Hypoxia/Reoxygenation increases the permeability of endothelial cell monolayers: Role of oxygen radicals

Inauen, W.; Payne, D. Keith; Kvietys, Peter R.; Granger, D. Neil

doi:10.1016/0891-5849(90)90031-d

Cited by 106 publications

(48 citation statements)

References 27 publications

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“…Xanthine oxidase (XO) is an endothelial cell (EC)-associated enzyme that is purported to be a key source of toxic 02 metabolites in acute reperfusion injury of ischemic tissues (1). This possibility is supported by studies showing that in cultured EC exposed to brief periods of anoxia, endogenous XO generates increased levels of superoxide anion (O-f), resulting in cell injury and dysfunction (2)(3)(4). In this acute setting, conversion of xanthine dehydrogenase (XD) to XO occurs and accounts for increases in XO, but total activity (XD plus XO) does not increase (3).…”

mentioning

confidence: 62%

Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity.

Terada

Guidot

Leff

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

177

104

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Exposure to decreasing oxygen tensions progressively increased xanthine dehydrogenase (XD) and xanthIne oxidase (XO) activities over 48 hr in cultured pulmonary artery endothelial cells (EC) without altering XD/XO ratios. Increases in XD and XO activity in EC induced by hypoxia were associated upon reoxygenation with increased (P < 0.05) extraceflular superoxide anion (O2 ) levels that were inhibited by treatment with XO inhibitors (tungsten, allopurinol) or an anion-channel blocker (4,4'-diisothiocyanatoslbene-2,2'-disulfonic acid). EC monolayers subjected to hypoxia/reoxygenation also leaked more preioaded 51Cr, were more adherent to neutrophils, and permitted greater albumin transit than control monolayers.Treatment with tungsten, aflopurinol, and/or superoxide dismutase decreased (P < 0.05) 5'Cr release, neutrophil adherence, and albumin transit in EC monolayers exposed to hypoxia/reoxygenation. We conclude that prolonged hypoxia increases both XO and XD activity in EC and may predispose the endothelium to oxidative and inflammatory damage. Recently, we found a marked suppression of XD and XO activity in EC exposed to high oxygen tensions (6). Accordingly, we examined the effects of prolonged hypoxia on EC XD and XO activities. We found that prolonged hypoxia increased both XD and XO activities in cultured EC and, further, that increases in XD and XO activities contributed to 0j -mediated injury and neutrophil adherence to EC. Measurement of XD and XO Activity. EC were grown in 75-cm2 flasks (Falcon, Becton Dickinson). Medium was removed from confluent EC monolayers and replaced with Hanks' balanced salt solution/5% fetal calf serum; then flasks were placed in sealed chambers, flushed with 5% C02/0-95% 02 and 95-0o N2, and incubated at 370C. 02 tensions of parallel saline samples measured with a Clark electrode were 15, 33, 66, 120, and 660 torr (1 torr = 133.3 Pa) after 48-hr incubations in 0, 3, 10, 21, and 95% 02, respectively, at Denver altitude. In some experiments, EC were grown for three-passages in sodium tungstate (10 ppm final) before use; or allopurinol (100 AM), 4,4'-diisocyanatostilbene-2,2'-disulfonic acid (DIDS, 100 ,AM), or SOD (10 jug/ml) was added to EC before incubation. Monolayers of EC were triply washed with phosphate-buffered saline (20 mM, pH 7.4), mechanically harvested with a plastic policeman in 50 mM phosphate buffer, pH 7.8/1 mM EDTA/1 mM phenylmethylsulfonyl fluoride/1 mM dithioerythritol/SOD at 20 ,ug/ml/catalase at 20 ,ug/ml and sonicated on ice. Nuclei were removed by centrifugation, and supernatants were desalted by using a Centricon filter system. Aliquots (0.25 ml) were incubated aerobically at 370C for 3 hr with xanthine (200 AuM) to measure XO activity or xanthine plus NAD' (300 AM) to measure XO plus XD activity in a final volume of 0.5 ml. Lactic dehydrogenase (70 units per ml) and pyruvate (1.75 mM) were added to tubes containing NAD' to prevent inhibition of XD by NADH (9). Baseline values were obAbbreviations: XO, xanthine oxidase; O-., superoxide anion; XD,...

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mentioning

confidence: 62%

Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity.

Terada

Guidot

Leff

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

177

104

View full text Add to dashboard Cite

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“…Endothelial cells produce ROS in response to cytokines (Matsubara and Ziff, 1986) and to hypoxia/re-oxygenation (Ratych et al, 1987;Inauen et al, 1990;Lum et al, 1992;Zweier et al, 1988Zweier et al, , 1994aTerada, 1996). ROS have been implicated in the mechanism of damage following ischaemia and during low-flow hypoxia (McCord, 1985).…”

Section: Discussionmentioning

confidence: 99%

“…Angiogenesis involves intense endothelial cell cytokine-dependent proliferation and hypoxia/re-oxygenation. Cultured endothelial cells produce reactive oxygen species (ROS) spontaneously (Sundqvist, 1991;Terada et al, 1991) and this is augmented by hypoxia/reoxygenation (Ratych et al, 1987;Inauen et al, 1990;Lum et al, 1992;Zweier et al, 1988Zweier et al, , 1994aTerada, 1996) as well as by cytokines (Matsubara and Ziff, 1986). In vivo, oxygen radicals are also produced as by-products of normal oxidative metabolism (Malmstrom, 1982).…”

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confidence: 99%

Thiram inhibits angiogenesis and slows the development of experimental tumours in mice

Marikovsky

2002

Br J Cancer

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Thiram-tetramethylthiuram disulphide -a chelator of heavy metals, inhibited DNA synthesis and induced apoptosis in cultured bovine capillary endothelial cells. Bovine capillary endothelial cells were 10 -60-fold more sensitive to thiram than other cell types. These effects were prevented by addition of antioxidants, indicating involvement of reactive oxygen species. Exogenously added Cu 2+ impeded specifically and almost completely the inhibitory effect of thiram for bovine capillary endothelial cells. Moreover, thiram had markedly inhibited human recombinant Cu/Zn superoxide dismutase enzymatic activity (85%) in vitro. Moreover, PC12-SOD cells with elevated Cu/Zn superoxide dismutase were less sensitive to thiram treatment than control cells. These data indicate that the effects of thiram are mediated by inhibition of Cu/Zn superoxide dismutase activity. Oral administration of thiram (13 -30 mg mouse 71 ), inhibited angiogenesis in CD1 nude mice. Tumour development is known to largely depend on angiogenesis. We found that oral administration of thiram (30 mg) to mice caused significant inhibition of C6 glioma tumour development (60%) and marked reduction (by 3 -5-fold) in metastatic growth of Lewis lung carcinoma. The data establish thiram as a potential inhibitor of angiogenesis and raise the possibility for its use as therapy in pathologies in which neovascularisation is involved, including neoplasia.

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“…Epithelial injury can for example be mimicked by addition of detergents like Triton X-100 for a short amount of time [32]. Ischemia can also be modeled by replacing O 2 by N 2 [22]. Psychological stress can also be mimicked by adding into the chambers the neurotransmitters released after a stress like CRH, norepinephrine or ACTH [36].…”

Section: Type Of Experiments Performed With Ussing Chambersmentioning

confidence: 99%

The Ussing chamber technique to evaluate alternatives to in-feed antibiotics for young pigs

Boudry

2005

Anim. Res.

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-The recent ban of in-feed antibiotics in animal production has brought scientists to find alternatives to in-feed antibiotics. The Ussing chamber is an in vitro technique in which intestinal tissue is collected and immediately mounted as a flat sheet between two half-chambers, establishing a luminal and a serosal side. This technique allows the measurement of actively transported ions as well as the permeability of the tissues, two parameters relevant for the evaluation of gut health. Ussing chambers have been used to describe the changes of intestinal physiology occurring at weaning. We observed that weaning induces an acute alteration of the epithelial barrier function and electrolyte transport in the first days. Long-lasting modifications (15 days after weaning) have also been observed. Alternatives to in-feed antibiotics can be evaluated by studying the intestinal physiology of animals treated with those alternatives. A benefic effect of pro-and pre-biotics on the rat intestine, human biopsies or epithelial cell monolayers has been demonstrated using Ussing chambers. In pigs, the influence of the source of dietary ingredients or the physical form of the diet seems limited. A few results obtained in pigs with pre-or probiotics appear less convincing than in laboratory animals. Another way to use Ussing chambers is to incubate the intestinal tissue with different substances added directly into the chambers. Some substances can be detrimental to the intestine, inducing electrolyte secretion or decreasing barrier function. However, some substances show a beneficial effect. Although this approach has limits and should be combined with in vivo measurements, it constitutes a rapid way to evaluate the effect of substances on intestinal physiology and can also, at least partly, elucidate the mechanisms of action of those alternatives. perturbations intestinales au moment du sevrage. Nous avons ainsi pu montrer que le sevrage induit des altérations transitoires de la physiologie intestinale ainsi que des modifications à plus long terme. Les alternatives aux antibiotiques peuvent être évaluées par l'étude de la physiologie intestinale des animaux traités avec ces alternatives. Un effet bénéfique des pro-et prébiotiques sur l'intestin de rat, des biopsies intestinales humaines ou des monocouches de cellules épithéliales intestinales a été démontré en chambres d'Ussing. Chez le Porc, l'influence des sources de matières premières ou de la forme physique de l'aliment semble limitée. De plus, les rares résultats obtenus chez le porc quant à l'effet des pré-et probiotiques semblent moins convaincants que chez les animaux de laboratoire. Une autre façon d'utiliser les chambres d'Ussing est d'incuber l'intestin avec différentes substances ajoutées directement dans les chambres. Certains composés semblent avoir un effet délétère sur l'intestin, induisant des sécrétions d'électrolytes ou des modifications de la barrière épithéliale. Cependant, certaines substances ont montré un effet bénéfique. Bien que cette technique...

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Hypoxia/Reoxygenation increases the permeability of endothelial cell monolayers: Role of oxygen radicals

Cited by 106 publications

References 27 publications

Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity.

Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity.

Thiram inhibits angiogenesis and slows the development of experimental tumours in mice

The Ussing chamber technique to evaluate alternatives to in-feed antibiotics for young pigs

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