Purified XO, XO bound to heparin-Sepharose 6B and XO sequestered by endothelial cell glycosaminoglycans (GAGs) was used to examine reductase activity as assessed by several detection platforms including enhanced chemiluminescence (NO), EPR spin trapping and amperometric O2 detection. Male C57Blk/j6 mice were subjected to a HFD (60 % calories derived from fat) for 20 weeks and treated (drinking water) with the XO-specific inhibitor febuxostat (0.5 mg/L) or NaNO2 (100 mg/L) or both for the final 6 weeks. Age-matched controls were maintained on a standard rodent chow diet consisting of 15 % of adjusted calories from fat. After 20 weeks on diets mice were analyzed for right ventricular (RV) pressure volume (PV) relationship using a PV conductance catheter under steady state conditions.
Whereas reductase activity of XO free in solution is only operative under anoxia, sequestration of XO on endothelial cell GAGs confers the capacity for substantive NO formation in the presence of O2 (1-2 % or ¡«13-26 ¦ÌM) with concomitant diminution of XO-derived ROS production (¡ý40 % ). Treatment with febuxostat: reduced fasting blood glucose (223 vs. 181 mg/dL), improved (39 % ) impaired glucose tolerance and diminished oxidative stress in lung, liver, heart and skeletal muscle. Febuxostat also improved indices related to obesity-mediated right ventricular (RV) dysfunction and the onset of pulmonary arterial hypertension including RV end systolic pressure (RVESP) (41.2 ¡À 7.3 vs. 27.2 ¡À 5.2 mmHg), RV contractility index, pulmonary vascular resistance (PVR) (2.51 ¡À 0.47 vs. 1.96 ¡À 0.24 mmHg/mL/min), mean pulmonary artery pressure (mPAP) (25.2 ¡À 3.7 vs. 18.5 ¡À 3.0 mmHg) and Tau (diastolic function) (6.8 ¡À 1.3 vs. 4.5 ¡À 0.9). Febuxostat significantly reduced RV hypertrophy (Fulton Index), pulmonary arteriole smooth muscle (SM) remodeling (¦Á-SM actin staining) and pulmonary tissue macrophage infiltration. Treatment of HFD mice with produced similar yet more pronounced beneficial effects than febuxostat in all the parameters listed above. However, combined treatment with + febuxostat completely abolished these protective effects, suggesting the salutary actions of were mediated by XO.
These data demonstrate that under hypoxic/inflammatory conditions where vascular XO levels and subsequent XO-GAG interactions are enhanced, XO-catalyzed reduction increases NO generation, decreases ROS production and serves to diminish indices of obesity-mediated pathology.
Supported by AHA National Scientist Development-10SDG3560005 (E.E.K.).