The proverbial chicken or the egg? Dissection of the role of cell-free hemoglobin versus reactive oxygen species in sickle cell pathophysiology

Abstract

SICKLE CELL PATHOPHYSIOLOGY comprises a complex interplay of episodic vasoocclusive events, ischemia-reperfusion injury, overproduction of reactive oxygen species (ROS), inflammation, endothelial activation, and hemolysis, all somehow driven by a single amino acid substitution in the ␤-globin chain of hemoglobin. Hemolysis and oxidative stress act synergistically to promote vascular dysfunction in sickle cell disease (SCD). As a result of chronic hemolysis, levels of free plasma hemoglobin are increased at baseline and nitric oxide (NO) bioavailability is diminished, producing endothelial dysfunction that has been linked to chronic vasculopathic complications of SCD such as pulmonary hypertension, cutaneous leg ulceration, priapism, and sudden death (14,18,19,22,23,25).NO regulates vasorelaxation and also possesses antioxidant, antiadhesive, and antithrombotic properties (33). NO is produced from the substrate L-arginine by endothelial nitric oxide synthase (eNOS) and mediates vasorelaxation through a paracrine action on vascular smooth muscle cells underlying the endothelium. Endothelial dysfunction, characterized by impaired vascular responsiveness resulting from decreased NO bioavailability, is associated with atherosclerosis, diabetes mellitus, hypertension, hypercholesterolemia, smoking, and obesity, illustrating the central importance of NO in the physiological regulation of vasomotor activity (3, 6).Unlike coronary artery disease and its risk factors, which are associated with an impaired production of NO, SCD and other hemolytic diseases are characterized by a primary resistance to the action of NO (10,13,25,29). The NO resistance state observed in SCD is multifaceted, with at least two major mechanisms contributing to impaired NO homeostasis: 1) scavenging of NO by cell-free plasma hemoglobin, and 2) oxidant stress due to the generation of ROS by both enzymatic and nonenzymatic pathways (11,13,19,25). Hemolysis "unpackages" the red blood cell (RBC), releasing free hemoglobin into the plasma. No longer compartmentalized by the intact cell membrane, cellfree plasma hemoglobin rapidly reacts with and scavenges endothelial NO. Hemolysis further impairs NO bioavailability through the release of arginase from the RBC, which competes with NO synthase (NOS) for the substrate arginine. Arginase I levels and activity correlate with measures of intravascular hemolysis in patients with SCD, and notably the lowest ratios of arginine to ornithine are associated with pulmonary hypertension and prospective mortality (19,20). The depletion of arginine leads to the functional uncoupling of NOS, whereby superoxide is preferentially formed over NO, amplifying the conditions of oxidant stress (33).In their study, Kaul and colleagues (16) examine the mechanism of sickle cell vasculopathy in a transgenic mouse model of severe SCD and find robust correlations between in vivo NO resistance, measured by vasodilatory response to topical application of the NO donor sodium nitroprusside (SNP), hemolytic rate, and ROS generation. The...

Antisickling property of fetal hemoglobin enhances nitric oxide bioavailability and ameliorates organ oxidative stress in transgenic-knockout sickle mice

“…HbS polymerization and sickling) lead to increased organ oxidative stress. Oxidative damage in SCD and mouse models of SCD has been attributed to oxygen free radicals resulting from reperfusion injury and heme-mediated peroxidase chemistry (22,26,32,54). BERK mice, expressing exclusively human ␣-and ␤ S -globins, show maximal intravascular sickling that can intermittently disrupt blood flow to the tissues and result in reperfusion injury and oxidant generation.…”

Heme Degradation and Vascular Injury

“…Cell-free plasma hemoglobin in the ferrous (Fe 2þ ) valence state, is readily available to participate in Fenton-based redox reactions. In addition, hemolysis further impairs NO bioavailability through the release of arginase from the erythrocyte, which competes with NO synthase (NOS) for the substrate arginine (75). Combined, these factors contribute to the overall reduced bioavailability of NO in hemolytic diseases such as sickle cell disease, which contributes to pulmonary hypertension and other comorbidities (69).…”

Pulmonary hypertension and nitric oxide depletion in sickle cell disease

“…The high rate of red cell destruction in sickle cell patients releases erythrocyte arginase into the plasma possibly lowering levels of arginine, 14 the substrate for NO synthase. An alternate mechanism by which hemolysis might deplete NO has been proposed and vigorously promoted in multiple review articles/opinion pieces [15][16][17][18][19][20][21][22] and educational programs. 23,24 These reports advance the hypothesis that depletion of NO by elevated levels of free hemoglobin in the plasma is an important determinant of the clinical manifestations of SCD, particularly pulmonary hypertension which has been deemed a critical risk factor, greatly limiting life expectancy.…”