Reaping of nitric oxide by sickle cell disease

Sickle cell disease (SCD) is the most common genetic disease among African Americans, with an 8% incidence of the trait among this population. This autosomal recessive disorder involves a single amino acid substitution in the beta subunit of hemoglobin, forming an abnormal protein (hemoglobin S) that after deoxygenation results in polymerization and the consequent characteristic sickle-like shape of the erythrocytes. The pathogenesis of this disease has classically been attributed to the effects of passage of the rigid malformed cells through the vasculature resulting in abnormal blood flow caused by physical trapping or increased adhesion of the sickled cell to the microvascular endothelium, commonly referred to as vaso-occlusive crisis. Perfusion abnormalities are well documented in the clinical literature, and there is good evidence that a complex array of inflammatory events are also involved, including increased levels of circulating cytokines, up-regulation of cellular adhesion-related receptors, and appearance of activated macrophages and leukocytes, which results in increased production of reactive oxygen species (ROS) (1). Recognition of the importance of these events has lead to the recent inclusion of SCD as a chronic inflammatory disease (2). It has been speculated that an important consequence of this sequence of events is a defect in the actions of nitric oxide (nitrogen monoxide, NO), which will further exacerbate the syndrome by means of an array of effects (1–3). In a recent issue of PNAS, a report from B. Freeman and collaborators at the Center for Free Radical Biology at the University of Alabama at Birmingham presents a surprising and important finding regarding the mechanism of scavenging of NO in SCD (4).

Aslan et al. begin by reporting that ROS formation is in fact not increased in sickle red cells.

Among the most important of the multiple biological actions of NO in the cardiovascular system are the stimulation of vasodilation and the inhibition of vascular cell adhesion and aggregation. Thus, under conditions of attenuated endothelial NO production, blood flow will be restricted because of constriction and also vascular coagulation. The finding that disturbances in the vascular circulation can cause dysfunction in endothelial function, specifically related to NO production, dates back to the very beginning of the endothelium-derived relaxing factor (EDRF)/NO field. For example, Lefer and Lefer (5) demonstrated attenuated EDRF formation induced by ischemia/reperfusion injury, and similar dysfunction has since been reported in other vascular pathologies, including atherosclerosis, sepsis, diabetes, and hypertension (6–8). Although the mechanistic basis for attenuated NO activity is complex, one major contributory factor is an increased inactivation of NO, and the main culprit seems to be superoxide anion (O). This ROS is produced in heightened levels under many conditions involving inflammation and disturbances in oxygen metabolism, and its reaction with NO is extremely rapid, near the diffusion limit (9). In addition to decreased NO formation, the product of this reaction, peroxynitrite, is a highly reactive oxidant and thus is capable of causing cell and tissue injury. There is evidence that in SCD vascular production of NO is elevated to maintain vasodilation, and Aslan et al. (4) now have delineated a major mechanism by which NO activity is attenuated in this disease.

One of the principal culprits in cell and tissue injury from ROS is the enzyme xanthine oxidoreductase (XOR), which normally catalyzes the oxidation of the purine xanthine with electron donation to NAD⁺. However, many pathological conditions cause increase in a form of the enzyme that donates electrons to oxygen, producing superoxide. This form is called xanthine oxidase (XO), and a multitude of work has identified XO as a major contributory factor to vascular dysfunction in a variety of pathological states. Previous work by Freeman and others has demonstrated that in addition to causing injury from O within a cell, during pathological conditions such as reperfusion injury, hepatitis, adult respiratory distress syndrome, and atherosclerosis, human plasma XO concentrations increase dramatically; this is most likely a result of the capacity for the splanchnic system and both normoxic and hypoxic vascular endothelial cells to express and release XO into the circulation (8). As characterized recently by the Freeman group (10), this circulating XO can bind to vascular cells and be taken up by them, impairing cell function and also blunting endothelium-driven NO-mediated formation of the second messenger cGMP within the smooth muscle cell, which is responsible for vascular relaxation. Thus, circulating XO contributes to the pathogenesis of a collection of diverse forms of vascular disease, and Aslan et al. (4) here add SCD to this list.

A hallmark of SCD is increased tissue rates of ROS production, and prior attention has been directed to the sickle red cell itself, where studies have claimed increased formation of O and other ROS, as well as lipid oxidation products, in these cells compared with normal. However, Aslan et al. (4) begin by reporting that ROS formation is in fact not increased in sickle red cells; perhaps more importantly, the rates of consumption of NO by normal and sickle erythrocytes are similar, directing attention elsewhere in the search for the source for ROS and for increased NO disappearance.

A major strength of the work by Aslan et al. (4) is that it utilizes both human patients with SCD and an animal model; the animal experimentation involves a knockout-transgenic mouse model, where the normal gene for murine hemoglobin is disrupted and the gene for hemoglobin S is expressed. Thus, in this model, the mice express hemoglobin S exclusively, and previous work has shown that many of the major features of human SCD are recapitulated in this murine model (11). In both human patients and the knockout-transgenic animals there is significantly increased plasma XO activity, accompanied by a decrease in liver XO (both protein and also activity) in the animals. In addition, increased XO protein and activity is present in the aortas of the mice with SCD, and perhaps most importantly increased O production, which is enhanced by addition of xanthine. Finally, NO-dependent vessel relaxation is severely impaired in the mice with SCD, which is significantly restored by treatment with a compound that removes O. The important conclusion is that a major source of the vascular dysfunction in SCD is the result of intraorgan (most notably the liver) injury that results in release of XO, which then avidly binds to vessel luminal cells, creating an oxidative milieu and consuming NO.

Evidence for defective NO action in patients with SCD includes reduced plasma levels of l-arginine (the substrate for enzymatic NO production) (12) and increased plasma concentrations of NO metabolites (13) (apparently caused by chronic activation to maintain dilation), a tendency for priapism (14), and altered vascular reactivity in response to endogenous angiotensin II (15). In addition, much attention has been directed to the possible beneficial effects of inhaled NO administration in patients with SCD (16, 17), and therapeutic benefits are observed in patients treated with hydroxyurea, a drug that not only induces fetal hemoglobin but also is metabolized to NO (18). The important insights provided by Aslan et al. (4) are sure to open up new and exciting possible avenues for therapeutic development and also provide a common link between this disease and a host of other pathologies involving inflammation- and oxidation-induced dysfunction in vascular NO activity.