Nitrite reductase activity is elevated in atherosclerotic mice: role for xanthine oxidoreductase

K.S. Rathod, R.S. Khambata, S.M. Ghosh, A. Ahluwalia. William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK

Background

Xanthine Oxidoreductase (XOR) has been identified as an important vascular nitrite reductase in CVD. Our previous evidence suggests elevation of XOR expression and activity in hypertension underlies the improved potency of nitrite in that condition. Since recent evidence intimates that nitrite might also be useful in atherosclerotic disease (1), (2), we have investigated whether XOR expression and activity is upregulated in a pre-clinical model of atherosclerosis and whether this might be associated with elevated nitrite reductase activity.

Methods

Liver, mesentery and red blood cells (RBCs) were collected from male ApoE knockout (KO) and wild type (WT) littermate mice (12 weeks of age) fed a chow diet. These tissues were chosen since the liver represents the major source of XOR in the body, the mesenteric vasculature informs on vascular XOR expression and the RBC represents a major site for localisation of circulating XOR (3). XOR protein expression was determined using Western blotting and analysis conducted using Image J (NIH, USA). Levels of XOR expression were normalised to actin. XOR activity within these tissues was ascertained using a xanthine-based colourimetric XO activity assay (abcam, UK). The nitrite reductase activity of liver homogenates bubbled with nitrogen was determined using gas phase ozone chemiluminescence in the presence of nitrite (10-300 μmol/L) and under pH 7.4 and 6.8. To ascertain the role of XOR in any activity seen samples were incubated with the XOR inhibitor allopurinol (100 μmol/L) for 30 min. Results are expressed as mean±s.e.m and statistical analysis conducted using Graphpad Prism 5.0.

Results

Western blot analysis demonstrated a ~44% increase in XOR expression in the of livers ApoE KO mice compared to the WT (n = 8, p < 0.05). Similarly in the mesenteric vascular tissue, there was an ~48% increase in the ApoE KO mice compared to the WT (n = 8, p < 0.05). Furthermore, in the RBCs, there was an ~61% increase in XOR expression in the ApoE KO mice compared to the WT (n = 8, p < 0.05). In the liver this difference in expression was associated with significantly higher levels of XOR activity in liver homogenates of ApoE KO compared to WT mice (0.25±0.01 mU/mg vs. 0.20±0.02 mU/mg, n=8, p < 0.05). Nitrite-derived (10-300 μmol/L) NO formation was concentration-dependent in liver homogenates of both ApoE KO (n = 5) and WT mice (n = 5) at both pH 6.8 and 7.4. However, allopurinol (100 μmol/L) caused a ~28% reduction in nitrite-derived (300 μmol/L) NO formation by liver homogenates of ApoE KO mice at pH 6.8 (n = 5, p <0.01) and a ~23% reduction at pH 7.4 (n = 5, p <0.01 not seen in tissues of WT mice). These differences were significant using 2-way ANOVA followed by Bonferroni post-tests.

Conclusions

These findings suggest that in ApoE KO mice, XOR expression and activity is raised and that this is associated with increased XOR and nitrite reductase activity. Further investigation of the implications of this enhanced activity of nitrite in atherosclerosis is warranted.

References

(1) Alef et al., (2011). J Clin Invest 121(4):1646-1656;

(2) Stokes et al., (2009). Am J Physiol Heart Circ Physiol 296(5):H1281-H1288

(3) Ghosh et al., (2013). Hypertension 61(5):1092-1102.