Published online September 1, 2006
PEDIATRICS Vol. 118 No. 3 September 2006, pp. 1056-1064 (doi:10.1542/peds.2006-0195)
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ARTICLE

Pulmonary Nitric Oxide Synthases and Nitrotyrosine: Findings During Lung Development and in Chronic Lung Disease of Prematurity

Mark Sheffield, MD, Sherry Mabry, MS, Donald W. Thibeault, MD and William E. Truog, MD

Children's Mercy Hospitals and Clinics, Section of Neonatology, Department of Pediatrics, University of Missouri-Kansas City School of Medicine, Kansas City, Missouri


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND. Nitric oxide mediates and modulates pulmonary transition from fetal to postnatal life. NO is synthesized by 3 nitric oxide synthase isoforms. One key pathway of nitric oxide metabolism results in nitrotyrosine, a stable, measurable marker of nitric oxide production.

OBJECTIVE. The purpose of this study was to assess, by semiquantitative immunohistochemistry, nitric oxide synthase isoforms and nitrotyrosine at different airway and vascular tree levels in the lungs of neonates at different gestational ages and to compare results in control groups to those in infants with chronic lung disease.

DESIGN/METHODS. Formalin-fixed, paraffin-embedded, postmortem lung blocks were prepared for immunohistochemistry using antibodies to each nitric oxide synthase isoform and to nitrotyrosine. Blinded observers evaluated the airway and vascular trees for staining intensity (0–3 scale) at 5 levels and 3 levels, respectively. The control population consisted of infants from 22 to 42 weeks' gestation who died in <48 hours. Results were compared with gestation-matched infants with varying severity of chronic lung disease.

RESULTS. In control and chronic lung disease groups, 22 to 42 weeks' gestation, staining for all 3 of the nitric oxide synthase isoforms was found in the airway epithelium from the bronchus to the alveolus or distal-most airspace. The abundance or distribution of nitric oxide synthase-3 staining in the airways did not show significant correlation with gestational age or severity of chronic lung disease. In the vascular tree, intense nitric oxide synthase-3 and moderate nitric oxide synthase-2 staining was found; nitric oxide synthase-1 was not consistently stained. Nitrotyrosine did stain in the pulmonary tree. Compared with controls where nitrotyrosine staining was minimal, regardless of gestation, in infants with chronic lung disease there was more than fourfold increase between severe chronic lung disease (n = 12) and either mild chronic lung disease or control infants (n = 16).

CONCLUSIONS. All 3 of the nitric oxide synthase isoforms and nitrotyrosine are detectable by immunohistochemistry early in lung development. Nitric oxide synthase ontogeny shows no significant changes in abundance or distribution with advancing gestational age nor with chronic lung disease. Nitrotyrosine is significantly increased in severe chronic lung disease.

Key Words: pulmonary nitric oxide synthases • lung development • nitrotyrosine

Abbreviations: NO—nitric oxide • NOS—nitric oxide synthase • CLD—chronic lung disease • INO—inhaled nitric oxide • NOS-2—inducible nitric oxide synthase • NOS-3—endothelial cell nitric oxide synthase • NOS-1—neuronal nitric oxide synthase • 3-NT—3-nitrotyrosine

Nitric oxide (NO) mediates and modulates successful transition from intrauterine to extrauterine life through its participation in complex physiologic processes including vasodilatation.1–3 Although endogenous NO production has been studied in pediatric asthma,4 relatively little is known regarding ontogeny of the 3 NO synthases (NOS) and subsequent production of NO in the developing or prematurely born human lung. Also relatively unstudied in humans is the potentially modifying effect on NOS expression of developmentally related pulmonary disorders, particularly chronic lung disease (CLD), of prematurity.

Several recent large clinical trials have tested the possibility that inhaled nitric oxide (INO), administered beginning in the first few days after birth to very premature infants requiring assisted ventilation, would reduce the risk of development of or severity of CLD of prematurity.5–7 These well designed trials have reported different results. One surprising finding that emerged from the study of Schreiber et al5 is that the less severely ill infants at the time of initiation of NO therapy seemed to benefit more than the more severely ill infants. This finding was confirmed by Van Meurs et al7 and Field et al,6 who showed no benefit, overall, in a population of critically ill, very low birth-weight infants. Not assessed in any of these trials was baseline NO production, baseline presence of pulmonary NOSs, or the capacity to upregulate endogenous pulmonary NO production. Administration of additional, exogenous NO would be expected to be of little benefit in infants whose pulmonary NOS was already upregulated, providing substantial endogenous NO. Also not assessed was quantitation of pulmonary nitrotyrosine. Nitrotyrosine is formed from the reaction of oxidant and nitrosyl compounds and serves as a marker for NO metabolism.8,9

Because of the public health importance of CLD of prematurity,10,11 with an increasing population of infants at risk for its development,12 and because of the paucity of established therapies for CLD of prematurity after the recommendation for withdrawal of postnatal high-dose corticosteroids for treatment of CLD,13 INO has been intensely studied. Given the mixed results to date of the clinical trials, it is important to use all of the available information in understanding pulmonary NO biology to evaluate specific indications and contraindications for NO administration in very preterm infants with, or at risk for, pulmonary insufficiency.

The first goal of this study was to assess the presence and the relative density of the 3 NOS isoforms at specific predefined levels of the airway and the vascular bed in lungs of infants who were either stillborn or who died very shortly after birth. The second goal was to quantitate the presence of pulmonary nitrotyrosine in the same lung tissues.

The null hypothesis tested was that there would be no relationship between the presence or density of any 1 of the 3 NOS isoforms or of nitrotyrosine with gestational age or, separately, with the severity of CLD in infants who died with, or of, this disorder. To test these hypotheses, we characterized the gestational age-dependent distribution of the 3 NOS isoforms and of nitrotyrosine. We also evaluated the relationship between the location and the quantity of each of the 3 NOS isozymes and of nitrotyrosine between gestational age and postconceptional age and severity of CLD in the infants who died. We used a library of postmortem human pulmonary tissue. The details of this library have been described previously.14–16


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Approval
The Institutional Review Board of Children's Mercy Hospitals and Clinics reviewed the following study and granted exemption status, because we used existing specimens and because the subjects' identities were separated from the data analyzed.

Tissue Samples
The tissues evaluated were drawn from a bank of previously collected, postmortem, paraffin-embedded, infant lung tissue blocks. Samples were obtained at autopsy, conducted within 8 to 24 hours of death. Lungs, collected over a 16-year period (1985–2001), were perfused and fixed using a standard method.17 The left lung was inflated to 24-cm H2O pressure with 10% formaldehyde, via the trachea, and fixed for 72 hours.

Inclusion criteria for controls were that the infant lived <48 hours before significant pulmonary rearchitecturing had occurred or died from nonlung complications. Exclusion criteria included prolonged rupture of fetal membranes (>48 hours), multiple congenital anomalies, inability to inflate the lung postmortem, or extensive pulmonary hemorrhage seen histologically. None of the infants was treated with NO.

Tissues were available from 56 infants with postconceptional age 22 to 42 weeks. Four to 15 samples in each of 3 respiratory score ([score] respiratory score = integrated area under the curve of average daily fraction of inspired oxygen [FIO2] x mean airway pressure [cm H2O] over the number of days lived)17 observation groups were compared with 25 gestation-matched controls.

Definition of Pulmonary Disease
Patients were placed into groups based on score and gestational age. The score for each infant was determined by multiplying the average daily FIO2, recorded hourly, by the average daily mean airway pressure in centimeters of H2O, recorded hourly, and integrating the area under the curve using the trapezoidal rule, for the total number of days lived. Infants not receiving assisted ventilation were assigned scores of 0. Score values of <20, 21 to 69, and 70 to 300 correlated with mild, moderate, and severe CLD of prematurity, respectively. During tissue analysis, investigators were unaware of the infants' medical history, gestational age, and score group.

Immunohistochemical Assessment
Formalin-fixed, paraffin-embedded tissue blocks were cut into 5 µM sections. Samples were mounted on positively charged glass microscope slides and kept in a 60°C oven overnight. Sections were deparaffinized and rehydrated. Staining was enhanced by steaming slides in a target retrieval solution (DAKO, Carpinteria, CA) bath for 20 minutes. After incubation with blocking serum (DAKO, Carpinteria, CA) for 10 minutes, slides were incubated with one of the following primary antibodies for 1 hour: inducible NOS (NOS-2) or endothelial cell NOS (NOS-3) monoclonal antibody, neuronal NOS (NOS-1) polyclonal antibody (Transduction Laboratories, Lexington, KY), or nitrotyrosine polyclonal antibody (Upstate Biotechnology, Inc, Lake Placid, NY). Sections were then immunohistochemically stained using a modified avidin-biotin-peroxidase method.18 Slides were incubated for 30 minutes with biotinylated immunoglobulin G and 30 minutes with avidin-biotin-peroxidase complex (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA). Antigenic sites were visualized by the addition of the chromogen 3, 3' diaminobenzidine. Slides were counterstained with Harris hematoxylin (Fisher HealthCare, Houston, TX). Negative control slides were stained using the same procedure, omitting the primary antibody.

The 3 NOS isoforms were semiquantitatively evaluated based on a 0 to 3 grading of the intensity and numbers of cells stained. Observers were blinded to patient group. Five airway levels (bronchus, proximal bronchiole, terminal bronchiole, respiratory bronchiole, and alveolar septum) and 3 vascular tree levels (large vessels, >90 µM; terminal bronchiolar vessels; and preseptal vessels) were evaluated.

The grading scale used was: 0 for no staining, 1 for rare or faint intensity, 2 for moderate staining, and 3 for intense staining. For all of the NOS isoforms, a minimum of 5 random areas of each patient slide were evaluated and graded for each of the airway and vascular levels. The only exception was when there were not 5 of the desired structures on a slide. Ratings at each airway and vascular level on each slide were averaged. Negative control slides revealed no specific staining.

Nitrotyrosine staining was evaluated as follows: a randomly selected group of tissues from control infants, and 5 to 6 infants in each score group had computerized, quantitative image analysis (AnalySIS Soft Imaging Systems, Lakewood, CO) performed for nitrotyrosine immunostaining. Data were expressed as the percentage of area of lung tissue demonstrating nitrotyrosine immunostaining, separate from air spaces.

Statistical Evaluation
Ordinal data were assessed statistically using {chi}2. Continuous data (nitrotyrosine staining quantity) were evaluated using Student's t test. All of the analyses were conducted with SPSS 3.0 (SPSS, Chicago, IL). Analysis of variance was used to test for gestational age effects. Results were corrected for multiple comparisons.

NOS staining was evaluated independently and without knowledge of the clinical outcome. Evaluations from independent observers were averaged.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NOS Immunostaining
Immunohistochemical staining for each of the 3 NOS isoforms was found in the lungs at all of the gestations from the youngest (22 weeks) to the oldest (42 weeks) (Fig 1 A–C). Staining for all 3 of the NOS isoforms was found in all of the lungs from the bronchiolar epithelium to the most distal alveoli.


Figure 1
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  		FIGURE 1 Examples of NOS stain ratings. A, NOS-1 stain rating 0; B, NOS-1 stain rating 2.2; C, NOS-2 stain rating 0; D, NOS-2 stain rating 3.0; E, NOS-3 stain rating 0; F, NOS-3 stain rating 2.5. Magnification, x400.

 
The amount and distribution of NOS-3 staining in the airways did not correlate significantly with gestational age or CLD severity (Table 1 and Fig 2). The only statistically significant difference was at the respiratory bronchiolar level, at which NOS-3 staining was decreased in the severe CLD group compared with 22- to 29-week gestational age infants.


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  		TABLE 1 Stain Scores for NOS-1, NOS-2, and NOS-3 at 5 Airway Levels in Control Infants (22 Weeks' Gestational Age to Term) and Severe CLD Infants

 

Figure 2
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  		FIGURE 2 Stain scores (0–3) throughout the respiratory tree in control infants, (22 weeks' gestation to term) and severe CLD infants. A, NOS-1; B, NOS-2; C, NOS-3.

 
In the vascular endothelium, immunostaining was found for each NOS isoform. Intense NOS-3 staining, average grade 2, and moderate NOS-2, average grade 1.5, staining were found at all vascular levels (Table 2 and Fig 3 A–C). NOS-1 was not consistently stained in vascular endothelium, with grading 0 to 2 (Fig 3A), and was less intensely stained than in the airway (Fig 2A).


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  		TABLE 2 Stain Scores for NOS-1, NOS-2, and NOS-3 at 3 Vascular Levels in Control Infants (22 Weeks' Gestational Age to Term) and Severe CLD Infants

 

Figure 3
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  		FIGURE 3 Stain scores (0–3) throughout the vascular tree in control infants, (22 weeks' gestation to term) and severe CLD infants. A, NOS-1; B, NOS-2; C, NOS-3. Note different scales than in Fig 2.

 
Nitrotyrosine
Nitrotyrosine staining was found in the pulmonary tree from 22 to 42 weeks. Examples of nitrotyrosine staining are shown (Fig 4). Staining was found mostly in the nuclear/perinuclear areas of the airway epithelium. Mean area of staining reveals large differences in the different score groups (Table 3). In controls, the mean area of nitrotyrosine staining was 2.3%, and in the score <20 group, it was 2.4%. The moderate score group, 20 to 69, was 3.7%. However, the severe pulmonary disease group, with the score range of 70 to 300, had a mean area of intensity more than fourfold higher at 20.8% (P < .001) versus the other groups (Table 3).


Figure 4
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  		FIGURE 4 Nitrotyrosine staining. A, Control lung showing little staining. B, Severe CLD demonstrating heavy nitrotyrosine staining. Magnification, x400.

 

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  		TABLE 3 Mean Percent Tissue Area of Nitrotyrosine Immunostaining of Distal Lung Parenchyma

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Summary of Results
The current findings provide evidence for expression of all 3 of the NOS isoforms at all of the airway levels studied in the developing lung and in all of the levels studied of the developing vascular tree from 22 weeks to term. The abundance and distribution of NOS-1, NOS-2, and NOS-3 in the airways did not correlate significantly with gestational age or with severity of CLD, as measured by our score. NOS-1 was diminished in the vascular tree compared with its staining in the airways. In contrast, nitrotyrosine, which was also identified immunohistochemically at all gestational ages from 22 to 42 weeks, was positively correlated with severity of CLD (Table 3). As noted, immunohistochemical staining seemed to be localized primarily to the nuclear and perinuclear areas of many cells in the airway epithelium.

NOS Expression
The site of NOS expression has been different in different models and species. Abman and colleagues,19,20 in 2 studies, found NOS-3 in vascular endothelium but not in the bronchial epithelium regardless of fetal age. Similarly, Black et al21 confined NOS-3 to the vascular epithelium in fetal and newborn lambs. However, NOS-3 and NOS-2 were detected in all arteriole generations and all airway levels in a study showing reduced NOS-3 in chronically ventilated lungs of preterm lambs.22 Localization of NOS-3, NOS-2, and NOS-1 was described in the developing lamb lung by Sherman et al.23 These investigators did not detect NOS-3 in the terminal bronchiole or more distal parts. Xue et al24,25 found no messenger RNA NOS-3 in the fetal rat airway epithelium, but did detect NOS-3 protein in the lung within 2 hours of birth. In a preterm lamb model of CLD ventilated with or without INO for 3 weeks, Bland et al26 found reduced NOS-3 protein in distal airways and vasculature. In a baboon model of CLD, Afshar et al27 found reduction in NOS-3 in lungs exposed to assisted ventilation for 14 days compared with gestational age-matched controls. Our finding of decreased vascular and perhaps distal airway NOS-3 in severe CLD in humans is consistent with these findings. There is little information in the developing human neonate as to the location and expression of NOS, NO function and metabolic pathways, and its modulation in CLD.

Aikio et al28 studied neonates, but mostly term and near-term infants, seeking to investigate the influence of INO on NO production in respiratory failure. Only 6 CLD infants were studied. Another study evaluating NOS in early second trimester-aborted fetuses by Kobzik et al29 found NOS-1 and NOS-3 in substantially varying amounts in large vessel endothelium and NOS-2 in large cartilaginous bronchi and occasional alveolar macrophages. No CLD infants were studied.

Other studies of pulmonary NOS presence in humans have used adult lungs, usually resected from patients with lung cancer, or have used human pulmonary cell culture. Watkins et al30 did not find NOS-3 in airways in resected tissue or the BEAS-2B cell line. However, NOS-2 was found in 5 of 6 resected airways. These studies in adults have identified NOS, but the relevance to the developing human lung is unclear. We found all 3 of the NOS isoforms in the airway epithelium from the bronchus to the most distal levels at the alveolar septum in all of the gestations from 22 to 42 weeks, revealing the enzymatic potential for NO production in even the youngest gestations.

Nitrotyrosine Staining
One NO metabolic pathway is the radical-radical interaction with superoxide and NO to form peroxynitrite, a process that occurs at a near-diffusion-limited rate.8 Peroxynitrite is a reactive nitrogen species that can affect various biological compounds. Specifically, peroxynitrite can nitrate phenolic compounds, including proteins containing tyrosine. Nitrotyrosine residues are products of nitro groups added to the ortho position of the hydroxyl group of tyrosine. Nitrotyrosine is a stable and measurable end product of this reaction; it can serve as a footprint for the products of reactive nitrogen species.31,33 Nitrotyrosine has been detected in various studies in both humans and animals in diseases ranging from adult respiratory distress syndrome, tuberculosis, or acute lung injury to atherosclerosis and neurodegenerative diseases.34–36 There are few studies assessing nitrotyrosine in infants with lung injury. Haddad et al37 have shown a twofold increase in nitrotyrosine staining in lungs of children dying of adult respiratory distress syndrome, and Kooy et al36 demonstrated intense staining in lungs of children with acute lung injury versus slight staining in control samples. Banks et al38 demonstrated nitrotyrosine in the plasma of infants who develop bronchopulmonary dysplasia, and Lorch et al39 revealed a correlation with severity of BPD and increased plasma 3-nitrotyrosine (3-NT) with those having received exogenous NO, but the relationship between plasma 3-NT and lung immunostaining is unclear.39 The cause of the increased 3-NT staining in the infants with the most severe CLD is not clear. It is possible that there was greater inflammation in the lungs of these infants at death and that increased activity NOS-2 was contributing in the lungs of these infants to the apparent increase of NO production before death. This possibility would need to be confirmed with in vivo assessment of lung-specific endogenous NO production.

Limitations of the Methodology
There are several limitations to the methodology used in this study. Although there is a risk of postmortem changes in pulmonary tissue from the time of death to the time of tissue fixation, we sought to minimize them by excluding samples in which apparent postmortem autolysis had occurred. In addition, the time from death to autopsy was short and was within the range established by other postmortem human studies.28 A single individual (D.W.T.) prepared and fixed all of the lung tissue and was present to review the lung preparation at the time of the autopsy. Thus, there is a reasonable degree of standardization of the approaches. The relatively large size of the sample collection reduces the possibility of type I errors.

Immunohistochemical studies have specific limitations associated with them. For example, we performed the appropriate negative staining without relevant antibodies to confirm the absence of nonspecific staining. It is difficult in the lung, especially, to provide quantitative assessment of staining, especially when the specific staining is for an enzyme as opposed to a structural protein. Two observers read the slides independently and averaged their findings of the semiquantitative scoring for the NOS isozymes. Nonetheless, it is not possible to completely exclude a type II error in that there may have been either developmentally associated or disease-associated changes in expression of the protein.

Staining of the enzyme does not predict function. It is possible that there could be truncated proteins or that the protein may be quiescent even if present. In an additional effort to overcome these limitations, we used image software for quantitation of nitrotyrosine staining. Our scoring system demonstrated a dramatic increase in nitrotyrosine in infants with the most severe respiratory insufficiency. These infants had never received exogenous NO therapy. We conclude that there was substantial endogenous NO production and consequent interaction with reactive oxygen species producing the reactive nitrogenous species and, consequently, nitrotyrosine staining.

The strengths of this study are that it was performed in humans and has produced the most comprehensive assessment to date of the presence of pulmonary NOS isozyme proteins from 22 to 42 weeks' gestation. Thus, we were able to account for the relevant range of gestational age at the time of birth. Perhaps the most important finding is the intense nitrotyrosine staining correlating with severity of CLD.

Clinical Implications
The clinical implications of our findings may be that, in infants with very severe CLD, it may not always be useful to administer exogenous NO, because some of these infants have already demonstrated activation of their system. Our findings seem to be in contrast to those of Aikio et al,28 who did not find any change in nitrotyrosine staining in 3 infants with CLD who had received INO. None of the currently published reports of clinical trials of INO has reported on any postmortem findings that would address this issue in the subset of infants who received NO compared with those who received placebo. It should also be noted that the lungs from the infants studied in this report represented the subset of children who did not recover from CLD and whose potential response to exogenous NO was not tested. We speculate that it would be useful to identify in infants with early or established CLD an accurate, reproducible, quickly available, and relatively noninvasive marker of pulmonary NO activity. Evidence of activation of any of the NOS isozymes with subsequent production of NO may serve as a relative contraindication to the exogenous administration of NO and may represent a group in which the therapy would be contraindicated. Immunohistochemical staining of nitrotyrosine in lung tissue certainly does not qualify for that purpose, but we speculate that exhaled NO measurements may still provide a tool for more specific and precise use of INO, as has been performed in infants with RDS.40


   ACKNOWLEDGMENTS
 
This work was supported in part by Children's Mercy Hospital Research and Education Grant (Dr Sheffield) and by the National Heart, Lung, and Blood Institute R-01 70560 (Dr Truog).


   FOOTNOTES
 
Accepted Mar 28, 2006.

Address correspondence to William E. Truog, MD, Department of Pediatrics, Section of Neonatology, Children's Mercy Hospitals and Clinics, 2401 Gillham Rd, Kansas City, MO 64108. E-mail: wtruog{at}cmh.edu

The authors have indicated they have no financial relationships relevant to this article to disclose.


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PEDIATRICS (ISSN 1098-4275). ©2006 by the American Academy of Pediatrics




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