In patients with sickle cell disease (SCD), exposure to hypoxia and deoxygenation of intra-erythrocytic Hemoglobin S, or dehydration, results in hemoglobin polymerization, erythrocyte rigidity leading to microvascular occlusion, and end-organ damage. In addition, these individuals exhibit endothelial dysfunction that exists in the absence of overt atherosclerotic vascular disease. This vasculopathy is characterized by increased endothelial oxidant stress, decreased bioavailable nitric oxide (NO), and increased expression of adhesion molecules. While this phenomenon has been attributed, in part, to decreased NO owing to hemolysis and sequestration of NO by cell-free hemoglobin, accumulating evidence suggests that endothelial dysfunction in SCD may result from an aldosterone-mediated decrease in glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD, the first and rate-limiting enzyme of the pentose phosphate pathway, is the principal source of NADPH, a reducing equivalent and cofactor for the endothelial isoform of nitric oxide synthase. As such, G6PD regulates endothelial redox state and NO production. In SCD, dehydration due to renal dysfunction is a common phenomenon;(sub)acute or chronic dehydration activates the renin-aldosterone system and clinical studies 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 the endothelium to augment endothelial dysfunction. Hyperaldosteronism has been associated with endothelial 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 G6PD deficiency in cultured endothelial cells and in vivo, resulting in decreased G6PD expression and activity to increase oxidant stress, decrease bioavailable NO, and impair vascular reactivity. Although early studies reported no influence of G6PD deficiency on hemolysis in SCD patients, these studies examined 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 on endothelial function. We have found that an acquired G6PD deficiency, as occurs when aldosterone levels are mildly elevated, is associated with >80% reduction in G6PD activity, akin to a Class V deficiency, and this degree of G6PD deficiency is associated with significant endothelial dysfunction. As the central theme of this proposal, we, therefore, hypothesize that endothelial dysfunction associated with 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 in SCD endothelial cells in vitro. 2) Determine the influence of aldosterone and decreased G6PD activity on erythrocyte-endothelial cell interactions in SCD in vitro. 3) Evaluate the influence of aldosterone-mediated acquired G6PD deficiency on endothelial function and vascular reactivity in vivo in a mouse model of SCD.
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