Xanthine Oxidase Drives Hemolysis and Vascular Malfunction in Sickle Cell Disease

Abstract

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.

Keywords: anemia, sickle cell; bone marrow; endothelium; febuxostat; hemolysis; xanthine oxidase.

Figure 1.. Febuxostat treatment inhibits XO activity in chimeric SCD mice.

XO activity was assessed in A) human sickle cell disease patients compared to healthy controls and B) chimeric AA control, AS sickle trait, and SS sickle mice. C) Experimental design. D) XO activity of plasma, liver, lung, and kidney after 10 weeks of febuxostat treatment. LC/MS-MS was used for purine metabolite analysis of E) hypoxanthine, F) xanthine, and G) urate. H) A CBA assay was used to measure plasma oxidant load. I) Febuxostat treatment did not alter body weight. Values are mean ± SEM using an unpaired Student’s t test unless otherwise noted. ⁺Values are mean ± SEM using an unpaired Student’s t test with Welch’s correction. ^$Values are mean ± SEM using a one-way ANOVA with Dunnett’s multiple comparisons test. ^#Values are mean ± SEM using a Mann-Whitney test. XO, xanthine oxidase; SCD, sickle cell disease; CBA, coumarin boronate acid; WT, wild type; febux, febuxostat.

Figure 2.. XO inhibition improves hematological parameters.

A) Experimental design. B-E) Complete blood counts shown as a delta change from 0 to 10 weeks of treatment. F) Flow cytometry was used to measure the delta change of reticulocyte percentage from 0 to 10 weeks of treatment. Values are mean ± SEM using an unpaired Student’s t test. WT, wild type; febux, febuxostat; RBCs, red blood cells.

Figure 3.. XO inhibition decreases hemolysis.

A) Experimental design. B) UV-visible spectral deconvolution was used to measure plasma cell free hemoglobin, a combination of oxyhemoglobin and methemoglobin, after 10 weeks of treatment. C) An ELISA was used to measure plasma haptoglobin concentration after 10 weeks of treatment. D) UV-visible spectral deconvolution was used to measure plasma cell free hemin concentration after 10 weeks of treatment. E) An ELISA was used to measure plasma hemopexin concentration after 10 weeks of treatment. Values are mean ± SEM using an unpaired Student’s t test unless otherwise noted. ⁺Values are mean ± SEM using an unpaired Student’s t test with Welch’s correction. ^#Values are mean ± SEM using a Mann-Whitney test. WT, wild type; febux, febuxostat; ELISA, enzyme-linked immunosorbent assay.

Figure 4.. XO inhibition decreases constriction across multiple vascular beds, but only improves dilation in pulmonary arteries.

A) Ex vivo wire myography was used to asses vasoreactivity of pulmonary, mesenteric, and TDA arteries. B) Pulmonary (n=8), C) mesenteric (n=12), and D) TDA (n=6) constriction was measured by normalizing to maximum KCl response. Values are mean ± SEM using an unpaired Student’s t test. An acetylcholine dose response was used to measure dilation of E) pulmonary, F) mesenteric, and G) TDA arteries. Values are mean ± SEM using a two-way ANOVA with Sidak’s multiple comparisons test. **P<0.01. TDA, thoracodorsal; KPSS; potassium physiological salt solution; PSS, physiological salt solution; febux, febuxostat; max, maximum; PGF, prostaglandin F2α; XO, xanthine oxidase.

Figure 5.. XO inhibition did not affect pulmonary pressure or cardiac function.

A) Experimental design. Closed chest right heart catheterization was used to measure B) RV maximum pressure and, C) heart rate after 10 weeks of treatment. D) RV weight and E) LV + septum weight was normalized to tibia length. Echocardiogram was used to asses cardiac function as a delta change from 0 to 10 weeks of febuxostat treatment: F) systolic volume, G) diastolic volume, H) stroke volume, and I) cardiac output. Values are mean ± SEM using an unpaired Student’s t test. WT, wild type, febux, febuxostat; RV, right ventricle; max, maximum; LV, left ventricle; XO, xanthine oxidase.

Figure 6.. Hepatocyte-specific XO knockout did not decrease hemolysis or alter pulmonary vasoreactivity.

A) Experimental design. B) HPLC was used to measure plasma, liver, lung, and kidney XO activity. Complete blood counts show the hematological parameters C) hematocrit, D) hemoglobin, E) RBCs, and F) platelets, as a delta change from 0 to 10 weeks post-engraftment. G) An ELISA was used to measure plasma haptoglobin concentration 10 weeks post-engraftment. H) UV-visible spectral deconvolution was used to measure plasma cell free hemoglobin, a combination of oxyhemoglobin and methemoglobin, 10 weeks post-engraftment. Values are mean ± SEM using an unpaired Student’s t test unless otherwise noted. +Values are mean ± SEM using an unpaired Student’s t test with Welch’s correction. #Values are mean ± SEM using a Mann-Whitney test. I) An acetylcholine dose response was used to measure dilation of pulmonary arteries 10 weeks post-engraftment (Xdh^fl/fl n=8, Xdh^−/− n=7). Values are mean ± SEM using a two-way ANOVA with Sidak’s multiple comparisons test. XDH, xanthine dehydrogenase; XO, xanthine oxidase; RBCs, red blood cells; max, maximum; HPLC, high performance liquid chromatography ELISA, enzyme linked immunosorbent assay.