Hemolytic diseases are associated with elevated levels of circulating free heme that can mediate endothelial dysfunction directly via redox reactions with biomolecules or indirectly by upregulating enzymatic sources of reactive species. A key enzymatic source of these reactive species is the purine catabolizing enzyme, xanthine oxidase (XO) as the oxidation of hypoxanthine to xanthine and subsequent oxidation of xanthine to uric acid generates superoxide (O2•-) and hydrogen peroxide (H2O2). While XO has been studied for over 120 years, much remains unknown regarding specific mechanistic roles for this enzyme in pathologic processes. This gap in knowledge stems from several interrelated issues including: 1) lethality of global XO deletion and the absence of tissue-specific XO knockout models have coalesced to relegate proof-of-principle experimentation to pharmacology; 2) XO is mobile and thus when upregulated locally can be secreted into the circulation and impact distal vascular beds by high-affinity association to the glycocalyx on the endothelium; and 3) endothelial-bound XO is significantly resistant (> 50%) to inhibition by allopurinol, the principle compound used for XO inhibition in the clinic as well as the laboratory. While it is known that circulating XO is elevated in hemolytic diseases including sickle cell, malaria and sepsis, little is understood regarding its role in these pathologies. As such, the aim of this review is to define our current understanding regarding the effect of hemolysis (free heme) on circulating XO levels as well as the subsequent impact of XO-derived oxidants in hemolytic disease processes.

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Objective: Chronic hemolysis is a hallmark of sickle cell disease (SCD) and a driver of vasculopathy; however, the mechanisms contributing to hemolysis remain incompletely understood. Although XO (xanthine oxidase) activity has been shown to be elevated in SCD, its role remains unknown. XO binds endothelium and generates oxidants as a byproduct of hypoxanthine and xanthine catabolism. We hypothesized that XO inhibition decreases oxidant production leading to less hemolysis. Approach and Results: Wild-type mice were bone marrow transplanted with control (AA) or sickle (SS) Townes bone marrow. After 12 weeks, mice were treated with 10 mg/kg per day of febuxostat (Uloric), Food and Drug Administration–approved XO inhibitor, for 10 weeks. Hematologic analysis demonstrated increased hematocrit, cellular hemoglobin, and red blood cells, with no change in reticulocyte percentage. Significant decreases in cell-free hemoglobin and increases in haptoglobin suggest XO inhibition decreased hemolysis. Myographic studies demonstrated improved pulmonary vascular dilation and blunted constriction, indicating improved pulmonary vasoreactivity, whereas pulmonary pressure and cardiac function were unaffected. The role of hepatic XO in SCD was evaluated by bone marrow transplanting hepatocyte-specific XO knockout mice with SS Townes bone marrow. However, hepatocyte-specific XO knockout, which results in >50% diminution in circulating XO, did not affect hemolysis levels or vascular function, suggesting hepatocyte-derived elevation of circulating XO is not the driver of hemolysis in SCD. Conclusions: Ten weeks of febuxostat treatment significantly decreased hemolysis and improved pulmonary vasoreactivity in a mouse model of SCD. Although hepatic XO accounts for >50% of circulating XO, it is not the source of XO driving hemolysis in SCD.

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