Biology subjects

Khambata, R. S.

Publications and source records attributed to Khambata, R. S..

3 recordsLinked to original sources

bioRxiv2026

Hepatic Xanthine Oxidoreductase Sustains Antithrombotic Nitric Oxide Signalling Through the Nitrate-Nitrite-Nitric Oxide Pathway

Background: Tonic endothelium-derived nitric oxide (NO) suppresses platelet activation physiologically, and its loss underlies the thrombotic risk of endothelial dysfunction. Inorganic nitrate and nitrite provide an alternative NO source, and xanthine oxidoreductase (XOR) is a candidate nitrite reductase, but whether endogenous XOR sustains platelet NO signalling in vivo, and its source is unknown. Methods: Dietary nitrate (15 mmol/L KNO3) was given to eNOS-/- and ApoE-/- mice. XOR was interrogated pharmacologically (allopurinol) and genetically (global Xdh+/- and hepatocyte-specific XOR knockout, HXOR KO). Haemostasis and thrombosis were assessed by tail bleeding and intravital microscopy imaging of FeCl3-induced mesenteric arterial thrombosis, alongside aggregometry, flow cytometry, platelet VASPSer239 phosphorylation, cGMP and ozone chemiluminescence-based analysis of nitrate and nitrite. Results: Dietary nitrate raised plasma nitrate and nitrite levels in all genotypes. In eNOS-/- and ApoE-/- mice, it prolonged bleeding time and normalised thrombus burden. In ApoE-/- mice nitrate treatment improved vasorelaxation, without altering XOR or eNOS expression. Allopurinol suppressed nitrite reductase activity in liver and plasma but not aorta, elevated plasma nitrite, shortened bleeding time and reduced platelet P-VASPSer239 expression. Xdh+/- mice were spontaneously prothrombotic, showed reduced P-VASPSer239 and exaggerated calcium mobilisation, and were refractory to dietary nitrate treatment despite equivalent nitrite elevation. XOR was undetectable in platelets. HXOR KO mice phenocopied global deficiency, with blunted nitrite-induced vasorelaxation but preserved acetylcholine and spermine-NO responses, reduced P-VASPSer239, enhanced aggregation and shortened bleeding time. Conclusions: Hepatic XOR sustains platelet NO-cGMP signalling and thromboresistance through an inter-organ, endocrine-like axis. Dietary nitrate restores antithrombotic protection when endothelial NO generation fails, whereas XOR inhibition removes physiological tonic antithrombotic signal.

pharmacology and toxicology↗

bioRxiv2024

The nitrite reductase activity of xanthine oxidoreductase sustains cardiovascular health

BackgroundXanthine oxidoreductase (XOR) is a multi-functional enzyme that metabolises purines generating uric acid and is a generator of reactive oxygen species. Both functions have been implicated in the pathogenesis of cardiovascular disease. More recently, a third function of XOR as a nitrite reductase has been identified and been shown to play a key role in the benefits of targeting the non-canonical pathway for nitric oxide (NO) generation in the cardiovascular disease setting. This effect has been specifically attributed to XOR dependent recovery of NO levels. However, whether XOR derived NO plays any role in maintaining cardiovascular homeostasis in health is unknown. To explore this, we used global and hepatocyte-specific Xdh-deleted mice to assess cardiovascular homeostasis. MethodsXdh+/+ and Xdh+/-, Xdhfl/fl and Xdhfl/flAlbCre+/-(HXOR KO) mice littermates, matched for sex and age, were used for in vivo cardiovascular phenotyping; blood pressure, cardiac function, endothelial reactivity, and leukocyte trafficking. Tissues from these mice were used for biochemical measurements of nitrate, nitrite, and markers of NO downstream signalling. ResultsXdh+/- and HXOR KO mice expressed significantly attenuated liver and plasma nitrite reductase activity and platelet cGMP levels versus littermate controls. These effects were associated with increased systolic blood pressure, left ventricular remodelling, and increased leukocyte activation. These effects were associated with and likely driven by endothelial dysfunction evident in both mouse models. This dysfunction was reflected by increased endothelial adhesion molecule expression (P-selectin), increased ischaemia-induced vasoconstriction, during vessel occlusion, and an impaired flow-mediated dilation response of the iliac artery in vivo. ConclusionsIn summary, XOR derived NO is critical for maintaining vascular homeostasis under physiological conditions and is key in mediating the benefits of dietary nitrate regimes in cardiovascular pathology

pharmacology and toxicology↗

bioRxiv2023

Natural mutations of human XDH promote the nitrite (NO2-)-reductase capacity of xanthine oxidoreductase: a novel mechanism to promote redox health?

Several rare genetic variations of human XDH have been shown to alter xanthine oxidoreductase (XOR) activity leading to impaired purine catabolism. However, XOR is a multi-functional enzyme that depending upon the environmental conditions also expresses oxidase activity leading to both O {middle dot}- and H O and nitrite ({middle dot}NO -) reductase activity leading to NO. Since these products express important, and often diametrically opposite, biological activity consideration of the impact of XOR mutations in the context of each aspect of the biochemical activity of the enzyme is needed to determine the potential full impact of these variants. Herein, we show that known naturally occurring hXDH mutations do not have a uniform impact upon the biochemical activity of the enzyme in terms of uric acid (UA), reactive oxygen species (ROS) and nitric oxide ({middle dot}NO) formation. We show that the His1221Arg mutant, in the presence of xanthine, increases UA, O2{middle dot}- and NO generation compared to the WT, whilst the Ile703Val increases UA and {middle dot}NO formation, but not O2{middle dot}-. We speculate that this change in the balance of activity of the enzyme is likely to endow those carrying these mutations with a harmful or protective influence over health that may explain the current equipoise underlying the perceived importance of XDH mutations. We also suggest that targeting enzyme activity to enhance the NO2--reductase profile in those carrying such mutations may provide novel therapeutic options, particularly in cardiovascular disease. HighlightsO_LIMutations of xanthine oxidoreductase modulate both its expression and activity C_LIO_LIThe His1221Arg natural mutation increases xanthine oxidoreductase activity C_LIO_LIRaised xanthine oxidoreductase activity coupled with increased availability of nitrite substrate leads to increased NO provision C_LI

biochemistry↗