The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury

doi:10.1016/0003-9861(92)90743-G

Archives of Biochemistry and Biophysics

Volume 294, Issue 2, 1 May 1992, Pages 695-702

https://doi.org/10.1016/0003-9861(92)90743-G Get rights and content

Abstract

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O₂; 5% CO₂) for 0–48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH + XO activity decreased 98% after 48 h of 95% O₂ exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH + XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8-¹⁴C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH + XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K₃Fe(CN)₆). After 48 h of culture in air with allopurinol, cell XDH + XO activity was enhanced when assayed after reversal of inhibition with K₃Fe(CN)₆, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K₃Fe(CN)₆ oxidation, XDH + XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH + XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O₂⁻ and H₂O₂ during normoxia.

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XO is a source of ROS in hypoxia and ischemia/reperfusion injury, which increases the levels of xanthine and hypoxanthine and the conversion of XD to XO [96]. In these conditions, XO could be released in the blood, and bind glycosaminoglycans on endothelial cell surface, leading to NO inactivation and inhibition of NO-dependent SMC relaxation [92,97]. In humans, electron spin resonance studies allowed to show that endothelial dysfunction observed in CAD patients would be associated with an increased XO activity [98].
Atherosclerosis is a multifactorial chronic and inflammatory disease of medium and large arteries, and the major cause of cardiovascular morbidity and mortality worldwide. The pathogenesis of atherosclerosis involves a number of risk factors and complex events including hypercholesterolemia, endothelial dysfunction, increased permeability to low density lipoproteins (LDL) and their sequestration on extracellular matrix in the intima of lesion-prone areas. These events promote LDL modifications, particularly by oxidation, which generates acute and chronic inflammatory responses implicated in atherogenesis and lesion progression. Reactive oxygen species (ROS) (which include both free radical and non-free radical oxygen intermediates), play a key-role at each step of atherogenesis, in endothelial dysfunction, LDL oxidation, and inflammatory events involved in the initiation and development of atherosclerosis lesions. Most advanced knowledge supporting the “oxidative theory of atherosclerosis” i.e. the nature and the cellular sources of ROS and antioxidant defences, as well as the mechanisms involved in the redox balance, is based on the use of genetically engineered animals, i.e. transgenic, genetically modified, or altered for systems producing or neutralizing ROS in the vessels. This review summarizes the results obtained from animals genetically manipulated for various sources of ROS or antioxidant defences in the vascular wall, and their relevance (advance or limitation), for understanding the place and role of ROS in atherosclerosis.
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Experimental studies have revealed the possibility that circulating XO released from XO-rich organs under pathophysiological conditions is bound to glycosaminoglycans on the surface of endothelial cells and may be subsequently endocytosed into intracellular compartments, resulting in the inhibition of NO-dependent vascular smooth muscle cell relaxation [23]. These findings suggest that not only XO produced endogenously in endothelial cells but also inducible circulating XO released from XO-rich organs is an important source of ROS contributing to endothelial dysfunction [24]. Xanthine oxidoreductase has been shown to be involved in the transformation of macrophages into foam cells [25].
Uric acid is the end product of purine metabolism catalyzed by xanthine oxidase in humans. In the process of purine metabolism, reactive oxygen species, including superoxide, are generated concomitantly with uric acid production, which may deteriorate endothelial function through the reaction of superoxide with nitric oxide (NO), leading to decreased NO bioavailability and increased production of peroxynitrite, a reactive oxidant. Therefore, xanthine oxidase may be a therapeutic target in the treatment of endothelial dysfunction. Indeed, clinical studies have shown that endothelial dysfunction is restored by treatment with a xanthine oxidase inhibitor in patients with cardiovascular risk factors. However, it has not been fully determined whether uric acid per se is an independent causal risk factor of endothelial dysfunction in humans. Although experimental studies have indicated that uric acid absorbed into endothelial cells via the activation of uric acid transporters expressed in endothelial cells causes endothelial dysfunction through increased oxidative stress and inflammation, an actual biological effect of uric acid on endothelial function in vivo has not been fully elucidated, in part, because of the difficulty in investigating the effect of uric acid alone on endothelial function due to the close associations of uric acid with other conventional cardiovascular risk factors and the complicated relationship between uric acid and endothelial function attributed to the potent antioxidant properties of uric acid. In this review, we focus on the relationship between uric acid and endothelial function from molecular to clinical perspectives.
The treatment of hyperuricemia
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Indeed, the administration of febuxostat is able to reduce nitro-oxidative stress highlighted by nitrotyrosine formation, lipid peroxidation and over-expression of various inflammation-related molecules promoting the onset and the progression of the inflammatory state [20,24]. In addition, febuxostat inhibits circulating XO, preventing its binding to the surface of endothelial cells by glycosaminoglycans (GAGs), a process involved in the pathogenesis of endothelial injury [25]. This novel drug is also beneficial in the CHF as well as during the initial phase of left ventricular remodeling and cardiac functional deterioration after myocardial infarction.
Hyperuricemia has long been established as the major etiologic factor in gout. Alongside with an inflammatory state triggered by urate crystal deposition in the joints, hyperuricemia displayed additional pathophysiological consequences leading to tissue inflammation mainly in the vascular wall. Thus, therapeutic strategies used to treat hyperuricemia in the past decades have often been focused on limiting acute episodes. Recently, evidence has been accumulated suggesting that chronic urate deposition requires a correct treatment not limited to acute episodes based on the modulation of the activity of key enzymes involved in metabolism and excretion of urate including xanthine oxidoreductase (XO) and URAT1. The present review article will try to summarize the most recent evidences on the efficacy of XO inhibitors and uricosuric compounds in lowering uric acid levels in both the bloodstream and peripheral tissues. In particular, we will focus on the effect of novel XO inhibitors in counteracting uric acid overproduction. On the other hand, the effect of lowering uric acid levels via XO inhibition will be correlated with attenuation oxidative stress which leads to endothelial dysfunction thereby contributing to the pathophysiology of diabetes, hypertension, arteriosclerosis, and chronic heart failure. Hence, scavenging and prevention of the XO generated oxygen radical accumulation emerge as an intriguing novel treatment option to counteract uric acid-induced tissue damages.
Cellular distribution, metabolism and regulation of the xanthine oxidoreductase enzyme system
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Recently, it has been shown that cellular oxygen concentration may be an important physiological factor for XOR regulation. Hyperoxia has previously been shown to decrease XOR activity in cell culture and rat lungs [65,66], while hypoxia was shown to induce a gradual increase in XOR activity in endothelial cells [28,67]. More recent studies have shown that in acutely hypoxic (0–24 h) Swiss 3T3 cells, XOR activity increased without a concomitant increase in XOR de novo synthesis or mRNA levels.
Xanthine oxidase (EC 1.1.3.22) and xanthine dehydrogenase (EC 1.1.1.204) are both members of the molybdenum hydroxylase flavoprotein family and represent different forms of the same gene product. The two enzyme forms and their reactions are often referred to as xanthine oxidoreductase (XOR) activity. Physiologically, XOR is known as the rate-limiting enzyme in purine catabolism but has also been shown to be able to metabolize a number of other physiological compounds. Recent studies have also demonstrated its ability to metabolize xenobiotics, including a number of anticancer compounds, to their active metabolites. During the past 10 years, evidence has mounted to support a role for XOR in the pathophysiology of inflammatory diseases and atherosclerosis as well as its previously determined role in ischemia-reperfusion injury. While significant progress has recently been made in our understanding of the physiological and biochemical nature of this enzyme system, considerable work still needs to be done. This paper will review some of the more recent work characterizing the interactions and the factors that influence the interactions of XOR with various physiological and xenobiotic compounds.
Binding of xanthine oxidase to vascular endothelium: Kinetic characterization and oxidative impairment of nitric oxide-dependent signaling
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Concentrations of up to 1.5 milliunits/ml xanthine oxidase (XO) (1.1 μg/ml) are found circulating in plasma during diverse inflammatory events. The saturable, high affinity binding of extracellular XO to vascular endothelium and the effects of cell binding on both XO catalytic activity and differentiated vascular cell function are reported herein. Xanthine oxidase purified from bovine cream bound specifically and with high affinity (K _d = 6 nm) at 4 °C to bovine aortic endothelial cells, increasing cell XO specific activity up to 10-fold. Xanthine oxidase-cell binding was not inhibited by serum or albumin and was partially inhibited by the addition of heparin. Pretreatment of endothelial cells with chondroitinase, but not heparinase or heparitinase, diminished endothelial binding by ∼50%, suggesting association with chondroitin sulfate proteoglycans. Analysis of rates of superoxide production by soluble and cell-bound XO revealed that endothelial binding did not alter the percentage of univalent reduction of oxygen to superoxide. Comparison of the extent of CuZn-SOD inhibition of native and succinoylated cytochrome creduction by cell-bound XO indicated that XO-dependent superoxide production was occurring in a cell compartment inaccessible to CuZn-SOD. This was further supported by the observation of a shift of exogenously added XO from extracellular binding sites to intracellular compartments, as indicated by both protease-reversible cell binding and immunocytochemical localization studies. Endothelium-bound XO also inhibited nitric oxide-dependent cGMP production by smooth muscle cell co-cultures in an SOD-resistant manner. This data supports the concept that circulating XO can bind to vascular cells, impairing cell function via oxidative mechanisms, and explains how vascular XO activity diminishes vasodilatory responses to acetylcholine in hypercholesterolemic rabbits and atherosclerotic humans. The ubiquity of cell-XO binding and endocytosis as a fundamental mechanism of oxidative tissue injury is also affirmed by the significant extent of XO binding to human vascular endothelial cells, rat lung type 2 alveolar epthelial cells, and fibroblasts.
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¹: Permanent address: Department of Biochemistry, Faculty of Medicine, University of the Republic, Montevideo, Uruguay CP11800.

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The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury

Abstract

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Gastroenterology

Arch. Biochem. Biophys.

Biochem. Pharmacol.

J. Biol. Chem.

J. Surg. Res.

Arch. Biochem. Biophys.

Anal. Biochem.

Anal. Biochem.

Free Radicals Biol. Med.

J. Biol. Chem.

Free Radicals Biol. Med.

Arch. Biochem. Biophys.

Free Radicals Biol. Med.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Acta Physiol. Scand.

J. Clin. Invest.