A New Paradigm for XOR-Catalyzed Reactive Species Generation in the Endothelium

Abstract

A plethora of vascular pathologies 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 nitrite 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.

Introduction

In primates, the molybdopterin-flavin enzyme xanthine oxidoreductase (XOR) catalyzes the final steps in purine catabolism; oxidation of hypoxanthine to xanthine and xanthine to uric acid. Although XOR is reported to be expressed in numerous human tissues including the lung, kidney and myocardium, the greatest XOR specific activity is localized in the splanchnic system [1]. Several inflammatory cytokines (TNF-α, IL-1β, IFN-γ) as well as hypoxia are noted to induce XOR expression. In particular, vascular endothelial cells respond to hypoxia, cytokine stimulation and shear stress by upregulating XOR and exporting the enzyme to the circulation [1,2]. Structurally, XOR is a 295 kD homodimer with each subunit composed of four redox centers: a molybdopterin cofactor (Mo-co), a flavin adenine dinucleotide (FAD) and two iron-sulfur centers (Fe/S). The Mo-co is comprised of a pterin derivative and one Mo atom which is pentacoordinated with a dithiolene, two oxygen atoms and a sulfur atom. The Mo-co is the site of hypoxanthine and xanthine oxidation whereas NAD⁺ and O₂ reduction occur at the FAD. The two Fe/S clusters provide the conduit for electron flux between the Mo-co and the FAD. Distinguishable by their specific electron paramagnetic resonance (EPR) spectra, these Fe/S centers are both of the ferredoxin type but are not identical [3–5].

Xanthine oxidoreductase is transcribed as xanthine dehydrogenase (XDH) where its substrate-derived electrons reduce NAD⁺ to NADH, Fig. 1. However, during hypoxia/inflammation, reversible oxidation of critical cysteine residues (535 and 992) and/or limited proteolysis converts XDH to xanthine oxidase (XO) [6]. In the oxidase form, affinity for NAD⁺ at the FAD is greatly diminished while affinity for oxygen is enhanced resulting in univalent and divalent electron transfer to O₂ to generate O₂^•− and hydrogen peroxide (H₂O₂), respectively, Fig. 1 [7]. Therefore, when situated at critical sites in the tissue and vasculature, XO can serve as an abundant source of ROS mediating alterations in vascular function by reducing ^•NO bioavailability via direct reaction with O₂^•− (^•NO + O₂^•− → ONOO⁻) or indirectly, by mediating redox-dependent cell signaling reactions [8–10].

Figure 1. Xanthine Oxidoreductase Reactions.

For XDH, xanthine is oxidized to uric acid at the Mo-co and electrons transferred via 2 Fe/S centers to the FAD where NAD⁺ is reduced to NADH (left). For XO, xanthine is oxidized to uric acid at the Mo-co and electrons are transferred to the FAD where O₂ is reduced to O₂^•− and H₂O₂ (right). Conversion from XDH to XO is mediated by post-translational modification.

Oxidant Formation

In the literature XO is rarely recognized as a direct source of H₂O₂ and mainly referred to as a source of O₂^•− with H₂O₂ formation resulting from reaction of O₂^•− with superoxide dismutase (SOD) or spontaneous dismutation. However, XO is actually a H₂O₂-producing enzyme that also produces some O₂^•− under physiological conditions. For example, achievement of 100% O₂^•− production from XO requires 100% O₂ and pH 10 whereas at pH 7.0 and 21% O₂, XO generates ~25% O₂^•− and ~75% H₂O₂ [11]. At normal pH and hypoxia, XO-catalyzed H₂O₂ increases approaching 90–95% of total electron flux though the enzyme [12]. Thus, under conditions such as ischemia and/or hypoxia, where both O₂ tension and pH are reduced, H₂O₂ formation is favored suggesting that XOR may participate in the numerous signaling cascades where H₂O₂ has been implicated [13,14]. While the post-translational conversion of XDH to XO has become synonymous with the conversion from a “good” housekeeping enzyme to a “bad” ROS-producing enzyme, it is critical to recognize that XDH can also reduce O₂ and generate ROS. For example, ischemia/hypoxia either localized, regional or systemic mediate O₂-dependent alterations in cellular respiration leading to decreased mitochondrial NADH oxidation and thus significant decreases NAD⁺ concentration [15]. Although NAD⁺ is the preferred electron acceptor for XDH, when levels of this substrate are low XDH will utilize O₂ and thus care should be taken not to exclusively associate XDH as the form of XOR that does not produce ROS [16]. In the aggregate, XO is primarily a source of H₂O₂ that also produces O₂^•− while, under certain circumstances, both forms of the enzyme are capable of producing oxidants.

XOR-Endothelium Interaction

Intravenous administration of heparin in the clinic and in animal models of vascular disease results in elevated XO activity in the plasma suggesting XO is bound to the vascular endothelium [17–19]. During ischemic/hypoxic conditions XDH released into the circulation is rapidly (<1 min) converted to XO and subsequently binds to negatively charged glycosaminoglycans (GAGs) on the surface of endothelial cells [18,20]. Although XO exhibits a net negative charge at physiological pH, pockets of cationic amino acid motifs on the surface of the protein confer high affinity for GAGs (K_d = 6 nM) [18,21–23]. Sequestration of XO by vascular endothelial GAGs amplifies local XO concentration and significantly alters XO kinetic properties. For example, when compared to XO in free in solution, GAG-immobilized XO demonstrates an increased K_m for xanthine (6.5 vs. 21.2 μM) and an increased K_i for allo/oxypurinol (85 vs. 451 nM) [23]. In addition to affecting kinetic properties at the Mo-co, binding of XO to GAGs confers alterations to the FAD resulting in reduction of O₂^•− production by 34% and thus elevation of H₂O₂ formation [23]. Combined, XO-GAG interaction results in: 1) diminished affinity for hypoxanthine/xanthine, 2) resistance to inhibition by the pyrazalopyrimidine-based inhibitors allo/oxypurinol and 3) diminished O₂^•− production and thus enhanced H₂O₂ generation. This vascular milieu where XO is sequestered on the surface of the endothelium is prime for prolonged enhancement of oxidant formation that is partially resistant to inhibition by the most commonly prescribed clinical agents.

Nitrite Reductase Activity

For over 40 years, dogma dictates that elevation of XO activity during hypoxia/ischemia/inflammation equates to increased XO-derived ROS generation and ultimately to poor clinical outcomes. However, recent reports have proposed a paradigm shift by demonstrating XO-mediated formation of salutary ^•NO under similar pathologic conditions. Indeed, under anoxic conditions and acidic pH, XO demonstrates a nitrite reductase activity by catalyzing the reduction of NO₂⁻ to ^•NO in the presence of either xanthine or NADH as reducing substrates (sources of electrons) [24–27]. The Mo-co has been identified as the site of NO₂⁻ reduction where xanthine oxidation directly reduces the cofactor; alternatively, NADH can indirectly provide reducing equivalents via electron donation at the FAD with subsequent retrograde flow to the Mo-co, Fig. 2. This catalytic activity has also been demonstrated is tissue homogenates in the presence of xanthine or aldehyde [28]. In addition to in vitro/ex vivo biochemical studies, diminution or ablation of NO₂⁻-mediated beneficial effects upon co-treatment with allo/oxypurinol has been observed in vivo suggesting XOR involvement as a NO₂⁻ reductase. For example, XOR inhibition has diminished protective effects mediated by NO₂⁻ therapy both clinically and in animal models of intimal hyperplasia following vessel injury [29], acute lung injury and ventilator-induced pulmonary pathology [30,31], ischemia/reperfusion (I/R)-induced damage in lung transplantation [32], pulmonary hypertension [33], myocardial infarction [34] and renal [35], cardiac [36] and liver [37] I/R injury. It is also important to note that circulating NO₂⁻ concentration and subsequent ^•NO levels may be enhanced in an XOR-dependent manner by supplemental nitrate (NO₃⁻). In this case it is hypothesized that XOR serves first as a NO₃⁻ reductase (NO₃⁻ + 1e⁻ → NO₂⁻) and ultimately a NO₂⁻ reductase (NO₂⁻ + 1e⁻ → ^•NO) [38]. In these experiments, germ-free mice, void of bacterial NO₃⁻ reductases, were treated with NaNO₃⁻. While NO₃⁻ treatment resulted in elevation of plasma NO₂⁻ levels it was not seen when mice received co-treatment with allopurinol and thus is consistent with previous biochemical reports demonstrating NO₃⁻ reductase activity for XOR [39]. Taken together, these recent reports serve to promote a new paradigm where XOR, in the presence of supplemental NO₂⁻ or NO₃⁻, may adopt a protective role under conditions where it was previously assumed that inhibition therapy would provide optimal benefit.

Figure 2. Nitrite Reductase Activity of XOR.

(A) Under low O₂ tensions, NO₂⁻ undergoes a 1 electron reduction to ^•NO at the Mo-cofactor (electrons are donated directly to Mo by xanthine). The thickness of the arrows represents the degree of electron flux while the size of the font represents level of substrate or product formation. (B) Again, under low O₂ tensions, NO₂⁻ is reduced to ^•NO at the Mo-cofactor while electrons are supplied by NADH and transferred retrograde to reduce the Mo-co.

While the concept of XOR-derived ^•NO formation is enticing, it is remains underdeveloped for several reasons: 1) in vitro demonstration of ^•NO formation from XOR and NO₂⁻ has required anoxic conditions and concentrations of NO₂⁻ several orders of magnitude greater than those encountered in vivo, 2) other mechanisms for hypoxic and acidic reduction of NO₂⁻ to ^•NO including heme-based reactions [40–42] have not been clearly delineated from XO-mediated effects 3) reports have relied upon inhibition of XOR with allo/oxypurinol, an inhibitor known to have off target effects on alternative purine catabolism pathways including those affecting adenosine [43,44] or 4) by systemic molybdopterin enzyme inactivation by dietary supplementation with sodium tungstate (NaW), a process that mediates the replacement of the active site Mo with tungsten (W) rendering XOR as well as aldehyde oxidase (AO), sulfite oxidase (SO) and mitochondrial amidoxime reducing component or mARC inactive [45,46]. This is crucial as the molybdopterin enzymes AO, SO and mARC have also been reported to demonstrate NO₂⁻ reductase activity [47–50]. In toto, these issues reveal a current state of uncertainty regarding the biological relevance of XO-mediated ^•NO formation. The following will address these issues and generate a working hypothesis focused on a micro-environmental setting where XO-catalyzed NO₂⁻ reduction may be operative.

A significant obstacle to assessing of the biological relevance of XO-mediated ^•NO formation is the substantive difference in affinity between hypoxanthine/xanthine (K_m = 6.5 μM) and NO₂⁻ (K_m = 2.5 mM) for the Mo-co of XO [7,26]. At first glance it would be appropriate to conclude this reaction would never occur due to: 1) the vast difference in K_m values and 2) normal tissue NO₂⁻ concentration (~0.3 μM) is greater than an order of magnitude lower than the K_m-NO₂⁻ and 3) hypoxia results in diminution of NO₂⁻ levels [51]. However, hypoxia enhances NO₃⁻ levels from 30–40 μM to 120–150 μM depending upon the tissue [51]. This increase in NO₃⁻ is not trivial as reports indicate XOR-dependent reduction of organic NO₃⁻ to NO₂⁻ and then to ^•NO with K_m values ranging from 330–500 μM for glycerol trinitrate (GTN) to 32 mM for inorganic nitrate (NaNO₃) [52,53]. On the other hand, the K_m for xanthine is 6.5 μM and levels of xanthine above 20 μM, commonly seen under pathologic conditions, are reported to induce substrate-dependent inhibition of NO₂⁻ reduction (K_i = 55 μM) [26, 54–56]. Therefore, when assessed together, it would seem that the potential to encounter a scenario whereby reduction in xanthine concentration and elevation in NO₂⁻ + NO₃⁻ levels would overcome the difference in K_m for the Mo-co is, at best, improbable. However, it can be hypothesized that NADH, not hypoxanthine/xanthine, may be the major source of electrons necessary to reduce the Mo-co and drive NO₂⁻ reduction. Under ischemia or hypoxia, O₂-dependent alterations in cellular respiration lead to decreased mitochondrial NADH oxidation and thus significant elevation of NADH concentration [15]. Enhancement of NADH levels may serve to efficiently reduce the Mo-co via retrograde electron flow from the FAD, Fig. 2. This process would also impact affinity of xanthine at the Mo-co as xanthine-Mo-co reaction is dependent on the Mo-co being oxidized. In addition, immobilization of XO on GAGs confers a diminution in affinity for xanthine (see above) which could further facilitate capacity for NO₂⁻ + NO₃⁻ to compete for reaction. Regardless of the potential shifts in affinity away from xanthine and towards NO₂⁻, it remains unclear how such vast differences in K_m values can be surmounted to generate operative NO₂⁻ reductase activity from XO.

Another obstacle to the assessment of the biological relevance of XO-mediated ^•NO formation is oxygen tension. Nitrite reductase activity of XO is extremely oxygen sensitive [24,58]. This is primarily due to oxidation of the Mo-co via O₂-dependent electron withdrawal at the FAD. Therefore, the presence of O₂ results in a more electron deficient Mo-co resulting in lower capacity to donate an electron to NO₂⁻ (this concept is extensively reviewed [59]). The O₂ tension whereby the NO₂⁻ reductase activity of XOR may become effective is ultimately kinetically governed at the FAD cofactor. First, one must consider that O₂ tensions must drop to or below K_m for O₂ at the FAD (27 μM or ~2% O₂) in order to diminish inhibitory actions of O₂ and thus facilitate NO₂⁻ reaction at the Mo-co [59]. Second, local NADH levels must be considered as hypoxia-mediated elevation in NADH can serve to provide competition for O₂ at the FAD while concomitantly reducing the enzyme, Fig. 2. Third, pH is also crucial as hypoxanthine and xanthine oxidation at the Mo-co is a base-catalyzed process with a pH optimum of 8.9 [11]. The reduction of NO₂⁻ at the Mo-co is an acid-catalyzed process with a pH optimum of (6–6.5) [24]. Considering in vivo vascular microenvironments encountered during inflammatory conditions demonstrate pH’s that fall below 7.0, it is logical to assume that this setting would shift affinity away from xanthine and towards NO₂⁻ reaction at the Mo-co. This concept was observed when pH was sequentially lowered from 8.0→5.0 while using either NADH or xanthine as reducing substrates [58]. Thus, the combination of low O₂ tension, elevated NADH concentration and lower pH is conducive for XO-catalyzed NO₂⁻ formation. However, it must be considered that XDH and not XO may be the major XOR isoform driving NO₂⁻ reduction as the K_m O₂ for the FAD of XDH is 260 μM versus 27 μM for XO suggesting O₂-mediated inhibition would be insignificant for XDH [3,60].

Regardless of the recognized biochemical obstacles for XOR-derived ^•NO formation in purified enzyme preparations, the in vivo impact of NO₂⁻ supplementation has demonstrated significant XO dependence. For example, XOR inhibition has diminished or abolished salutary actions of NO₂⁻ therapy in a variety of vascular models including wire-induced vessel injury, lung injury, ischemia/reperfusion, pulmonary hypertension, myocardial infarction [29–37]. These studies have utilized varied pharmacologic approaches to diminish XO enzymatic activity including allo/oxypurinol, febuxostat and dietary supplementation with NaW (sodium tungstate) which serves to strengthen the argument for XO-dependent NO₂⁻ reduction to ^•NO as the central mechanism driving beneficial outcomes. In addition to NO formation, the presence of NO₂⁻ serves to “inhibit” XO-derived reactive species formation by diverting electrons destined for O₂ reduction to ^•NO generation and effectively providing a 2-fold beneficial impact (see Fig. 2). However, administration of NO₂⁻ is not uniform among all reports and must be further studied to optimize the beneficial impact while avoiding the potential for adventitious nitrosamine formation. This is especially relevant in the stomach where acid pH can facilitate the formation of these carcinogenic compounds. Future in vivo studies will not only define these optimal NO₂⁻ doses but reveal the potential for utilizing the enzymatic activity of XO, upregulated by inflammatory/hypoxic processes, to be quite promising.

Summary

Consideration of XOR as a significant source of ^•NO during ischemic/hypoxic conditions seems to countervail studies reporting XOR inhibition resulting in a reduction in symptoms and enhanced function. In addition, several substantive biochemical issues frustrate the designation of XOR as a biologically relevant source of ^•NO. Yet, in vivo XOR inhibition studies were not conducted in models where NO₂⁻ levels were elevated and in vitro biochemical studies have not considered all possible permutations, especially XO vs. XDH, whereby Mo-co and FAD kinetics may be altered to favor NO₂⁻ reduction. Herein, we have discussed several key factors (acidic pH, elevated levels of NADH, O₂ tension, immobilization-induced kinetic alterations and enzyme isoform) that contribute to a local milieu where XOR-mediated NO₂⁻ reduction may be relevant. A summary of our working hypothesis is represented in cartoon format, Fig. 3. Inflammatory conditions in the absence of hypoxia result in enhanced XDH expression and export to the circulation where XDH is rapidly converted to XO with subsequent sequestration by endothelial GAGs either locally or in a distal vascular beds. With abundant O₂ and xanthine, XO-dependent ROS formation is dominant while eNOS is the primary source of ^•NO. Under these conditions it is possible that, in the presence of elevated NO₂⁻, intracellular XDH could serve as a nitrite reductase. As tissue O₂ concentrations decrease, approaching and falling below the K_m for O₂ at the FAD (~27 μM or ~1.5% O₂), XO as well as XDH can assume NO₂⁻ reductase activity while eNOS-mediated O₂ formation is blunted by lack of requisite substrate (O₂) and may become uncoupled resulting in O₂^•− generation. At this point XO and/or XDH may become a significant source of ^•NO. It is interesting to note that the K_m O₂ for eNOS is 23 μM; a value very similar to the K_m O₂ for XO (27 μM) and thus may represent the O₂ tension where ^•NO production is transferred from eNOS to XOR [61]. The degree to which this hypothesis is accurate remains unclear; however, it is clear that further investigation is warranted to more precisely define the mechanisms underpinning XOR-mediated ^•NO formation and the potential impact this process may have on therapeutic strategies to address vascular disease.

Figure 3. XOR-Catalyzed NO Production in the Vasculature.

(top panel) Under oxygenated conditions, inflammation results in enhanced XDH expression and export to the circulation where XDH is rapidly converted to XO with subsequent sequestration by endothelial GAGs. With abundant O₂ and xanthine, XO-dependent ROS formation is dominant while eNOS is the primary source of ^•NO. XO-derived O₂^•− effectively reduces ^•NO bioavailability by its diffusion limited reaction with eNOS-derived ^•NO to form peroxynitrite (O=NOO⁻). Under these conditions it is possible that, in the presence of elevated NO₂⁻, intracellular XDH could serve as a NO₂⁻ reductase. (bottom panel) Under hypoxic conditions (O₂ tensions approaching and falling below the K_m for O₂ at the FAD (~27 μM or ~1.5% O₂), XO as well as XDH can assume NO₂⁻ reductase activity while eNOS-mediated ^•NO formation is diminished due to the absence of the substrate, O₂ (noted by a diminished arrow thickness). Under these conditions, eNOS may become uncoupled resulting in O₂^•− generation. It is at this point that XO and/or XDH may become a significant source of ^•NO.

Abbreviations

GAGs

glycosaminoglycans

GTN

glycerol trinitrate

H₂O₂

hydrogen peroxide

^•NO

nitric oxide

NOS

nitric oxide synthase

O₂^•

superoxide

ROS

reactive oxygen species

SOD

superoxide dismutase

XDH

xanthine dehydrogenase

XO

xanthine oxidase

XOR

xanthine oxidoreductase