 15-min incubation). The control preparations were incubated with PAF and Ringer-albumin. For experiments involving PNA, the vasculature was first treated with PAF and followed by infusion of PNA or control human serum albumin (HSA) (each 33 mg/ml) diluted in Ringer-albumin solution (incubation 30 min). In PAF or PAF/HSA-treated preparations, infusion of SS RBCs (Hct 30 in Ringer-albumin) caused widespread adhesion of these cells exclusively in postcapillary venules. The adhesion was inversely correlated with vessel diameter, resulting in frequent blockage of small-diameter venules. In contrast, preparations treated with PAF/SOD or PAF/catalase showed a marked decrease in adhesion in venules of all diameters resulting in a significantly lower Y-intercept of the regression line as compared with the control group (p<0.00001). On the other hand, PNA caused almost complete inhibition of SS RBC adhesion in PAF-treated preparation, demonstrating even a greater anti-adhesive efficacy as evidenced by lower Y-intercept (p<0.001–0.0001 vs. SOD and catalase). The decrease in adhesion was accompanied by a significantly lower peripheral resistance (p<0.03–0.018). These results show that antioxidants inhibit SS RBC adhesion to endothelium stimulated by PAF, probably by scavenging and/or blocking PAF-generated oxidants. Thus, blockade of oxidant generation may constitute an effective therapeutic strategy to prevent SS RBC adhesion.