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Introduction

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

In addition to its critical role in gout, hyperuricemia is increasingly being considered a potential pathogenic factor for hypertension, metabolic syndrome and type 2 diabetes, and renal disease, as well as atherosclerosis and several adverse consequences of vascular disease (stroke, myocardial infarction, and cardiovascular death) (1, 2). Uric acid is generated via the action, in the purine degradation pathway, of both the oxidized and reduced forms of xanthine oxidoreductase (XOR) (known as xanthine oxidase [XOD] and xanthine dehydrogenase [XDH], respectively) on hypoxanthine and xanthine (3–6). In addition to uric acid itself, increasing evidence implicates the oxidized form XOD in vascular diseases (3, 7). This is noteworthy, given the role of XOR inhibition as first-line urate-lowering pharmacologic therapy in gout (8).

At physiologic pH, uric acid exists as its ionized salt, monosodium urate. Humans and higher primates have higher serum urate levels than other mammals due to the loss in hominid evolution of the expression of an active form of the hepatic peroxisomal enzyme uricase (uric acid oxidase) (9). Uricase catalyzes the breakdown of uric acid to 5-hydroxyisourate. It has been reported that 5-hydroxyisourate is subject to hydrolysis by transthyretin-related protein (10) and degradation to allantoin in humans and higher primates; this is in contrast to the more rapid hepatic intracellular enzymatic chain of uric acid degradation to 5-hydroxyisourate by uricase, and of 5-hydroxyisourate to allantoin by 2 other enzymes, in lower mammals (9).

The limited solubility of urate predisposes to the deposition of monosodium urate crystals and thereby to the development of gout. Hyperuricemia (as defined by a single reading >7 mg/dl) was estimated to exist in ∼21.4% of adults in the US in 2007–2008, with the mean serum urate in US adults continuing to rise over the last few decades and attaining an estimated level of close to 5.5 mg/dl (11). The duration and level of hyperuricemia directly correlate with the risk of developing gout, with the risk particularly increased among those with serum urate levels >9 mg/dl (12). In 2007–2008, a self-reported diagnosis of gout was estimated to be present in ∼8.3 million Americans (∼3.9% of all adults [defined as age ≥20 years]; ∼5.9% of male adults and ∼2% of female adults) (11). Importantly, gout prevalence is substantially higher in elderly persons, a group disproportionately affected by vascular disease (13). With the rise over the last several decades in the prevalence of both gout and hyperuricemia, clarifying the potential adverse effects of hyperuricemia (in patients with and without gout) is of public health importance.

Since a minority of individuals with sustained hyperuricemia go on to develop gout as a clinical disease, asymptomatic hyperuricemia (i.e., in the absence of gout, urate nephrolithiasis, or tumor lysis syndrome) is considered neither a disease state nor an accepted indication for urate-lowering treatment. Reflecting this, serum urate levels are not part of the routine metabolic panel of blood tests, and asymptomatic hyperuricemia is not currently considered an issue for preventive clinical care. However, as reviewed here, asymptomatic hyperuricemia, and hyperuricemia in gout patients, from a vascular biology point of view alone, may not be benign.

Uric acid is not simply an inert end product of purine metabolism in humans, but rather has potential antioxidant, prooxidant, and proinflammatory effects. However, controversy remains as to which, if any, of these effects are of clinical relevance in the development and complications of human vascular diseases in gout and asymptomatic hyperuricemia. Further, it is not yet clear how to identify those at risk of developing adverse sequelae of hyperuricemia, whether gout or vascular disease. Additionally, studies of urate-lowering therapy with XOR inhibition or uricosuric agents have not been able to definitively identify whether any such effects may be mediated by uric acid versus XOD. Adequately sized prospective, randomized clinical trials of sufficient duration, and employing appropriate biomarkers, now appear critical to resolving the putative toxic roles of uric acid and XOD in the human arterial circulation.

Table 1 summarizes recent shifts in thinking on soluble urate and XOD actions pertinent to vascular disease as discussed in this review.

Table 1. Major hypotheses from new knowledge of uric acid, hyperuricemia, and XOD to be resolved in clinical investigation of gout and vascular disease in humans*
Recent research advances, principally from in vitro and animal modelsSpecific hypotheses suggested for testing in well-controlled, prospective clinical studies in humans and trials in patients with and without gout
  • *

    XOD = xanthine oxidase; NO = nitric oxide; EC = endothelial cell; XDH = xanthine dehydrogenase; XOR = xanthine oxidoreductase.

  • In humans, uric acid is not an inert end product of purine catabolism, since multiple uric acid byproducts, including oxidative catabolites (5-aminouracil, allantoin, triuret), are demonstrable in human urine

  • Uric acid has both antioxidant and prooxidant effects, the latter exerted by oxidative degradation of uric acid. This includes oxidative degradation of uric acid (in the presence of peroxide) by neutrophil-derived myeloperoxidase, a circulating biomarker (and proposed pathogenic factor) in atherosclerosis

  • Reactive oxygen species from uric acid degradation have biologic consequences (e.g., in inflammatory responses mediated by cell necrosis)

  • Soluble uric acid affects vascular cell functions, including promotion of degradation of the vasodilator NO by oxidation and effects on arginase expression, and promotion of activity of the renin–angiotensin–aldosterone axis

  • In humans, XOD expression is highly regulated and can be increased in macrophage-lineage cells and, in hypoxia and inflammatory states, on the EC surface

  • XOD, and not simply NADPH oxidase, can promote oxidative stress in ECs, and does so by generating 2 moles of superoxide per mole of uric acid generated

  • XOD affects endothelial function independent of uric acid generation XOD promotes inflammatory differentiation and function in macrophages, a cell type central to atherogenesis and atherosclerotic plaque complications

  • Do uric acid oxidation–derived reactive oxygen species, and other effects of uric acid on endothelial arginase, promote measurable NO degradation that can affect blood pressure and EC homeostasis?

  • Does oxidative stress–related XOD activity contribute to functionally significant changes in vascular tissue urate levels?

  • Is the source of serum and vascular tissue uric acid (i.e., generated by XOD or XDH) equally or more important than the serum urate level in vascular disease?

  • Does XOR inhibition inhibit atherogenesis and atherosclerotic plaque complications and progression of renal impairment, independently of urate lowering, in gout and asymptomatic hyperuricemia?

Uric acid is not an inert end product of purine metabolism in humans

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

Clearly, gout attacks would not occur if uric acid was simply an inert end product of purine metabolism in humans subject to simple disposition triggered via excretion unchanged into the urine and small intestine. Of relevance for the vasculature, uric acid interfaces with superoxide (O2), the major vasodilator nitric oxide (NO) (14, 15), the potent long-lived NO-derived oxidant peroxynitrite (ONOO) (16), and myeloperoxidase (MPO) (17) (Figure 1). Uric acid–derived end products of these reactions, including oxidative catabolites (6-aminouracil, allantoin, triuret), are demonstrable in human urine (18). It remains unclear whether there are biologically significant activities of these catabolites in humans with hyperuricemia, but they do have the potential to be used as biomarkers for the interface of oxidative stress with uric acid. For example, MPO potentially links the neutrophil activation–mediated pathology of gouty arthritis and “spillover” to systemic inflammation in gout, including leukocytosis, with vascular pathology.

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Figure 1. Uric acid is not an inert end product of purine catabolism and interacts with other molecules in alternative pathways of degradation that can modulate oxidative stress, as illustrated here and discussed in the text. NO = nitric oxide; O2 = superoxide anion; ONOO = peroxynitrite; MPO = myeloperoxidase; TRP = transthyretin-related protein.

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MPO is expressed by neutrophils and monocytes in azurophil granules and lysosomes, respectively, and is principally secreted by activated neutrophils (17). MPO-catalyzed uric acid oxidation in the presence of H2O2 generates 5-hydroxyisourate, urate radical, and hydroperoxide (17). Uric acid enhances MPO-dependent consumption of NO, and in the presence of uric acid, MPO accelerates the oxidation of the critical intracellular antioxidant glutathione to oxidized glutathione (17). MPO exerts multiple proinflammatory effects, impairs antiinflammatory function of high-density lipoprotein, and is a promoter and biomarker of atherosclerotic plaque instability and rupture (19). Neutrophil and monocyte adhesion and activation occur at sites of hypoxic injury to arteries and therapeutic arterial neovascularization. Hence, the potential for MPO-related effects on uric acid to affect endothelial cell (EC) homeostasis and blood pressure, as illustrated schematically in Figure 2, warrants assessment in clinical studies, particularly those of acute and chronic gouty arthritis.

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Figure 2. Model for uric acid and xanthine oxidase (XOD [XO]) interactions with vascular cells that affect oxidative stress and vascular pathophysiology (see also text and Table 1). In this model, uric acid turns on “inflammatory,” cytotoxic, and dysfunctional responses, including up-regulation of the renin–angiotensin–aldosterone system in cultured endothelial cells (ECs) and arterial smooth muscle cell (SMC) proliferation and migration. These effects are mediated by uric acid–induced oxidative stress in ECs, scavenging of nitric oxide (NO), and induction of EC arginase that reduces production of vasodilatory NO. There are additional adverse consequences for cell redox status and NO levels of oxidative degradation of uric acid (in the presence of peroxide) by neutrophil-derived myeloperoxidase (MPO). Soluble uric acid–induced promotion of NO degradation by oxidation and effects on arginase expression are illustrated, as are adverse effects of peroxynitrite (ONOO) whose oxidant effects are inhibited by uric acid. In this model, XOD expression is increased in macrophages as well as on EC surfaces by inflammatory conditions (e.g., gouty arthritis) and ischemia. Moreover, XOD promotes oxidative stress in ECs and impairs endothelial function independently of uric acid generation. XOD and uric acid also stimulate macrophage-mediated inflammation in the artery wall. Not depicted here but discussed in the text are potential effects of xanthine oxidoreductase (XOR) inhibition on the accumulation of upstream precursors such as inosine and adenosine that have antiinflammatory properties. GI = gastrointestinal; XDH = xanthine dehydrogenase; O2 = superoxide anion; eNOS = endothelial cell nitric oxide synthase; PPARγ = peroxisome proliferator–activated receptor γ; HIF-1α = hypoxia-inducible factor 1α.

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Uric acid is both an antioxidant and a prooxidant

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

Like other compounds with labile electrons and a redox potential, uric acid can act as a prooxidant or an antioxidant, depending on its redox potential relative to the donor or acceptor substrates undergoing oxidation or reduction. Hence, the simultaneous pro- and antioxidant actions of uric acid, which have been described in numerous and contradictory studies (20–34), have not been particularly informative. For example, administration of intravenous uric acid systemically in small controlled studies of healthy volunteers improved the serum antioxidant capacity at rest (32) and reduced exercise-induced oxidative stress (30). However, uric acid infusion had no acute effects, positive or negative, on endothelial function in another study (33).

Uric acid–derived free radicals include urate anion and aminocarbonyl, which is formed by interaction with peroxynitrite (27). In this context, a clinical study of hyperuricemic patients with gout treated with the recombinant PEGylated uricase pegloticase, which leads to marked lowering of serum urate, showed a trend toward a decrease in levels of some plasma oxidative stress markers, but the effect did not reach statistical significance (35).

Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

Evidence from in vitro and in vivo studies performed over the last decade has presented a cohesive model of how uric acid can act in arterial pathophysiology (1, 2). In this paradigm, originating largely from an impressive body of in vitro and in vivo work by Johnson and colleagues, uric acid turns on “inflammatory,” cytotoxic, and dysfunctional responses (including up-regulation of the angiotensin system) in cultured ECs and proliferation and migration of arterial smooth muscle cells (SMCs) (1, 2, 36–42) (Figure 2). Uric acid–induced oxidative stress in ECs and scavenging of NO and induction of EC arginase that reduces NO production form part of this model (14, 15), and triggering of activation of the renin–angiotensin–aldosterone axis has been implicated in some studies (1, 2, 36). Many of the in vitro findings in this model, including NO depletion (43) and activation of the renin–angiotensin–aldosterone axis, are complemented by in vivo studies in rats fed with a uricase inhibitor, oxonic acid, which produces an increase in the basal low level of serum urate of ∼1 mg/dl to sustained levels of 2–3 mg/dl and likely even higher transitory spikes in levels of serum urate (1, 2, 40).

Oxonic acid–induced hyperuricemia in rats is associated with worsening of renal function triggered by a variety of insults, including cisplatin and cyclosporine administration (1, 2, 40, 44, 45). In a translational context, recent small randomized clinical trials in humans evaluating efficacy of XOR inhibition in limiting renal progression have had positive results (46–48), supporting a potential role for urate lowering as a therapeutic target in human renal disease.

A prominent feature of the rat model of oxonic acid–induced hyperuricemia is hypertension with afferent glomerular arteriolopathy, which is reversed by XOR inhibitor and uricosuric treatments that resolve the hyperuricemia (1, 2, 40). Moreover, in this rat model, inhibition of angiotensin signaling by losartan inhibits the hypertension and renal pathology, whereas diuretic therapy that resolves the hypertension but not the elevated serum urate does not eliminate the renal vascular pathology (40).

These findings mirror findings in large-scale randomized trials in humans. In the Losartan Intervention For Endpoint reduction in hypertension (LIFE) study, approximately one-third of the improved cardiovascular mortality was attributed to an independent effect on serum urate levels in those who received losartan, a drug with uricosuric effects, as compared with those who received atenolol, a beta-blocker with no such effects (49). On the other hand, in the Systolic Hypertension in the Elderly Program (SHEP) trial, those with hypertension appropriately controlled with a thiazide diuretic but with a concomitant increase in serum urate levels did not have a reduced risk of coronary heart disease compared with placebo (50). Recently, a randomized, placebo-controlled, crossover trial of allopurinol versus placebo demonstrated significant efficacy of allopurinol among hyperuricemic adolescents with hypertension (51). Because these effects may be related to XOR inhibition rather than urate lowering, results from trials using uricosurics are awaited. A pathogenic role of hyperuricemia in hypertension in humans and therapeutic use of urate lowering in hypertension both merit further investigation.

An entire field has recently emerged linking fructose intake and metabolism to uric acid biology. It has been proposed that fructose intake, linked with hepatic ATP depletion that promotes uric acid generation and the development of hyperuricemia, influences the development of various features of the metabolic syndrome. This includes nonalcoholic hepatosteatosis “fatty liver” putatively mediated in part by hepatic ATP depletion promoted by dysregulated intrahepatic fructose metabolism (52). Furthermore, the metabolic syndrome, a frequent comorbidity among persons with gout, clearly influences cardiovascular disease susceptibility and pathogenesis by multiple direct and indirect effects of insulin resistance on the vasculature. The topic of fructose metabolism, hyperuricemia, and the metabolic syndrome is beyond the scope of this review and is addressed in depth elsewhere (53).

Critical appraisal of data regarding potential vascular toxicity of uric acid

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

One caution with interpreting the oxonic acid–induced hyperuricemia rat model is that oxonate alone can promote inflammatory differentiation of cultured macrophage-lineage cells (54). Moreover, oxonate potentially modulates renal transport of uric acid and thereby could affect intracellular handling and effects of uric acid in renal proximal tubular cells (55). Hence, some mechanisms of renal disease in the in vivo model of oxonic acid–induced hyperuricemia remain to be resolved; ideally, an alternative model of hyperuricemia would help in this task. In this context, as discussed above, intravenous infusions of urate either did not appear to impair forearm blood flow in healthy human adults or in fact seemed to improve endothelial function (30–33). This indicates that, at least acutely in these small studies, urate does not worsen endothelial function in humans. Moreover, short-duration urate lowering using a single dose of intravenous uricase had a neutral effect on endothelial function in a small (n = 10), randomized, single-blind, placebo-controlled crossover study in humans with type 2 diabetes mellitus (56). Whether long-term, sustained elevations in uric acid result in different effects cannot be discerned from these studies.

Not only cultured arterial cells, but also macrophage-lineage cells (54), mesangial cells, and adipocytes have been observed to respond to soluble uric acid in the hyperuricemic range (27, 57). As such, the reported ability of soluble uric acid to induce oxidative stress, inflammatory responses such as NF-κB and MAPK activation, and chemokine expression in cells, if valid, has huge potential ramifications. As one example, hyperuricemia in humans has been suggested to promote heightened ex vivo proinflammatory responses of neutrophils (58). However, the proposal that hyperuricemia predisposes to gouty arthritis by inflammatory effects beyond the promotion of monosodium urate crystal formation remains provocative, and gout could primarily be a signal of heightened inflammatory responses of phagocytes.

Collectively, results of studies of high ambient uric acid in cell culture and studies of oxonic acid–treated rats cannot be simply extrapolated to humans. Indeed, randomized trials in humans of therapeutic agents with uricosuric effects such as sulfinpyrazone and estrogen have had conflicting cardiovascular outcomes (59–63). Importantly, trials of the XOR inhibitor febuxostat have not demonstrated a cardiovascular benefit. In fact, an increase in cardiovascular events (although not statistically significant) in those who took febuxostat compared with allopurinol in the clinical trials program necessitated further study of the cardiovascular safety of febuxostat (64–67). Potential beneficial effects of serum urate lowering on vascular function have the potential to be outweighed by effects of increased flares of gout, which are well documented to occur with more intense serum urate–lowering regimens and can induce systemic inflammation, with release of proatherogenic cytokines (e.g., interleukin-1β [IL-1β], tumor necrosis factor α [TNFα], IL-6, IL-8) and other mediators. Further, in the LIFE study described above, losartan may have had other beneficial effects beyond those of beta-blockers, and in the SHEP trial, mitigation of beneficial effects for stroke or “any cardiovascular event” was not demonstrated, raising concerns about a false-positive result for the coronary heart disease outcome. Thus, it is not clear based on these collective human trials whether uric acid and/or arthritis-related inflammation have a pathogenic role or whether serum urate level is a biomarker or an epiphenomenon.

Acceptance of the aforementioned results for soluble uric acid effects on vascular cells in vitro also assumes that inflammatory artifacts of submicroscopic, cell-modulated urate crystallization have been adequately excluded, particularly when assessing results using uric acid concentrations well above 7 mg/dl. Vascular cell uptake of uric acid has been reported, and linkage with intracellular uric acid oxidation is plausible and has translational relevance, since vascular cells can express urate anion transporters and uricosurics inhibit the capacity of uric acid to activate vascular cells (38, 41). One can argue that the uptake kinetics of uric acid by cultured ECs and SMCs in studies published to date do not appear robust (38, 39, 41), and uricosurics have nonselective effects on ion transport and cell physiology. However, extracellular (and indirect) effects of uric acid on NO and on oxidative stress are likely sufficient to drive certain “inflammatory” effects of high levels of uric acid on vascular cells.

Our appraisal is that host factors, such as intrinsic inflammation and/or oxidative stress in gout, congestive heart failure (CHF), metabolic syndrome, diabetes, and chronic kidney disease, are likely major determinants of whether high levels of soluble uric acid are benign or promote pathology without crystallization. One such host factor to consider in this context is activity of the XOD form of XOR.

XOD, vascular oxidative stress, and inflammation

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

Irrespective of its role as an antioxidant, prooxidant, or both, the nature of localized vascular urate production (by XOD versus by XDH) could be more relevant than the systemic serum urate level, which reflects multiple influences on uric acid production and elimination. This may particularly be the case in atherosclerotic plaques, where substantial concentrations of both uric acid and allantoin have been found (68–70). This can be considered analogous to local microenvironment uric acid concentrations within joints affected by gout or areas of tophaceous deposits.

Local uric acid levels are influenced by XOR activity. XOR is widely distributed throughout various human organs including the liver, gut, lung, kidney, heart, and brain (3), with the highest levels in the gut and liver (5). In the myocardium, it is localized to the capillary ECs (7). Mammalian XOR is present in vivo as the dehydrogenase form (XDH) but is easily converted to XOR by oxidation of the sulfhydryl residues or by proteolysis (3). Although XDH has a much greater affinity for NAD+ compared to oxygen (and therefore is practically incapable of directly producing reactive oxygen species [ROS]), both XOR and XDH can oxidize NADH, which results in ROS formation (6). Fully reduced XOD contains 6 electrons, and its reoxidation involves electron transfer to oxygen molecules, which generates two H2O2 and two O2 species (71) for every fully reduced XOD molecule. XDH can theoretically produce more O2 per mole of oxygen during NADH oxidation than can XOR. However, studies using rat liver indicate that the rate of reaction is very slow (25% of XOD Vmax, where Vmax is the maximum reaction rate at which the enzyme catalyzes the reaction) (72). Unlike XDH, XOR has very little reactivity with NADH (73).

Regulation and functions of XOD

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

Variability in human XOR expression can be severalfold, and on average, expression is 20% higher in men than in women (74). Although basal expression of XOR is low in humans, XOR transcription can be increased by hypoxia, ischemia-reperfusion, IL-1, IL-6, TNFα, lipopolysaccharide, and corticosteroid treatment (4). XDH conversion to XOD is also accelerated in hypoxia (75). XOD is significantly elevated in a variety of conditions including limb ischemia (76), major surgery (77), coronary artery disease (CAD) (78), and heart failure (79). XOD is also up-regulated in chronic obstructive pulmonary disease (80) and by tobacco smoke in pulmonary artery ECs (81). NO is an endogenous suppressor of XOD (3); therefore, reduced tonic NO suppression of XOD promotes oxidative stress and endothelial dysfunction (82).

Circulating XOD binds to glycosaminoglycans on the surface of ECs, where it can acquire modified kinetics (higher Km and Ki and oxidant-producing capacity and increased stability) (83). This form of circulating and depositing XOD appears to be more important in the pathogenesis of endothelial injury than XOD constitutively produced from ECs (84).

The effects of XOD on both EC function and inflammation appear substantial. For example, when infused acutely, XOD produced a decrease in cardiac contractility, cardiac index, and left ventricular systolic pressure in anesthetized dogs (85). XOR and uric acid have both been implicated in evolution of innate immune inflammatory responses (86, 87). XOR expression is inducible by macrophage differentiation, the chemokine monocyte chemotactic protein 1, and Th1 cytokines in monocyte/macrophage-lineage cells (54). XOR promotes inflammatory differentiation, caspase 1 activation, IL-1β release, and chemokine expression in these cells, partly mediated by effects on hypoxia-inducible factor 1α and on peroxisome proliferator–activated receptor γ sumoylation (54, 88). Interestingly, forced expression of XOR, as well as exogenous uric acid and oxonate, appear to suppress alternative, antiinflammatory M2 macrophage differentiation (54).

Allopurinol as a “probe” into noxious XOD activity in arteries

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

Allopurinol has been demonstrated to improve endothelial function in humans in several studies, including 2 placebo-controlled crossover trials in CHF (89, 90) and a small randomized controlled trial (RCT) in patients with type 2 diabetes and mild hypertension (91). In an experimental murine model of myocardial infarction in which myocardial XOD levels increased, allopurinol significantly attenuated left ventricular dilatation, hypertrophy, fibrosis, and dysfunction (92). Allopurinol in combination with vitamins C and E appeared to be beneficial following coronary artery bypass surgery, where reduced ischemic events and less ST segment depression were noted with this regimen (93). Moreover, 600 mg/day of allopurinol significantly improved endothelial function as well as indices of vascular stiffness (measured by pulse wave analysis) in optimally treated patients with CAD (94). Compared with placebo, allopurinol at 600 mg/day also significantly increased time to chest pain and ST segment changes in CAD patients undergoing exercise electrocardiogram testing (95).

Other studies have demonstrated conflicting results. Oxypurinol administration (600 mg/day) improved left ventricular ejection fraction in a post hoc analysis of only a select subset of patients whose baseline ejection fraction was <40% in a small RCT (96); in another large RCT in CHF, no benefit was seen except for those whose serum urate levels were >9.5 mg/dl (97). Direct infusion of oxypurinol improved endothelial function in persons with hypercholesterolemia, but not in those with hypertension (98). Exercise capacity has also been evaluated as an end point in RCTs, with high-dose (600 mg/day) allopurinol being associated with improved exercise capacity in unstable angina (95), while 300 mg/day did not improve exercise capacity in CHF (99). It is not clear whether some of these differences are related to dose, formulation (allopurinol versus oxypurinol), or disease physiology, let alone to XOD or uric acid.

It is also not clear if positive effects of XOD inhibition on vascular function are mediated by “oxygen-sparing” effects (due to reduced consumption of molecular oxygen by XOD during periods of ischemia), a purine salvage mechanism due to reduced hypoxanthine breakdown, or other mechanisms. It is difficult to support a direct effect of lower circulating uric acid levels as an explanation, as the relationship of allopurinol to improved endothelial function has not been consistently associated with the extent of urate lowering (90). Moreover, the uricosurics probenecid (90) and benzbromarone (100) had neutral effects on endothelial function in studies of CHF. Compelling effects of XOD (and allopurinol) on myocardial oxygen consumption and mechanoergetics are being actively investigated (101–108). However, certain clinical studies in CHF that have actively lowered urate failed to demonstrate improvements either in New York Heart Association class (109), as in the Oxypurinol Therapy in Chronic Heart Failure trial (110, 111), or in other measures of clinical improvement, such as the 6-minute walk test (99). In contrast to these studies of urate lowering, direct uric acid infusion has also been studied as discussed above for its potential beneficial endothelial effects with conflicting results (either positive or neutral).

Currently, any evidence for beneficial effects of allopurinol in vascular disease need to be interpreted as potentially reflective of effects on XOR, uric acid levels, nonspecific drug effects on pyrimidine metabolism, or all of these. Allopurinol also has some direct antioxidant effects (112–115), although these may only be physiologically significant at high doses (116, 117). In addition, dosages of allopurinol that affect endothelial dysfunction may not be the same as those needed to potentially modulate inflammation or simply reduce uric acid. In this context, a statin, but not allopurinol at 300 mg/day, significantly suppressed circulating MPO in a small clinical study (118). Finally, effects of XOR inhibition on accumulation of upstream precursors such as inosine and adenosine (119–121) have also been proposed to contribute to beneficial effects of XOR inhibition in models of vascular disease and in pain. Adenosine has antiinflammatory properties and protects ECs from leukocyte-mediated injury, and inosine suppresses certain phagocyte functions and inhibits experimental inflammation (122–125).

Next steps needed in understanding the role of uric acid and XOD in vascular disease

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

Despite the substantial body of evidence regarding the role of uric acid and XOD in vascular disease summarized herein, this review emphasizes the conflicting results on the effects of uric acid and XOD in human vascular biology. We have underlined why speculation remains regarding direct roles of uric acid or XOD, or both (or neither), in human vascular pathology. Briefly, numerous observational epidemiologic studies have examined the association of serum urate levels with cardiovascular end points and proxies for those end points, with conflicting results. Moreover, some studies have demonstrated neutral or even positive effects of direct uric acid infusion, while others show a negative effect of elevated uric acid and/or XOD in humans.

Several methodologic challenges limit our ability to make definitive conclusions from these studies. It could be argued that there is sufficient clinical equipoise to justify a large-scale clinical trial to evaluate these effects. It would not be feasible to infuse uric acid in such a setting; instead, with the bulk of the evidence supporting a potential negative effect of uric acid or XOD, the effects of lowering uric acid and of inhibiting XOR need to be tested. A definitive evaluation is needed to disentangle the effects of uric acid lowering from those of XOR inhibition. This can only be achieved by testing mechanisms of lowering uric acid that do not rely solely on XOR inhibition, such as with use of uricosuric agents. A few small studies have provided intriguing preliminary data to suggest that it is XOR inhibition rather than uric acid lowering that is conferring benefit, but this needs to be rigorously studied in large RCTs. Should high-quality RCT(s) demonstrate a beneficial effect of XOR inhibition rather than simply the lowering of uric acid itself, uric acid would still play a role in clinical management of vascular disease as a biomarker of effective XOR inhibition. The need for such trials to definitively answer these questions needs to be balanced with the potential costs and adverse effects of these agents.

Does the lack of evidence of a protective cardiovascular effect from febuxostat trials in gout dampen the enthusiasm for testing the utility of either uric acid lowering or XOR inhibition in management of vascular disease? We would argue that it does not. These trials did not have sufficient power or durations needed to detect differences in cardiovascular outcomes. Additionally, it could be argued that patients with gouty arthritis may not be an optimal model in which to assess these effects. The inflammatory nature of early urate-lowering therapy–induced exacerbation of gouty arthritis may outweigh any potential benefit conferred by uric acid lowering or XOR inhibition and might be clouded by use of certain types of gout attack prophylaxis and treatment, including nonsteroidal antiinflammatory drugs (NSAIDs) and corticosteroids. Such effects of acute gouty arthritis could be analogous to the capacity of infectious disease stimuli to trigger vascular events (126). Nonetheless, evaluation of uric acid lowering by XOR inhibition and uricosurics is warranted in a population of persons at risk for vascular events, which includes those with gout. With a sufficiently powered trial of long enough duration, the theoretical confounding inflammatory effects of gout (which have not yet been established as a risk factor for cardiovascular events) and the effects of NSAIDs on inflammation may not be an issue.

In addition to vascular outcomes, trials adequately powered to assess hypertension as an outcome would also be useful. Finally, evaluation of specific biomarkers may help shed light on pathophysiologic mechanisms, including those related to uric acid oxidation and NO metabolism, by which uric acid–lowering agents, with and without modulation of gout inflammation, may be exerting their effects on vascular disease. Figure 3 provides a schematic of the currently available evidence base as well as a road map of the necessary components of future studies to enable more definitive evaluation of these unanswered questions in gout and asymptomatic hyperuricemia.

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Figure 3. Schematic of currently available clinical evidence base from which inferences regarding vascular effects of uric acid (UA) (and xanthine oxidase [XOD]) have been made. The schematic summarizes key needs and appropriate design considerations for future studies to enable truly definitive clinical evaluation of the role of uric acid and/or XOD in vascular disease. XOR = xanthine oxidoreductase; SUA = soluble uric acid; CV = cardiovascular; RCT = randomized controlled trial; NO = nitric oxide; CRP = C-reactive protein.

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Conclusions

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES

Uric acid is not an inert end product of purine catabolism in humans, and uric acid can act as an antioxidant or prooxidant. XOD, which generates uric acid, also induces oxidative stress, and both uric acid and XOD may promote inflammation. Large bodies of in vitro and animal model evidence implicate pathogenic effects of hyperuricemia and XOD that promote endothelial dysfunction and certain vascular pathologies. The bulk of epidemiologic evidence in humans also supports the hypothesis that hyperuricemia is an independent risk factor for certain vascular diseases and complications of atherosclerosis. Host factors, such as intrinsic inflammation and/or oxidative stress in gout, CHF, metabolic syndrome, diabetes, and chronic kidney disease, are likely major determinants of whether high levels of soluble uric acid are benign or promote pathology without monosodium urate crystallization. One such host factor is very likely XOD activity in the vasculature.

Given the prevalence of both gout and asymptomatic hyperuricemia, larger, randomized, well-controlled, and prospective clinical trials using XOR inhibition and other strategies to lower serum urate are urgently required. Future clinical trials in this area should be accompanied by appropriate monitoring of biomarkers (e.g., NO metabolism, allantoin, and renin–angiotensin–aldosterone axis activity) and changes in arterial pathology (e.g., atherosclerotic plaque size and stability) by sensitive, advanced imaging. Due to the huge scope of the problems of gout and hyperuricemia, such large, prospective, and well-designed clinical trials are urgently needed to resolve the putative toxic roles of hyperuricemia and XOD in the human arterial circulation.

REFERENCES

  1. Top of page
  2. Introduction
  3. Uric acid is not an inert end product of purine metabolism in humans
  4. Uric acid is both an antioxidant and a prooxidant
  5. Uric acid indirectly and directly regulates vascular cell functions in vitro, with apparent consequences in vivo
  6. Critical appraisal of data regarding potential vascular toxicity of uric acid
  7. XOD, vascular oxidative stress, and inflammation
  8. Regulation and functions of XOD
  9. Allopurinol as a “probe” into noxious XOD activity in arteries
  10. Next steps needed in understanding the role of uric acid and XOD in vascular disease
  11. Conclusions
  12. AUTHOR CONTRIBUTIONS
  13. REFERENCES
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