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Published ahead of print on April 29, 2004, doi:10.1164/rccm.200309-1282OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 313-318, (2004)
© 2004 American Thoracic Society

Original Article

Discordant Extracellular Superoxide Dismutase Expression and Activity in Neonatal Hyperoxic Lung

Lisa B. Mamo, Hagir B. Suliman, Brenda-Louise Giles, Richard L. Auten, Claude A. Piantadosi and Eva Nozik-Grayck

Departments of Pediatrics and Medicine, Duke University Medical Center, Durham, North Carolina; Department of Pediatrics, University of Manitoba and Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada

Correspondence and reprint requests should be addressed to Eva Nozik-Grayck, M.D., 4200 E. Ninth Avenue B131, University of Colorado Health Sciences Center, Denver, CO 80262. E-mail: eva.grayck{at}uchsc.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antioxidant defenses in the neonatal lung are required to adapt to the oxygen (O2)-rich postnatal environment, and oxidant/antioxidant imbalance is a predisposition to lung injury when high concentrations of inspired O2 are used in neonatal lung diseases. The lung's main extracellular enzymatic defense against superoxide, extracellular superoxide dismutase (EC-SOD), is closely regulated during development. In testing the hypothesis that developmental change in EC-SOD expression and activity in the immature lung would be disrupted by hyperoxia, we found a doubling of lung EC-SOD protein in newborn rats exposed to 95% O2 for 1 week. Furthermore, EC-SOD protein secretion increased, but EC-SOD enzyme activity did not change with O2 exposure. EC-SOD mRNA did not change at multiple points between 6 hours and 8 days. Lung EC-SOD recovered by immunoprecipitation after 1 week of O2 showed strong increases in protein nitrotyrosine and variable, nonsignificant differences in protein carbonyl content. These data provide the first direct evidence that EC-SOD is itself a target of nitration in hyperoxia, and offer a plausible explanation for low EC-SOD activity despite its increased secretion by O2-exposed neonatal lung.

Key Words: superoxide dismutase • antioxidant • oxygen • nitrotyrosine • protein carbonyl

The neonatal lung requires adequate antioxidant defenses to adapt to the oxygen (O2)-rich postnatal environment. In development, multiple antioxidant enzymes, including catalase, glutathione peroxidase, and superoxide dismutases (SOD), must be regulated in the lung (1–5). In premature infants, deficient antioxidant defenses contribute to the development of chronic lung disease of the newborn (6–8). In full-term infants, oxidative stress also contributes to lung injury due to diseases such as neonatal pneumonia or persistent pulmonary hypertension of the newborn, particularly to those in which treatment requires high concentrations of inspired O2 to support gas exchange. This condition increases production of reactive O2 species, which contribute to lung injury. However, general antioxidant therapy, i.e., antioxidant vitamins or enzyme replacement, has not been successful in preventing chronic lung disease after acute neonatal lung injury in baboon studies or human trials (9–11). Therefore, more specific manipulations of antioxidant/oxidant balance, or improved antioxidant targeting in the neonatal lung will be needed to improve therapy for these diseases.

Among the lung's major antioxidant defenses are the three known SOD isoforms, metalloproteins that catalyze the rapid dismutation of superoxide (O2–) to hydrogen peroxide (Kd ~3 x 109), each of which has a unique cellular distribution (12–16). SOD3 isoform, also termed extracellular (EC-) SOD, is the only known extracellular enzymatic defense against O2–, and its expression is tightly regulated in the developing lung (5, 17). Another known function of EC-SOD is to augment nitric oxide (NO) bioavailability by preventing the rapid reaction of NO with O2– (Kd ~7 x 109) in airways and pulmonary vasculature (18). EC-SOD is highly expressed in the extracellular matrix of blood vessels and lung parenchyma as well as in the extracellular fluids (15, 19–22). The localization of EC-SOD to the extracellular matrix requires the presence of a heparin-binding domain, which undergoes proteolytic cleavage during biosynthesis and under oxidant conditions (5, 23–28).

In experimental animals, including adult and neonatal mice, EC-SOD activity protects against the effects of pulmonary O2 toxicity. Adult mice that over-express human EC-SOD in alveolar type II cells and bronchial epithelial cells have decreased mortality after 3 days of hyperoxia, whereas EC-SOD knock-out mice exposed to hyperoxia have increased lung injury and mortality (29, 30). EC-SOD over-expression also protects the developing lung in newborn mice exposed to chronic hyperoxia (31). In adult mice, Oury and colleagues recently demonstrated that exposure to hyperoxia disrupts the oxidant/antioxidant balance in the lung by depleting EC-SOD in the alveolar region (27). In this study, we hypothesized that hyperoxia disrupts normal developmental changes in EC-SOD expression and activity in the immature lung. We tested this hypothesis in newborn rat pups administered greater than 95% O2 continuously for 1 week, and measured lung EC-SOD gene and protein expression and activity. EC-SOD limits the reaction of O2– with NO, therefore, the disruption of EC-SOD expression in hyperoxia would promote both oxidative and nitrosative stress in the lung. To determine if EC-SOD protein is modified in hyperoxia, we isolated the enzyme from lungs of O2-exposed and control rat pups, and evaluated EC-SOD function and protein oxidation and nitration.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Model and O2 Exposure
Animal studies were approved by the Duke University Institutional Animal Care and Use Committee. Timed pregnant Sprague-Dawley rats (250–300 g) were obtained during late gestation (Charles River Laboratories, Charles River, NJ). Newborn rats were assigned randomly to receive air or 95% O2 exposure for up to 1 week. Nursing dams were alternated between air and O2-exposed litters every 24 hours to prevent maternal O2 toxicity (31). After the exposures, animals were killed with intraperitoneal pentobarbital (150 mg/kg). Lungs from control and O2-exposed pups were harvested at 6 and 24 hours, 2 days, and 1 week. Control lung tissue was also obtained from adult male rats.

EC-SOD Expression
Protein (40 µg) from lung homogenates was analyzed under denaturing and reducing conditions by immunoblotting using a polyclonal EC-SOD peptide antibody, as previously described (5). Blots were reprobed with ß-actin to confirm equal protein loading. Immunohistochemical analysis for EC-SOD localization using the same EC-SOD antibody was performed on inflation-fixed paraffin-embedded lung tissue from 1-week-old control and O2-exposed rat pups (5). Tissue sections were photographed using a 40x objective for a final magnification of 100x. Immunoblots were also probed with NO synthase (NOS) antibodies, mouse monoclonal endothelial NOS (termed eNOS or NOS III) or inducible NOS (termed iNOS or NOS II) IgG antibody (1:500) (BD, Lexington, KY), to examine changes in NOS expression; and rabbit polyclonal EC-SOD (Cu,ZnSOD) or mouse monoclonal intracellular SOD (termed iSOD or MnSOD) IgG antibody (1:1,000) (RDI, Flanders, NJ) to measure changes in intracellular SOD isoforms.

EC-SOD mRNA Expression
Total RNA was isolated from the lung and EC-SOD mRNA amplified by reverse-transcriptase polymerase chain reaction, as described previously (32). Rat EC-SOD sense and anti-sense primers were designed using MacVector (Accelrys, San Diego, CA) to span the intron–exon 3 coding region of rat EC-SOD (sense, 5' GAA CCT CAG CCA TGG TGG 3'; anti-sense, 5' GCT TAA GTG GTC TTG CAC 3') (Roche Molecular Biochemicals, Indianapolis, IN). EC-SOD amplicons were normalized to 18S ribosomal RNA amplicons (Quantum RNA 18S; Ambion, Austin, TX).

EC-SOD Activity Levels
EC-SOD was separated from intracellular SOD (Cu,Zn and MnSOD) by concavalin A-sepharose chromatography, and EC-SOD activity was measured by inhibition of cytochrome C reduction at pH 10.0, as described (13). Lungs of three pups were pooled for each value.

Oxidation of EC-SOD Protein
EC-SOD was immunoprecipitated from 1-week air- and O2-exposed lungs using a protein G immunoprecipitation kit, (Kirkegaard and Perry, Gaithersburg, MD), and 20 µg of the immunoprecipitated EC-SOD protein was treated with 2,4-dinitrophenylhydrazine and the derived protein was analyzed for carbonyl content (OxyBlot; Intergen, Purchase, NY). Negative controls were not exposed to 2,4-dinitrophenylhydrazine. Samples were transferred to polyvinylidene fluoride membrane using a slot blot manifold system or analyzed by immunoblotting (Amersham BioSciences, Piscataway, NJ) with anti-2,4-dinitrophenylhydrazone antibody. Details are outlined in the online supplement.

Nitration of EC-SOD Protein
EC-SOD was immunoprecipitated from 1-week air- and O2-exposed lungs and transferred to polyvinylidene difluoride membrane using a slot blot manifold system. Nitration of immunoprecipitated EC-SOD (5 ug) was determined by detection of nitrotyrosine using a polyclonal anti-nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY). MnSOD and Cu,ZnSOD were immunoprecipitated from the lung tissue of 1-week-old animals exposed to air or O2 and probed for nitrotyrosine, as described above.

Reagents
Reagents, unless specified, were obtained from Sigma Chemical Co. (St Louis, MO).

Statistical Analysis
Data were expressed as means ± SEM. Comparisons were made by unpaired t test for reverse-transcriptase polymerase chain reaction and Western blot data, or by two-way analysis of variance for activity data, followed by a Fisher PLSD test using Statview software (SAS Institute, Cary, NC).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Hyperoxia on EC-SOD Expression in Immature Rat Lungs
EC-SOD protein migrates as an approximately 30 kD doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two bands represent full-length, uncleaved EC-SOD monomer and cleaved EC-SOD after proteolytic removal of the heparin-binding domain. The immature rat lung contained predominantly cleaved EC-SOD, and protein expression increased greatly in control rats from 2 days of age to adult (Figure 1A) . Lung EC-SOD protein expression was similar at 2 days in neonatal control and O2-treated rats (p > 0.05 by unpaired t test, n = 4) (Figure 1B), but levels were increased twofold over age-matched control rats after 1 week of O2 exposure (p < 0.05 by unpaired t test, n = 4) (Figure 1C). Following 1 week of O2 exposure, levels of both the full-length and cleaved EC-SOD monomer increased. A small amount of EC-SOD (< 10%) remained as an approximately 60 kD dimer despite strong reducing conditions. EC-SOD dimer, like EC-SOD monomer, was increased after 1 week of O2 (not shown). In contrast to EC-SOD, lung eNOS protein expression of 1-week-old control and O2-exposed rat pups was variable with no statistical change relative to baseline expression (p > 0.05 by unpaired t test, n = 4) (see Figure E1 in the online supplement). iNOS was not detectable in rat pup lung (data not shown). MnSOD tended to increase after 2 days of O2 exposure (p = 0.12, n = 4), but levels were not statistically different from those of control lungs after 8 days (p > 0.05 by unpaired t test, n = 4) (see Figure E2 in the online supplement). Cu,ZnSOD expression did not increase with O2 exposure at 2 or 8 days (p > 0.05 by unpaired t test, n = 4) (see Figure E3 in the online supplement). Immunohistochemistry revealed that EC-SOD of lung tissue from 1-week-old control rat pups localized predominantly to the intracellular compartment of airway epithelial and alveolar epithelial cells (arrowheads). However, after 1 week of O2 exposure, there was also prominent staining of the extracellular matrix (long arrows) (Figure 2) . Although 1 week of O2 exposure increased EC-SOD protein expression and its extracellular secretion, there was no corresponding increase in lung EC-SOD mRNA expression. EC-SOD mRNA expression was similar after 6 and 24 hours, 2 days, and 1 week of O2 exposure (Figure 3) .



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  		Figure 1. Effects of hyperoxia on extracellular superoxide dismutase (EC-SOD) expression in immature rat lungs. (A) EC-SOD protein expression increased greatly in control rats from age 2 days to adult. (B) EC-SOD protein expression was similar at 2 days of age in neonatal control and oxygen (O2)-treated rats but increased significantly after 1 week of oxygen exposure. (C) Both full-length and cleaved EC-SOD monomer were increased in 1-week-old lungs. The corresponding densitometry for each blot shows the significant increase in protein expression at 1 week of hyperoxia. (p > 0.05 by unpaired t test, n = 4).

 


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  		Figure 2. Effects of hyperoxia on localization of EC-SOD in lung tissue. Lung sections from 1-week-old (A) control and (B) O2-treated rats. In control lungs at 1 week of life, EC-SOD was predominantly localized by immunohistochemistry to the intracellular compartment of airway epithelial and alveolar cells (arrowheads). After 1 week of O2 exposure, the extracellular matrix (long arrows) exhibited strong staining as well. Tissue sections were photographed at x100.

 


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  		Figure 3. Effects of hyperoxia on EC-SOD mRNA expression. EC-SOD mRNA expression, determined by semiquantitative reverse transcriptase-polymerase chain reaction, was measured at (A) 6 hours, (B) 24 hours, (C) 2 days, and (D) 1 week in control and O2-exposed (hyperoxia) rats. mRNA from 2 representative rat lungs at each age are shown for control and hyperoxia conditions. EC-SOD was standardized to its corresponding 18s rRNA. No significant increase in mRNA transcripts was evident.

 
Effects of Hyperoxia on EC-SOD Protein Activity in Immature Rat Lungs
EC-SOD activity was measured in control and O2-treated neonatal lungs. The rat lung showed a small but significant increase in EC-SOD activity between 2 days of age and adulthood (Table 1) . Although lung EC-SOD protein expression and secretion increased in neonatal lungs after 1 week of hyperoxia, O2 exposure did not increase EC-SOD enzyme activity in the lung (Figure 4) .


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  		TABLE 1. Extracellular superoxide dismutase activity in developing rat lungs

 


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  		Figure 4. Effects of hyperoxia on EC-SOD activity. EC-SOD activity levels in age-matched control lungs (white bars) remained stable in neonatal rat lung after O2 exposure for 2 days or 1 week (black bars) (p > 0.05 by analysis of variance, n = 3). Activity levels are expressed as U/mg protein.

 
Modification of EC-SOD Protein by Hyperoxia
We examined whether a lack of increased EC-SOD activity in the setting of increased protein levels following 1 week of O2 exposure was associated with EC-SOD protein modification by oxidation, nitration, or both. Lung EC-SOD protein isolated by immunoprecipitation was analyzed for protein carbonyl and nitrotyrosine content. Analysis of protein carbonyl content using the Oxyblot kit demonstrated statistically similar signals in EC-SOD protein with or without hyperoxia due to wide variability in O2-treated animals (Figure 5A) . The relative densitometry of the protein carbonyl signal was 1.00 ± 0.03 for control lung versus 1.32 ± 0.27 for lung from 1-week O2-exposed rats (p > 0.1 by unpaired t test, n = 3). Protein carbonyl formation was also not statistically different in adult rats treated with O2 and control rats (n = 3, data not shown). Western blot analysis of protein carbony content in EC-SOD from O2-exposed rat pups showed a strong approximately 30 kD doublet, indicating oxidation of EC-SOD monomers, and a faint signal at 60 kD, representing oxidized EC-SOD dimer (Figure 5B). This confirmed that immunoprecipitation of EC-SOD did not coprecipitate other proteins that could interfere with the signal for EC-SOD on the slot blot. While oxidation of EC-SOD did not consistently increase with hyperoxia, EC-SOD was a target for protein nitration. A strong increase in nitrotyrosine staining occurred in EC-SOD immunoprecipitated from O2-exposed lungs (Figure 6) . The relative densitometry of the nitrotyrosine signal was 0.99 ± 0.15 for control lung versus 1.50 ± 0.02 for lung from 1-week O2-exposed rats (p < 0.05 by unpaired t test, n = 3). Cu,ZnSOD and MnSOD of lungs of 1-week O2-exposed rat pups showed nitrotyrosine staining that was highly variable with no statistical differences in signal increases compared with that of control lungs (see Figures E4 and E5 in online supplement).



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  		Figure 5. Protein carbonyl detection in immunopreciptated EC-SOD. (A) Protein carbonyl content in EC-SOD protein from lungs was variable, although it tended to increase after 1 week of O2 exposure. Data from 2 representative lungs obtained after 1 week of control conditions or hyperoxia are shown. (B) Protein carbonyls in EC-SOD immunoprecipitated from a 1-week-O2-exposed rat pup lung showed a strong approximately 30 kD doublet, indicating oxidation of EC-SOD monomers with a faint signal at 60 kD representing oxidized EC-SOD dimer.

 


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  		Figure 6. Nitrotyrosine detection in immunopreciptated EC-SOD. Nitrotyrosine staining increased in immunoprecipitated EC-SOD following 1 week of O2 exposure. The blot shows the nitrotyrosine signal from 2 representative rat lungs at 1 week of age for both control and hyperoxia-treated rats.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We tested the hypothesis that hyperoxia disrupts developmental changes in EC-SOD expression and activity in the immature rat lung, and found new evidence that this extracellular antioxidant enzyme protects against pulmonary O2 toxicity. It is known that neonatal animals are more resistant to O2 toxicity than adults (33), and that EC-SOD over-expression protects against pulmonary O2 toxicity in adult and neonatal mice (29, 31). We know that active EC-SOD secretion is regulated in developing lung, and that after prenatal hypoxia EC-SOD activity is decreased by a posttranslational mechanism(s) (5, 32). In this study, we report that although EC-SOD protein content and extracellular secretion increase in neonatal rat lung with O2 exposure, the enzymatic activity does not increase. The discordance between EC-SOD expression and activity is accompanied by an increase in nitration of EC-SOD protein. This provides new insight into the role of EC-SOD in the pathophysiology of pulmonary O2 toxicity in the developing lung.

EC-SOD expression is regulated in the developing rat lung in a manner similar to that reported for immature rabbits (5). EC-SOD content and activity are low, and EC-SOD expression is localized intracellularly until birth. Increased active EC-SOD secretion occurs early in postnatal development. This is consistent with other antioxidant responses of late gestation, including those of glutathione, vitamins A and E, catalase, glutathione peroxidase, and Cu,ZnSOD (1–4, 34). These defenses are upregulated in the lung at the time of birth in preparation for the increased oxidant stress of an O2-rich environment. The increase in EC-SOD activity in the postnatal period suggests that EC-SOD provides extracellular antioxidant defenses in response to the transition from the hypoxic intrauterine environment to the relative hyperoxia of air breathing. EC-SOD may also help maintain low pulmonary vascular tone in the transition from fetal to adult circulation by preserving pulmonary vascular NO bioactivity.

Prolonged exposure of rat pups to hyperoxia for 1 week greatly increased EC-SOD protein content in the lung, and enhanced EC-SOD expression in the extracellular compartment, as determined by immunohistochemistry. Both cleaved and uncleaved EC-SOD monomers were detected in the lungs. In contrast, exposure to O2 for shorter periods did not alter EC-SOD protein content of neonatal rat lungs. The changes in EC-SOD protein expression with hyperoxia appear to be specific for neonatal lung, as adult mice exposed to O2 for 72 hours had decreased EC-SOD protein content in both lung and bronchoalveolar lavage fluid (27). Furthermore, EC-SOD protein expression also decreased in the lungs of adult rats exposed for 1 week to 95% O2 (data not shown). Age-dependent responses to hyperoxia also have been reported for other antioxidant enzymes. We did not detect changes in MnSOD or Cu,ZnSOD protein expression after 2 or 8 days of O2 exposure. In other studies, it was determined that adult rats exhibit a transient increase in lung catalase expression after 24 hours of O2 exposure, while neonates exposed to the same O2 treatment show a delayed but sustained increase in catalase activity at 72 hours (35). The increase in EC-SOD expression in neonatal animals likely contributes to the increased resistance to O2 toxicity of the immature lung.

In this study, EC-SOD protein content in neonatal lung increased with 1 week of hyperoxia, but neither neonatal nor adult animals showed changes in EC-SOD mRNA expression at early or late time points. While O2 upregulates mRNA expression for some genes by activating O2-sensitive transcription factors, this mechanism does not appear to regulate EC-SOD gene expression in hyperoxic rat lung. The increase in protein expression without concomitant changes in mRNA may be the result of an increase in mRNA stability, translation, or protein stability, factors that we did not investigate.

Despite the increase in EC-SOD protein content and extracellular secretion in the O2-exposed neonatal rat lung, it was notable that EC-SOD activity did not increase in the whole lung. Interestingly, we previously noticed discordance between EC-SOD expression and enzymatic activity in neonatal rabbit lung. In the developing rabbit lung, EC-SOD mRNA decreased with age, while EC-SOD activity increased sixfold (5, 32). In addition, total EC-SOD protein levels were stable while secretion of EC-SOD to the extracellular matrix and EC-SOD activity increased. In 1-week-old rabbits, secretion of lung EC-SOD was delayed by prenatal hypoxia (32), while in this study, postnatal hyperoxia enhanced extracellular EC-SOD secretion in 1-week-old rat lungs. However, the ability to increase extracellular secretion of EC-SOD in response to hyperoxia was not accompanied by greater whole-lung enzyme activity. The mechanism regulating O2-dependent secretion of EC-SOD has not yet been determined. In adult mice, discordance between stable mRNA and decreased protein expression and activity after hyperoxia was attributed to posttranslational proteolytic cleavage of EC-SOD protein at the heparin-binding domain with depletion of EC-SOD in airways (27). Exposure of mice to bleomycin, which produces pulmonary fibrosis, also enhances proteolysis of the EC-SOD heparin-binding domain with loss of protein from the lung (28). In this study, however, the latter mechanism did not appear to account for low EC-SOD activity, because EC-SOD protein expression increased with hyperoxia, and the increase included both cleaved- and full-length uncleaved EC-SOD monomers. Therefore, we evaluated EC-SOD for posttranslational chemical modifications and found that EC-SOD in O2-exposed lungs contained higher levels of nitrotyrosine, suggesting that nitration of EC-SOD could impair enzyme activity despite increased protein accumulation in whole lung.

Since EC-SOD functions to scavenge O2– and prevent O2– reactions with NO, extracellular O2– production in excess of the EC-SOD antioxidant capacity promotes formation of both reactive O2 and nitrogen species (16). Hyperoxia increases reactive O2 species production, which will enhance reactive nitrogen species production, such as peroxynitrite. This can occur due to loss of SOD activity or to an increase in NO production (18). Different NOS isoforms appear to respond differently to hyperoxia. In two models of immature lung, 3-day-old and 3-week-old rats exposed to 95% O2 showed increased lung eNOS and iNOS (36, 37). In adult rats, long exposures (28 days) increased eNOS and iNOS expression, while short exposures (4–72 hours) did not change eNOS expression (38, 39). In our neonatal rat study, NOS expression did not increase, suggesting new nitrotyrosine formation was due primarily to inadequate O2-scavenging.

Reactive nitrogen species can inhibit activity of multiple antioxidants, including SOD (Cu,Zn and Mn), catalase, and glutathione peroxidase (40). The mitochondrial SOD, MnSOD, is inactivated in vitro and in disease by specific tyrosine nitration (41–45). However, SOD nitration, as seen with bacterial FeSOD, does not always alter enzyme function (46); therefore, the effects of EC-SOD nitration site on enzyme activity will require further study. Our data provide the first direct evidence that EC-SOD itself is a target of nitration in hyperoxia, which provides a plausible explanation for low neonatal lung activity despite increased production and secretion of EC-SOD with O2 exposure.

The evidence that EC-SOD protects the lung against O2 toxicity is substantial, and multiple studies have shown that neonatal lungs are relatively resistant to such toxicity (29, 31, 33, 47, 48). The distinct differences we found in EC-SOD response of neonatal rats in hyperoxia could contribute significantly to this relative resistance. In adults, lung EC-SOD protein expression decreases with O2 exposure (27), while in neonatal rat lung O2 enhances EC-SOD protein secretion. EC-SOD activity did not increase with greater protein expression, but the lung did not lose EC-SOD activity as it does in adult mice, and, thus, it may be able to better provide protection against O2 toxicity. These findings have important implications for understanding extracellular oxidative stress, and factors that influence differential susceptibility to lung disease in developing and mature mammals.


   Acknowledgments
 
The authors thank Owen Doar, Craig Marshall, and S. Nicholas Mason for excellent technical assistance, and Tim D. Oury for helpful discussions.


   FOOTNOTES
 
Supported by the Duke Neonatal Perinatal Research Institute (E.N-G. and R.L.A.), National Heart, Lung, and Blood Institute grant PO1 HL42444 (C.A.P.), and by the American Lung Association of North Carolina (R.L.A.).

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

Conflict of Interest Statement: L.B.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; H.B.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; B.L.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; R.L.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; C.A.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; E.N.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this article.

Received in original form September 15, 2003; accepted in final form April 25, 2004


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