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(The FASEB Journal. 1999;13:1025-1030.)
© 1999 FASEB

Isoprostanes and PGE2 production in human isolated pulmonary artery smooth muscle cells: concomitant and differential release

KAREN B. JOURDAN, TIMOTHY W. EVANS, PETER GOLDSTRAW and JANE A. MITCHELL1

Unit of Critical Care, National Heart and Lung Institute at Imperial College of Science, Technology and Medicine, Royal Brompton Hospital, London, SW3 6NP, U.K.

1Correspondence: Unit of Critical Care. Royal Brompton Hospital, Sydney St., London, SW3 6NP U.K. E-mail: j.a.mitchell{at}ic.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The isoprostanes are a group of biologically active arachidonic acid metabolites initially thought to be formed under conditions of oxidative stress and independently of cyclooxygenase. However, recent studies have demonstrated isoprostane production under conditions in which cyclooxygenase is intentionally activated/induced. Here we describe for the first time formation of isoprostanes by human vascular cells via independent pathways of oxidative stress and cyclooxygenase induction. We compared the release of the isoprostane with that of the traditional prostaglandin, prostaglandin E2. Cyclooxygenase-2 induction was confirmed by Western blot. When cells were stimulated with cytokines, the release of isoprostanes was inhibited by the cyclooxygenase-1 and -2 inhibitor indomethacin as well by as the cyclooxygenase-2 selective inhibitor L-745,337. However, treatment of cells with the superoxide-producing enzyme xanthine oxidase also resulted in isoprostane release, which was not affected by cyclooxygenase inhibition, unlike PGE2 release under the same condition. Thus, two independent pathways relating to oxidative stress and cyclooxygenase-2 induction form isoprostanes. These findings may have particular importance in diseases such as sepsis and ARDS in which oxidant stress occurs and cyclooxygenase is induced.—Jourdan, K. B., Evans, T. W., Goldstraw, P., Mitchell, J. A. Isoprostanes and PGE2 production in human isolated pulmonary artery smooth muscle cells: concomitant and differential release.


Key Words: 8-iso PGF2{alpha}a • indomethacin • L-745,337 • sepsis • lung vasculature


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE ACUTE RESPIRATORY distress syndrome (ARDS)2 is characterized by increased alveolar capillary permeability leading to pulmonary edema formation. The associated refractory hypoxemia is attributable to a loss of pulmonary vascular control with associated ventilation-perfusion mismatch, probably due to endothelial damage leading to changes in the production of vasomotor substances. The underlying vascular smooth muscle is emerging as a significant autocrine organ, producing a variety of vasoactive substances under inflammatory conditions. Isoprostanes are a group of prostaglandin (PG) -like compounds formed independently of cyclooxygenase, by oxidative modification of esterified arachidonic acid in precursor phospholipids (1, 2) . Traditional cyclooxygenase products have established inflammatory and vasoactive properties. Isoprostanes, specifically 8-iso PGF2{alpha}, also have potent effects of vasoreactivity. 8-iso PGF2{alpha} is an agonist or partial agonist of thromboxane receptors and causes smooth muscle contraction (3 4 5 6 7 8 9) , platelet aggregation (10, 11) , and endothelin-1 release (12) through this receptor. 8-iso PGF2{alpha}a has also been hypothesized to act on putative isoprostane selective receptors (3, 9, 13, 14) for some of its activity, including smooth muscle relaxation (3) .

Isoprostanes were initially thought to be formed in plasma in vitro (15) and have since been found during the oxidative modification of isolated low density lipoprotein (16) . Although first demonstrated as in vitro-generated products, there is now increasing evidence that isoprostanes, particularly the F2 isoprostanes, are formed in vivo during conditions associated with oxidative stress. 8-iso PGF2{alpha} is among the most abundant isoprostanes formed in vivo and is increased in rats by oxidant-induced injury with carbon tetrachloride (17) , iron overload (18) , diquat poisoning (19) , copper deficiency (20) , or vitamin E and selenium deficiency (21) . Levels of 8-iso PGF2{alpha}a have also been shown to be increased in humans by smoking (22, 23) , during coronary reperfusion (24) , and in individuals with non-insulin-dependent diabetes mellitus (25) or familial hypercholesterolemia (26) , all conditions associated with oxidant stress as is ARDS. Although there is compelling evidence that isoprostanes are produced directly as a result of oxidant stress, there is increasing controversy in the field surrounding their possible dependence on cyclooxygenase during production. Cyclooxygenase is the first enzyme catalyzing the formation of traditional prostaglandins (e.g., PGE2) from arachidonic acid. Cyclooxygenase exists in cells either constitutively (cyclooxygenase-1) or after induction with inflammatory stimuli (cyclooxygenase-2; see ref 27 ). The involvement of cyclooxygenase-1 or cyclooxygenase-2 in the production of isoprostanes remains controversial. Nevertheless, human platelets (cyclooxygenase-1) (28, 29) and monocytes (cyclooxygenase-2) (30, 31) release 8-iso PGF2{alpha}, which is blocked by inhibitors of cyclooxygenase. Moreover, we have shown recently that agents that induce cyclooxygenase-2 in human blood vessels (32) also release 8-iso PGF2{alpha} (33) , an effect that was blocked by the cyclooxygenase-1/-2 inhibitor indomethacin.

Most of the studies investigating the mechanism of release of isoprostanes have addressed their formation by either oxidant stress (15) or cyclooxygenase activation (29) , but not both. Thus, the question of cyclooxygenase involvement in isoprostane production remains unresolved. Since isoprostanes are able to modulate vascular function, particularly in pulmonary vessels, further understanding of how they are formed and the conditions under which nonsteroidal antiinflammatory drugs would or would not block their production is clinically relevant. We have demonstrated that the smooth muscle component of human blood vessels can be stimulated to produce 8-iso PGF2{alpha} (33) . In the current study, we therefore addressed the role of cyclooxygenase in the release of 8-iso PGF2{alpha} by human pulmonary artery smooth muscle cells under two conditions: cyclooxygenase induction by interleukin-1ß (IL-1ß) and after treatment with xanthine oxidase to induce an oxidant stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Human pulmonary artery (mean diameter ~8 mm) was obtained from surgically resected lung. Under sterile tissue culture conditions, specimens were cleaned of connective tissue and the endothelium was gently removed with a rounded scalpel blade. The artery was cut into pieces and placed in a flask with Dulbecco's modified Eagle medium containing 1 mM sodium pyruvate and phenol red, supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), amphotericin B (2.5 µg/ml), a mixture of nonessential amino acids [L-alanine, L-asparagine, L-aspartate, L-glutamate, glycine, L-proline, and L-serine at the manufacturer's recommended concentration (Life Technologies, Paisley, U.K.)[, and 16% heat-inactivated fetal calf serum. The flasks were placed in a cell culture incubator (37°C, 5%CO2 and 95% air) and smooth muscle cells were explanted to form a confluent layer in 4–8 wk. For experiments, cells were passaged into 96-well plates with 100 µl medium containing drugs and/or cytokines (10 ng/ml for each) or xanthine oxidase (1 U/ml). The medium was removed from the cells after 24 h of treatment and split into two aliquots, one stored at -20oC until PGE2 was measured and the other stored at -80oC until 8-iso PGF2{alpha} was measured. This time point was chosen after preliminary studies showed that the rate of both PGE2 and 8-iso PGF2{alpha} release was optimum at 24 h.

Western blotting
Pulmonary artery smooth muscle cells were seeded in 6-well plates. They were either left untreated or treated for 24 h with cytokines. The medium was then removed and the cells were lysed with Tris buffer (50 mM; pH7.4) containing 1%v/v Triton X-100, EDTA (10 mM), PMSF (1 mM), pepstatin (0.05 mM), and leupeptin (0.2 mM). Extracts were boiled at a 1:1 ratio with Tris (50 mM; pH 6.8; 4% w/v sodium dodecyl sulfate; 10% v/v glycerol; 4% v/v 2-mercaptoehanol; 2 mg/ml bromphenol blue). Samples of equal protein were loaded onto 7.5% Tris-glycine sodium dodecyl sulfate gels and separated by electrophoresis. After transfer to nitrocellulose, the blots were primed with a specific anti-human cyclooxygenase-2 antibody (34) (Merck Frosst, Montreal, Canada) raised in rabbit. The blots were then incubated with anti-rabbit immunoglobulin G (raised in goat), conjugated to horseradish peroxidase, and developed by enhanced chemiluminescence (Amersham International, Ltd., Bucks, U.K.). Rainbow markers (14–200 kDa; Amersham) were used for molecular weight determinations.

Xanthine oxidase preparation
The enzyme used in this study was grade 1 xanthine oxidase extracted from buttermilk and had a nominal specific activity of 0.5 U/mg protein. The enzyme was suspended in 2.3M (NH4)2SO4 (ammonium sulfate) containing 1 mM sodium salicylate. For experiments, the enzyme was diluted 10-fold in sodium phosphate buffer 0.1 M (pH 7.4) and passed over a 1 ml column of Sephadex G25 to remove salts and salicylate. The conversion of xanthine to uric acid, determined spectrophotometrically at 295 nm, was used to estimate the activity of the desalted enzyme.

Cell respiration
The ability of cells to oxidize 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide (MTT) was used as an indicator of cell respiration. After 24 h of treatment fresh medium containing 1 mg/ml MTT was added, incubated for 15 min at 37°C and carefully removed. The formazan product of MTT was dissolved with 100 µl DMSO and 15 min of shaking. The absorbency was read at 550 nm in a plate reader.

Prostanoid determination
PGE2 was measured by radioimmunoassay using commercial antibodies and tritiated prostanoids, as described previously (35) . The cross-reactivity of 8-iso PGF2{alpha}a with PGE2 antibodies is ~4%. 8-Isoprostanes were measured using an enzyme immunoassay kit from Cayman Chemical (Ann Arbor, Mich.; purchased through R & D Systems Europe Ltd., Abingdon, Oxfordshire, U.K.) that was previously used to measure levels of 8-iso PGF2{alpha}a immunoreactivity in porcine vascular smooth muscle cells (36) , human lung (37) , and rat lung (38) . Levels detected with the enzyme immunoassay are comparable to those measured using gas chromatography/mass spectrometry (39) . The cross-reactivity of PGE2 with the 8-iso PGF2{alpha} antibodies is 0.02%.

Materials
Tritiated PGE2 was obtained from Amersham). IL-1ß and interferon-{gamma} (IFN{gamma}) were purchased from Boehringer Mannheim (Boehringer Mannheim, Lewes, East Sussex, U.K.) and tumor necrosis factor {alpha} (TNF-{alpha}) from R & D Systems Europe Ltd. Amphotericin B and nonessential amino acids were purchased from Life Technologies. L-745, 337 was a gift from Merck. All other materials were purchased from Sigma Chemical Company (Poole, U.K.).

Statistical analysis
All data are the mean ± standard error of the mean. Data was analyzed by one-way analysis of variance. Statistical significance (represented by an asterisk) was taken to be P < 0.05. IC50 values were calculated using GraphPad Prism.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Release of PGE2 and 8-iso PGF2{alpha} from human pulmonary artery smooth muscle cells after stimulation with inflammatory mediators
The inflammatory cytokines IL-1ß (10 ng/ml) and, to a lesser extent, TNF-{alpha} (10 ng/ml), stimulated the release of both 8-iso PGF2{alpha}a and PGE2 from human pulmonary artery smooth muscle cells, but neither IFN{gamma} (10 ng/ml) nor lipopolysaccharide (LPS; 10 µg/ml) increased the release of either eicosanoid (Fig. 1 ). When cells were stimulated with IL-1ß, TNF-{alpha}, INF-{gamma}, and LPS in combination, levels of 8-iso PGF2{alpha}a and PGE2 were released similar to those seen in the presence of IL-1ß alone, indicating no synergy between cytokines. In each case, ~50 fold more PGE2 was released than 8-iso PGF2{alpha}a.



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  		Figure 1. Shows the release of 8-iso PGF2{alpha}a (filled columns) and PGE2 (open columns) from human pulmonary artery smooth muscle cells treated with inflammatory cytokines TNF-{alpha}, IL-1ß, IFN{gamma} (each 10 ng/ml), and LPS (10 µg/ml) or in combination (MIX). Data are the mean ± SE mean (n=5–12). *P<0.05 compared to basal (analysis of variance).

Induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells
Human pulmonary artery smooth muscle cells treated with IL-1ß (lane 4) or a mixture of IL-1ß, TNF-{alpha}, IFN{gamma}, and LPS (lane 7) for 24 h expressed detectable levels of cyclooxygenase-2 protein determined by Western blot analysis (Fig. 2 ). TNF-{alpha}, IFN{gamma}, or LPS alone did not induce cyclooxygenase-2 in human pulmonary artery smooth muscle cells (Fig. 2) .



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  		Figure 2. Western blot of cyclooxygenase-2 protein from human pulmonary artery smooth muscle cells. Lane 1, standard cyclooxygenase-2 protein; lane 2, protein extract from untreated cells; lane 3, protein extract from cells stimulated with TNF-{alpha} (10 ng/ml); lane 4, protein extract from cells stimulated with IL-1ß (10 ng/ml); lane 5, protein extract from cells stimulated with IFN{gamma} (10 ng/ml); lane 6, protein extract from cells stimulated with LPS (10 µg/ml); lane 7, protein extract from cells treated with a mixture of all three cytokines and LPS.

Inhibition of isoprostane production and PGE2 release by inhibitors of cyclooxygenase-1 and -2
Release of PGE2 from human pulmonary artery smooth muscle cells stimulated with IL-1ß was inhibited by the mixed cyclooxygenase-1 and cyclooxygenase-2 inhibitor, indomethacin, and the selective cyclooxygenase-2 inhibitor, L-745,337 (34) . Indomethacin (100 pM to 100 µM) was 122-fold more potent than L-745,337, with mean IC50 values of 8.2 ± 3 nM and 1.0 ± 0.5 µM, respectively. Both cyclooxygenase inhibitors also inhibited 8-iso PGF2{alpha} release from IL-1ß-stimulated human pulmonary artery smooth muscle cells. The IC50 values for 8-iso PGF2{alpha} inhibition were very similar to those for PGE2 (indomethacin, 1.37 ±0.4 nM, and L-745,337, 408 ±62 nM).

Release of PGE2 and 8-iso PGF2{alpha}a from human pulmonary artery muscle cells under oxidant stress conditions
Xanthine oxidase reduces its substrate hypoxanthine to xanthine, then uric acid. Superoxide is released at each step. Superoxide directly, and via the formation of hydroxyl radicals in cells, causes oxidant stress resulting in impaired respiration. When cells were treated with xanthine oxidase, with or without its substrate hypoxanthine, cellular respiration was reduced in a concentration-dependent manner (data not shown). When respiration was reduced by 50% or more, 8-iso PGF2{alpha} was released by human pulmonary artery smooth muscle cells (Fig. 3 A). In parallel experiments, cells treated with xanthine oxidase also released increased levels of PGE2 (Fig. 3B ). Similar to observations made with IL-1ß-stimulated cells, PGE2 release from cells treated with xanthine oxidase was greatly reduced by indomethacin (Fig. 4 B) and partially inhibited by L-745,337 (1 µM; inhibited by 49 ±15%). However, in contrast to release by cells stimulated with IL-1ß, 8-iso PGF2{alpha} release from cells treated with xanthine oxidase was not significantly inhibited by indomethacin (Fig. 4A ) or L-745,337 (1 µM; data not shown).



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  		Figure 3. Shows the release of (A) 8-iso PGF2{alpha} and (B) PGE2 from human pulmonary artery smooth muscle cells under basal conditions (control) and after treatment with xanthine oxidase (XO; 1 U/ml), hypoxanthine (HX; 10 µM), and enzyme and substrate together. Data are the mean ± SE mean (n=3–15).



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  		Figure 4. Shows the effect of indomethacin (filled bars) on the release of PGE2 and 8-iso PGF2{alpha} from human pulmonary artery smooth muscle cells after treatment with xanthine oxidase (XO; 1 U/ml) or IL-1ß (10 ng/ml). Data are the mean ± SE mean (n=3–9).

In separate experiments, oxidant stress induced by xanthine oxidase stimulated the release of 8-iso PGF2{alpha} by vascular smooth muscle cells cultured from the systemic artery, internal mammary artery (control 9.9 ±4.5 pg/ml; plus xanthine oxidase, 36.5 ±2.02 pg/ml), radial artery (control 57.1±5.0 pg/ml; plus xanthine oxidase, 258.5±67.2 pg/ml), and vein saphenous vein (control 26.4±5.8; plus xanthine oxidase, 92.5±4.0 pg/ml).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we demonstrated that vascular cells can be stimulated to release isoprostanes via two independent mechanisms. Stimulation of cells with IL-1ß or a mixture of proinflammatory cytokines for 24 h induced cyclooxygenase-2 protein formation and the release of PGE2, a typical cyclooxygenase metabolite, and the isoprostane 8-iso PGF2{alpha}. The ratio of release of the two prostanoids was ~50:1 PGE2 to 8-iso PGF2{alpha}. The release of both PGE2 and 8-iso PGF2{alpha} under these conditions was inhibited by the cyclooxygenase-2 inhibitor L-745,337 (34) in a concentration-dependent fashion. The potency of L-745,337 as well as the cyclo-oxygenase-1 inhibitor indomethacin (35) to reduce the formation of each eicosanoid were similar, indicating a comparable pathway of formation for PGE2 and 8-iso PGF2{alpha}a. Similar observations have recently been made using rat mesangial cells stimulated to express cyclooxygenase-2 using IL-1ß or purified forms of cyclooxygenase; in both cases, 8-iso PGF2{alpha} was blocked with indomethacin (39) . In addition, human monocytes stimulated with LPS corelease PGE2 and 8-iso PGF2{alpha} at levels and proportions similar to those released by human pulmonary artery smooth muscle cells in the current study. Moreover, 8-iso PGF2{alpha} release by LPS-stimulated monocytes was completely blocked by L-745,337 (31) . Thus, from our current and previous work (33) as well as that of others (40) , we can conclude that 8-iso PGF2{alpha} is synthesized along with other cyclooxygenase-2 products. Cyclooxygenase-2 is induced in a number of inflammatory conditions, including rheumatoid arthritis (41) and sepsis (42) . Since we may expect that the cyclooxygenase production of traditional products and isoprostanes would be reduced to similar extents by nonsteroidal antiinflammatory drugs, it is not yet possible to elucidate a role for isoprostanes in modulating inflammatory or vascular diseases.

In addition to IL-1ß (39) or LPS (30, 33) , we found that oxidant stress stimulated cells to release 8-iso PGF2{alpha}. However, in contrast to observations made in protocols where cyclooxygenase is either activated (28) or induced (29, 30, 39) , we found that when cells were subjected to oxidant stress, 8-iso PGF2{alpha} release was not blocked by cyclooxygenase inhibition. We found that cells subjected to oxidant stress also released PGE2. However, the release of PGE2 in these experiments, unlike that of 8-iso PGF2{alpha}, was greatly reduced by indomethacin. In contrast, PGE2 release by cells treated with xanthine oxidase was only partially inhibited by L-745,337. This observation suggests that activation of cyclo-oxygenase-1, and not induction of cyclo-oxygenase-2, is primarily responsible for PGE2 release under these conditions. Xanthine oxidase breaks down hypoxanthine to uric acid in two steps. Each stage leads to the production of superoxide, which will react quickly to form other reactive oxygen species, including hydrogen peroxide and the highly damaging hydroxyl radical. Cyclo-oxygenase is activated by hydroperoxides under certain conditions. Thus, some of the breakdown products of superoxide anions may directly activate cyclo-oxygenase-1, leading to the formation of PGE2 seen in this study by cells stimulated with xanthine oxidase.

Addition of hypoxanthine, a substrate of xanthine oxidase, did not further increase the reduction cell respiration or release of eicosanoids elicited by xanthine oxidase alone, suggesting an endogenous substrate is present in excess in these cells. Under these conditions, it is likely that isoprostanes are formed by lipid peroxidation of arachidonic acid in situ at phospholipid membranes. Our studies using oxidant irritants in vitro are in line with a number of early (43) and recent publications (44) by Morrow and co-workers, who have specifically investigated the release of isoprostanes in conditions of oxidant stress in vivo. Moreover, we are able to demonstrate that the release of isoprostanes by oxidant stress is of a magnitude similar to that occurring after cyclooxygenase activation/induction.

Although isoprostanes are found in low concentrations in plasma and urine, there are likely to be high concentrations at their site of synthesis, where they may act in an autocrine fashion. Both we and others have shown that 8-iso PGF2{alpha} induces vasoconstriction (3 4 5 6 7 8 9) and vasodilation (3) in pulmonary blood vessels. Thus, isoprostanes could contribute significantly to vascular dysfunction in diseases where either cyclooxygenase-2 is expressed or oxidant stress occurs, such as sepsis and ARDS. Since nonsteroidal antiinflammatory drugs do not block isoprostane release during oxidant conditions, formation by this route may be of particular importance in diseases where these drugs do not afford therapeutic benefits.


   ACKNOWLEDGMENTS
 
The authors would like to thank Nick Lamb for his help with Western blots and Cathy Ratcliffe for her help with collection of the human pulmonary artery. K.B.J. is an MRC research assistant. J.A.M. is a Wellcome Trust career development fellow.


   FOOTNOTES
 
2 Abbreviations: ARDS, acute respiratory distress syndrome; IL-1ß, interleukin-1ß; IFN{gamma}, interferon-{gamma}; MTT, 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide; LPS, lipopolysaccharide; PG, prostaglandin; TNF-{alpha}, tumor necrosis factor {alpha}.

Received for publication September 15, 1998. Revision received January 25, 1999.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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