Cliff R. Stevens

Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions

Xanthine oxidoreductase (XOR) catalyses the reduction of the therapeutic organic nitrate, nitroglycerin (glyceryl trinitrate, GTN), as well as inorganic nitrate and nitrite, to nitric oxide (NO) under hypoxic conditions in the presence of NADH. Generation of nitric oxide is not detectable under normoxic conditions and is inhibited by the molybdenum site‐specific inhibitors, oxypurinol and (−)BOF 4272. These enzymic reactions provide a mechanism for generation of NO under hypoxic conditions where nitric oxide synthase does not function, suggesting a vasodilatory role in ischaemia.

A Reappraisal of Xanthine Dehydrogenase and Oxidase in Hypoxic Reperfusion Injury: the Role of NADH as an Electron Donor

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophoresis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodonium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopurinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.

Xanthine oxidoreductase is asymmetrically localised on the outer surface of human endothelial and epithelial cells in culture

Subcellular localisation of xanthine oxidoreductase (XOR) was determined by indirect immunofluorescence using confocal microscopy in human endothelial and epithelial cell lines and in primary cultures of human umbilical vein endothelial cells. XOR was diffusely distributed throughout the cytoplasm but with higher intensity in the perinuclear region. In non‐permeabilised cells, XOR was clearly seen to be asymmetrically located on the outer surfaces, showing, in many cases, a higher intensity on those faces apposed by closely neighbouring cells. Such specific distribution suggests a functional role for the enzyme in cell‐cell interactions, possibly involving signalling via reactive oxygen species

Hypoxia in Abdominal Aortic Aneurysm Supports a Role for HIF-1α and Ets-1 as Drivers of Matrix Metalloproteinase Upregulation in Human Aortic Smooth Muscle Cells

Background/Aims: We sought to determine whether hypoxia is an initiating factor in the matrix metalloproteinase-2 (MMP-2) up-regulation observed in abdominal aortic aneurysm (AAA) and whether hypoxia-inducible factor-1α (HIF-1α) or Ets-1 are mediating factors. Methods: Human AAA and normal aorta were analysed for MMP-2, HIF-1α and Ets-1 by immunohistochemistry. Human aortic smooth muscle cell (HASMC) cultures exposed to experimental hypoxia were analysed for hypoxia-induced proteins using gelatin zymography and immunoblotting. Multiplex PCR was used to detect MMP-1, membrane-type (MT)-MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9. Results: AAA tissues expressed HIF-1α, MMP-2 and Ets-1 strongly within smooth muscle cells and inflammatory infiltrate of the tunica media. Up-regulated MMP-2 was detected in hypoxia-exposed HASMC (p < 0.05), with MMP-9 elevations after exposure to sequential O2 decreases (p < 0.05). Immunoblotting confirmed HIF-1α, Ets-1, VEGF and MMP-2 are up-regulated in HASMC exposed to hypoxia (p < 0.05), while transcription for MMP-1, MT-MMP-1, MMP-9, MMP-2 and MMP-7 (p < 0.05) increased in hypoxic HASMCs. Conclusion: Hypoxia facilitates HIF-1α, Ets-1 and VEGF up-regulation in addition to driving enhanced secretion of MMP-2 and MMP-9 by HASMC. Enhanced transcription of factors relevant to aneurysmal disease in hypoxia indicates possible roles in disease progression and potential targets for therapeutic intervention.

Reactive oxygen/nitrogen species and acute inflammation: A physiological process

There is an increasing body of experimental evidence implicating partially reduced forms of oxygen in a wide variety of pathological states and xenobiotic metabolism and associated toxicity. What is often dismissed, however, is the role of such species in physiology. Relatively recent evidence showing the signalling capacity of many forms of “reactive oxygen species” (ROS) has strengthened the view that they are not only associated with toxicity and pathology but have significant controlling influences in many physiological processes such as the acute inflammatory response. Recently, the biological signalling functions of nitric oxide, a “reactive nitrogen species” (RNS) have come to the fore. In this chapter, we summarise the characteristics of well known ROS and RNS, discuss the endogenous sources of such species in different environmental settings and describe how such species can bring about a physiological defence mechanism such as the acute inflammatory response.

Xanthine oxidase is a peroxynitrite synthase: newlyidentified roles for a very old enzyme

Xanthine oxidase (XO) was first identified in 1902 by the German scientist Schardinger in bovine milk by following the hydroxylation of hypoxanthine to xanthine. 1 Following this first discovery, the history of xanthine oxidase has followed the usual routes of investigation such as they were at the start of the 20th century. The electron donors and acceptors were identified and the constituents of the active sites were deduced. It turns out to be a classic, multicentred, redox enzyme. For many years the function of the enzyme in milk was questioned and various theories were proffered. These ideas ranged from being a carrier of iron and molybdenum to the infant, to purely an additional dietary protein, a notion still prevalent in the medical profession today. XO was thought not to have an enzymic role in milk. However, further thought on the matter brings to light the high, almost neutral pH of the neonatal gut in the first few weeks post partum. The acid pH, which develops later in life, acts as the primary defence to infective organisms via ingestion. In this time before the acid develops, other systems must be in place. The role of a radical generating enzyme may, therefore, be in the reduction of infective bacteria by the generation of superoxide and hydrogen peroxide. Unfortunately, the potency of these reactive oxygen species is dependent on a number of factors including detoxification enzymes in bacteria, particularly superoxide dismutases and catalase, and the availability of oxygen in the environment. In 1996, we began to study the nitrate reductase activity of XO, a largely ignored phenomenon, which culminated in the first description of nitrate and nitrite reductase activities with the formation of nitric oxide (NO) as the end product. 2‐4 In the light of these findings and our further studies, we have developed and tested the theory that XO-generated reactive molecules have antibacterial activities in the neonatal gut (see Fig. 1). It raises the possibility that, in areas where breast-feeding is not possible due to HIV infection in the mother, an alternative formula can be employed to reduce life-threatening diarrhoeal disease. This system acts as a low-cost, naturally occurring antibiotic for the reduction of infective bacteria. But first some background and comparative biochemistry.

Xanthine Oxidase Mediates Cytokine-induced, but not Hormone-induced Bone Resorption

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have been implicated as mediators of osteoclastic bone resorption. Xanthine oxidase (XO) a ubiquitous enzyme is widely known for its production of these ROS. We therefore evaluated the potential of XO as a source of ROS in cytokine- and hormone-induced bone resorption. XO activity in rat calvarial osteoblasts was found to be significantly elevated upon stimulation by the cytokines, TNF f and IL-1 g . These cytokines also caused a dose related increase in bone resorption of mouse calvariae, which was significantly inhibited by catalase (10 IU/ml). Allopurinol, the competitive inhibitor of XO, also caused a dose related (1-50 w M) inhibition of TNF f (20 ng/ml) and (0.01-10 w M) IL-1 g (50 IU/ml)-induced bone resorption, respectively. PTH- and 1,25-(OH)2 Vitamin D3-induced bone resorption could also be inhibited by catalase (100 IU/ml) but was unaffected by allopurinol, indicating that another mediator, other than XO, is required for hormone-induced bone resorption. These results demonstrate, that modulation of the redox balance in the bone microenvironment, which contains XO, can affect the bone resorbing process. Therefore, XO may play a pivotal role in cytokine-induced bone resorption and, if manipulated appropriately, could show a therapeutic benefit in inflammatory bone disorders such as RA.

Vascular physiology and pathology of circulating xanthine oxidoreductase: from nucleotide sequence to functional enzyme

Abstract The evolutionarily conserved, cofactor-dependent, enzyme xanthine oxidoreductase exists in both cell-associated and circulatory forms. The exact role of the circulating form is not known; however, several putative physiological and pathological functions have been suggested that range from purine catabolism to a mediator of acute respiratory distress syndrome. Regulation of gene expression, cofactor synthesis and insertion, post-translational conversion, entry into the circulation, and putative physiological and pathological roles for human circulating xanthine oxidoreductase are discussed.

Porcine-Derived Collagen as a Scaffold for Tissue Engineering

Porcine-derived dermal collagen has been studied as a potential scaffold for the production of tissue engineered products. The surface structure of the collagen has been examined by low temperature-scanning electron microscopy and reveals an open fibrous network. Human endothelial, fibroblast and smooth muscle cells were seeded, at densities of up to 1 × 10 6 cell ml -1 , onto 10 × 10 mm collagen sections or 16 mm diameter (2 cm 2 ) discs of either 0.75 or 1.5 mm thickness. The seeded collagen was incubated at 37°C and 5% CO 2 . Adhesion of the cells to the collagen matrix was observed, but this was not as high as in control experiments on tissue culture plastic. After washing of the collagen matrix, in PBS and culture media, endothelial and smooth muscle cells were observed to strongly adhere and proliferate on the matrix. A perfusion bioreactor was used for the controlled cultivation of human fibroblast cells. Cells were seeded onto collagen sheets and transferred to the bioreactor, where they were maintained for up to three weeks.

A novel method for quantifying cell migration through tissue engineering scaffolds: evaluation of modified collagen matrices

A novel method to quantify cell migration through potential tissue engineering 3-d scaffolds is described. The migration assay uses a dot-blotting apparatus into which the tissue engineering matrix is placed on top of a nitrocellulose membrane. This assay was used to evaluate human dermal fibroblast migration through four porcine collagen matrices with varying pore diameters and pitch lengths. Fibroblasts were placed on the matrix surface, at between 1 ×103–3 × 103 cells mm−2, and left for 18 h to allow migration. The nitrocellulose membrane was stained with haematoxylin, the membrane digitised and the pixel intensity of the stained cells quantified. We showed that for all matrix variants, migration was more effective with a higher initial seeding density. The application of varying initial cell densities resulted in the greatest extent of cell migration through the matrix variant with pores of 30 μm diameter and 400 μm pitch length (i.e. 10.3% migration at 1 ×103 cells mm−2). This method was coupled with confocal microscopy to evaluate the depth of cell migration within the matrix. At a depth of 20 μm cell numbers were similar to those on the matrix surface: at a depth of 100 μm only a few cells were observed.