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Am J Physiol Lung Cell Mol Physiol 291: L119-L127, 2006. First published January 27, 2006; doi:10.1152/ajplung.00395.2005
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Inhaled NO restores lung structure in eNOS-deficient mice recovering from neonatal hypoxia

Vivek Balasubramaniam, Anne M. Maxey, Danielle B. Morgan, Neil E. Markham, and Steven H. Abman

Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado

Submitted 23 August 2005 ; accepted in final form 25 January 2006


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously shown that neonatal mice deficient in endothelial nitric oxide synthase (eNOS–/–) are more susceptible to hypoxic inhibition of alveolar and vascular growth. Although eNOS is downregulated, the role of nitric oxide (NO) during recovery after neonatal lung injury is poorly understood. We hypothesized that lung vascular and alveolar growth would remain impaired in eNOS–/– mice during recovery in room air and that NO therapy would augment compensatory lung growth in the eNOS–/– mice during recovery. Mice (1 day old) from heterozygous (eNOS+/–) parents were placed in hypobaric hypoxia (FIO2= 0.16). After 10 days, pups were to recovered in room air (HR group) or inhaled NO (10 parts/million; HiNO group) until 3 wk of age, when lung tissue was collected. Morphometric analysis revealed that the eNOS–/– mice in the HR group had persistently abnormal lung structure compared with eNOS-sufficient (eNOS+/+) mice (increased mean linear intercept and reduced radial alveolar counts, nodal point density, and vessel density). Lung morphology of the eNOS+/– was not different from eNOS+/+. Inhaled NO after neonatal hypoxia stimulated compensatory lung growth in eNOS–/– mice that completely restored normal lung structure. eNOS+/– mice (HR group) had a 2.5-fold increase in lung vascular endothelial growth factor (VEGFR)-2 protein compared with eNOS+/+ (P < 0.05). eNOS–/– mice (HiNO group) had a 66% increase in lung VEGFR-2 protein compared with eNOS–/– (HR group; P < 0.01). We conclude that deficiency of eNOS leads to a persistent failure of lung growth during recovery from neonatal hypoxia and that, after hypoxia, inhaled NO stimulates alveolar and vascular growth in eNOS–/– mice.

nitric oxide; endothelial nitric oxide

PREMATURE BIRTH AND the treatment of neonatal respiratory distress syndrome contribute to the development of chronic lung disease of infancy, or bronchopulmonary dysplasia (BPD; see Ref. 35). BPD is characterized by persistent abnormalities of lung structure, including dysmorphic vascular growth and impaired alveolarization (12, 22). Structural lung abnormalities in BPD include a reduction of lung surface area that results in abnormal gas exchange, exercise intolerance, and pulmonary hypertension (1, 12, 20, 23, 44, 45). Mechanisms that either disrupt alveolarization or angiogenesis during the development of BPD or enhance lung growth during recovery after neonatal lung injury are poorly understood.

The saccular and alveolar phases of lung development are characterized by rapid increases in vessel growth and septation of the distal air spaces. These phases of lung development occur from the 24th wk of gestation through the first 3 yr of life in humans, and from fetal day 17 through the first 3 wk of postnatal life in rodents (10, 31, 47). Experimental models of BPD include exposure of neonatal rodents to hyperoxia or hypoxia (31, 46, 54–56). Exposure of rats to severe hypoxia (10% oxygen) during the perinatal period impairs alveolar and vascular growth (46). Abnormalities of lung structure can persist into adulthood, suggesting that lung injury during a critical period of lung development results in the persistence of abnormal lung structure (46). Furthermore, recovery of lung structure may not be possible after a certain period of lung development (40, 46). These studies suggest that exposure during critical periods of lung development impairs alveolarization and angiogenesis and that lung injury during the neonatal period may impair the capacity for compensatory lung growth during infancy, leading to a persistence of abnormal lung structure into adulthood. Chronic ventilation of premature lambs and baboons impairs distal lung growth (3, 7, 13, 36). In these models of BPD, lung endothelial nitric oxide synthase (eNOS) protein is reduced, suggesting that eNOS is important in the regulation of lung alveolar and vascular growth (2, 28, 42). However, the role of eNOS in the pathogenesis of abnormal lung structure in BPD is unknown.

We have previously reported that mice genetically deficient in eNOS are susceptible to mild neonatal hypoxia, with evidence of alveolar simplification and a reduction in vascular volume that is not seen in wild-type mice (5). Recently, it has been reported that inhaled nitric oxide (NO) may protect against changes in lung structure in baboon (32), lamb (8), and rat models (27) of BPD. Whether inhaled NO can enhance lung structure after injury to the developing lung has not been studied.

Therefore, we hypothesized that a genetic deficiency in eNOS will impair the recovery of lung structure in infant mice after exposure to mild hypoxia in the neonatal period. Furthermore, we hypothesized that treatment with inhaled NO (iNO) would enhance alveolarization and angiogenesis during recovery from neonatal lung injury. To test this hypothesis, we examined changes in lung structure in eNOS-deficient (eNOS–/–) mice that were allowed to recover from mild neonatal hypoxia (16% oxygen) in room air compared with the structure of mice that were chronically treated with iNO [10 parts/million (ppm)] after neonatal hypoxia. We report that eNOS–/– mice lacked compensatory lung growth during room air recovery after exposure to mild neonatal hypoxia. In addition, mice heterozygous for eNOS (eNOS+/–) that recovered in room air were able to completely restore their lung structure. We also report that iNO treatment during room air recovery after hypoxia was able restore lung structure in the eNOS–/– mice.


   METHODS
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 ABSTRACT
 METHODS
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Study animals and protocols. All procedures and protocols were reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Mice (C57BL/6J-nos3tm1Unc) that were genetically engineered to be heterozygous for a deficiency of eNOS (19) were obtained from Jackson Laboratories. Genomic DNA was isolated from tails of experimental mice. Genotyping was done by PCR, using previously described primers to identify the presence of the Neo gene insert in the eNOS gene (19).

Exposure of neonatal mice to mild hypoxia. Males and females that were known to be heterozygous for eNOS deficiency were bred to ensure the generation of litters that consisted of pups that were homozygous and heterozygous for eNOS deficiency and the wild type. The litters of heterozygote x heterozygote matings were allowed to deliver and recover in room air for 24 h. Mothers and litters were placed in hypobaric chambers at a simulated altitude of 12,300 feet (16% O2; PO2 = 90 mmHg) for 10 days or maintained in room air. Exposure to hypoxia was continuous, with a brief interruption for animal care (<1 h/day). After 10 days, litters were allowed to recover either in room air (hypoxia-room air: HR; Fig. 1) or in chambers in which iNO was delivered and maintained at a level of 10 ppm (hypoxia-inhaled NO: HiNO) until the mice were 3 wk of age. Another set of litters was kept in room air until 3 wk of age (room air-room air: RR). The concentration of NO gas was continuously measured and delivered by an INOVent (iNO Therapeutics, Clinton, NJ). The concentration of NO2 was also monitored by the INOVent and was maintained <3 ppm. NO gas and INOVent were kindly provided by iNO Therapeutics. At 3 wk of age, lung tissue was collected from at least five animals of each genotype in each of the three groups for histology and a similar number for each group for biochemical studies described below.


Figure 1
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  		Fig. 1. Study design. On postnatal day 1, litters from eNOS heterozygote x heterozygote matings were placed in hypobaric hypoxia (FIO2 = 0.16; 12,300 ft.). One group of litters remained at room air for the entire study period (RR group). At the end of 10 days (postnatal day 11), the mice were recovered either in room air (HR group) or in room air + 10 parts/million (ppm) inhaled nitric oxide (HiNO group) until 21 days of age. iNO, inhaled nitric oxide.

 
Tissue for histological analysis. Lungs were prepared for histology as previously described (4). Animals were killed with intraperitoneal injections of pentobarbital sodium (100 mg/kg). A catheter was placed in the trachea, and the lungs were inflated at 30 cmH2O pressure with 4% paraformaldehyde in PBS and maintained under constant pressure for at least 45 min. A ligature was tightened around the trachea to maintain pressure, and then the tracheal cannula was removed. The lungs were immersed in paraformaldehyde solution overnight. The left lower lobe was embedded in paraffin, and sections were cut with a microtome set at 5 µm and mounted on RNase-free slides for histochemical analysis.

Morphometric analysis. Six lung sections from each animal were selected for study in an unbiased fashion. The orientation of these samples was at random, creating isotropic uniform random plane sections of the lung tissue (5). Each section was stained with hematoxylin and eosin. Images of each section were captured on a Zeiss Axioscope2, using the x20 objective, as a high-resolution PICT image by a QICAM digital camera (1,392 x 1,040 pixel resolution; Qimaging, Burnaby, Canada) and was analyzed with the use of Stereology Toolbox software (Davis, CA). The intra-alveolar distance was measured as the mean linear intercept (MLI) by standard methods, as previously described (5, 49, 50). Briefly, MLI was determined by dividing the total length of 42 lines drawn across the lung section by the number of intercepts encountered as determined by the principal investigator (V. Balasubramaniam; see Ref. 50). The investigator was blinded to the identity of the sections at the time of analysis. Lines that crossed large airways or vessels were excluded from analysis. MLI is inversely proportional to the surface area of the lung (49, 50). Radial alveolar counts (RAC) were assessed by standard methods, as previously described (5, 14).

For assessment by skeletonization (51), six lung sections were selected in unbiased fashion. Images of each section were captured with a QICAM digital camera on a Zeiss Axioscope2 with x20 objective and were saved as TIFF files. These images were processed with a plug in that was written by V. Balasubramaniam and Dr. Christopher Coulon, to utilize ImageJ, a public-domain Java image processing program created by Wayne Rasband at the Research Services Branch, National Institute of Mental Health, Bethesda, MD (http://rsb.info.nih.gov/ij). The user is prompted either to select an image of a scale taken at the same magnification as the image set to be processed or to input the number of micrometers per pixel. With the scale image, the plug in thresholds the image, uses the ImageJ Analyze Particles routine to segment the scale and find the centers of each scale bar, and then takes the mean distance between centers as the calibration distance. This number is used to calibrate all subsequent measurements. The user is next prompted to navigate to the directory containing the images to be processed and select the first image. Processing continues automatically after this action. Images are opened, processed, and closed. Images are acquired in red-green-blue (RGB) color format and converted to an RGB stack having eight bits (256 levels of gray) in each of the red, green, and blue slices of the stack. Because the green slice gives the best contrast for regions of interest, the green slice is selected and duplicated as a single eight-bit gray scale image, and the original image is closed without alteration. The image is bitmapped, and the lung parenchyma is transformed into a skeleton of curved and straight line segments with nodal and end points, as described by Tschanz and Burri (51), and the number of all end points, intersections (nodes), and internode distances are calculated and returned as a results table.

Sections were stained for the presence of factor VIII (von Willebrand factor; DAKO), an endothelium-specific marker. For assessment of vessel density, images of factor VIII-stained slides were captured with the x20 objective. The number of factor VIII-positive vessels (20–80 µm in size) was counted per each high-power field.

Western blot analysis. Frozen lung samples were homogenized in ice-cold buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.1% beta-mercaptoethanol, 1 mM 4-(2-aminoethyl)benzenesulfoyl fluoride, 1 mM leupeptin, and 1 µM pepstatin A. The samples were centrifuged at 1,500 g for 20 min at 4°C to remove cellular debris. Protein content in the supernatant was determined by the Bradford (9) method, using BSA as the standard. Briefly, 25 µg protein sample/lane were resolved by SDS-PAGE, and proteins from the gel were transferred to polyvinylidine difluoride membranes. Blots were blocked for 1 h in 5% nonfat dry milk in TBS with 0.1% Tween 20. These blots were incubated for 1 h at room temperature with either rabbit anti-human polyclonal vascular endothelial growth factor (VEGF) antibody (sc-152; 1:500; Santa Cruz Biotechnology), rabbit anti-human polyclonal VEGF receptor (VEGFR)-2 antibody (KDR/flk-1; sc-504; 1:250; Santa Cruz Biotechnology), or goat anti-mouse polyclonal platelet/endothelial cell adhesion molecule-1 (PECAM; sc-1506; 1:200; Santa Cruz Biotechnology) diluted 1:200 in 5% nonfat dry milk in TBS with 0.1% Tween 20. Blots were incubated for 1 h at room temperature with a goat anti-rabbit IgG-horseradish peroxidase (HRP) antibody (sc-2054; 1:5,000; Santa Cruz Biotechnology) or donkey anti-goat IgG-HRP antibody (sc-2020; 1:15,000; Santa Cruz Biotechnology). After being washed, bands were visualized by enhanced chemiluminescence (ECL+ kit; Amersham Pharmacia Biotech, Buckinghamshire, UK). Adult mouse lung homogenate was run as a control, and the band that comigrated with the molecular size as identified by the manufacturer for the protein of interest was quantified by densitometry for VEGFR-2 and PECAM. For Western analysis of VEGF, recombinant mouse VEGF was used as a control. Densitometry was performed using NIH Image (version 1.61).

Immunohistochemical staining. Lungs were fixed in 4% paraformaldehyde/PBS for 24 h and then stored in 70% ethanol. The left lower lobe was embedded in paraffin, cut into 5-µm-thick sections, and mounted on "plus" slides. Slides were deparaffinized in HemoDe and rehydrated by serial immersions in 100% ethanol, 95% ethanol, 70% ethanol, and 100% water. Proteinase K (50 µg/ml) was placed on the sections for 5 min. The sections were washed with 1x PBS (in mM: 2.7 KCl, 1.2 KH2PO4, 138 NaCl, and 8.1 Na2HPO4). Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide in methanol, and rinsed with PBS. The sections were incubated with 10% goat/2% mouse serum and rabbit anti-human polyclonal factor VIII antibody (A0082: DAKO), tropoelastin antibody (or mouse IgG diluted 1:1,000 in 1x PBS with 1% BSA), and 0.1% sodium azide for 1 h at room temperature. After incubation, the sections were rinsed with PBS, incubated in 10% goat/2% mouse serum for 5 min, and then incubated with biotin-labeled goat anti-mouse secondary antibody diluted 1:200 in 10% goat/2% mouse serum for 15 min at room temperature. After incubation with the secondary antibody, sections were rinsed with PBS. Sections were incubated with ABC complex (Vector) for 30 min at room temperature, rinsed in PBS, and developed with diaminobenzidine (DAB; Vector) and hydrogen peroxide. Washing with water stopped the DAB reaction. A light hematoxylin counterstain was applied. Sections were dehydrated by sequential immersion in 70% ethanol, 95% ethanol, 100% ethanol, and then HemoDe before a coverslip was placed on the section.

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed with the Prism software package (GraphPad Software, San Diego, CA). Statistical comparisons were made using ANOVA and Tukey's post hoc test. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Body weight. At 21 days postnatal age, there was no difference in body weight between the wild-type, eNOS+/–, and eNOS–/– mice raised in room air, hypoxia followed by room air, and hypoxia followed by iNO.

eNOS deficiency impairs recovery of lung structure from neonatal hypoxia. There was no difference in lung histology between wild-type, eNOS+/–, and eNOS–/– mice raised in room air at 3 wk of age (RR group). Wild-type mice exposed to neonatal hypoxia and recovered in room air (HR group) had no difference in RAC, MLI, or nodal point density compared with wild-type RR controls. At 3 wk of age, after recovery from neonatal hypoxia in room air, there was no change in RAC, nodal point density, or vessel density in the wild-type mice (Figs. 2–4). MLI increased by 9% in the wild-type HR mice during the room air recovery that was similar to the changes in MLI observed in RR mice from 11 to 21 days of age. Vessel density in the wild-type HR mice was reduced by 25% compared with wild-type RR mice (Fig. 4; P < 0.01). In contrast to wild-type mice, the eNOS+/– HR mice demonstrated a 23% increase in RAC and a 44% increase in nodal point density by 3 wk of age (Figs. 2 and 3). This increase in lung growth resulted in absolute values of RAC, MLI, and nodal point density that are similar to the measurements of the wild-type mice (Figs. 2, 3, and 5). Vessel density in the eNOS+/– HR mice was reduced by 33% compared with both wild-type RR and eNOS+/– RR mice (P < 0.01).


Figure 2
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  		Fig. 2. Radial alveolar counts (RAC) remain reduced in the eNOS–/– mice despite recovery in room air but increase during iNO therapy. Data are shown for RAC immediately after hypoxia at 11 days of age, at 21 days of age with room air recovery, at 21 days of age with iNO recovery and for animals raised in room air for 21 days. As previously reported (5), exposure to neonatal hypoxia in the 11-day-old eNOS–/– animal and eNOS heterozygote (+/–) reduces RAC compared with the wild-type mice (*P < 0.01 compared with wild type). In the wild-type mice, RAC did not increase from 11 days of age to 21 days of age. RAC increased during recovery in room air in the eNOS heterozygote but not the eNOS–/– (**P < 0.01 compared with 11 day eNOS+/– exposed to hypoxia). iNO therapy increased RAC in the eNOS–/– mice to levels seen in the wild-type mice. ({dagger}P < 0.01 compared with 11 day eNOS–/– exposed to hypoxia and 21 day eNOS–/– with RA recovery; n = 5 animals of each genotype in each group). +/+, eNOS-sufficient group.

 

Figure 4
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  		Fig. 4. Vessel density is reduced in eNOS–/– mice after recovery in room air and increases in mice recovered with iNO therapy. Data are shown for vessel density immediately after hypoxia at 11 days of age, at 21 days of age with room air recovery, at 21 days of age with iNO recovery, and for animals raised in room air for 21 days. Vessel density is reduced by exposure to neonatal hypoxia in the eNOS–/– mice compared with 11-day-old wild-type hypoxic controls (*P < 0.01). Recovery from neonatal hypoxia in room air results in a persistent reduction in vascular density compared with room air-raised animals in all genotypes (**P < 0.01). iNO during the recovery increases vessel density in all genotypes, including a 2-fold increase in the eNOS–/– mice ({dagger}P < 0.01). Recovery from hypoxia in iNO increases vascular density in all genotypes to levels greater than that in room air-raised animals (#P < 0.05; n = 5 animals of each genotype in each group).

 

Figure 3
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  		Fig. 3. Nodal point density is reduced in eNOS–/– mice after recovery in room air and increases with iNO therapy. Data are shown for nodal point density immediately after hypoxia at 11 days of age, at 21 days of age with room air recovery, at 21 days of age with iNO recovery, and for animals raised in room air for 21 days. Exposure to neonatal hypoxia reduces nodal point density in the eNOS–/– and eNOS+/– mice at 11 days of age (*P < 0.01). Recovery in room air resulted in a significant %increase in nodal density in the eNOS+/– mice (**P < 0.01) but no change in eNOS–/– animals. Recovery in iNO resulted in a significant increase in the nodal point density in the eNOS–/– mice to levels seen in the wild-type mice and eNOS–/– mice raised in room air ({dagger}P < 0.01; n = 5 animals of each genotype in each group). hpf, High-power field.

 

Figure 5
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  		Fig. 5. Mean linear intercept (MLI) remains increased in eNOS–/– mice after recovery in room air, but normalizes with iNO therapy. Data are shown for MLI immediately after hypoxia at 11 days of age, at 21 days of age with room air recovery, at 21 days of age with iNO recovery, and for animals raised in room air for 21 days. After exposure to neonatal hypoxia, the eNOS–/– animal has an elevated MLI, as previously reported (Ref. 5; *P < 0.01). During recovery in room air, the MLI remained persistently elevated compared with the 11-day-old eNOS–/– mice (**P < 0.01). iNO resulted in a significant decrease in MLI ({dagger}P < 0.01) in the eNOS–/– mice compared with mice recovered in room air. The MLI of the eNOS–/– mice recovered in iNO were not different from wild-type or room air-raised eNOS–/– mice; n = 5 animals of each genotype in each group.

 
Lung histology of eNOS–/– mice that recovered in room air after neonatal hypoxia (Fig. 6D) shows enlarged airspaces compared with wild-type mice (Fig. 6C). RAC did not increase in eNOS–/– mice by 3 wk of age (Fig. 2). MLI of eNOS–/– mice during room air recovery from neonatal hypoxia increased by 25% at 3 wk of age, but remained elevated in comparison with wild-type HR mice (P < 0.01; Fig. 5). The nodal point density of the eNOS–/– mice during room air recovery after neonatal hypoxia decreased by 8% (Fig. 3) by 3 wk of age and remained significantly reduced compared with wild-type HR mice (P < 0.01; Fig. 3). Although vessel density increased by 26% in eNOS–/– mice after room air recovery from hypoxia at 3 wk of age, the absolute number remained lower in the eNOS–/– HR mice than wild-type HR mice (P < 0.05; Fig. 4).


Figure 6
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  		Fig. 6. iNO restores lung structure in the eNOS–/– mouse after neonatal hypoxia. Lung structure is markedly abnormal with enlarged air spaces and a simplified architecture in the eNOS–/– mice compared with the wild type after 10 days of neonatal mild hypoxia (A and B). In C and D, the simplified air space structure persists in eNOS–/– mice exposed to neonatal hypoxia despite recovery in room air. In eNOS–/– mice that were treated with iNO (10 ppm) during recovery, lung structure appears indistinguishable from the lungs of wild-type mice (E and F). All images taken with a x10 objective; n = 5 animals of each genotype in each group.

 
iNO restores normal lung structure in eNOS–/– mice exposed to neonatal hypoxia. Lung histology from eNOS–/– mice that were exposed to neonatal hypoxia and recovered in 10 ppm inhaled NO (HiNO group; Fig. 6F) shows normal lung structure that is difficult to distinguish from wild-type mice (Fig. 6, C and E). During recovery in iNO, the MLI was reduced by 17% in eNOS–/– HiNO mice to a measurement that was not different from wild-type HR mice (Fig. 5). Recovery of eNOS–/– mice in iNO increased RAC by 70% to a count not different from that of wild-type HR mice (Fig. 5). Nodal point density in eNOS–/– mice treated with iNO after neonatal hypoxia increased by 60%, a level greater than wild-type HR mice (P < 0.01; Fig. 3). Recovery in iNO after neonatal hypoxia resulted in an increased vessel density in all of the eNOS genotypes (Fig. 4). There was a twofold increase in vessel density in the eNOS–/– animals to levels that were similar to the HiNO wild-type mice, and greater than values in the eNOS–/– RR mice (P < 0.05; Fig. 4).

Changes in lung VEGF and VEGFR-2 protein during the recovery from neonatal hypoxia in room air or inhaled NO. As determined by Western blot analysis at 3 wk of age, lung VEGF protein content was not different between wild-type RR, HR, and HiNO groups. Lung VEGF protein was increased in both the heterozygote (88%)and knockout mice (57%) in the HR group at 3 wk of age compared with wild-type mice (Fig. 7A). In the HiNO group, lung VEGF protein content was not different in the wild-type, eNOS+/–, and eNOS–/– mice. Wild-type mice in the RR, HR, and HiNO groups had no difference in lung VEGFR-2 protein. In the HR group, eNOS+/– mice had a 264% increase in lung VEGFR-2 protein compared with both the wild-type and eNOS–/– mice (Fig. 7B). The HiNO eNOS–/– mice had no change in lung VEGF levels (Fig. 8A). In the HiNO eNOS–/– mice, lung VEGFR-2 protein content was increased by 66% compared with the HR eNOS–/– mice (Fig. 8B). There was no change in lung VEGFR-2 with iNO treatment in wild-type or eNOS+/– mice (data not shown).


Figure 7
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  		Fig. 7. Lung vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR)-2 protein expression in mice recovering from mild neonatal hypoxia in room air. Lung VEGF protein levels are increased in both eNOS+/– and eNOS–/– mice during the recovery from mild neonatal hypoxia (A). Lung VEGFR-2 protein was increased in the eNOS+/– mice but not in the eNOS–/– mice during recovery from mild neonatal hypoxia in room air (B). *P < 0.05; n = 5 animals in each group.

 

Figure 8
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  		Fig. 8. Lung VEGF and VEGFR-2 expression in the eNOS–/– animal during recovery from mild neonatal hypoxia in iNO. Lung VEGF protein did not change in eNOS–/– mice exposed to mild neonatal hypoxia that were recovered in 10 ppm iNO compared with room air (A). Lung VEGFR-2 expression increased by 66% in the eNOS–/– mice exposed to mild neonatal hypoxia that recovered in iNO compared with room air (B). *P < 0.01; n = 5 animals in each group.

 
Lung PECAM protein is not different between wild-type mice in the RR and HR groups. There was no difference in lung PECAM protein between wild-type, eNOS+/–, and eNOS–/– mice in the HR group. Inhaled NO increased lung PECAM by twofold in wild-type mice but not in the eNOS+/– or eNOS–/– mice (P < 0.01; data not shown).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously reported that neonatal exposure to mild hypoxia (16% oxygen) impairs lung growth and structure in eNOS–/– mice but not in wild-type mice (5), suggesting that eNOS deficiency increases the susceptibility for impaired alveolar and vascular growth to mild reductions in oxygen tension. In this study, we now report that abnormal lung structure persists in eNOS–/– mice despite prolonged recovery in room air. In contrast, eNOS+/– mice demonstrate a capacity for compensatory lung growth during recovery in room after mild neonatal hypoxia. These findings suggest that endogenous NO is essential for compensatory lung growth during the recovery after neonatal lung hypoxia.

In addition, we further report that treatment of eNOS–/– mice with low doses of inhaled NO (10 ppm) during the recovery period after neonatal hypoxia improves lung structure. A recent study in the preterm baboon model of BPD has suggested that there is abnormal deposition of elastin that leads to abnormalities in lung compliance in these animals (32). This same study showed a more normally distributed elastin location with the use of inhaled NO, although they did not see quantifiable changes in alveolar structure (32). In the present study, we looked for the presence of myofibroblasts by staining for tropoelastin, the soluble precursor for elastin, and found that in the eNOS–/– mice in the HR group tropoelastin deposition was shifted from the septal tips to tufts in the alveolar walls (Fig. 9). similar to the pattern reported in the baboon model of BPD. Inhaled NO therapy during recovery in the eNOS-deficient animal shifts tropoelastin deposition from the alveolar wall back to the septal tips, similar to the pattern observed in wild-type mice. Previous studies have suggested that NO has a direct effect on myofibroblasts (52). These findings suggest that inhaled NO promotes alveolar and vascular growth during the recovery from neonatal lung injury. These findings also show that the developing lung has the capacity for compensatory growth during late infancy and that this potential for postnatal lung growth is dependent, at least partially, on NO.


Figure 9
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  		Fig. 9. Immunohistochemical staining for tropoelastin. Neonatal hypoxia does not affect tropoelastin localization in the eNOS+/+ animal (A), but the staining pattern in the eNOS–/– mice shows localization at the septal tips and positively stained cells in the alveolar walls (arrowheads, B). iNO treatment restores the pattern of tropoelastin staining to the septal tips of the eNOS–/– mice (D), similar to the eNOS+/+ mouse (C). All images were taken with a x20 objective.

 
Cross talk between the developing blood vessels of the lung and air spaces is essential for normal alveolarization (6, 17, 41). Stress to the lung during this period of development can lead to changes in lung structure and function that resemble the changes seen in BPD (13, 31, 37, 38, 46, 54, 56). In large animal models of BPD, injury to the lung reduces lung eNOS protein content (2, 13, 28). The role of eNOS during the recovery after lung injury is unclear. These studies suggest that decreased eNOS expression is associated with the impaired recovery of lung structure after neonatal injury. The mechanism by which a deficiency in endothelium-derived NO impairs lung growth is unclear.

Angiogenesis and alveolarization are complex processes that are intertwined, since an interruption of one process can lead to abnormalities in the other. Treatment of neonatal rats with nonspecific inhibitors of angiogenesis, thalidomide and fumagillin, have resulted in abnormalities in alveolarization and vascular growth without recovery of lung structure in adulthood (21). VEGF is a potent growth factor for endothelial cell growth, survival, and angiogenesis (34). Specific inhibition of VEGF during the neonatal period impairs alveolar and vascular growth that, into adulthood, is precluded by concurrent iNO therapy. NO is a downstream effector of VEGF via VEGF/VEGFR-2 phosphorylation and activation of eNOS (15, 16, 24, 43). In addition, NO upregulates VEGFR-2 expression in isolated endothelial cells in vitro (53) and in vivo (18). In the systemic circulation of the eNOS–/– mouse, VEGF-mediated angiogenesis in wound healing is impaired, illustrating that VEGF-mediated angiogenesis is dependent on functional eNOS (16). The ischemic hindlimb model of angiogenesis in the eNOS–/– mouse showed no increase in angiogenesis in response to VEGF, further demonstrating that eNOS is a downstream effector of VEGF signaling (33). Previously, we have reported a reduction in lung VEGFR-2 protein in the infant eNOS–/– and eNOS+/– mouse exposed to mild neonatal hypoxia (5). In our present study, VEGF expression increases during recovery in room air (HR group) in the eNOS+/– and eNOS–/– mouse lung, but VEGFR-2 expression increases only in eNOS+/– HR mice and not in the eNOS–/– HR mice. Inhaled NO therapy increases lung VEGFR-2 protein in the eNOS–/– mice and enhances distal lung growth. Interestingly, iNO during recovery from neonatal hypoxia resulted in an increase in vessel density in all three genotypes to levels that were greater than room air-raised control animals. These findings suggest that NO, either from endogenous sources such as the pulmonary endothelium (as in the eNOS+/– animals) or as inhaled gas, results in an increase in lung growth, angiogenesis, and alveolarization after neonatal stress. One possible mechanism for the action of NO is by increasing or sustaining VEGFR-2 expression and thus preserving VEGF signaling to promote angiogenesis.

Potential limitations of our study include the fact that other growth factor signaling pathways may be affected by eNOS deficiency and that these may contribute to abnormalities in compensatory lung growth and structure. We did not measure absolute changes in NO production in these mice, and it is possible that, in the eNOS+/– mice, the other two NOS isoforms (types I and II) were increased and may have compensated for the decrease in eNOS. We also did not quantify any changes in epithelial cells that may have contributed to the recovery of lung structure in the eNOS+/– mice and the inhaled NO-treated mice. We were not able to measure changes in elastic recoil and lung compliance in these mice, and changes in these could lead to abnormal air space size. Future studies will need to assess measures of lung function in these mice. Whether inhaled NO has effects on other organs besides the lung is unknown. Recently, abnormalities of lung vascular and airway structure were observed in fetal eNOS–/– mice (18). We did not observe similar abnormalities in the eNOS–/– mice, which may be because of the use of eNOS–/– parents in the previous report (18).

In summary, a deficiency of eNOS during the recovery from mild neonatal hypoxia results in a persistent impairment of lung growth. Furthermore, even a reduced level of eNOS expression, as in the eNOS+/– mice, or treatment with inhaled NO during the recovery from neonatal hypoxia is sufficient to allow catch-up lung growth. The deficiency of eNOS results in an impairment of vascular growth that is reversed during recovery from neonatal hypoxia in the eNOS+/– and with inhaled NO in the eNOS–/– animal. NO may act by promoting angiogenesis, in part by increasing VEGFR-2 expression and preserving VEGF signaling, in addition to other possible downstream effects. We speculate that NO is essential for lung alveolar and vascular growth during the recovery from neonatal stress and that inhaled NO during infancy may augment lung growth after neonatal injury.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant 1 K08 HL-073893 and an unrestricted grant from iNO Therapeutics.


   ACKNOWLEDGMENTS
 
We thank Dr. Christopher Coulon for assistance in the automated image analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Balasubramaniam, Pediatric Pulmonary Medicine, Dept. of Pediatrics, UCHSC at Fitzsimmons, Pediatrics 8317, PO Box 6511, Aurora, CO 80045 (e-mail: vivek.balasubramaniam{at}uchsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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