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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 208-214, (2002)
© 2002 American Thoracic Society

Original Article

Thromboxane A2 Receptors Mediate Pulmonary Hypertension in 60% Oxygen–exposed Newborn Rats by a Cyclooxygenase-independent Mechanism

Robert P. Jankov, Rosetta Belcastro, Emira Ovcina, Julia Lee, Hamid Massaeli, Stephen J. Lye and A. Keith Tanswell

Canadian Institutes of Health Research (CIHR) Group in Lung Development, Lung Biology Programme; Division of Cardiovascular Research, Hospital for Sick Children Research Institute; CIHR Group in Developmental and Fetal Health, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital; and Departments of Obstetrics and Gynaecology, Paediatrics, and Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada

Correspondence and requests for reprints should be addressed to Dr. A. Keith Tanswell, Division of Neonatology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: keitht{at}sickkids.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelin-1 (ET-1) mediates the development of pulmonary hypertension (PHT) in newborn rats exposed to 60% O2 for 14 days, a model for human chronic neonatal lung injury. ET-1 production by d-14 rat pulmonary artery smooth muscle cells in vitro was markedly increased by thromboxane (TX) A2 receptor agonists and inhibited by a competitive antagonist. We hypothesized that stimulation of the TX A2 receptor contributed to O2-mediated PHT in vivo. Newborn rat pups received daily intraperitoneal injections of L670596, a competitive TX A2 receptor antagonist, or 5,5-dimethyl-3-(3-fluorophenyl)4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU), a cyclooxygenase-2 inhibitor, during 14 days of 60% O2 or air exposure. L670596, but not DFU, prevented 60% O2–mediated right ventricular and small pulmonary vessel smooth muscle hypertrophy. Lung ET-1 content was significantly reduced by L670596 in 60% O2–exposed animals. We conclude that TX A2 receptor activation, though not by TX A2, caused upregulation of ET-1 and PHT in this model. A likely mediator is the stable lipid peroxidation product, 8-iso-prostane, which acts as an incidental ligand of the TX A2 receptor and is a potent inducer of ET-1 production by cultured d-14 rat pulmonary artery smooth muscle cells in vitro.

Key Words: endothelin-1 • 8-isoprostane • pulmonary oxygen toxicity • reactive oxygen species


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sustained exposure to an elevated concentration of O2 is a necessary clinical intervention for preterm infants with respiratory failure, which is believed to contribute to the development of chronic lung injury, or bronchopulmonary dysplasia (BPD). Recent studies on preterm infants with early respiratory failure (1), or established BPD (2, 3), have demonstrated a high incidence of pulmonary hypertension (PHT), which is recognized to contribute to poor outcome. Our lack of understanding of the mechanisms that lead to the development of PHT and BPD in infants is reflected in the fact that there are no effective therapeutic interventions currently available.

Endothelin-1 (ET-1) is indirectly implicated as a mediator of PHT in adult humans (4), a role that is supported by models of chemical- and hypoxia-induced PHT (5, 6). Though there is little such information available for newborns, an increase in ET-1 in tracheal aspirates is an early marker for the development of BPD in human infants (7). Because reactive oxygen species (ROS) generated under excess of antioxidant defenses are also implicated in the pathogenesis of BPD and its complications (8), we hypothesized that an oxidant-mediated induction of ET-1 expression is causal in the development of PHT due to chronic hyperoxia. This hypothesis was tested in a model of BPD in which neonatal rats were exposed to 60% O2 for 14 days (9). Manifestations of PHT in this model include right ventricular hypertrophy (RVH) and pulmonary vascular smooth muscle hyperplasia (10–12). ET-1 was increased in the lungs of 60% O2–exposed animals (10, 11). Both the O2-dependent PHT and increase in ET-1 expression were attenuated by an antioxidant intervention (11). The development of PHT was also attenuated by ET receptor blockade (10), confirming that ET-1 is a critical mediator of PHT in our newborn rat model of BPD. However, the mediators of ET-1 release during O2 exposure are not known.

Possible upstream mediators that are known to be elevated by oxidant stress include the cyclooxygenase (COX) product, thromboxane (TX) A2 (13–15), and 8-isoprostane (10, 11, 16, 17), which both bind to and activate the thromboxane A2/prostaglandin H2 (TP) receptor.

We hypothesized that chronic exposure of newborn rats to 60% O2 led to ET-1 upregulation and PHT through activation of the TP receptor. This hypothesis was based on several observations. First, a 21-aminosteroid antioxidant both inhibited 8-isoprostane formation and prevented PHT in 60% O2–exposed newborn rats (11). Second, 8-isoprostane caused marked upregulation of prepro-ET-1 mRNA in rat fetal lung cells (11). Finally, as described herein, the TX analog, U46619, and 8-isoprostane caused marked increases in ET-1 release in vitro by cultured d-14 rat pulmonary artery smooth muscle cells (PASMC). These effects were inhibited by blockade of the TP receptor. We subsequently examined the in vivo effects of L670596 (18), a competitive TP receptor antagonist, and 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone (DFU), a selective COX-2 inhibitor (19) in newborn rats exposed to chronic hyperoxia.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Interventions
All procedures involving animals were conducted according to criteria established by the Canadian Council for Animal Care. Rat pups were maintained in paired chambers (air and 60% O2) for a 14-day exposure period. Daily intraperitoneal injections of L670596 (0.5 mg/kg) or DFU (10 mg/kg) were given from the day of birth, as described for other compounds (10, 11, 20, 21).

Right Ventricular Hypertrophy
Right ventricular hypertrophy (RVH), a well-established index of PHT (12, 22), was measured as previously described (10–12).

Lung TX B2, Total (Free and Esterified) 8-Isoprostane and ET-1 Measurement
Lungs were flushed of blood and homogenized in phosphate-buffered saline and spiked with 5,000 cpm of [3H]TXB2 or [3H]8-iso-prostaglandin F2{alpha}. Samples were purified as previously described (21). For ET-1, lungs were homogenized in cell lysis buffer (10 mM NaPO4, 0.3 M NaCl, 0.1% [wt/vol] sodium dodecyl sulfate, 1% [vol/vol] NP-40, 1% [vol/vol] deoxycholate, 2 mM ethylenediamine tetraacetic acid, pH 7.2) with protease inhibitors. Samples and standards were analyzed in duplicate using commercially available enzyme immunoassay kits. Values were normalized to total lung protein.

Western Blot Analyses
Lung tissue lysates containing 20 µg ({alpha}-smooth muscle actin) or 40 µg (COX-1 and -2) protein were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with nonfat milk and incubated with primary antibody (1 µg/ml for COX-1 and -2; 0.04 µg/ml for {alpha}-smooth muscle actin) overnight at 4° C followed by secondary antibody for more than 1 hour at room temperature. The protein bands were imaged and analyzed as described previously (10).

Immunohistochemistry
Lung tissue sections from two animals per group were prepared as previously described (9–11). Immunohistochemical studies for COX-2 (1 µg/ml), von Willebrand Factor (5 µg/ml), and prepro-ET-1 (1 µg/ml) were performed by an avidin-biotin-peroxidase complex method (23).

PASMC-enriched Culture
PASMC were isolated using explants from d-14 rats by a modification of a previously described method (24). Pulmonary arteries were minced with scissors and washed in phosphate-buffered saline with antibiotic–antimycotic solution. Tissue was resuspended in medium with 20% (vol/vol) heat-inactivated fetal bovine serum and antibiotic–antimycotic solution and incubated for 1–2 weeks until the cells had attached. Cells were routinely maintained in medium with 10% (vol/vol) fetal bovine serum and antibiotic–antimycotic solution. Cells were passaged by trypsinization using 0.05% (wt/vol) trypsin/ethylenediamine tetraacetic acid and centrifugation at 300 x g for 5 minutes. PASMC were identified by their characteristic hill-and-valley morphology and positive immunostaining for {alpha}-smooth muscle actin. All experiments were performed in quadruplicate using cells from the first passage.

Effect of TP Receptor Agonists on ET-1 and Big ET-1 Production by PASMC
PASMC were treated with U46619 (1 µM) or 8-isoprostane (1 µM) with and without the TP receptor antagonist, L670596 (0.1 µM), for 6 hours. Culture medium and standards were analyzed in duplicate using commercially available enzyme immunoassay kits. Values were expressed as picograms per 106 cells.

Statistical Analyses
Values are expressed as mean ± SEM. Statistical significance (p < 0.05) was determined by one-way analysis of variance (ANOVA) followed by assessment of differences using Duncan's multiple range test (25).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Studies
Cells cultured from the pulmonary arteries of d-14 rats were immunostained for the presence of {alpha}-smooth muscle actin and von Willebrand Factor (data not shown) to identify smooth muscle cells and to exclude significant contamination by endothelial cells. No cells stained positively for von Willebrand Factor, a marker for endothelial cells. Of the total nuclei counted, 87 ± 4.3% coincided with cells that stained positively for {alpha}-smooth muscle actin. Effects of 8-isoprostane (1 µM) and U46619 (1 µM), a TP receptor agonist, on ET-1 and Big ET-1 release from cultured PASMC are shown in Figures 1A and 1B. Compared with untreated control cells, both compounds caused similar and significant increases in the ET-1 and Big ET-1 (p < 0.05) content of culture media after 6 hours of exposure. Increased production of ET-1 and Big ET-1, induced by 8-isoprostane and the TP receptor agonist, U46619, was prevented by the addition of 0.1 µM of the competitive TP receptor antagonist, L670596 (p < 0.05 when compared with cells treated with 8-isoprostane or U46619 alone; p > 0.05 for 8-isoprostane when compared with control cells).



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  		Figure 1. Effects of 8-isoprostane and U46619 on ET-1 and Big ET-1 production by PASMC. (A) ET-1 (closed bar) and Big ET-1 (open bar) in culture medium after a 6-hour incubation with Dulbecco's modified Eagle's medium (DMEM) alone (control), DMEM with 1 µM 8-isoprostane or 8-isoprostane, and 0.1 µM L670596 (+ L670596), a competitive TP receptor antagonist. Bars represent mean ± SEM for four experiments per group. *p < 0.05, by one-way ANOVA, for 8-isoprostane–treated cells compared with both other groups. (B) ET-1 (closed bar) and Big ET-1 (open bar) in culture medium after a 6-hour incubation with DMEM alone (control), DMEM with 1 µM U46619, a TX analog, or U46619 with 0.1 µM L670596 (+ L670596). Bars represent mean ± SEM for 4 experiments per group. *p < 0.05, by one-way ANOVA, for U46619-treated cells compared with both other groups. #p < 0.05, by one-way ANOVA, for Big ET-1 measured in U46619 + L670596–treated cells compared with control cells.

 
In Vivo Studies
As shown in Figure 2A, lung COX-1 expression assessed by Western blot analysis was unaffected by exposure to 60% O2 for 14 days (4.0 ± 0.3 arbitrary units in air-exposed animals versus 3.6 ± 0.3 arbitrary units in 60% O2–exposed animals; p > 0.05). In contrast, as shown in Figure 2B, lung COX-2 was markedly increased by exposure to 60% O2 (2.8 ± 0.28 arbitrary units) compared with air controls at 14 days (0.93 ± 0.33 arbitrary units; p < 0.05). Lung COX-2 expression is also shown by immunohistochemistry in Figure 3. Compared with air controls (Figure 3A), 60% O2–exposed animals had a marked increase in COX-2 expression (Figure 3B) throughout the lung parenchyma and around airways. Addition of COX-2 blocking peptide (inset, Figure 3B) to sections from 60% O2–exposed animals inhibited immunoreactivity, confirming antibody specificity for COX-2. Sections from air- (Figure 3C) and 60% O2–exposed (Figure 3D) animals treated with DFU showed an almost complete lack of immunoreactivity, indicating that the "enzyme–inhibitor" complex prevented binding of COX-2 antibody. In keeping with increased COX-2 expression, vehicle-treated 60% O2–exposed animals had significantly increased (2,302 ± 301 pg/ml) total lung TX B2, a stable hydrolysis product of TX A2, compared with air controls (1,306 ± 42 pg/ml; p < 0.05). As shown in Figure 4, administration of the COX-2 inhibitor, DFU, to 60% O2–exposed animals completely attenuated the increase in lung TX B2 (1,185 ± 74 pg/ml; p < 0.05 compared with 60% O2–exposed, vehicle-treated control animals).



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  		Figure 2. Effect of exposure to 60% O2 on COX expression in whole lung. Western blot analysis of (A) COX-1 (70 kD) and (B) COX-2 (72 kD) expression in lung tissue homogenates from three separate litters after exposure to air (AIR) or 60% O2 (60%) for 14 days. Antibody specificity was verified by preincubation with the appropriate blocking peptide (+BP). Protein size is in kilodaltons.

 


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  		Figure 3. COX-2 immunohistochemistry. Immunohistochemistry for COX-2 (brown stain using 3,3'-diaminobenzidine and light blue counterstain using Carazzi hematoxylin) in lung tissue after exposure to air or 60% O2 for 14 days. Newborn rats received daily intraperitoneal injections of the COX-2 inhibitor, DFU (10 mg/kg), or vehicle alone. Bar length = 250 µm. (A) Air-exposed pups that received vehicle had negligible COX-2 expression. (B) The 60% O2–exposed pups that received vehicle had abundant expression of COX-2 in lung parenchyma and around vessels and airways. Inset: Staining was markedly reduced when the antibody was preincubated with blocking peptide. (C) Air-exposed pups that received DFU had negligible COX-2 expression. (D) The 60% O2–exposed pups that received DFU had markedly reduced COX-2 immunoreactivity compared with vehicle-treated control animals.

 


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  		Figure 4. Effect of COX-2 inhibitor (DFU) on lung TX B2 content. TX B2 in lung homogenates after exposure to air (open bars) or 60% O2 (closed bars) for 14 days. Newborn rats received daily intraperitoneal injections of DFU (10 mg/kg) or vehicle alone (Vehicle). Bars represent mean ± SEM for four litters per group. *p < 0.05, by one-way ANOVA, for vehicle-treated, 60% O2–exposed animals compared with air controls. #p < 0.05, by one-way ANOVA, for DFU-treated animals compared with 60% O2–exposed, vehicle-treated control animals.

 
Vehicle-treated, 60% O2–exposed animals had significant RVH (RV/LV + S dry weight ratio of 0.29 ± 0.013), an index of PHT, compared with air-exposed animals (0.207 ± 0.0009; p < 0.05; Figure 5). RVH induced by 60% O2 was completely attenuated by the TP receptor antagonist, L670596 (0.22 ± 0.008; p < 0.05 compared with 60% O2–exposed vehicle controls), but not by the COX-2 inhibitor, DFU (0.30 ± 0.014; p > 0.05 compared with 60% O2–exposed vehicle controls). Lung smooth muscle mass was assessed by Western blot analysis of {alpha}-smooth muscle actin (Figure 6B). Compared with air-exposed control animals (Figure 6A), 60% O2–exposed animals showed an increase in {alpha}-smooth muscle actin content (11.1 ± 0.9 versus 17.8 ± 2.1 arbitrary units; p < 0.05), which was inhibited by treatment with the TP receptor antagonist, L670596 (10.1 ± 0.6 arbitrary units; p < 0.05 compared with 60% O2–exposed vehicle controls; p > 0.05 compared with air-exposed, L670596-treated animals). Coimmunostaining of sections for von Willebrand Factor and prepro-ET-1, an inactive intracellular precursor of ET-1 (Figure 7), was used to localize de novo production of ET-1 to the media of small pulmonary arteries. When compared with air-exposed vehicle-treated control animals (Figure 7A), 60% O2–exposed animals (Figure 7B) showed medial thickening with intense positive staining for prepro-ET-1 where smooth muscle cells are localized. These changes were not evident in either air- (Figure 7C) or 60% O2–exposed (Figure 7D) animals treated with the TP receptor antagonist, L670596.



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  		Figure 5. RVH. Right ventricular dry weight (RV) compared with that of the left ventricle and septum (LV+S), as an index of RVH, after exposure to air (open bars) or 60% O2 (closed bars) for 14 days. Newborn rats received daily intraperitoneal injections of L670596 (0.5 mg/kg), a competitive TP receptor antagonist, DFU (10 mg/kg), a COX-2 inhibitor, or vehicle alone (Vehicle). Bars represent mean ± SEM for four litters per group. *p < 0.05, by one-way ANOVA, for 60% O2–exposed animals compared with air controls in the same treatment group. #p < 0.05, by one-way ANOVA, for L670596-treated animals compared with 60% O2–exposed, vehicle-treated control animals.

 


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  		Figure 6. Effect of L670596 on total lung {alpha}-smooth muscle actin content. (A) Western blot analysis of {alpha}-smooth muscle actin (43 kD) expression in lung tissue homogenates after exposure to air (open bar) or 60% O2 (closed bar) for 14 days. Newborn rats received daily intraperitoneal injections of L670596 (0.5 mg/kg), a competitive TP receptor antagonist or vehicle alone. Bars represent mean ± SEM for three litters per group. *p < 0.05, by one-way ANOVA, for 60% O2–exposed animals compared with air controls in the same treatment group. #p < 0.05, by one-way ANOVA, for L670596-treated animals compared with 60% O2–exposed, vehicle-treated control animals. (B) Representative Western blot for {alpha}-smooth muscle actin after exposure to air or 60% O2 for 14 days and treatment with either L670596 or vehicle alone (VEHICLE). Protein size is in kilodaltons.

 


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  		Figure 7. Effect of 60% O2 exposure and L670596 treatment on prepro-ET-1 expression in the media of small pulmonary vessels. Coimmunostaining for von Willebrand Factor (black stain using 3,3'-diaminobenzidine nickel) as an endothelial cell marker, and prepro-ET-1 (brown-red stain using 3-amino-9-ethylcarbazole and light blue counterstain using Carazzi hematoxylin) in small (50 µm diameter) pulmonary arteries after exposure to air or 60% O2 for 14 days. Newborn rats received daily intraperitoneal injections of L670596 (0.5 mg/kg), a competitive TP receptor antagonist, or vehicle alone. Bar length = 30 µm. (A) Vessel (v) from an air-exposed animal that received vehicle. Minimal staining for prepro-ET-1 is seen. (B) Vessel (v) from a 60% O2–exposed, vehicle-treated animal. Arrow points to an endothelial cell staining positive for von Willebrand Factor. Inset: Subendothelial and medial localization of immunoreactivity for prepro-ET-1 (arrowheads) is seen. (C) Vessel (v) from an air-exposed animal that received L670596. (D) Staining for prepro-ET-1 in a vessel (v) from a 60% O2–exposed, L670596-treated animal is comparable to that seen in air-exposed animals.

 
Lung content of ET-1 was measured in animals treated with the TP receptor antagonist, L670596 (Figure 8). Vehicle-treated animals exposed to 60% O2 had a significantly increased lung content of ET-1 (578 ± 159 pg/mg protein) compared with air controls (203 ± 7.4 pg/mg protein; p < 0.05), as previously reported (15, 16). Animals treated with the TP receptor antagonist, L670596, did not have a 60% O2–mediated increase in lung ET-1 (148 ± 32 pg/mg protein; p < 0.05 compared with 60% O2–exposed, vehicle controls). To rule out an unanticipated effect of either L670596 or DFU, the lung content of total (free and esterified) 8-isoprostane was measured in animals treated with the TP receptor antagonist, L670596, and the COX-2 inhibitor, DFU, as shown in Figure 9. Compared with air-exposed control animals, 60% O2–exposed animals in all treatment groups had significantly and equivalently elevated (p < 0.05) concentrations of lung 8-isoprostane, similar to those from vehicle-treated control animals.



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  		Figure 8. Effect of L670596 on lung ET-1 content. ET-1 in lung homogenates after exposure to air (open bar) or 60% O2 (closed bar) for 14 days. Newborn rats received daily intraperitoneal injections of L670596 (0.5 mg/kg), a competitive TP receptor antagonist, or vehicle alone (Vehicle). Bars represent mean ± SEM for four litters per group. *p < 0.05, by one-way ANOVA, for 60% O2–exposed animals compared with air controls in the same treatment group. #p < 0.05, by one-way ANOVA, for 60% O2–exposed, L670596-treated animals compared with vehicle-treated control animals.

 


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  		Figure 9. 8-Isoprostane content in lung tissue. Total (free and esterified) 8-isoprostane in lung tissue after exposure to air (open bars) or 60% O2 (closed bars) for 14 days. Newborn rats received daily intraperitoneal injections of L670596 (0.5 mg/kg), a competitive TP receptor antagonist, DFU (10 mg/kg), a COX-2 inhibitor, or vehicle alone (Vehicle). Bars represent mean ± SEM for four liters per group. *p < 0.05, by one-way ANOVA, for 60% O2–exposed animals compared with air controls in the same treatment group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results indicate that ET-1 upregulation and PHT in newborn rats exposed to 60% O2 for 14 days were mediated by the TP receptor. We have also shown that pulmonary vascular smooth muscle cells from neonatal rats are capable of synthesizing ET-1 both in vitro and in vivo, which may contribute to the observed elevation of ET-1 during O2 exposure. In cultured PASMC, 8-isoprostane caused release of ET-1 with similar potency to the TP agonist, U46619, which were both inhibited by a TP receptor antagonist. This 8-isoprostane–induced production of ET-1 by PASMC occurred de novo rather than by release of ET-1 stores, as demonstrated by the concurrent increase of Big ET-1 (the inactive precursor of ET-1) in culture medium. In vivo activation of the TP receptor, leading to PHT, was not explained by the observed increased production of TX A2, as demonstrated by the lack of effect of the COX-2 inhibitor, DFU. An alternative explanation, supported by our data, is that 8-isoprostane, which was increased in the lung by O2 exposure, may have acted in vivo as a ligand of the TP receptor. Studies with human monocytes have suggested that COX-2 may at least partially mediate isoprostane production (26). This pathway is not active in the neonatal rat lung, in that COX-2 inhibition had no effect on 8-isoprostane content.

The putative importance of isoprostane generation, mediated by ROS, to the development of PHT, is supported by data from our previous studies (10, 11). Inhibition of O2-mediated 8-isoprostane production by an antioxidant approach (11) or by prevention of macrophage accumulation in the lung (10) were both effective in preventing PHT. The isoprostanes are a series of stable, prostaglandin-like compounds that are produced nonenzymatically by ROS-mediated reactions with arachidonic acid (17). The differing biologic properties of some of the numerous isoforms of isoprostanes are only just beginning to be appreciated (27). To date, the best-characterized isoform is 8-isoprostane (now classified as ipF2{alpha}-III), which is abundantly produced in vivo during oxidant stress in humans. Indeed, increased levels of 8-isoprostane in tracheal aspirate fluid have been found in term infants with severe pulmonary disease (28). In animals, 8-isoprostane causes vasoconstriction in the lung (29–31) and has mitogenic effects on vascular smooth muscle (32). These biologic effects of 8-isoprostane were consistently found to be inhibited by TP receptor antagonists in lung (15, 31) and other organs (33–38), indicating that 8-isoprostane may be acting as an incidental ligand of the TP receptor. Some evidence points to the existence of a distinct "TP-like" isoprostane receptor in certain cell types (29, 32, 39). Until a distinct 8-isoprostane receptor is characterized, however, the precise mechanism of its effect will remain speculative.

The TP receptor is abundantly expressed in the lung, where it has been localized to bronchial and vascular smooth muscle cells, endothelial cells, and myofibroblasts (40). The natural ligand of the TP receptor is the COX-derived prostanoid, TXA2, which was increased in the lungs of animals exposed to 60% O2. Two COX enzymes are known to exist in mammalian species. COX-1 is constitutively expressed, whereas COX-2 activity is inducible by a number of stimuli, including oxidant stress (15). The preventive effect of the TP receptor antagonist on the development of PHT, and our finding that COX-2 expression and lung TXB2 content were greatly increased by O2 exposure, led us to assess the effect of COX-2 inhibition in vivo. As we have described, COX-2 inhibition was not effective in preventing PHT, despite successful inhibition of the O2-mediated increase in lung TX production. One possible explanation is that any beneficial effect of COX-2 inhibition may have been offset by the attenuation of the vasodilator prostaglandin, prostacyclin (41–43). We propose several alternative explanations for these findings. First, local concentrations of 8-isoprostane in the lung and plasma are likely to be several orders of magnitude higher than COX-derived prostanoids (44). Second, 8-isoprostane is highly stable in vivo (45), in contrast to prostanoids, and therefore may be expected to have a cumulative effect.

Striking similarities exist between the biologic effects of 8-isoprostane and ET-1 on vascular smooth muscle cells. The effects of ET-1 on vascular smooth muscle include contraction, mitogenesis (46–48), and inhibition of apoptosis (49). Vascular smooth muscle cells have been shown to produce ET-1 de novo under a variety of stimuli, including serum (48), angiotensin II (50), platelet-derived growth factor-AA (50), and tumor necrosis factor-{alpha} (51). To our knowledge, our study is the first to demonstrate an 8-isoprostane–mediated release of ET-1 in smooth muscle cells, an effect that has only been shown previously in cultured endothelial cells (16, 52). These data raise the possibility that the effects of 8-isoprostane in vivo may be largely mediated by release of ET-1, which in turn may have an autocrine effect on smooth muscle cell proliferation via the ETA receptor. Studies of ET receptor expression in animal models of PHT have demonstrated relatively greater expression of the ETA receptor (53–55), which is critical to the development of pulmonary vascular remodeling (6, 56, 57).

In summary, we have extended our previous observations on 60% O2–mediated PHT in newborn rats to include a critical role for the TP receptor. Our findings are consistent with activation of the TP receptor being an important mechanism of ET-1 release during chronic hyperoxia, leading to the development of PHT and pulmonary vascular remodeling. The ligand of the TP receptor was not found to be a COX-2–derived product. Rather, TP receptor activation may have at least partly been due to the ROS-derived product of lipid peroxidation, 8-isoprostane.


   Acknowledgments
 
The authors wish to thank Ms. Leslie Johnstone for technical assistance.

Supported by Canadian Institutes of Health Research (Group grant), Merck and Merck Frosst, Canada (provision of pharmacologic compounds), and the Ontario Thoracic Society (an equipment grant). A. K. T. holds the Women's Auxiliary Chair in Neonatal Medicine. R. P. J. is supported by Postdoctoral Fellowships from the CIHR and Canadian Lung Association and a Clinician-Scientist Training Fellowship from the Hospital for Sick Children Research Institute.


   FOOTNOTES
 
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form December 12, 2001; accepted in final form February 6, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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