Role of RBC Reactive Oxygen Species and Their Vicious Cycle in Sickle Vasculopathy Abstract: In sickle cell disease (SCD), red blood cells (RBCs) play an active role in vasoocclusion, largely through adhesion to endothelium and activation of leukocytes. While it is established that excessive production of reactive oxygen species (ROS) in sickle RBCs leads to oxidative damage and accelerated hemolysis, the contribution of RBC ROS to RBC adhesion and leukocyte activation remains unclear. Here, we provide evidence for a novel pathogenic mechanism of sickle RBC ROS that promotes adhesion and vasoocclusion in SCD. We show that NADPH oxidase (Nox)-dependent ROS production in sickle RBCs mediates adhesion to the vascular endothelium. This ROS production is regulated by MEK/ERK and G-protein coupled-receptor kinase 2 (GRK2), a mechanism that can itself be stimulated by inflammatory cytokines and external ROS. Consequently, sickle RBC ROS form a positive feedback loop with MEK/ERK and GRK2. These findings suggest that ROS, MEK/ERK and GRK2 create an intracellular cycle regulating sickle RBC-induced vasoocclusion. We also have implicated small nucleolar RNAs (snoRNAs) as regulators of RBC ROS. Specifically, these unusual regulators of cellular ROS are present in sickle RBCs at high levels that correspond to elevated ROS levels and RBC adhesion. We hypothesize that this deleterious cycle in sickle RBCs acts to promote vasoocclusion and drive SCD pathophysiology. We further hypothesize that disrupting components of this cycle can prevent vasoocclusion. Accordingly, we have recently shown that blocking MEK/ERK with MEK inhibitors reduces vasoocclusion in SCD mouse models. We also now demonstrate that scavenging ROS in sickle RBCs ex vivo suppresses ERK activation and sickle RBC-induced vasoocclusion in vivo. Additionally, we show that non-coding snoRNAs regulate pathologic RBC ROS production, since silencing these snoRNAs in mice reduces ROS levels in normal and sickle RBCs. We propose to further validate dynamic contributions of the RBC ROS cycle to vasoocclusive events and SCD pathophysiology, with the following Specific Aims. In SA1, we will show that the RBC ROS cycle is critical in controlling the cellular processes of vasoocclusion, and that disrupting this loop in vivo by scavenging ROS prevents vasoocclusion; in SA2, we will test the hypothesis that sickle RBC GRK2 is a druggable target to suppress this vicious cycle leading to cell activation; and in SA3, we will determine the role of the non-coding snoRNAs in ROS-stimulated sickle RBC pathology. Our long-term goal is to identify remediable sickle RBC abnormalities to reduce oxidative damage and vasoocclusion. We strongly believe that our studies will not only provide novel insights into the exact mechanisms of vasoocclusion and SCD pathophysiology, but will also lay the foundation for more rational approaches to therapies that better prevent oxidative damage and vasoocclusion in SCD.
Relevance Sickle cell disease (SCD) affects approximately 100,000 individuals in the USA and several million worldwide. Development of new effective therapies to curtail painful vascular occlusion is critical to reducing the disease's morbidity. We propose to test the hypothesis, supported by our preliminary data that reactive oxygen species create a vicious loop in sickle red cells. This loop is required for cell adherence and vessel obstruction, and disrupting this loop in sickle red cells pharmacologically or genetically may reduce the events of vessel occlusion.