Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity.

Abstract

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,...

Contribution of xanthine oxidase-derived superoxide to chronic hypoxic pulmonary hypertension in neonatal rats

“…Our observation of an increase in expression of XOR in the hypoxia-exposed lung, which was attenuated by Allopurinol, is in keeping with reports by other groups demonstrating hypoxia-induced upregulation of XO activity and XOR expression in vascular endothelium in vitro (34,62,63) and in the adult lung in vivo (25). Conversion of XOR to the oxidase form is believed to occur through two mechanisms: direct exposure to hypoxia through an adenosine-dependent process (34) and endothelial binding of circulating XO (26) secreted by other organs, such as the gut (5) and the liver (66).…”

Sequential activation of JAKs, STATs and xanthine dehydrogenase/oxidase by hypoxia in lung microvascular endothelial cells

“…We found that hypoxia-mediated XDH/XO activation is dependent on the JAK/ STAT pathway in primary cultures of LMVEC. We demonstrated that the XDH/XO activity significantly increased by hypoxia, consistent with the increased phosphorylation or activation of XDH/XO in response to hypoxia in other cells (Terada et al, 1992;Dupont et al, 1992;Kayyali et al, 2001;Mervaala et al, 2001;Terada et al, 1997;Poss et al, 1996;Sohn et al, 2003;Kang et al, 2006). Interestingly, our data demonstrated that XDH/XO activation induced by could be inhibited by either depletion of IL6 using IL6 antibodies, pretreatment with the JAK2 inhibitor AG490 or transient expression of the JAK-STAT pathway blocker SOCS3 and their inhibitory effect on the XDH/XO activation was at a similar magnitude.…”

“…It has been demonstrated that hypoxia activates XDH/XO, an important source of reactive oxygen species (ROS) (Terada et al, 1992;Dupont et al, 1992;Kayyali et al, 2001;Mervaala et al, 2001;Terada et al, 1997;Poss et al, 1996;Sohn et al, 2003;Kang et al, 2006). However, the molecular mechanism underlying the hypoxia-mediated activation of XDH/XO remains largely unknown.…”

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

“…XO was found to be much higher in capillary endothelial cells (Jarasch et al, 1986). Moreover, hypoxia was shown to injure endothelial cells by increasing xanthine oxidase activity (Terada et al, 1992). Another mechanism generating ROS that could preferentially affect endothelial cells has recently been identified (Beckman et al, 1990).…”