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We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 μM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 μU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control μU/mg). Interestingly, free iron (Fe 2+ and Fe 3+ ) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20 μM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

A number of molybdopterin enzymes, including xanthine oxidoreductase (XOR), aldehyde oxidase (AO), sulfite oxidase (SO), and mitochondrial amidoxime reducing component (mARC), have been identified as nitrate and nitrite reductases. Of these enzymes, XOR has been the most extensively studied and reported to be a substantive source of nitric oxide (NO) under inflammatory/hypoxic conditions that limit the catalytic activity of the canonical NOS pathway. It has also been postulated that XOR nitrite reductase activity extends to red blood cell (RBCs) membranes where it has been immunohistochemically identified. These findings, when combined with countervailing reports of XOR activity in RBCs, incentivized our current study to critically evaluate XOR protein abundance/enzymatic activity in/on RBCs from human, mouse, and rat sources. Using various protein concentrations of RBC homogenates for both human and rodent samples, neither XOR protein nor enzymatic activity (xanthine → uric acid) was detectable. In addition, potential loading of RBC-associated glycosaminoglycans (GAGs) by exposing RBC preparations to purified XO before washing did not solicit detectable enzymatic activity (xanthine → uric acid) or alter NO generation profiles. To ensure these observations extended to absence of XOR-mediated contributions to overall RBC-associated nitrite reduction, we examined the nitrite reductase activity of washed and lysed RBC preparations via enhanced chemiluminescence in the presence or absence of the XOR-specific inhibitor febuxostat (Uloric®). Neither addition of inhibitor nor the presence of the XOR substrate xanthine significantly altered the rates of nitrite reduction to NO by RBC preparations from either human or rodent sources confirming the absence of XO enzymatic activity. Furthermore, examination of the influence of the age (young cells vs. old cells) of human RBCs on XO activity also failed to demonstrate detectable XO protein. Combined, these data suggest: 1) that XO does not contribute to nitrite reduction in/on human and rodent erythrocytes, 2) care should be taken to validate immuno-detectable XO by demonstrating enzymatic activity, and 3) XO does not associate with human erythrocytic glycosaminoglycans or participate in nonspecific binding., (Copyright © 2021. Published by Elsevier Inc.)

Cardiovascular disease is the leading cause of death and disability worldwide with increased oxidative stress and reduced NO bioavailability serving as key risk factors. For decades, elevation in protein abundance and enzymatic activity of xanthine oxidoreductase (XOR) under hypoxic/inflammatory conditions has been associated with organ damage and vascular dysfunction. Recent reports have challenged this dogma by identifying a beneficial function for XOR, under similar hypoxic/acidic conditions, whereby XOR catalyzes the reduction of nitrite (NO2 - ) to nitric oxide (NO) through poorly defined mechanisms. We previously reported that hydrogen sulfide (H 2 S/sulfide) confers significant vascular benefit under these same conditions via NO2 - mediated mechanisms independent of nitric oxide synthase (NOS). Here we report for the first time the convergence of H 2 S, XOR, and nitrite to form a concerted triad for NO generation. Specifically, hypoxic endothelial cells show a dose-dependent, sulfide and polysulfide (diallyl trisulfide (DATS)-induced, NOS-independent NO 2 - reduction to NO that is dependent upon the enzymatic activity of XOR. Interestingly, nitrite reduction to NO was found to be slower and more sustained with DATS compared to H 2 S. Capacity for sulfide/polysulfide to produce an XOR-dependent impact on NO generation translates to salutary actions in vivo as DATS administration in cystathionine-γ-lyase (CSE) knockout mice significantly improved hindlimb ischemia blood flow post ligation, while the XOR-specific inhibitor, febuxostat (Febx), abrogated this benefit. Moreover, flow-mediated vasodilation (FMD) in CSE knockout mice following administration of DATS resulted in greater than 4-fold enhancement in femoral artery dilation while co-treatment with Febx completely completely abrogated this effect. Together, these results identify XOR as a focal point of convergence between sulfide- and nitrite-mediated signaling, as well as affirm the critical need to reexamine current dogma regarding inhibition of XOR in the context of vascular dysfunction., Competing Interests: Declaration of competing interest C.G.K. has provisional patents regarding nitric oxide/nitrite and hydrogen sulfide chemistry and is a co-founder of Innolyzer LLC. X.S. has intellectual property regarding hydrogen sulfide chemistry., (Copyright © 2020. Published by Elsevier B.V.)

Systemic hyperuricemia (HyUA) in obesity/type 2 diabetes facilitated by elevated activity of xanthine oxidoreductase (XOR), which is the sole source of uric acid (UA) in mammals, has been proposed to contribute to the pathogenesis of insulin resistance/dyslipidemia in obesity. Here, the effects of hepatocyte-specific ablation of Xdh , the gene encoding XOR (HXO), and whole-body pharmacologic inhibition of XOR (febuxostat) on obesity-induced insulin resistance/dyslipidemia were assessed. Deletion of hepatocyte Xdh substantially lowered liver and plasma UA concentration. When exposed to an obesogenic diet, HXO and control floxed (FLX) mice became equally obese, but systemic HyUA was absent in HXO mice. Despite this, obese HXO mice became as insulin resistant and dyslipidemic as obese FLX mice. Similarly, febuxostat dramatically lowered plasma and tissue UA and XOR activity in obese wild-type mice without altering obesity-associated insulin resistance/dyslipidemia. These data demonstrate that hepatocyte XOR activity is a critical determinant of systemic UA homeostasis, that deletion of hepatocyte Xdh is sufficient to prevent systemic HyUA of obesity, and that neither prevention nor correction of HyUA improves insulin resistance/dyslipidemia in obesity. Thus, systemic HyUA, although clearly a biomarker of the metabolic abnormalities of obesity, does not appear to be causative., (© 2019 by the American Diabetes Association.)

Inhibition of xanthine oxidoreductase (XOR) has proven beneficial in a plethora of inflammatory disease processes due to a net reduction in pro-inflammatory oxidants and secondary nitrating species. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO 2 ) are also noted to display a broad spectrum of anti-inflammatory effects via interaction with critical signaling pathways. An alternative process in which nitrated fatty acids may extend anti-inflammatory actions is via inactivation of XOR, a process that is more effective than allo/oxypurinol-mediated inhibition. Herein, we describe the molecular aspects of nitrated fatty acid-associated inactivation of XOR, identify specificity via structure function relationships and discuss XOR as a crucial component of the anti-inflammatory portfolio of nitrated fatty acids.

Xanthine oxidoreductase (XOR), the molybdoflavin enzyme responsible for the terminal steps of purine degradation in humans, is also recognized as a significant source of reactive species contributory to inflammatory disease. In animal models and clinical studies, inhibition of XOR has resulted in diminution of symptoms and enhancement of function in a number of pathologies including heart failure, diabetes, sickle cell anemia, hypertension and ischemia-reperfusion injury. For decades, XOR involvement in pathologic processes has been established by salutary outcomes attained from treatment with the XOR inhibitor allopurinol. This has served to frame a working dogma that elevation of XOR-specific activity is associated with enhanced rates of reactive species generation that mediate negative outcomes. While adherence to this narrowly focused practice of designating elevated XOR activity to be "bad" has produced some benefit, it has also led to significant underdevelopment of the processes mediating XOR regulation, identification of alternative reactants and products as well as micro-environmental factors that alter enzymatic activity. This is exemplified by recent reports: (1) identifying XOR as a nitrite reductase and thus a source of beneficial nitric oxide ((•)NO) under in vivo conditions similar to those where XOR inhibition has been assumed an optimal treatment choice, (2) describing XOR-derived uric acid (UA) as a critical pro-inflammatory mediator in vascular and metabolic disease and (3) ascribing an antioxidant/protective role for XOR-derived UA. When taken together, these proposed and countervailing functions of XOR affirm the need for a more comprehensive evaluation of product formation as well as the factors that govern product identity. As such, this review will critically evaluate XOR-catalyzed oxidant, (•)NO and UA formation as well as identify factors that mediate their production, inhibition and the resultant impact on inflammatory disease.

A plethora of vascular pathology is associated with inflammation, hypoxia and elevated rates of reactive species generation. A critical source of these reactive species is the purine catabolizing enzyme xanthine oxidoreductase (XOR) as numerous reports over the past 30 years have demonstrated XOR inhibition to be salutary. Despite this long standing association between increased vascular XOR activity and negative clinical outcomes, recent reports reveal a new paradigm whereby the enzymatic activity of XOR mediates beneficial outcomes by catalyzing the one electron reduction of nitrite (NO2(-)) to nitric oxide (NO) when NO2(-) and/or nitrate (NO3(-)) levels are enhanced either via dietary or pharmacologic means. These observations seemingly countervail numerous reports of improved outcomes in similar models upon XOR inhibition in the absence of NO2(-) treatment affirming the need for a more clear understanding of the mechanisms underpinning the product identity of XOR. To establish the micro-environmental conditions requisite for in vivo XOR-catalyzed oxidant and NO production, this review assesses the impact of pH, O2 tension, enzyme-endothelial interactions, substrate concentrations and catalytic differences between xanthine oxidase (XO) and xanthine dehydrogenase (XDH). As such, it reveals critical information necessary to distinguish if pursuit of NO2(-) supplementation will afford greater benefit than inhibition strategies and thus enhance the efficacy of current approaches to treat vascular pathology., (Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H2O2, 0.15%) or allopurinol (30 μg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H2O2 and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H2O2 restored wound healing in tungsten-fed mice. In vitro, nitrite and H2O2 both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.

Numerous inflammatory disorders are associated with elevated levels of xanthine oxidoreductase (XOR) and allied enhancement of reactive species formation contributory to systemic pathology. Despite a long standing association between increased XOR activity and negative clinical outcomes, recent reports describe a paradigm shift where XOR mediates beneficial actions by catalyzing the reduction of NO2(-) to NO. While provocative, these observations contradict reports of improved outcomes in similar models upon XOR inhibition as well as reports revealing strict anoxia as a requisite for XOR-mediated NO formation. To garner a more clear understanding of conditions necessary for in vivo XOR-catalyzed NO production, this review critically analyzes the impact of O2 tension, pH, substrate concentrations, glycoaminoglycan docking and inhibition strategies on the nitrite reductase activity of XOR and reveals a hypoxic milieu where this process may be operative. As such, information herein serves to link recent reports in which XOR activity has been identified as mediating the beneficial outcomes resulting from nitrite supplementation to a microenvironmental setting where XOR can serve as substantial source of NO., (2013 Elsevier Inc. All rights reserved)

Nearly 30 years have passed since the discovery of xanthine oxidoreductase (XOR) as a critical source of reactive species in ischemia/reperfusion injury. Since then, numerous inflammatory disease processes have been associated with elevated XOR activity and allied reactive species formation solidifying the ideology that enhancement of XOR activity equates to negative clinical outcomes. However, recent evidence may shatter this paradigm by describing a nitrate/nitrite reductase capacity for XOR whereby XOR may be considered a crucial source of beneficial (•)NO under ischemic/hypoxic/acidic conditions; settings similar to those that limit the functional capacity of nitric oxide synthase. Herein, we review XOR-catalyzed reactive species generation and identify key microenvironmental factors whose interplay impacts the identity of the reactive species (oxidants vs. (•)NO) produced. In doing so, we redefine existing dogma and shed new light on an enzyme that has weathered the evolutionary process not as gadfly but a crucial component in the maintenance of homeostasis.

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 microM, limiting both purine oxidation and formation of superoxide (O2.). Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, beta-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent O2. production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation.

Recent studies have identified skeletal muscle as a tissue compartment where nitrate and nitrite can be stored and utilized to potentially maintain nitric oxide (NO) homeostasis. Given its capacity to reduce nitrate and nitrite, the molybdopterin-containing enzyme, xanthine oxidoreductase (XOR) has been suggested as a key enzyme within skeletal muscle which catalytically reduces these N-oxides; however, there remains limited insight into the role of XOR in this process as well as how different conditions (e.g. health vs disease and rest vs exercise) may determine when and where, within skeletal muscle, XOR could serve as a significant source of NO. A key factor that determines the extent by which XOR may or may not contribute to NO generation in a biologically relevant manner is the biochemical landscape (e.g. oxygen tension, pH, isoform of XOR (XDH vs. XO) and substrate levels of the microenvironment in normal versus stressed skeletal muscle. As such, a critical focus of this review is the evaluation of the biochemical and physiologic data supporting the role of XOR within skeletal muscle for supplying nitrite and/or NO from endogenous and exogenous sources during pathophysiologic conditions and/or exercise stress. • Inorganic nitrate is believed to improve exercise performance in health and disease. • Skeletal muscle seems to play a pivotal role in maintaining NO bioavailability. • XOR by reducing nitrate and nitrite might be key in maintaining NO homeostasis dependent on biochemical conditions. • XOR's activity in its different isoforms at rest-exercise or health-disease conditions within muscle is not fully understood. • XOR's role during exercise and if it is pivotal to exercise performance after inorganic nitrate supplementation is yet unknown. [ABSTRACT FROM AUTHOR]