In patients with sickle cell disease (SCD), exposure to hypoxia and deoxygenation of intra-erythrocyticHemoglobin S, or dehydration, results in hemoglobin polymerization, erythrocyte rigidity leading tomicrovascular occlusion, and end-organ damage. In addition, these individuals exhibit endothelialdysfunction that exists in the absence of overt atherosclerotic vascular disease. This vasculopathy ischaracterized by increased endothelial oxidant stress, decreased bioavailable nitric oxide (NO), andincreased expression of adhesion molecules. While this phenomenon has been attributed, in part, todecreased NO owing to hemolysis and sequestration of NO by cell-free hemoglobin, accumulatingevidence suggests that endothelial dysfunction in SCD may result from an aldosterone-mediateddecrease in glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD, the first and rate-limitingenzyme of the pentose phosphate pathway, is the principal source of NADPH, a reducing equivalentand cofactor for the endothelial isoform of nitric oxide synthase. As such, G6PD regulates endothelialredox state and NO production. In SCD, dehydration due to renal dysfunction is a commonphenomenon; (sub)acute or chronic dehydration activates the renin-aldosterone system and clinicalstudies have shown that SCD patients have elevated levels of renin and aldosterone. Furthermore,elevated levels of aldosterone have been shown to increase intracellular Ca2+ stores and, thereby,may lead to Gardos channel activation, dehydration of erythrocytes, and increased adhesion to theendothelium to augment endothelial dysfunction. Hyperaldosteronism has been associated withendothelial dysfunction and vascular inflammation in the absence of erythrocyte adhesion, and,recently, we have shown that mildly elevated levels of aldosterone cause an acquired form of G6PDdeficiency in cultured endothelial cells and in vivo, resulting in decreased G6PD expression and activityto increase oxidant stress, decrease bioavailable NO, and impair vascular reactivity. Although earlystudies reported no influence of G6PD deficiency on hemolysis in SCD patients, these studiesexamined individuals with the G6PD A- variant, who had only modest reductions in G6PD activity(Class III), and did not examine the consequences of concomitant genetic G6PD deficiency onendothelial function. We have found that an acquired G6PD deficiency, as occurs when aldosteronelevels are mildly elevated, is associated with >80% reduction in G6PD activity, akin to a Class Vdeficiency, and this degree of G6PD deficiency is associated with significant endothelial dysfunction. Asthe central theme of this proposal, we, therefore, hypothesize that endothelial dysfunction associatedwith SCD results from aldosterone-mediated acquired G6PD deficiency.To examine this hypothesis, we propose the following specific aims:1) Characterize the functional consequences of aldosterone-mediated acquired G6PD deficiency inSCD endothelial cells in vitro. 2) Determine the influence of aldosterone and decreased G6PD activity on erythrocyte-endothelial cellinteractions in SCD in vitro. 3) Evaluate the influence of aldosterone-mediated acquired G6PD deficiency on endothelial functionand vascular reactivity in vivo in a mouse model of SCD.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Specialized Center--Cooperative Agreements (U54)
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