Proc. Natl. Acad. Sci. USA Vol. 89, pp. 3362-3366, April 1992 Cell Biology
Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity (oxygen radicals/vascular injury/lung/superoxide anion/neutrophils)
LANCE S. TERADA*t, DAVID M. GuIDOT*, JONATHAN A. LEFF*, IRENE R. WILLINGHAM*, MICHAEL E. HANLEY*, DALE PIERMATTEI*, AND JOHN E. REPINE* Departments of *Medicine and SNeurosurgery at the University of Colorado Health Sciences Center and the Webb-Waring Lung Institute, Denver, CO 80262
Communicated by David W. Talmage, January 6, 1992
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.
MATERIALS AND METHODS Source of Reagents. Cytochrome c (horse heart, type VI) purchased from Sigma and succinoylated with succinic anhydride (7). Catalase (bovine liver, 81,536 units per mg) was purchased from Worthington. Xanthine sodium was obtained from Serva. Chromium isotope (51Cr, 545.2 mCi/ mg, 1.0 mCi/ml; 1 Ci = 37 GBq) was purchased from NEN. Superoxide dismutase (SOD, bovine erythrocyte, 3000 units per mg), lactate dehydrogenase (rabbit muscle type II, 1100 units per mg), and all other reagents were obtained from Sigma. Purification and Culture of EC. EC were harvested from bovine pulmonary arteries (8) and initially grown in 20%16 fetal calf serum with D-valine Eagle's minimal essential medium to eliminate fibroblast contamination. After the third passage, factor VIII and low density lipoprotein receptor staining revealed abluminal) by adjusting the relative heights of columns of medium in polyethylene tubing made continuous with the respective chambers. The apparatus was then incubated at 370C in room air/5% CO2. Aliquots were removed from luminal and abluminal compartments at t = 0, 30, 60, and 120 min and analyzed for albumin with bromcresyl green (Sigma). The permeability coefficient (Pd for albumin) for each filter was calculated (14). Statistical Analysis. Multiple test groups were compared by one-way analysis of variance (ANOVA) with StudentNewman-Keuls multiple comparison test. O- scavenging curves were compared with two-way ANOVA and also separately for each riboflavin dose by Student's t test.
RESULTS Effect of Oxygen Tension on XO and XD Activity in EC. Exposure to decreasing concentrations of oxygen for 48 hr led to a graded increase (P < 0.05) in XO, XD, and XD plus XO activities in cultured EC (Fig. 1). XO, XD, and XD plus XO activities in EC negatively correlated with 02 tension over a broad range of oxygen concentrations. EC exposed to hypoxia for 24 or 48 hr increased (P < 0.05) XD plus XO activities compared with control EC (Fig. 2). Effect of Hypoxia/Reoxygenation on Detection of 2 *from EC. More (P < 0.05) 0- was detected from EC monolayers exposed to hypoxia for 48 hr and then reoxygenated for 4 hr than EC monolayers exposed to normoxia for 48 hr (Fig. 3). After hypoxia/reoxygenation, less (P < 0.05) 0- was detected from EC grown in tungsten-supplemented medium than from control cells. Addition of allopurinol, an XO 150
._c o
x
. 100
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0 0,'.0) o C D
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Co 0 £__0_10_2 21 0
10,
95
Ambient fraction of 02, %
FIG. 1. Effect of oxygen tension on EC XO and XD activity after a 48-hr incubation. XO and XD activity increased (P < 0.05) in a
graded fashion with each successive decrease in 02 tension. Each value is the mean + SEM of four individual determinations. mU, milliunits.
3364
Cell Biology: Terada et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
tungsten, allopurinol, and/or SOD did not significantly alter (P > 0.05) baseline albumin transit. However, EC exposed to hypoxia for 24 hr and then reoxygenated had increased (P < 0.05) albumin transit (Pd) compared with normoxia-exposed EC (Fig. 4c). Treatment with tungsten, allopurinol, and/or SOD decreased (P < 0.05) albumin transit through EC monolayers exposed to hypoxia/reoxygenation.
150. ._c
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0 C
0-E
DISCUSSION
w., 50. .C 0
0
48
12 24 Time in hypoxia, hr
FIG. 2. Time course of increase in EC XO and XD activity in hypoxia (0o ambient 02). Total (XO + XD) activity increased (P < 0.05) after 24 and 48 hr in hypoxia-exposed EC compared to normoxia-exposed EC. Each value is the mean + SEM of four individual determinations. mU, milliunits.
inhibitor, or DIDS, an anion-channel blocker, also decreased (P < 0.05) O°- detection from hypoxia/reoxygenated EC compared with levels seen in untreated EC subjected to hypoxia reoxygenation. By comparison, exposure of EC to normoxia resulted in only minimal O2* detection, which was not significantly altered by tungsten, allopurinol, or DIDS treatment. Neither allopurinol (100 jkM) nor DIDS (100 uM) scavenged O-j generated in vitro at rates from 0 to 0.40 ,uM/min. Furthermore, tungsten or allopurinol effectively inhibited (98 ± 2% and 62 ± 12% inhibition, P < 0.05), whereas DIDS or SOD did not change (6 ± 10% and 8 ± 3% inhibition, P > 0.05) XO activity in normoxia-exposed EC. Effect of Hypoia/Reoxygenation on 51Cr Release, Neutrophil Adherence, and Albumin Passage by EC. EC exposed to hypoxia/reoxygenation released more (P < 0.05) preloaded 51Cr than EC exposed to normoxia (Fig. 4a). Treatment with tungsten, allopurinol, and/or SOD decreased (P < 0.05) 51Cr release from EC exposed to hypoxia/reoxygenation. In addition, more (P < 0.05) neutrophils adhered to EC monolayers that had been exposed to hypoxia/reoxygenation than to normoxia (Fig. 4b). Pretreatment with tungsten or coaddition of allopurinol and/or SOD decreased (P < 0.05) neutrophil adherence compared with control EC exposed to hypoxia/ reoxygenation. Control EC monolayers effectively resisted albumin transit even against a 10-cm head of pressure (Pd for bare filters was 8.15 x 10-4 + 2.16 x 10-4 cm/sec) and
Prolonged hypoxia increased both XO and its precursor XD in cultured lung endothelial cells. Along with the observation that hyperoxia decreases XO and XD activities in EC (6), this finding indicates that oxygen tension negatively modulates XO and XD activities in EC. The effect of oxygen tension on XD and XO activity was continuous from 95% to 01% ambient 02 and was most pronounced in the range of 21% to 0%o, emphasizing its potential physiologic importance. Importantly, irreversible conversion of XD to XO did not occur in the presence of hypoxia-a finding that contrasts with what has been observed acutely in completely ischemic, isolated organs ex vivo (15). Because hypoxia-induced increases in XO activity did not involve XD to XO conversion, but rather an increase in total XD plus XO activity, the observation suggests a fundamentally different mechanism by which chronic hypoxia per se may predispose EC to oxygen metabolite-related injury. Hypoxia-induced increases in EC XO activity was associated with an increase in release of O- by EC. O- was measured extracellularly by reduction of the large impermeant derivatized cytochrome c molecule. Although it is possible that O- was formed extracellularly, for instance through autooxidation ofthiols or proteins (16) that may have leaked out of EC, the decrease in O- caused by addition of the anion-channel blocker DIDS implies an efflux of O° from the intracellular compartment after hypoxia/reoxygenation. Moreover, the inhibitory effect of DIDS was relatively specific, as DIDS did not scavenge O- in vitro or inhibit XO in EC. Similarly, the lower Oj Ievels associated with tungsten or allopurinol treatment suggest that XO was in some way responsible for either generation or extracellular release of O-j, especially because allopurinol had no appreciable O°scavenging ability in vitro. Although relatively unreactive, O- may result in cell demise through several mechanisms. For example, O2- can directly inactivate glutathione peroxidase and catalase, two enzymes that constitute an important line of defense against oxidant insults (17, 18). In addition, °2- can interfere with
90
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0
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None
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Normoxia
DIDS
None
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Hvpoxia -_--
FIG. 3. Effect of hypoxia on 0°* release from EC. EC were exposed to normoxia (21% ambient 02) or hypoxia (0%6 ambient Oz) for 48 hr, and the rate of O° appearance was then measured in the extracellular medium of both groups under normoxic conditions. More (P < 0.05) 0°was measured in medium of EC preexposed to hypoxia than to normoxia. Pretreatment with tungsten (Tung, 10 ppm) or coaddition of allopurinol (Allo, 100 ,uM) or DIDS (100 ,uM) decreased (P < 0.05) the rate of °j appearance in medium of hypoxia-preexposed EC but did not (P > 0.05) alter 0-' levels in the medium of normoxia-preexposed EC. Each value is the mean ± SEM of six individual determinations.
Cell
Biology: Terada et al. 50
Proc. Natl. Acad. Sci. USA 89 (1992)
3365
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Exposure:
Allo SOD
None Tung Allo SOD Allo
Hypoxia
SOD
FIG. 4. Effect of hypoxia on 51Cr leak from EC, neutrophil adherence to EC, and albumin transit across EC. EC were exposed to normoxia (21% ambient 02 at Denver altitude, 122 torr) or hypoxia (0%6 ambient 02) for 48 hr, and both groups were assayed under normoxic conditions. Hypoxic preexposure increased (P < 0.05) leak of preloaded 51Cr from EC (a), adherence of neutrophils to EC (b), and flux of albumin across EC monolayers (c), when compared with normoxic preexposure. Pretreatment with tungsten (Tung, 10 ppm) or cotreatment of EC with allopurinol (Allo, 100 /AM) and/or SOD (100 ,ug/ml) decreased (P < 0.05) 51Cr leak, neutrophil adherence, and albumin transit in EC preexposed to hypoxia. Each value is the mean SEM of six to eight determinations. ±
important metabolic intermediates such as NADH (19). Fiis an important precursor of the more reactive nally, hydroxyl radical ( OH). Intracellular * OH is a product of XO-derived 2- in EC following acute hypoxia/reoxygenation (2), and 07 can also enhance the Haber-Weiss reaction by mobilizing ferritin iron (20). Evidence for the toxicity of XO-derived 0- was derived from three independent findings. (i) 51Cr leak, widely used as an index of oxidative cellular injury (21, 22), was increased in EC exposed to hypoxia/reoxygenation relative to normoxic EC. Both XO inhibitors (tungsten and allopurinol) and SOD decreased 51Cr release, suggesting that XO-derived 0° contributed to cellular injury. (ii) More neutrophils adhered to EC subjected to hypoxia/reoxygenation than to those exposed to normoxia. Tungsten, allopurinol, and SOD attenuated neutrophil adherence, again suggesting a role for XOderived 0-7. This result parallels prior findings implicating O°- in causing neutrophil margination in the reperfused mesenteric vascular bed in vivo (23) and neutrophil adherence to cultured EC in vitro (24). The precise mechanism for increased neutrophil adherence remains unknown, although specific neutrophil receptors may be elaborated or expressed by EC (25), or released 0- may react with extracellular serum components to form neutrophil chemotaxins (26). Ultimately, the close approximation of neutrophil to EC,
required for neutrophils to exert their deleterious effects (27), may amplify posthypoxic injury sustained by EC. Treatment with SOD or suppression of XO activity by allopurinol or tungsten did not completely abrogate the posthypoxic increase in either 51Cr release or neutrophil adherence, suggesting that mechanisms independent of XO are likely to be operative in mediating these alterations. (iii) Tungsten, allopurinol, and SOD treatment decreased albumin transit across EC monolayers exposed to hypoxia/reoxygenation. Resistance to albumin passage is a basic function of the vascular endothelium, and maintenance of this albumin gradient appears to be an active process (28). Oxidative injury to EC by XO may facilitate albumin passage by depleting intracellular ATP levels (29), by altering Ca2` homeostasis and cytoskeletal architecture (30), and/or by frank cell lysis (8). Increases in endogenous XO and XD may be germane to vascular conditions that render tissues chronically hypoxic. In addition, tissues or species that have low basal levels of XO may be prone to XO-related vascular injury and inflammation under hypoxic or other conditions that increase XO activity. This work was supported, in part, by grants from the National Institutes of Health (K08 HL02375, P50 HL40784), American Lung
3366
Cell Biology: Terada et al.
Association, American Heart Association, Swan, Hill, Kleberg and American Express Foundations. Dr. Leff and Dr. Guidot were Will Rogers Fellowship recipients. 1. McCord, J. M. (1985) N. Engl. J. Med. 312, 159-163. 2. Zweier, J. L., Kuppusamy, P. & Lutty, G. A. (1988) Proc. Nadl. Acad. Sci. USA 85, 4046-4050. 3. Ratych, R. E., Chuknyiska, R. S. & Bulkley, G. B. (1987) Surgery 102, 122-131. 4. Inauen, W., Payne, D. K., Kvietys, P. R. & Granger, D. N. (1990) Free Radical Biol. Med. 9, 219-224. 5. Jarasch, E.-D., Bruder, G. & Heid, H. W. (1986) Acta Physiol. Scand. Suppl. 548, 39-46. 6. Terada, L. S., Beehler, C. J., Banedjee, A., Brown, J. M., Grosso, M. A., Harken, A. H., McCord, J. M. & Repine, J. E. (1988) J. Appl. Physiol. 65, 2349-2353. 7. Kuthan, H. & Ullrich, V. (1982) Biochem. J. 203, 551-558. 8. Bowman, C. M., Butler, E. N. & Repine, J. E. (1983) Am. Rev. Respir. Dis. 128, 469-472. 9. DellaCorte, E. & Stirpe, F. (1970) Biochem. J. 117, 97-100. 10. Schacterle, G. R. & Pollack, R. L. (1973) Anal. Biochem. 51, 654-655. 11. Massey, V. (1959) Biochim. Biophys. Acta 34, 255-256. 12. Terada, L. S., Leff, J. A., Guidot, D. M., Shibao, G. A. & Repine, J. E. (1990) Inflammation 14, 217-221. 13. Hoover, R. L., Briggs, R. T. & Karnovsky, M. J. (1978) Cell 14, 423-428. 14. Kim, K. J., Critz, A. M. & Crandall, E. D. (1979) Am. Rev. Respir. Dis. 120, 883-892. 15. Engerson, T. D., McKelvey, T. G., Rhyne, D. B., Boggio,
Proc. Natl. Acad. Sci. USA 89 (1992)
16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26.
27. 28. 29. 30.
E. B., Snyder, S. J. & Jones, H. P. (1987) J. Clin. Invest. 79, 1564-1570. Misra, H. P. (1974) J. Biol. Chem. 249, 2151-2155. Blum, J. & Fridovich, I. (1985) Arch. Biochem. Biophys. 240, 500-508. Kono, Y. & Fridovich, I. (1982) J. Biol. Chem. 257, 5751-5754. Chan, P. C. & Bielski, B. H. J. (1974) J. Biol. Chem. 249, 1317-1319. Thomas, C. E., Moorehouse, L. A. & Aust, S. D. (1985) J. Biol. Chem. 260, 3275-3280. Ager, A. & Gordon, J. L. (1984) J. Exp. Med. 159, 592-603. Sacks, T., Moldow, C. F., Craddock, P. R., Bowers, T. K. & Jacob, H. S. (1978) J. Clin. Invest. 61, 1161-1167. Granger, D. N., Benoit, J. N., Suzuki, M. & Grisham, M. B. (1989) Am. J. Physiol. 257, G683-G688. Suzuki, M., Inauen, W., Kvietys, P. R., Grisham, M. B., Meininger, C., Schelling, M. E., Granger, H. J. & Granger, D. N. (1989) Am. J. Physiol. (Suppl.) 257, H1740-H1745. Patel, K. D., Zimmerman, G. A., Prescott, S. M., McEver, R. P. & McIntyre, T. M. (1991) J. Cell Biol. 112, 749-759. Petrone, W. F., English, D. F., Wong, K. & McCord, J. M. (1980) Proc. Natl. Acad. Sci. USA 77, 1159-1163. Shasby, D. M., Shasby, S. S. & Peach, M. J. (1983) Am. Rev. Resp. Dis. 127, 72-76. Shasby, D. M. & Shasby, S. S. (1985) Circ. Res. 57, 903-908. Spragg, R. G., Hinshaw, D. B., Hyslop, P. A., Schraufstatter, I. U. & Cochrane, C. G. (1985) J. Clin. Invest. 76, 1471-1476. Shasby, D. M., Lind, S. E., Shasby, S. S., Goldsmith, J. C. & Hunninghake, G. W. (1985) Blood 65, 605-614.
Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity (oxygen radicals/vascular injury/lung/superoxide anion/neutrophils)
LANCE S. TERADA*t, DAVID M. GuIDOT*, JONATHAN A. LEFF*, IRENE R. WILLINGHAM*, MICHAEL E. HANLEY*, DALE PIERMATTEI*, AND JOHN E. REPINE* Departments of *Medicine and SNeurosurgery at the University of Colorado Health Sciences Center and the Webb-Waring Lung Institute, Denver, CO 80262
Communicated by David W. Talmage, January 6, 1992
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.
MATERIALS AND METHODS Source of Reagents. Cytochrome c (horse heart, type VI) purchased from Sigma and succinoylated with succinic anhydride (7). Catalase (bovine liver, 81,536 units per mg) was purchased from Worthington. Xanthine sodium was obtained from Serva. Chromium isotope (51Cr, 545.2 mCi/ mg, 1.0 mCi/ml; 1 Ci = 37 GBq) was purchased from NEN. Superoxide dismutase (SOD, bovine erythrocyte, 3000 units per mg), lactate dehydrogenase (rabbit muscle type II, 1100 units per mg), and all other reagents were obtained from Sigma. Purification and Culture of EC. EC were harvested from bovine pulmonary arteries (8) and initially grown in 20%16 fetal calf serum with D-valine Eagle's minimal essential medium to eliminate fibroblast contamination. After the third passage, factor VIII and low density lipoprotein receptor staining revealed abluminal) by adjusting the relative heights of columns of medium in polyethylene tubing made continuous with the respective chambers. The apparatus was then incubated at 370C in room air/5% CO2. Aliquots were removed from luminal and abluminal compartments at t = 0, 30, 60, and 120 min and analyzed for albumin with bromcresyl green (Sigma). The permeability coefficient (Pd for albumin) for each filter was calculated (14). Statistical Analysis. Multiple test groups were compared by one-way analysis of variance (ANOVA) with StudentNewman-Keuls multiple comparison test. O- scavenging curves were compared with two-way ANOVA and also separately for each riboflavin dose by Student's t test.
RESULTS Effect of Oxygen Tension on XO and XD Activity in EC. Exposure to decreasing concentrations of oxygen for 48 hr led to a graded increase (P < 0.05) in XO, XD, and XD plus XO activities in cultured EC (Fig. 1). XO, XD, and XD plus XO activities in EC negatively correlated with 02 tension over a broad range of oxygen concentrations. EC exposed to hypoxia for 24 or 48 hr increased (P < 0.05) XD plus XO activities compared with control EC (Fig. 2). Effect of Hypoxia/Reoxygenation on Detection of 2 *from EC. More (P < 0.05) 0- was detected from EC monolayers exposed to hypoxia for 48 hr and then reoxygenated for 4 hr than EC monolayers exposed to normoxia for 48 hr (Fig. 3). After hypoxia/reoxygenation, less (P < 0.05) 0- was detected from EC grown in tungsten-supplemented medium than from control cells. Addition of allopurinol, an XO 150
._c o
x
. 100
-.
0 0,'.0) o C D
C).¢t~50 .)
Co 0 £__0_10_2 21 0
10,
95
Ambient fraction of 02, %
FIG. 1. Effect of oxygen tension on EC XO and XD activity after a 48-hr incubation. XO and XD activity increased (P < 0.05) in a
graded fashion with each successive decrease in 02 tension. Each value is the mean + SEM of four individual determinations. mU, milliunits.
3364
Cell Biology: Terada et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
tungsten, allopurinol, and/or SOD did not significantly alter (P > 0.05) baseline albumin transit. However, EC exposed to hypoxia for 24 hr and then reoxygenated had increased (P < 0.05) albumin transit (Pd) compared with normoxia-exposed EC (Fig. 4c). Treatment with tungsten, allopurinol, and/or SOD decreased (P < 0.05) albumin transit through EC monolayers exposed to hypoxia/reoxygenation.
150. ._c
0o O n. 100. . O
0 C
0-E
DISCUSSION
w., 50. .C 0
0
48
12 24 Time in hypoxia, hr
FIG. 2. Time course of increase in EC XO and XD activity in hypoxia (0o ambient 02). Total (XO + XD) activity increased (P < 0.05) after 24 and 48 hr in hypoxia-exposed EC compared to normoxia-exposed EC. Each value is the mean + SEM of four individual determinations. mU, milliunits.
inhibitor, or DIDS, an anion-channel blocker, also decreased (P < 0.05) O°- detection from hypoxia/reoxygenated EC compared with levels seen in untreated EC subjected to hypoxia reoxygenation. By comparison, exposure of EC to normoxia resulted in only minimal O2* detection, which was not significantly altered by tungsten, allopurinol, or DIDS treatment. Neither allopurinol (100 jkM) nor DIDS (100 uM) scavenged O-j generated in vitro at rates from 0 to 0.40 ,uM/min. Furthermore, tungsten or allopurinol effectively inhibited (98 ± 2% and 62 ± 12% inhibition, P < 0.05), whereas DIDS or SOD did not change (6 ± 10% and 8 ± 3% inhibition, P > 0.05) XO activity in normoxia-exposed EC. Effect of Hypoia/Reoxygenation on 51Cr Release, Neutrophil Adherence, and Albumin Passage by EC. EC exposed to hypoxia/reoxygenation released more (P < 0.05) preloaded 51Cr than EC exposed to normoxia (Fig. 4a). Treatment with tungsten, allopurinol, and/or SOD decreased (P < 0.05) 51Cr release from EC exposed to hypoxia/reoxygenation. In addition, more (P < 0.05) neutrophils adhered to EC monolayers that had been exposed to hypoxia/reoxygenation than to normoxia (Fig. 4b). Pretreatment with tungsten or coaddition of allopurinol and/or SOD decreased (P < 0.05) neutrophil adherence compared with control EC exposed to hypoxia/ reoxygenation. Control EC monolayers effectively resisted albumin transit even against a 10-cm head of pressure (Pd for bare filters was 8.15 x 10-4 + 2.16 x 10-4 cm/sec) and
Prolonged hypoxia increased both XO and its precursor XD in cultured lung endothelial cells. Along with the observation that hyperoxia decreases XO and XD activities in EC (6), this finding indicates that oxygen tension negatively modulates XO and XD activities in EC. The effect of oxygen tension on XD and XO activity was continuous from 95% to 01% ambient 02 and was most pronounced in the range of 21% to 0%o, emphasizing its potential physiologic importance. Importantly, irreversible conversion of XD to XO did not occur in the presence of hypoxia-a finding that contrasts with what has been observed acutely in completely ischemic, isolated organs ex vivo (15). Because hypoxia-induced increases in XO activity did not involve XD to XO conversion, but rather an increase in total XD plus XO activity, the observation suggests a fundamentally different mechanism by which chronic hypoxia per se may predispose EC to oxygen metabolite-related injury. Hypoxia-induced increases in EC XO activity was associated with an increase in release of O- by EC. O- was measured extracellularly by reduction of the large impermeant derivatized cytochrome c molecule. Although it is possible that O- was formed extracellularly, for instance through autooxidation ofthiols or proteins (16) that may have leaked out of EC, the decrease in O- caused by addition of the anion-channel blocker DIDS implies an efflux of O° from the intracellular compartment after hypoxia/reoxygenation. Moreover, the inhibitory effect of DIDS was relatively specific, as DIDS did not scavenge O- in vitro or inhibit XO in EC. Similarly, the lower Oj Ievels associated with tungsten or allopurinol treatment suggest that XO was in some way responsible for either generation or extracellular release of O-j, especially because allopurinol had no appreciable O°scavenging ability in vitro. Although relatively unreactive, O- may result in cell demise through several mechanisms. For example, O2- can directly inactivate glutathione peroxidase and catalase, two enzymes that constitute an important line of defense against oxidant insults (17, 18). In addition, °2- can interfere with
90
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0
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Normoxia
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None
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FIG. 3. Effect of hypoxia on 0°* release from EC. EC were exposed to normoxia (21% ambient 02) or hypoxia (0%6 ambient Oz) for 48 hr, and the rate of O° appearance was then measured in the extracellular medium of both groups under normoxic conditions. More (P < 0.05) 0°was measured in medium of EC preexposed to hypoxia than to normoxia. Pretreatment with tungsten (Tung, 10 ppm) or coaddition of allopurinol (Allo, 100 ,uM) or DIDS (100 ,uM) decreased (P < 0.05) the rate of °j appearance in medium of hypoxia-preexposed EC but did not (P > 0.05) alter 0-' levels in the medium of normoxia-preexposed EC. Each value is the mean ± SEM of six individual determinations.
Cell
Biology: Terada et al. 50
Proc. Natl. Acad. Sci. USA 89 (1992)
3365
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SOD
FIG. 4. Effect of hypoxia on 51Cr leak from EC, neutrophil adherence to EC, and albumin transit across EC. EC were exposed to normoxia (21% ambient 02 at Denver altitude, 122 torr) or hypoxia (0%6 ambient 02) for 48 hr, and both groups were assayed under normoxic conditions. Hypoxic preexposure increased (P < 0.05) leak of preloaded 51Cr from EC (a), adherence of neutrophils to EC (b), and flux of albumin across EC monolayers (c), when compared with normoxic preexposure. Pretreatment with tungsten (Tung, 10 ppm) or cotreatment of EC with allopurinol (Allo, 100 /AM) and/or SOD (100 ,ug/ml) decreased (P < 0.05) 51Cr leak, neutrophil adherence, and albumin transit in EC preexposed to hypoxia. Each value is the mean SEM of six to eight determinations. ±
important metabolic intermediates such as NADH (19). Fiis an important precursor of the more reactive nally, hydroxyl radical ( OH). Intracellular * OH is a product of XO-derived 2- in EC following acute hypoxia/reoxygenation (2), and 07 can also enhance the Haber-Weiss reaction by mobilizing ferritin iron (20). Evidence for the toxicity of XO-derived 0- was derived from three independent findings. (i) 51Cr leak, widely used as an index of oxidative cellular injury (21, 22), was increased in EC exposed to hypoxia/reoxygenation relative to normoxic EC. Both XO inhibitors (tungsten and allopurinol) and SOD decreased 51Cr release, suggesting that XO-derived 0° contributed to cellular injury. (ii) More neutrophils adhered to EC subjected to hypoxia/reoxygenation than to those exposed to normoxia. Tungsten, allopurinol, and SOD attenuated neutrophil adherence, again suggesting a role for XOderived 0-7. This result parallels prior findings implicating O°- in causing neutrophil margination in the reperfused mesenteric vascular bed in vivo (23) and neutrophil adherence to cultured EC in vitro (24). The precise mechanism for increased neutrophil adherence remains unknown, although specific neutrophil receptors may be elaborated or expressed by EC (25), or released 0- may react with extracellular serum components to form neutrophil chemotaxins (26). Ultimately, the close approximation of neutrophil to EC,
required for neutrophils to exert their deleterious effects (27), may amplify posthypoxic injury sustained by EC. Treatment with SOD or suppression of XO activity by allopurinol or tungsten did not completely abrogate the posthypoxic increase in either 51Cr release or neutrophil adherence, suggesting that mechanisms independent of XO are likely to be operative in mediating these alterations. (iii) Tungsten, allopurinol, and SOD treatment decreased albumin transit across EC monolayers exposed to hypoxia/reoxygenation. Resistance to albumin passage is a basic function of the vascular endothelium, and maintenance of this albumin gradient appears to be an active process (28). Oxidative injury to EC by XO may facilitate albumin passage by depleting intracellular ATP levels (29), by altering Ca2` homeostasis and cytoskeletal architecture (30), and/or by frank cell lysis (8). Increases in endogenous XO and XD may be germane to vascular conditions that render tissues chronically hypoxic. In addition, tissues or species that have low basal levels of XO may be prone to XO-related vascular injury and inflammation under hypoxic or other conditions that increase XO activity. This work was supported, in part, by grants from the National Institutes of Health (K08 HL02375, P50 HL40784), American Lung
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Association, American Heart Association, Swan, Hill, Kleberg and American Express Foundations. Dr. Leff and Dr. Guidot were Will Rogers Fellowship recipients. 1. McCord, J. M. (1985) N. Engl. J. Med. 312, 159-163. 2. Zweier, J. L., Kuppusamy, P. & Lutty, G. A. (1988) Proc. Nadl. Acad. Sci. USA 85, 4046-4050. 3. Ratych, R. E., Chuknyiska, R. S. & Bulkley, G. B. (1987) Surgery 102, 122-131. 4. Inauen, W., Payne, D. K., Kvietys, P. R. & Granger, D. N. (1990) Free Radical Biol. Med. 9, 219-224. 5. Jarasch, E.-D., Bruder, G. & Heid, H. W. (1986) Acta Physiol. Scand. Suppl. 548, 39-46. 6. Terada, L. S., Beehler, C. J., Banedjee, A., Brown, J. M., Grosso, M. A., Harken, A. H., McCord, J. M. & Repine, J. E. (1988) J. Appl. Physiol. 65, 2349-2353. 7. Kuthan, H. & Ullrich, V. (1982) Biochem. J. 203, 551-558. 8. Bowman, C. M., Butler, E. N. & Repine, J. E. (1983) Am. Rev. Respir. Dis. 128, 469-472. 9. DellaCorte, E. & Stirpe, F. (1970) Biochem. J. 117, 97-100. 10. Schacterle, G. R. & Pollack, R. L. (1973) Anal. Biochem. 51, 654-655. 11. Massey, V. (1959) Biochim. Biophys. Acta 34, 255-256. 12. Terada, L. S., Leff, J. A., Guidot, D. M., Shibao, G. A. & Repine, J. E. (1990) Inflammation 14, 217-221. 13. Hoover, R. L., Briggs, R. T. & Karnovsky, M. J. (1978) Cell 14, 423-428. 14. Kim, K. J., Critz, A. M. & Crandall, E. D. (1979) Am. Rev. Respir. Dis. 120, 883-892. 15. Engerson, T. D., McKelvey, T. G., Rhyne, D. B., Boggio,
Proc. Natl. Acad. Sci. USA 89 (1992)
16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26.
27. 28. 29. 30.
E. B., Snyder, S. J. & Jones, H. P. (1987) J. Clin. Invest. 79, 1564-1570. Misra, H. P. (1974) J. Biol. Chem. 249, 2151-2155. Blum, J. & Fridovich, I. (1985) Arch. Biochem. Biophys. 240, 500-508. Kono, Y. & Fridovich, I. (1982) J. Biol. Chem. 257, 5751-5754. Chan, P. C. & Bielski, B. H. J. (1974) J. Biol. Chem. 249, 1317-1319. Thomas, C. E., Moorehouse, L. A. & Aust, S. D. (1985) J. Biol. Chem. 260, 3275-3280. Ager, A. & Gordon, J. L. (1984) J. Exp. Med. 159, 592-603. Sacks, T., Moldow, C. F., Craddock, P. R., Bowers, T. K. & Jacob, H. S. (1978) J. Clin. Invest. 61, 1161-1167. Granger, D. N., Benoit, J. N., Suzuki, M. & Grisham, M. B. (1989) Am. J. Physiol. 257, G683-G688. Suzuki, M., Inauen, W., Kvietys, P. R., Grisham, M. B., Meininger, C., Schelling, M. E., Granger, H. J. & Granger, D. N. (1989) Am. J. Physiol. (Suppl.) 257, H1740-H1745. Patel, K. D., Zimmerman, G. A., Prescott, S. M., McEver, R. P. & McIntyre, T. M. (1991) J. Cell Biol. 112, 749-759. Petrone, W. F., English, D. F., Wong, K. & McCord, J. M. (1980) Proc. Natl. Acad. Sci. USA 77, 1159-1163. Shasby, D. M., Shasby, S. S. & Peach, M. J. (1983) Am. Rev. Resp. Dis. 127, 72-76. Shasby, D. M. & Shasby, S. S. (1985) Circ. Res. 57, 903-908. Spragg, R. G., Hinshaw, D. B., Hyslop, P. A., Schraufstatter, I. U. & Cochrane, C. G. (1985) J. Clin. Invest. 76, 1471-1476. Shasby, D. M., Lind, S. E., Shasby, S. S., Goldsmith, J. C. & Hunninghake, G. W. (1985) Blood 65, 605-614.
