Sickle cell disease subjects and mouse models have elevated nitrite and cGMP levels in blood compartments

Highlights

• Sickle cell disease (SCD) is thought to be associated with nitric oxide (NO) deficit.

• SCD subjects and SCD mouse models have elevated blood nitrite and cGMP levels.

• Nitrite supplementation increases nitrite levels but does not change pain phenotype in mice.

• SCD mice have increased bleeding time, which are decreased with NO scavengers.

• These findings indicate that clinical trials seeking to increase nitrite in SCD should to be reconsidered.

Abstract

The hypothesis of decreased nitric oxide (NO) bioavailability in sickle cell disease (SCD) proposes that multiple factors leading to decreased NO production and increased consumption contributes to vaso-occlusion, pulmonary hypertension, and pain. The anion nitrite is central to NO physiology as it is an end product of NO metabolism and serves as a reservoir for NO formation. However, there is little data on nitrite levels in SCD patients and its relationship to pain phenotype. We measured nitrite in SCD subjects and examined its relationship to SCD pain. In SCD subjects, median whole blood, red blood cell and plasma nitrite levels were higher than in controls, and were not associated with pain burden. Similarly, Townes and BERK homozygous SCD mice had elevated blood nitrite. Additionally, in red blood cells and plasma from SCD subjects and in blood and kidney from Townes homozygous mice, levels of cyclic guanosine monophosphate (cGMP) were higher compared to controls. In vitro, hemoglobin concentration, rather than sickle hemoglobin, was responsible for nitrite metabolism rate. In vivo, inhibition of NO synthases and xanthine oxidoreductase decreased nitrite levels in homozygotes but not in control mice. Long-term nitrite treatment in SCD mice further elevated blood nitrite and cGMP, worsened anemia, decreased platelets, and did not change pain response. These data suggest that SCD in humans and animals is associated with increased nitrite/NO availability, which is unrelated to pain phenotype. These findings might explain why multiple clinical trials aimed at increasing NO availability in SCD patients failed to improve pain outcomes.

Introduction

Pre-clinical studies have generated the hypothesis that hemolytic anemias including sickle cell disease (SCD) are associated with decreased nitric oxide (NO) bioavailability [[1], [2], [3]]. In SCD, according to this hypothesis, repeated cycles of sickle hemoglobin polymerization, hemolysis, and release of free hemoglobin and arginase in plasma contribute to increased consumption and decreased production of NO. Free hemoglobin reacting with and scavenging NO [4,5] together with arginase competing with NO synthases (NOS) for their substrate, arginine, limit NO production by the endothelium [1,3]. In turn, NO depletion in the circulation, possibly by inducing endothelial injury, leukocyte adhesion, platelet activation and production of oxygen free radicals, would induce vasoconstriction and contribute to vaso-occlusion, the hallmark of SCD [1,3,[6], [7], [8], [9]]. However, conclusive evidence that NO bioavailability is decreased in SCD is lacking and the hypothesis that a NO deficit contributes to SCD pathobiology has been challenged [10,11]. In fact, results of clinical trials of therapeutic interventions aimed at increasing NO bioavailability (arginine administration, inhaled NO or administration of the phosphodiesterase-5 inhibitor sildenafil) have failed to provide reproducible clinical improvements in cardiovascular and pulmonary performances and SCD-related pain. Therefore these clinical trial findings, question the validity of the NO-deficit hypothesis in SCD [12].

Amid this controversy, some argue that rather than using strategies aimed at acute or chronic administration of agents that increase NO availability/activity, a better alternative would be nitrite supplementation [13,14]. Nitrite can serve as a storage pool for NO activity as it can be reduced into NO by deoxyhemoglobin, xanthine oxidoreductase (XOR), and NO synthase (NOS), thus enabling the delivery of NO and increased NO activity directly to hypoxic areas [[15], [16], [17], [18], [19]]. In humans, nitrite-generated NO has been shown to improve blood flow, especially under hypoxic conditions [16,18,20] and in subjects with SCD, intravascular nitrite infusions were shown to improve regional blood flow without complications [21]. In humanized SCD mice, nitrite supplementation was shown to decrease hemolysis and inflammation-induced leukocyte and platelet adhesion [13] and to improve grip force, a marker for muscle hyperalgesia [22]. Importantly, these reported anti-adhesion and vasodilatory properties under physiological hypoxic conditions are thought to be desirable in SCD.

In SCD patients, acute pain crises are the main reason for emergency room visits and hospitalizations [[23], [24], [25]]. While the triggers and mechanisms underlying SCD acute pain crises are incompletely understood, many believe that these crises result from tissue ischemia and reperfusion caused by vaso-occlusion and obstruction of the microvascular bed [26]. Therefore, the putative NO bioavailability deficit in SCD could be a contributing factor for vaso-occlusion, tissue ischemia, and pain. In turn, therapeutic strategies capable of increasing vascular NO levels in hypoxic areas could have a role in preventing and/or treating SCD pain crises. Here we investigated the association between nitrite, NO availability, and pain phenotype in SCD subjects and humanized SCD mouse models [27,28].