Pediatric Pulmonology 49:991–1002 (2014)

Cyclooxygenase-2 Inhibition Partially Protects Against 60% O2-Mediated Lung Injury in Neonatal Rats Azhar Masood, MD, PhD,1,2 Man Yi, MD, PhD,3 Mandy Lau, MSc,1,2 Rosetta Belcastro, BSc,1 Jun Li, MD, PhD,1 Crystal Kantores, BSc,1 Cecil R. Pace-Asciak, PhD,1,4 Robert P. Jankov, MD, PhD, FRACP,1,2,5,6 and A. Keith Tanswell, MB, MRCP(UK), FRCP(C)1,2,5* Summary. Rationale Use of the anti-inflammatory agent dexamethasone in premature infants with bronchopulmonary dysplasia has been curtailed, and no alternative anti-inflammatory agents are approved for this use. Our objective was to use a neonatal rat model of bronchopulmonary dysplasia to determine if an highly selective cyclooxygenase-2 inhibitor, 5,5-dimethyl-3-(3fluorophenyl)4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU; 10 mg/g body weight), could prevent inflammatory cell influx and protect against lung injury. Methods Neonatal rats exposed to air or 60% O2 for 14 days from birth either received daily i.p. injections of (i) vehicle or DFU or (ii) vehicle or an EP(1) receptor antagonist, SC-19220. Results DFU attenuated the lung macrophage and neutrophil influx, prevented interstitial thickening and prevented the loss of peripheral blood vessels induced by 60% O2, but did not protect against the variance in alveolar diameter induced by 60% O2. Exposure to 60% O2 caused both an increase in lung prostaglandin E2 content and a reduction in lung mesenchymal cell mass which was reversed by DFU. Prostaglandin E2 binding to the EP(1) receptor inhibited DNA synthesis in cultures of lung fibroblasts in a dose dependent fashion. Treatment with SC-19220 attenuated the reduction in lung mesenchymal mass observed following exposure of rat pups to 60% O2. Conclusions An highly selective cyclooxygenase-2 inhibitor is an effective anti-inflammatory substitute for dexamethasone for preventing phagocyte influx into the neonatal lung during 60% O2-mediated lung injury, and can modify the severity of that injury. Pediatr Pulmonol. 2014; 49:991–1002. ß 2013 Wiley Periodicals, Inc. Key words: bronchopulmonary dysplasia; chronic neonatal lung injury; alveolar formation; PGE2. Funding source: Canadian Institutes of Health Research (CIHR), Numbers: MGC-25029, MOP-84290, MOP-93596, MOP-86472, MOP-15276

INTRODUCTION

Chronic neonatal lung injury, or bronchopulmonary dysplasia (BPD), is a common and serious complication of extreme prematurity.1 The major pathological features of BPD, as seen in the current era, are an inhibition or arrest of alveolar formation from the in-growth of secondary crests into larger precursor saccules, and an associated

thickening of the interstitium.2 In the rat, a species in which alveolar formation is entirely a postnatal event,3 it is possible to replicate these histological changes by exposing pups to 60% O2 for the first 14 days of life.4 Recent observations suggest that the inhibited or arrested alveolarization of BPD may last many years, even into adult life,5,6 yet there is currently no safe or effective preventive therapy for BPD. Systemic

1

Lung Biology Programme, Physiology and Experimental Medicine, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.

6 Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada.

2 Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

Conflict of interest: None.


3

Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Manitoba, Canada. 4 Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada. 5

Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada.

ß 2013 Wiley Periodicals, Inc.

Correspondence to: A. Keith Tanswell, Division of Neonatology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8. E-mail: [email protected] Received 29 March 2013; Accepted 30 August 2013. DOI 10.1002/ppul.22921 Published online 23 November 2013 in Wiley Online Library (wileyonlinelibrary.com).

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dexamethasone, once widely used for its ability to produce a rapid improvement in pulmonary status, is now used sparingly because of associated adverse neurodevelopmental outcomes.7 It is unknown which of dexamethasone’s multiple actions accounted for its beneficial effects on the lung. Our hypothesis is that its effect is mediated, in major part, by its highly selective inhibition of the cyclooxygenase-2 (COX-2) isoform.8 A non-specific COX blockade, using indomethacin, is commonly used in the early care of premature infants to stimulate closure of a patent ductus arteriosus, with a reduced incidence of severe intra-ventricular brain hemorrhage being an additional benefit.9 In the rat model of neonatal lung injury induced by exposure to 60% O2, we have previously reported a 60% O2-dependent up-regulation of COX-2 expression, whereas the expression of COX-1 was unchanged.10 Although the specific contribution of COX-2 activity to the pathogenesis of hyperoxic lung injury is not known, the ability of alveolar epithelial cells to induce prostaglandin E2 (PGE2) synthesis, and to suppress fibroblast growth, has been observed to be COX-2 dependent.11 This consequence of COX-2 activation may contribute to the apparent preferential increase in the number of epithelial cells, relative to fibroblasts, that is observed in both the premature baboon12 and neonatal rat13 models of BPD. Both beneficial14–16 and detrimental17,18 effects of COX-2 inhibition have been observed in animal models of neonatal and adult acute lung injury. We hypothesized that one or more COX-2-mediated products play a key role in the development of the signature lung parenchymal changes seen in the 60% O2exposed neonatal rat, and that chronic administration of the highly selective COX-2 inhibitor, DFU,19 which we have previously demonstrated to effectively block synthesis of a COX-2-dependent prostanoid,10 would attenuate these changes. MATERIALS AND METHODS In Vivo Interventions

Animal experiments were conducted according to requirements established by the Canadian Council on Animal Care. Approval was obtained from the Animal Care Review Committee of the Hospital for Sick Children Research Institute. Sprague–Dawley dams delivered naturally at term gestation (Day 22). Rat pups (between 10 and 12 per litter) were maintained in paired exposure chambers (air or 60% O2) for 14 days from birth, as described previously.4,10,13,20–24 Dams were rotated between air and O2 groups each day to ensure continuity of lactation. Paired litters received daily i.p. injections of either DFU (10 mg/g: 5 ml/g) or normal saline vehicle containing 1% (v/v) DMSO via a 30-gauge needle into the right iliac fossa.10 DFU was provided by Merck Research Pediatric Pulmonology

Laboratories, Rahway, NJ. This compound was selected for use over other COX-2 inhibitors based on our previous experience with its efficacy, and it being well tolerated by rat pups.10 In other studies, paired litters received daily i.p. injections of either the prostaglandin E2 receptor antagonist SC-19220 (Cayman Chemical Company, Ann Arbor, MI) at a dose of 10 mg/g, as defined by others, in 5 ml/g of normal saline vehicle or vehicle alone via a 30 gauge needle into the right iliac fossa.25 Dosimetry

The dose of DFU required to reduce COX-2-derived products in the lung tissue of 60% O2-exposed pups to values similar to those of air-exposed pups was determined using an ELISA to PGFM (Cayman Chemical Company), the stable metabolite of PGF2a. Histology and Immunohistochemistry

Lungs were initially cleared of blood by flushing the pulmonary circulation with PBS containing 1 U/ml heparin. Lungs were then perfusion-fixed over 12 hr with 4% (w/v) freshly dissolved paraformaldehyde, while air-filled under a constant airway pressure of 20 cm water, for preparation and mounting of 5 mm sections as previously described,26 then stained with hematoxylin and eosin. The central left lung was randomly oriented for sectioning. An avidin–biotin–peroxidase complex method27 was used for immunostaining. Slides were incubated with the primary antibody overnight at 48C. After incubating with biotin-conjugated secondary antibody for 1 hr, the labeled Vectastain ABC system (Vector Laboratories, Bulingame, CA) was used with 3, 3diaminobenzidine (Peroxidase Substrate kit, DAB, Vector Laboratories) as a substrate. Slides were mounted in Permount mounting medium. Immunostaining for myeloperoxidase to identify neutrophils21 was with 1:1,000 primary rabbit polyclonal anti-human antibody (Dako Canada, Mississauga, ON; catalog # A 0398), with a 1:100 mouse anti-rat CD-68 antibody (Serotec, Oxford, UK; catalog # MCA341R) to identify macrophages28 and with a 1:2,000 monoclonal mouse anti-human vimentin (Scy Tek, Logan, UT; catalog # A9090) to identify mesenchymal cells.13 Secondary antibody concentrations were each 1:200. Counterstaining was with Nuclear Red for CD68, with Methyl Green for myeloperoxidase and with Carazzi’s hematoxylin for vimentin studies. Morphometric Analyses

All morphometric analyses were performed on the central left lung opposite the hilum to control for regional variations.29 Post-fixation lung volumes were measured by water displacement. Morphometric assessments were

COX-2 in Neonatal Lung Injury 13,21–23,30

performed as previously described on coded images (magnification ¼ 200) of 5-mm hematoxylin and eosin-stained sections. Each image was acquired randomly from non-overlapping fields. Ten fields were captured from each slide, with 3 slides per animal and 5 animals per treatment group. A 130-point contiguous counting grid superimposed on each (200) image was used to calculate the tissue fraction/image, by counting the proportion of total grid points which fell on tissue. The number of points that fell on secondary crests and all tissue were expressed as secondary crest/tissue ratios. Mean linear intercepts (Lm) were measured as described by Dunnill.31 The number of alveoli per field were counted manually without overlaying the grid. Estimated total alveolar numbers were calculated as described by Weibel and Gomez.32 We acknowledge that this approach lacks the elegance and precision of more sophisticated approaches currently in use,33 but has the advantage of simplicity and is well suited to demonstrating large differences between experimental groups. CD68- and MPO-positive cell numbers were similarly derived from coded photomicrographs of the same magnification and expressed as number per mm2. Hart’s Elastin Staining

To identify arterioles, by the presence of both inner and outer elastin lamina, paraffin-embedded tissue sections were stained using Weigert’s resorcin-fuchsin (Elastin Products Co., Owensville, MO) diluted in acidic 70% (v/ v) ethanol overnight at room temperature, followed by a 10-min wash with tap water.34 Slides were counterstained with 0.5% (w/v) tartrazine in 0.25% (v/v) acetic acid for 10 min, then rinsed in distilled water for 1.5 min. For peripheral vessel density assessments, random images were captured as above, except that all fields were within 435 mm of the lung edge. Peripheral pulmonary vessels per unit area were counted from within these fields. Only vessels with both inner and outer elastin bands, to identify arterioles, and outer diameters of 20–65 mm, as assessed by superimposed concentric rings, were counted as previously described.22,23,35

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secondary horseradish peroxidase-conjugated goat antirabbit antibody (Cell Signaling Technology, Beverly, MA; catalog # 7074S) for 90 min. Protein bands were quantified by enhanced chemiluminescence detection, and integrated band densities were calculated after subtraction of background values, as previously described.23 Lung PGE2 Content

PGE2 in whole lung tissue was measured by tandem mass spectrometry, as described elsewhere.37 In Vitro Assays

Fibroblasts were purified from the lungs of 6-day-old rats by differential adherence, as previously described.38 Cytotoxicity was assayed by measurement of the release of pre-incorporated [14C]adenine, and DNA synthesis was measured by the uptake of [3H]thymidine into DNA, as previously described.38,39 In a subsequent experiment, the inhibition of [3H]thymidine into DNA by PGE2 was measured in the presence or absence of various concentrations of the PGE2 EP(1) receptor antagonist SC-19220 (Cayman Chemical Company). Data Presentation

All values are presented as means  SE. One-way ANOVA was used to determine statistical significance

Western Analyses

Western blots of lysates from perfused lung tissue were performed as previously described.23 Lysates were prepared from pooled lungs from four average-sized pups in each litter. Protein content was measured by the Bradford assay.36 Membranes were incubated overnight at 48C with a 1:600 dilution of rabbit antibody to human vimentin (Santa Cruz Biotechnology, Santa Cruz, CA; catalog sc-7557-R) or 1:500 antibody to GAPDH (Santa Cruz Biotechnology; catalogue # sc-25578) for normalization, in blocking solution. After a thorough wash, the membranes were incubated for 1 hr at room temperature with 1:3,000

Fig. 1. Rat pups were exposed to air (open symbols) or 60% O2 (solid symbols) for 7 days and treated with daily i.p. vehicle (0) or 5, 7.5, or 10 mg/g of DFU in vehicle. The lung content of PGFM was measured by ELISA. At a dose of 10 mg/g the 60% O2induced increase in PGFM was attenuated by DFU.
P < 0.05 by one-way ANOVA compared to values for vehicle-treated airexposed pups. Values are means  SE for lung homogenates from four average-sized pups, each from different litters.

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(P < 0.05), followed by post hoc multiple comparison analyses using the Tukey test when significant differences were found between groups. RESULTS

significantly different from those in vehicle-treated, airexposed pups was 10 mg/g (Fig. 1). This dose was used for all subsequent experiments in which DFU was used. Lung Histology

Dosimetry

The minimum dose of DFU required to reduce lung PGFM content in 60% O2-exposed pups to values not

The histopathological changes typical of this lung injury model4 were observed in the sections of vehicleinjected, 60% O2-exposed pups: prominent areas of

Fig. 2. Histological appearance of lung tissue from rat pups exposed to air (A, C) or 60% O2 (B, D) for the first 14 days of life. Animals received daily i.p. injections of vehicle (A, B) or DFU (C, D). Tissue from vehicle-injected (v) pups exposed to 60% O2 (o) had apparent marked interstitial thickening, relative to tissue from air-exposed (a) pups, that appeared to be partially attenuated by treatment with DFU (d). Exposure to 60% O2 also seemed to induce a large variance in alveolar diameters in both vehicle- and DFU-treated pups. Bar ¼ 200 mm. Measurement of (E) tissue fraction confirmed an increase following exposure to 60% O2. Concurrent treatment with DFU reduced this increase such that it was no longer significantly different from values for airexposed pups. Measurement of (F) mean linear intercept variance confirmed a 60% O2-induced increased variance in alveolar diameters which was unaffected by concurrent treatment with DFU.
P < 0.05 by one-way ANOVA compared to both air groups. Values are means  SE for lungs from five average-sized pups from separate litters.

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interstitial thickening and small airspaces interspersed with areas of airspace enlargement (Fig. 2B), relative to air-exposed pups (Fig. 2A). DFU had no obvious effect on air-exposed pups (Fig. 2C), but the interstitial thickening observed in 60% O2-exposed pups appeared to be partially attenuated by 14 days of treatment with DFU (Fig. 2D). These observations were confirmed by measurement of the tissue fraction that, while significantly increased in response to exposure to 60% O2, was restored by concurrent DFU administration to a level which, while elevated, was not significantly different from that observed in vehicle-injected, air-breathing pups (Fig. 2E). Exposure to 60% O2 also appeared to result in increased variability in airspace diameters which did not appear to be improved

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by treatment with DFU (Fig. 2). This impression was verified upon measurements of mean linear intercepts, in which their variances were significantly increased in both groups exposed to 60% O2 (Fig. 2F). Inflammatory Cell Populations

As previously described20–23 a 14-day exposure to 60% O2 caused an influx of both macrophages and neutrophils into lung tissue. Treatment with DFU appeared to reduce the influx of both macrophages, as assessed by CD-68immunoreactivity (Fig. 3A–D) and neutrophils, as assessed by myeloperoxidase-immunoreactivity (Fig. 4A–D). Significant DFU-mediated reductions in the influx of both macrophages and neutrophils induced by 60% O2 were

Fig. 3. Rat pups were exposed to air (a) or 60% O2 (o) for 14 days and injected daily with vehicle (v) or DFU (d). Macrophages were identified by CD68 immunoreactivity (brown stain). Relative to lungs exposed to (A) air for 14 days, exposure to (B) 60% O2 resulted in an apparent increased macrophage influx. Concomitant administration of DFU had no obvious effect on (C) air-exposed lungs, but caused an apparent reduction in the macrophage influx in (D) 60% O2-exposed lungs. Bar ¼ 100 mm. Counts of CD68-positive cells (E) confirmed that exposure to 60% O2 resulted in an influx of macrophages into the lung, relative to those in the lung of air-exposed pups. Concomitant treatment with DFU had no effect on values for air-exposed pups, but attenuated the increase in macrophages in the pups exposed to 60% O2.
P < 0.05 by one-way ANOVA compared to all other groups. Values are means  SE for lungs from six average-sized pups, each from different litters.

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Fig. 4. Rat pups were exposed to air (a) or 60% O2 (o) for 14 days and injected daily with vehicle (v) or DFU (d). Neutrophils were identified by myeloperoxidase (MPO) immunoreactivity (brown stain). Relative to lungs exposed to (A) air for 14 days, exposure to (B) 60% O2 resulted in an apparent increased neutrophil influx. Concomitant administration of DFU had no obvious effect on (C) air-exposed lungs, but caused an apparent reduction in the neutrophil influx in (D) 60% O2exposed lungs. Bar ¼ 100 mm. Counts of MPO-positive cells confirmed that exposure to 60% O2 resulted in an influx of neutrophils into the lung, relative to those in the lung of air-exposed pups. Concomitant treatment with DFU had no effect on values for air-exposed pups, but attenuated the increase in neutrophils in the pups exposed to 60% O2.
P < 0.05 by one-way ANOVA compared to all other groups. Values are means  SE for lungs from 6 average-sized pups, each from different litters.

confirmed by counting the density of immunoreactive cells (Figs. 3E and 4E). Indices of Alveolar and Vessel Formation

The major index of early alveolar formation, namely, secondary crest-to-tissue ratio (Fig. 5A), as well as peripheral vessel density (Fig. 5B), were both decreased by exposure to 60% O2. Both these reductions were attenuated by concurrent treatment with DFU. The Pediatric Pulmonology

alveolar density per unit area (Fig. 5C) and estimated total lung alveoli (Fig. 5D) were both decreased by exposure to 60% O2, but these reductions did not achieve statistical significance. Concurrent administration of DFU resulted in significantly increased alveolar density and estimated alveolar number, relative to those values for vehicle-treated and air-exposed pups. Treatment with DFU had no significant effects on lung or body weights or post-fixation lung volumes (Table 1).

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Fig. 5. Rat pups were exposed to air (a) or 60% O2 (o) for 14 days and injected daily with vehicle (v) or DFU (d). (A) Exposure to 60% O2 resulted in a reduced secondary crest-to-tissue ratio, which was attenuated by concurrent treatment with DFU. (B) Exposure to 60% O2 resulted in a reduction in peripheral vessels, which was attenuated by concurrent treatment with DFU. (C) Concurrent exposure to 60% O2 and DFU resulted in an increase in alveolar density. (D) Concurrent exposure to 60% O2 and DFU resulted in an increase in estimated total alveolar numbers.
P < 0.05 by one-way ANOVA compared to all other groups. Values are means  SE for lungs from five average-sized pups, each from different litters.

Influence of PGE2 on Fibroblast DNA Synthesis and Cytotoxicity

We have previously noted an apparent relative increase in distal lung epithelial cells, and an apparent relative decrease in distal lung fibroblasts, following a 14-day exposure to 60% O2.13 Once again we observed a reduced intensity of immunostaining for vimentin, a marker for mesenchymal cells, following exposure of lungs to 60% O2, which appeared to be reversed by concurrent treatment with DFU (Fig. 6A–D). These qualitative

assessments were confirmed by quantitative assessments using Western analyses (Fig. 6E–F). This led us to speculate that a product of cyclooxygenase-2 could be inhibiting fibroblast proliferation following exposure to 60% O2. A likely candidate, as alluded to above, would be PGE2. We first confirmed that lung PGE2 content was elevated in response to exposure to 60% O2 (Fig. 7), and observed a significant increase at Day 4–7 of exposure, relative to air-exposed control pups. As shown in Figure 8A, PGE2 was cytotoxic to Day-6 neonatal rat lung fibroblasts at concentrations of 1 mg/ml. When

TABLE 1— Lung and Body Weights and Post-fixation Lung Volumes of Vehicle- or DFU-Treated Rat Pups Exposed to Air or 60% O2 for 14 Days From Birth Analysis

Air þ vehicle

Air þ DFU

60% O2 þ vehicle

60% O2 þ DFU

Lung weight (mg) Body weight (gm) Lung volume (ml)

480  16 30.70  0.24 900  4

447  14 29.90  0.47 885  8

430  17 31.53  0.35 880  8

474  13 30.31  0.35 873  11

Values are means  SE, n ¼ 4 average-sized pups from different litters in each group.

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Fig. 7. Rat pups were exposed to air (open symbols) or 60% O2 (solid symbols) for 14 days. The lung content of PGE2 was significantly increased after 4 and 7 days of exposure to 60% O2.
P < 0.05 by one-way ANOVA compared to values for pups exposed to air for the same duration. Values are means  SE for lung homogenates from six litters in each group.

Fig. 6. Rat pups were exposed to air (a) or 60% O2 (o) for 14 days and injected daily with vehicle (v) or DFU (d). Vimentin immunoreactivity (brown stain) was used to identify mesenchymal cells. Relative to air-exposed lung tissue (A), exposure to 60% O2 resulted in a reduced intensity of vimentin immunoreactivity (B). DFU had no obvious effect on air-exposed lung tissue (C), but appeared to attenuate the 60% O2-induced reduction of vimentin immunoreactivity (D). Bar ¼ 100 mm. (E) Exemplars of Western analyses of lung vimentin content normalized to GAPDH. (F) Densitometric analyses confirmed that treatment of air-exposed pups with DFU had no significant effect on lung vimentin content, when compared to vehicle-treated pups also exposed to air. Exposure of vehicle-treated pups to 60% O2 caused a significant reduction in lung vimentin content, relative to vehicle-treated air-exposed pups. This reduction was attenuated by concurrent treatment with DFU.
P < 0.05 by one-way ANOVA compared to air-exposed and vehicle-treated pups and to 60% O2-exposed and DFU-treated pups. Values are means  SE for lung homogenates from 3 litters in each group.

only non-cytotoxic concentrations of PGE2 were assessed for their effect on DNA synthesis in Day-6 neonatal rat lung fibroblasts, we observed a significant inhibition of DNA synthesis at concentrations of 100 pg–100 ng/ml (Fig. 8B). An in vivo concentration of 75 pg/mg tissue, assuming a tissue specific density of 1.0, converts to 7.5 ng/ml, which is within the range demonstrated to inhibit fibroblast DNA synthesis in vitro. Addition of the PGE2 EP(1) receptor antagonist SC-19220 attenuated the inhibition of DNA synthesis induced by 100 ng/ml PGE2 in a dose-dependent manner, and at a concentration of 100 mM fibroblast DNA synthesis was not significantly different from control values (Fig. 8C). Influence of the PGE2 EP(1) Receptor Antagonist on Mesenchymal Cell Mass In Vivo

The reduction of mesenchymal cell mass, as assessed by lung tissue vimentin content, induced by exposure to 60% O2 was completely attenuated by daily treatment with the PGE2 EP(1) receptor antagonist SC-19220 (Fig. 9). This treatment had no effect on lung or body weights (Table 2). DISCUSSION

Despite recommendations restricting the use of postnatal dexamethasone for evolving BPD, a safe and effective alternative pharmacological approach has yet to Pediatric Pulmonology

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Fig. 8. Fibroblasts were isolated from neonatal rat lungs on Day-6 of life for primary culture. (A) PGE2 was cytotoxic at concentrations of 1 mg/ml, as assessed by the release of preincorporated [14C]adenine. (B) Using a non-cytotoxic range of PGE2 concentrations, inhibition of DNA synthesis was observed at concentrations of 100 pg/ml, as assessed by [3H]thymidine incorporation into DNA, normalized to values for fibroblasts with no added PGE2. (C) Addition of the EP(1) receptor antagonist, SC-19220, attenuated the inhibition of DNA synthesis induced by 100 ng/ml PGE2 in a dose-dependent manner. NA ¼ control cells with no added PGE2.
P < 0.05 by one-way ANOVA compared to control values. Values are means  SE for lung cells from four wells per concentration tested.

Fig. 9. Rat pups were exposed to air (a) or 60% O2 (o) for 14 days and injected daily with vehicle (v) or SC-19220 (s). (A) Exemplars of Western analyses of lung vimentin content normalized to GAPDH. (B) Densitometric analyses confirmed that treatment of air-exposed pups with SC-19220 had no significant effect on lung vimentin content, when compared to vehicle-treated pups also exposed to air. Exposure of vehicle-treated pups to 60% O2 caused a significant reduction in lung vimentin content, relative to vehicle-treated air-exposed pups. This reduction was attenuated by concurrent treatment with SC-19220.
P < 0.05 by oneway ANOVA compared to air-exposed and vehicle-treated pups and to 60% O2-exposed and SC-19220-treated pups. Values are means  SE for lung homogenates from three litters in each group.

be devised. The use of a non-steroidal anti-inflammatory agent, such as a COX-2 inhibitor, seemed an attractive alternative approach worthy of testing in an animal model. For this purpose, we used the neonatal rat model of exposure to 60% O2, which results in a lung histopathology with features similar to those observed in human infants with BPD,4 and allows for interventions to be given during a critical period of alveolar formation involving secondary crest formation.40 In this model, exposure to 60% O2 up-regulates pulmonary COX-2 independently of COX-1.10 Previous studies employing this model have demonstrated that inhibition of a neutrophil influx protected against interstitial thickening and promoted alveolar formation,21 whereas selective macrophage depletion prevented the development of pulmonary hypertension.20 It was our hope that DFU, a non-steroidal agent with an high COX-2 selectivity akin to that of dexamethasone8,19, would confer similar benefits by modulation of prostanoid production and attenuation of inflammatory cell recruitment. As described above, treatment with DFU successfully limited the development of interstitial thickening, as reflected in the tissue fraction, following exposure to 60% O2, presumably as a result of the observed inhibition of a neutrophil influx.21 As reported elsewhere,23 exposure to 60% O2 does not affect mean alveolar diameters, but does have a major effect on the variance of these diameters. We have no explanation at this time for why DFU modulated other morphological changes induced by exposure to 60% O2, yet had no effect on the increased variance in alveolar diameter. DFU was, however, protective against the inhibition of secondary crest formation normally observed following exposure to 60% O2.22,23,41 We have previously reported that the pruning of vessels observed Pediatric Pulmonology

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TABLE 2— Lung and Body Weights of Vehicle- or SC-19220-treated Rat Pups Exposed to Air or 60% O2 for 14 Days From Birth Analysis

Air þ vehicle

Air þ SC-19220

60% O2 þ vehicle

60% O2 þ SC-19220

Lung weight (mg) Body weight (gm)

500  8 31.21  0.26

485  17 29.78  0.62

477  13 29.44  0.37

473  13 29.50  0.47

Values are means  SE, n ¼ 4 average-sized pups from different litters in each group.

following exposure to 60% O2 is mediated by peroxynitrite,23 and the likely explanation for the protective effect of DFU against 60% O2-mediated vessel pruning is a reduction in macrophage influx and macrophage-derived nitric oxide for peroxynitrite formation. The specific product(s) of COX-2 responsible for the influx of inflammatory cells has yet to be elucidated. The ability of DFU to suppress prostaglandin production and prevent inflammatory cell influx, without significantly affecting body weight or lung weight, suggests that a specific drug effect was seen, and that drug toxicity did not contribute significantly to the histological changes described. Concurrent exposure to 60% O2 and treatment with DFU resulted in an increased alveolar density and estimated alveolar number relative to control air-exposed pups. We have previously observed the same phenomenon when interventions have prevented inflammatory cell influx.21,22 That postfixation lung volumes were similar between experimental groups excludes inflation artefact contributing to the above findings. Exposure to 60% O2 results in parenchymal thickening. This is, in major part, due to an inhibition of postnatal lung cell apoptosis.24 It is likely that this inhibition of apoptosis mostly affects epithelial cells, since exposure to 60% O2 appears to result in an apparent preferential increase in the number of epithelial cells, relative to fibroblasts,13 a phenomenon also observed in the premature baboon model of BPD.12 The increase in epithelial cells could not be attributed to increased epithelial cell proliferation since, as assessed by [3H] thymidine autoradiography, there is a global inhibition of DNA synthesis affecting all cell types induced by 60% O2.4 That this reduction in fibroblasts was reversible by DFU led us to speculate that this suppression of fibroblast growth was mediated by PGE2, as described by others.11 In support of this hypothesis, we observed a 60% O2mediated increase in lung tissue PGE2 to values which suppress isotype age-matched lung fibroblast DNA synthesis in vitro. This inhibition of fibroblast DNA synthesis by PGE2 was attenuated by the concurrent exposure to the PGE2 EP(1) receptor antagonist SC19220. This led us to use SC-19220 for in vivo studies, in which we observed that the PGE2 EP(1) antagonist attenuated the loss of lung mesenchymal cell mass induced by 60% O2. As stated earlier, both beneficial14–16 and detrimental17,18 effects of COX-2 inhibition have been observed in Pediatric Pulmonology

animal models of neonatal and adult acute lung injury. It is worth noting that prophylactic indomethacin therapy in human infants has also been identified as a possible risk factor for BPD.42 An inhibition of alveolar formation in neonatal rat lungs by the non-selective COX inhibitors, indomethacin and ibuprofen, has also been reported. 18,43 We did not observe such an inhibition with the highly selective COX-2 inhibitor used in this study, nor did a 5day course of ibuprofen in immature baboons impair alveolarization.44 That an highly selective COX-2 inhibitor is partially protective against neonatal lung injury in this rat model of BPD offers hope for alternate therapeutic strategies in human BPD. ACKNOWLEDGMENTS

This study was supported by a group [A.K.T. (MGC25029)] and operating [R.P.J. (MOP-84290 and -93596), C.R.P.-A.(MOP-86472), A.K.T. (MOP-15276)] grants from the Canadian Institutes of Health Research (CIHR), and an infrastructure grant from the Canada Foundation for Innovation New Opportunities Fund (R.P.J). R.P.J. is supported by a CIHR Independent Investigator award. REFERENCES 1. Jobe AH, Bancalari E. Bronchopulmonary dysplasia. Am J Respir Crit Care Med 2001;163:1723–1729. 2. Coalson JJ. Pathology of chronic lung disease of early infancy. In: Bland RD, Coalson JJ, editors. Chronic lung disease in early infancy. New York: Dekker; 1999. pp 85–124. 3. Burri PH. Structural aspects of prenatal and postnatal development and growth of the lung. In: McDonald JA, editor. Lung growth and development. New York: Dekker; 1997; pp 1–35. 4. Han RNN, Buch S, Tseu I, Young J, Christie NA, Frndova H, Lye SJ, Post M, Tanswell AK. Changes in structure, mechanics, and insulin-like growth factor-related gene expression in the lungs of newborn rats exposed to air or 60% oxygen. Pediatr Res 1966;39:921–929. 5. Cutz E, Chaisson D. Chronic lung disease after premature birth. N Eng J Med 2008;358:743–746. 6. Wong PM, Lees AN, Louw J, Lee FY, French N, Gain K, Murray CP, Wilson A, Chambers DC. Emphysema in young adult survivors of moderate-to-severe bronchopulmonary dysplasia. Eur Respir J 2008;32:321–328. 7. Shinwell ES, Karplus M, Reich D, Weintraub Z, Blazer S, Bader D, Yurman S, Dolfin T, Kogan A, Dollberg S, Arbel E, Goldberg M, Gur I, Naor N, Sirota L, Mogilner S, Zaritsky A, Barak M, Gottfreid E. Early postnatal dexamethasone treatment and increased incidence of cerebral palsy. Arch Dis Child 2000;88: F177–F181.

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