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Published ahead of print on February 16, 2006, doi:10.1165/rcmb.2005-0254OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 738-745, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0254OC

Nitric Oxide Donor Restores Lung Growth Factor and Receptor Expression in Hyperoxia-Exposed Rat Pups

Emmanuel Lopez, Olivier Boucherat, Marie-Laure Franco-Montoya, Jacques R. Bourbon, Christophe Delacourt and Pierre-Henri Jarreau

Institut National de la Santé et de la Recherche Médicale U651, Université Paris XII, Faculté de Médecine, Créteil; Université Paris Descartes, Faculté de Médecine; Assistance Publique-Hôpitaux de Paris; Hôpital Cochin; Service de Médecine Néonatale de Port-Royal, Paris, France

Correspondence and requests for reprints should be addressed to Pierre-Henri Jarreau, Service de Médecine Néonatale de Port-Royal, Centre Hospitalier Cochin-Saint-Vincent-de-Paul-La Roche-Guyon 123, Bd de Port-Royal, 75014 Paris, France. E-mail: pierre-henri.jarreau{at}cch.ap-hop-paris.fr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Exposure of newborn rats to hyperoxia impairs alveolarization. Nitric oxide (NO) may prevent this evolution. Angiogenesis and factors involved in this process, but also other growth factors (GFs) involved in alveolar development, are likely potential therapeutic targets for NO. We studied the effects of the NO donor, [Z]-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)aminio]diazen-1-ium-1, 2-diolate, also termed DETANONOate (D-NO), on hyperoxia-induced changes in key regulatory factors of alveolar development in neonatal rats, and its possible preventive effect on the physiologic consequences of hyperoxia. Newborn rat pups were randomized at birth to hyperoxia (> 95% O2) or room air exposure for 6 or 10 d, while receiving D-NO or its diluent. On Day 6, several GFs and their receptors were studied at pre- and/or post-translational levels. Elastin transcript determination on Day 6, and elastin deposition in tissue and morphometric analysis of the lungs on Day 10, were also performed. Hyperoxia decreased the expression of vascular endothelial growth factor (VEGF) receptor (VEGFR) 2, fibroblast growth factor (FGF)-18, and FGF receptors (FGFRs) FGFR3 and FGFR4, increased mortality, and impaired alveolarization and capillary growth. D-NO treatment of hyperoxia-exposed pups restored the expression level of FGF18 and FGFR4, induced an increase of both VEGF mRNA and protein, enhanced elastin expression, and partially restored elastin deposition in alveolar walls. Although, under the present conditions, D-NO failed to prevent the physiologic consequences of hyperoxia in terms of survival and lung alveolarization, our findings demonstrate molecular effects of NO on GFs involved in alveolar development that may have contributed to the protective effects previously reported for NO.

Key Words: alveolarization • angiogenesis • bronchopulmonary dysplasia • lung development, vascular endothelial growth factor


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Despite recent major advances in the management of premature infants, including surfactant administration and noninvasive mechanical ventilation, the most immature neonates (often weighing < 1,000 g at birth) remain exposed to lung sequelae, called bronchopulmonary dysplasia (BPD). BPD is now thought to be mainly related to arrested lung development, with a decreased expression of key factors regulating alveolar and vascular development (1, 2). New therapeutic strategies are therefore necessary to maintain a harmonious alveolar development and to prevent BPD. Among these strategies, early treatment with inhaled nitric oxide (NO) was demonstrated to improve long-term pulmonary outcome in premature infants (3). Experimentally, inhaled NO was also demonstrated to attenuate pulmonary hypertension and to improve lung growth in rat pups after neonatal treatment with a vascular endothelial growth factor receptor (VEGFR) inhibitor (4) and, more recently, to improve lung growth after exposure to hyperoxia (5). However, the pathways mediating NO prevention of lung growth alteration have not been fully elucidated.

A likely potential therapeutic target for NO prevention of BPD is angiogenesis. Lung alveolar development is intimately related to vascular growth (6), and NO has been shown to enhance angiogenesis in ischemic tissues (7). In ischemic brain, this effect was demonstrated to be partly mediated via the synthesis of VEGF (8). Moreover, recombinant VEGF treatment has recently been shown to enhance alveolarization after hyperoxia (9).

Compensatory mechanisms other than angiogenesis may also contribute to prevention of BPD by NO. For instance, NO has been recently shown to improve elastin deposition in a baboon model of BPD (10). Growth factors and receptors other than VEGF and VEGFR are also potential targets. Several growth factors and their receptors are known to be involved in alveolar development, and their altered expression may play a key role in the pathogenesis of BPD (11–13). These factors include members of the fibroblast growth factor (FGF) family and their receptors (14–16), and platelet-derived growth factor-A (PDGFA) (17). More specifically, alveolar septation has been shown to be suppressed in mice lacking either both FGF receptors (FGFRs) 3 and FGFR4 (18), or PDGFA (17). FGF18 expression is upregulated throughout the period of alveolar secondary septation, and FGF18 stimulates elastogenesis in lung fibroblasts (16), an event closely related to the surge of new septa (2). Despite the potential role of these factors in the pathophysiology of BPD, little is known about their changes in the injured neonatal lung, and even less about the possible preventive effects of NO on these changes. Hyperoxia has been shown to decrease mRNA expression of FGFR4 (19) and tropoelastin (2), whereas PDGFA did not change in a model of hyperoxia in piglet (20). Although no data are available in lung, NO has been shown to regulate basic FGF (bFGF) in muscle cells (21).

The present study was designed to determine the hyperoxia-induced changes in key regulatory factors of alveolar development in neonatal rats, and their possible prevention by exogenous NO provided by the NO donor, [Z]-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)aminio]diazen-1-ium-1,2-diolate, also termed DETANONOate (D-NO). In parallel, we evaluated the effects of exogenous NO on the consequences of hyperoxia in terms of survival, body and lung weight, inflammation, alveolarization, and capillary growth.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
Pregnant Sprague-Dawley rats were purchased from Charles River Laboratories (Saint Germain sur l'Arbresle, France). The albino rat pups were born in the laboratory, and were divided in equal numbers and body weights between each experimental group (i.e., room air or O2 exposure), and kept on a 12:12-h light-dark cycle. Food pellets and water were given ad libitum to the dams.

Hyperoxic Exposure
Litters of randomly divided rat pups and their dams were placed in Plexiglas exposure chambers (Charles River) and run in parallel with either > 95% or 21% (room air) fraction of inspired oxygen, as previously reported (22), from Day 0 to Days 6 or 10. O2 concentrations were monitored regularly. Because adult rats have limited resistance to high O2, the dams were exchanged daily between O2-exposed and room air-exposed litters. Chambers were opened for 20 min every day to switch dams between air and O2 environments, to treat rat pups, and to clean cages.

DETANONOate Administration
Daily, from Day 0 to Days 6 or 10 of postnatal life, one half of rat pups received an intraperitoneal injection of 0.4 mg/kg of D-NO (Cayman Chemical, Ann Arbor, MI). This NO donor has a half-life of 20 h at 37°C. The administration protocol has been previously described (8). Control groups, either in O2 or room air, were obtained by giving daily equal amounts of D-NO vehicle (NaOH 0.01M, hereafter referred to as the diluent) to the pups. For the bronchoalveolar lavage (BAL) study (see below), a higher supplementary dose of D-NO (4 mg/kg/d), was used.

Sample Collection
On Day 6 or 10, rat pups were killed by an intraperitoneal overdose of sodium pentobarbital (70 mg/kg; Ceva, Libourne, France) and were exsanguinated by aortic transection. Lungs were either immediately lavaged, fixed for morphometric/morphologic analysis, or dropped in liquid nitrogen and kept frozen at –80°C until further RNA extraction or VEGF immunoassay.

BAL and Cell Count in Lung Fluid
Pups were placed in a supine position, and tracheas were cannulated. Isotonic saline was gently instilled with a syringe and then withdrawn. BAL was performed 12 times with 0.33 ml sterile saline, and the 12 lavage samples were pooled. Total cell counts were performed with a hemocytometer, then samples were centrifuged at 300 x g for 7 min. Cell pellets were resuspended in adequate volume to obtain 106 cells/ml, and differential cell counts were performed on cytospin preparations stained with Diff-Quik (Dade Behring, La Défense, France). A blinded observer counted a minimum of 300 cells to establish the differential cell count. Lavaged lung tissues were discarded.

RNA Extraction
Total RNA was extracted from frozen lung tissue using Trizol reagent (1 ml of Trizol reagent per 50–100 mg of tissue; Invitrogen, Cergy-Pontoise, France) according to the manufacturer's instructions. The quantity of RNA in each sample was determined by absorption at 260 nm (Biophotometer; Eppendorf, Hamburg, Germany). Purity of the total RNA extracted was evaluated by the 260:280 nm ratio, with expected values between 1.8 and 2.0. Integrity and quality of each RNA sample were estimated by visualization of clear 18S and 28S ribosomal RNA bands after electrophoresis of 1 µg RNA of each sample in 1.5% agarose gel.

Reverse Transcription and Real-Time Quantitative PCR
RNAs from each extraction sample were reverse-transcribed into cDNA using 2 µg of total RNA, Superscript II reverse transcriptase, and random hexamer primers (Invitrogen), according to the manufacturer's protocol. Real-time PCR was conducted for quantitative analysis of steady-state mRNA expression level using sequence-specific primers and Taqman MGB probe for the target genes VEGFA, VEGFR2, and housekeeping gene 18S rRNA (Assays-on-Demand, Gene Expression Products; Applied Biosystems, Courtaboeuf, France) and performed using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Reaction mixture for real-time PCR had a final volume of 20 µl, consisting of 5 µl of cDNA, 10 µl of TaqMan Universal PCR Master Mix 2x, and 1 µl of 20x Assays-on-Demand Gene Expression Assay Mix (Applied Biosystems). Amplification conditions were identical for all samples: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s each at 95°C, and 1 min at 60°C. The endpoint used in the real-time PCR quantification is defined as the PCR cycle number that crosses the signal threshold (Ct). After validation of amplification efficiencies of the two genes, quantification of target gene expression was performed using the comparative Ct method (Sequence Detector User Bulletin no. 2; PE Biosystems, Foster City, CA), and reported as the fold difference relative to the housekeeping gene. To calculate the fold change (increase or decrease), the Ct of the housekeeping gene was subtracted from the Ct of the target gene to yield the {Delta}Ct; the change in expression of the normalized target gene as a result of an experimental sample was then expressed as 2–{Delta}{Delta}Ct, where {Delta}{Delta}Ct = {Delta}Ct sample – {Delta}Ct calibrator (sample used as internal control during the PCR).

Northern Blot Analysis
Northern blot analysis was used to determine the steady-state expression level of those genes for which cDNA probes were available in the lab. Twenty micrograms of RNA were fractionated by electrophoresis through 1.2% agarose, 2.2 M formaldehyde gels, then blotted onto nylon membranes (Gene Screen; PerkinElmer, Courtaboeuf, France). The membranes were preincubated and successively probed for FGFR3, FGFR4, FGF18, elastin, and PDGFA transcripts, and 18S rRNA in a hybridization buffer containing 50% formamide, 50 mM Tris-HCl (pH 7.5), 0.8 M NaCl, 10% dextran sulfate, 0.1% sodium pyrophosphate, 5x Denhardt solution, 0.1% SDS, and 75 µg/ml denatured salmon sperm DNA. The rat cDNA probes consisted of a 904-bp sequence for FGF18 (gift from Dr. N. Itoh, Kyoto, Japan), a 1,100-bp sequence for elastin (gift from Dr. C. Rich, Boston, Massachussetts), a 501-bp sequence for FGFR3, a 501-bp sequence for PDGFA, and a 451-bp sequence for FGFR4, all three designed from Primer Express software (version 1.5; Applied Biosystems), respectively. Probes were labeled with (alpha-32P)-deoxycytidine triphosphate (NEN; PerkinElmer, Wellesley, MA) using the Rediprime DNA labeling system (Amersham, Orsay, France) and purified on G-50 probe purification columns (Amersham). The blots were exposed to X-OMAT Kodak Scientific imaging films (Eastman Kodak Co., Rochester, NY) for a suitable exposure time at –80°C. Autoradiographic signals were quantified by densitometry using the image analysis software NIH Image (National Institutes of Health, Bethesda, MD), and normalized to the relative amount of 18S rRNA.

VEGF Immunoassay
Frozen lung samples were ground and homogenized in lysis buffer CHAPS (Sigma, L'Isle d'Abeau, France). Assays were performed on 5-fold diluted supernatants using Quantikine Mouse VEGF Immunoassay kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. This assay recognizes both 164- and 120-aminoacyl residue forms of VEGF. Results are expressed as pg VEGF/g wet lung weight.

Lung Fixation and Morphometry
As previously detailed (23), lungs were gently extracted and fixed with 4% paraformaldehyde through a polyethylene tracheal cannula at a constant pressure of 20 cm H2O. The trachea was then ligated, and the lung was immersed in 4% paraformaldehyde for 24 h. Lung volumes were measured by the displacement method in the fixative solution. After fixation, lungs were embedded in paraffin, and 3-µm-thick tissue slices were cut throughout the entire lung sample and stained with hematoxylin, phloxine, and safran. Morphometric assessment of the lung parenchymal tissue by light microscopy was performed in blind fashion on coded slides by the same operator (E.L.), and repeated by a second observer (M.-L.F.-M.) to test reproducibility. A random examination of 10 fields for each right and left lung was performed. Images of histologic specimens observed with the light microscope (Laborlux D, Leitz, Wetzlar, Germany) were captured by a digital camera (CCD-IRIS, Sony Corp., Tokyo, Japan) and observed on a monitor with a line grid matrix. Alveolar surface density (Svap) was measured using point counting and mean linear intercept methods described by Weibel and Cruz-Orive (24). Absolute surface area (Sa) per lung was calculated by multiplying the surface density by the lung volume.

Platelet–Endothelial Cell Adhesion Molecule-1 Immunohistochemistry
Lungs were extracted as described previously here and fixed through a polyethylene tracheal cannula with OCT (Tissue-Tek, Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) embedding medium for frozen tissue specimens mixed with PBS, then frozen in liquid nitrogen and stored at –80°C. Seven–micrometer-thick tissue slices were cut with a cryostat (Jung CM3000; Leica Microsystems GmbH, Wetzlar, Germany). The platelet–endothelial cell adhesion molecule (PECAM)-1 labeling method was the same as in previous studies (25). Slides were rinsed in PBS, then incubated with H2O2 in methanol for 20 min for endogenous peroxidase blockade. Slides were then incubated in PBS containing 2% goat serum to block nonspecific binding. The primary antibody, mouse anti-rat CD 31 (PECAM-1) monoclonal antibody (BD Pharmingen, San Diego, CA) diluted to 1:200 in antibody diluent was added and incubated for 1 h at room temperature. The secondary antibody, biotinylated goat anti-mouse IgG (Anti-Ig HRP Detection Kits; BD Pharmingen,), diluted to 1:50, was added for 30-min. Streptavidin–horseradish peroxidase was applied to the tissue sections for 30 min, then diaminobenzidine was added for 5 min for staining. Slides were lightly counterstained in methyl green before being dehydrated and mounted. Negative control was mouse IgG in antibody diluent. Capillary density was quantified by measuring the proportion of PECAM-1 immunostaining relative to the whole parenchymal cell population using the point counting method (24). In practice, a random examination of 10 fields for each right and left lung was performed. A visual count of points matching with brown staining (PECAM-1–positive capillary endothelial cells) and with methyl green staining (parenchymal tissue) was done. The relative surface of capillary bed was expressed as the percentage of points on brown-stained areas relative to the total number of counted points.

Histochemical Analysis of Elastin
Paraffin-embedded sections of lung tissue were stained for elastin using Hart's method (10). Sections were deparaffinized and hydrated, then soaked overnight in Hart's solution. After being washed in 95% ethanol, then differentiated with 1% acid-alcohol, sections were dehydrated in 100% ethanol, cleared in xylene, and mounted. Sections were viewed by light microscopy (Laborlux D, Leitz), and images were captured with a digital camera (CCD-IRIS, Sony Corp.).

Statistical Analysis
Values are expressed as means ± SEM. Differences between three or more groups were evaluated using an ANOVA or Kruskall–Wallis test, as appropriate. Differences between two groups were evaluated using a Fisher's post hoc test or Mann–Whitney test, as appropriate. Overall survival in relation to treatment was evaluated by Kaplan–Meier survival function, and the log-rank test was used for comparisons between treatment groups. All calculations were performed with Statview software version 5.0 (SAS Institute Inc, Cary, NC). A P value < 0.05 was considered to be statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Growth Factors and Elastin Expression
Analyses were performed on lungs collected on Day 6 from rat pups exposed to room air or hyperoxia from birth and which received either D-NO or diluent.

VEGFR2 and VEGF. On Day 6, VEGFR2 mRNA expression was decreased by 30% in pups exposed to hyperoxia, and was restored to control level by D-NO administration (Figure 1A). VEGF mRNA and VEGF protein were unchanged under hyperoxic exposure, but were significantly increased by D-NO in hyperoxic pups only (Figures 1B and 1C).


Figure 1
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  		Figure 1. Effects of hyperoxia and D-NO on VEGFR2 and VEGF. Rat pups were placed under normoxia (room air, left bars) or hyperoxia (> 95% O2, right bars) from Day 0 to Day 6, while receiving D-NO (hatched bars) or diluent (solid bars). VEGF and VEGFR2 mRNAs were analyzed by real-time RT-PCR. Results are expressed as the percentage of mean control group values (normoxia, no D-NO). VEGF protein was determined by ELISA and expressed in pg/mg proteins. Data are mean ± SEM of determinations in six individual lungs for each group. VEGFR2 mRNA (A) was decreased by hyperoxia to 72% of the level of the control group, whereas VEGF mRNA level (B) and protein amount (C) were not changed by hyperoxia. In hyperoxia, D-NO restored VEGFR2 mRNA expression to control values and increased VEGF mRNA and protein, but had no effect in normoxia. #Significant difference (P < 0.05) between hyperoxia- and air-exposed pups treated with diluent. *Significant difference (P < 0.05) between D-NO– and diluent-treated pups with the same gas exposure (air or O2).

 
FGFRs and FGF18. In the hyperoxic environment, FGFR3 and FGFR4 mRNA levels were markedly decreased to about one-third those of the room-air control (Figures 2A and 2B). D-NO administration did not change FGFR3 mRNA expression under any of the experimental conditions (Figure 2A), but induced a 3-fold increase in FGFR4 mRNA in room air (P < 0.01), and restored it to control level under hyperoxia (Figure 2B). The expression level of FGF18 mRNA was decreased by 40% under hyperoxia compared with that of the room-air control group. D-NO administration significantly increased FGF18 mRNA in both normoxia and hyperoxia to about twice that of the control group (Figure 2C).


Figure 2
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  		Figure 2. Effects of hyperoxia and D-NO on FGF18, FGFR3, and FGFR4 transcripts. Rat pups were placed under normoxia (room air, left bars) or hyperoxia (> 95% O2, right bars) from Day 0 to Day 6, while receiving D-NO (hatched bars) or diluent (solid bars). mRNAs were analyzed by Northern blot. Results are expressed as the percentage of mean control group values (normoxia, no D-NO). Data are mean ± SEM of determinations in four individual lungs for each group. Hyperoxia decreased both FGFR3 (A) and FGFR4 (B) by 60%, and decreased FGF 18 mRNA (C) to 64% of the level of the control group. D-NO increased FGF18 mRNA to a similar degree under both conditions. D-NO had no effect on FGFR3 expression under any condition, but increased FGFR4 mRNA 3.5-fold in room air, and restored levels to normal in hyperoxia. #Significant difference (P < 0.05) between hyperoxia- and air-exposed pups treated with diluent. Significant difference (*P < 0.05; ***P < 0.001) between D-NO- and diluent-treated pups with the same gas exposure (air or O2).

 
PDGFA. PDGFA mRNA level was not significantly affected by hyperoxia as compared with room air, or by D-NO as compared with diluent under either hyperoxic or normoxic conditions (data not shown).

Elastin. Although hyperoxia did not change the elastin mRNA expression level at Day 6 (Figure 3), a marked decrease of septal elastin content was observed in hyperoxia-exposed lungs at Day 10 (compare Figures 4A and 4C). D-NO administration induced about a 2-fold increase in elastin mRNA expression level under both conditions (Figure 3), and restored some elastin deposition in alveolar septa of hyperoxia-exposed pups, although their appearance remained different from that observed under normoxic conditions (Figure 4D).


Figure 3
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  		Figure 3. Effects of hyperoxia and D-NO on elastin mRNA expression. Rat pups were placed under normoxia (room air, left bars) or hyperoxia (> 95% O2, right bars) from Day 0 to Day 6 while receiving D-NO (hatched bars) or diluent (solid bars). mRNA was analyzed by Northern blot. Results are expressed as the percentage of mean control group value (normoxia, no D-NO). Data are mean ± SEM of measurements in four lungs for each group. Hyperoxia did not change the expression level of elastin mRNA. D-NO increased elastin mRNA level under both conditions. Significant difference (*P < 0.05; **P < 0.01) between D-NO– and diluent-treated pups with the same gas exposure (air or O2) are indicated.

 

Figure 4
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  		Figure 4. Hart's resorcin–fuchsin stain for elastin in tissue sections. Rat pups were placed under normoxia (room air; A and B) or hyperoxia (> 95% O2; C and D) from Day 0 to Day 10, while receiving D-NO (B and D) or diluent (A and C). Photographs of the alveolar region, taken at the same magnification, are presented for each treatment group (bar in [C] represents 50 µm). Inserts are enlargements of the areas shown by dotted lines. Elastin staining was mainly located at the tips of secondary septal crests (arrows) in neonates exposed to room air with (B) or without (A) D-NO administration, with no apparent change in the presence of D-NO. In lungs exposed to hyperoxia (C), elastin deposition was markedly reduced; only focal aggregates of elastin were observed in alveolar walls. In lungs of rats placed under hyperoxia and treated with D-NO (D), elastin deposition was higher than with hyperoxia alone, although remaining lower than in controls and displaying a disorganized appearance (arrowheads). Note decreased lung septation and enlargement of distal airspace in (C) and (D) (hyperoxia).

 
Consequences of Hyperoxia and D-NO on Survival and Lung Development
Survival. Hyperoxia induced high mortality mainly on Days 5 and 6 of exposure. D-NO induced no mortality in pups breathing room air. Despite an apparent difference at Day 6, D-NO did not significantly alter the mortality rate or timing in O2-exposed pups (Figure 5).


Figure 5
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  		Figure 5. Survival of untreated and D-NO–treated rat pups in hyperoxia. Kaplan–Meier curve representation of survival to hyperoxia (> 95% O2) of rats treated by D-NO (n = 21; gray line) and their littermate controls treated by diluent alone (n = 20; black line). No significant difference by Log Rank test was observed between the two groups. No mortality was observed in animals breathing room air with or without D-NO treatment (data not shown).

 
Body-weight gain. Hyperoxia slightly impaired the weight gain of pups during the first 6 days of life; the difference was significant from Day 4 (mean weight 10.0 ± 0.1 g in hyperoxia versus 10.9 ± 0.2 g under air at Day 4, P < 0.01; and 12.2 ± 0.2 g versus 13.8 ± 0.2 g at Day 6, P < 0.01). D-NO administration did not affect weight gain under either hyperoxic or normoxic conditions.

Inflammatory-cell count in lung lavage fluid. Hyperoxia did not change the absolute number of inflammatory cells in BAL fluid, but induced a preferential recruitment of neutrophils in aispaces (P < 0.001). D-NO did not change these parameters significantly either at 0.4 mg/kg/d or at 4 mg/kg/d (Table 1).


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  		TABLE 1. EFFECTS OF HYPEROXIA FROM DAY 0 TO DAY 6 AND DETANONOate ON BRONCHOALVEOLAR LAVAGE CELL COUNT

 
Capillary growth. In hyperoxia, the capillary density, as evaluated by PECAM-1 immunostaining (Figure 6), was significantly decreased compared with normoxia (61.9 ± 3.5% versus 80.1 ± 3.7%, respectively; P < 0.01). D-NO did not modify the capillary density under either hyperoxic or normoxic conditions (51.0 ± 3.6% and 81.8 ± 2.0%, respectively).


Figure 6
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  		Figure 6. PECAM-1 immunostaining. Rat pups were placed under normoxia (room air; [A] and [B]) or hyperoxia (> 95% O2 [C] and [D]) from Day 0 to Day 6, while receiving D-NO (B and D) or diluent (A and C). The negative control (E) was obtained with mouse IgG in antibody diluent. Photographs of the alveolar region were taken at the same magnification (bar in [C] represents 50 µm). PECAM-1 immunostaining is clearly denser for rat pups in normoxia. D-NO had no visible effect in either normoxia or hyperoxia.

 
Alveolarization. As expected, hyperoxia induced a significant decrease of Svap and Sa at Day 10, and an increase of mean linear intercept. D-NO had no effect on these alterations of lung development (Table 2).


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  		TABLE 2. MORPHOMETRIC ANALYSIS OF RAT PUP LUNGS EXPOSED TO HYPEROXIA AND DETANONOate FROM DAY 0 TO DAY 10

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In this study, we evaluated the effects of hyperoxia and the NO donor, D-NO, on growth factors and receptors involved in lung development, and the possible protective effect of D-NO on the physiologic consequences of hyperoxia. NO is known to play a critical role in the lung, particularly in the developing lung (13), and its pharmacologic use to prevent progression to BPD, although controversial (26), appears to be a promising field of research (3, 10). Given the crucial role reported for several growth factors in lung development, we questioned whether NO could act via enhancement of the synthesis of these factors. As expected, hyperoxia decreased the expression of the VEGF receptor VEGFR2 transcript (19), and the expression of the transcripts of FGF18, its putative receptor FGFR3, and FGFR4, which had not previously been evaluated. These effects went along with the known physiologic consequences of hyperoxia on lung development. Treatment of hyperoxia-exposed pups with D-NO restored the expression level of FGF18 and growth factor receptors, except FGFR3, and induced an increase of both VEGF mRNA and peptide. It also enhanced elastin expression and deposition in alveolar walls in the presence of hyperoxia. However, it failed to prevent the physiologic consequences of hyperoxia in terms of survival and lung growth. To our knowledge, this is the first time that such effects of NO on these factors have been demonstrated in the developing lung.

The VEGF/VEGFR2 signaling pathway is critical for normal lung vascularization during development (27), as demonstrated by inhibiting VEGFR2 (28, 29). Several animal models of altered lung development have implicated VEGF (22, 30), and, in clinical studies, VEGF level was found to be decreased in infants with BPD. (31). In our study, hyperoxia did not decrease VEGF level at Day 6, which is consistent with previous data showing that VEGF expression decreased later during hyperoxic exposure (22). By contrast, hyperoxia decreased VEGFR2 expression, which is also consistent with previous findings (5, 19). D-NO administration increased the levels of VEGF mRNA and protein and VEGFR2 mRNA expression in pups exposed to hyperoxia, but not in those exposed to air. This result is in disagreement with those of a recent study (5) in which inhaled NO did not change VEGF expression, but, in this instance, NO was administered later, after hyperoxia exposure. The relationship between NO and VEGF is nevertheless complex, as NO is generally considered to be a downstream mediator of VEGF (4), and some data suggest that NO production may enhance VEGF generation in vitro as well as in vivo (8, 32, 33). The lack of effect of D-NO in air may indicate a specific role for NO under pathologic conditions.

FGFs and their receptors are involved in the various steps of lung development, including alveolarization (11, 14–16), in which their altered expression may play a key role in the pathogenesis of BPD (12). These effects are mediated by specific receptors (FGFR1 to 4), and gene targeting studies have demonstrated that simultaneous deletion of FGFR3 and FGFR4 suppresses alveolar septation (18). FGF18 is abundantly expressed in mouse embryonic lungs, and the lungs of FGF18-deficient mouse fetuses at term exhibit reduced saccular space (15). Although FGF18–/– mice do not survive beyond birth, FGF18 also appears to be involved in postnatal lung development, as it is upregulated throughout the period of alveolar secondary septation in rats, and is able to stimulate elastin expression (16). Elastin deposition in primary septa is an essential event for alveolar septation (2). Taken together with these previous data, the present finding that FGF18, FGFR3, and FGFR4 expression was decreased in hyperoxia suggests that hyperoxia-induced impairment of alveolar development may partly result from changes in FGF signaling. D-NO restored FGF18 in addition to increasing FGFR4 mRNA expression, and consistently, with an increase in elastin mRNA. Elastin labeling indicated that, despite the unchanged whole-lung elastin expression after hyperoxia exposure, a marked reduction of elastin was observed at the tip of alveolar septa. It also demonstrated that, although D-NO was unable to restore a normal appearance, the increased expression of elastin transcript induced by D-NO went concurrently with enhanced elastin deposition over that seen in hyperoxia alone. By contrast, hyperoxia and D-NO did not affect PDGFA expression, consistent with a recent report that PDGF mRNA level was unchanged in newborn rats exposed to 60% O2 (34).

A second part of this study evaluated the effects of exogenous NO on the physiologic consequences of hyperoxia. We did not observe any effect of D-NO on the various parameters examined, despite its biological effects on growth factors/receptors. Hyperoxia induced high mortality in rat pups, consistent with previous findings (19). In a different model, IL13 was described as having a protective effect partly mediated by VEGF (35)—an effect that was not observed in the present study, despite the increase of VEGF/VEGFR2 level. Although previous studies reported that inhaled NO reduced pulmonary leukocyte infiltration (36, 37), we did not observe any significant effect on this parameter with D-NO treatment, even with a dose enhanced to 10 times that reported here (Table 1). The difference in route of administration may be critical. It is possible that administering the NO donor intravenously did not allow a sufficient NO concentration in the lung, which was, by contrast, achieved when NO was inhaled. Finally, oxygen toxicity in neonatal rat lungs also resulted in inhibition of alveolarization with a decreased alveolar surface area (38) and disrupted microvascular development, as reflected by decreased PECAM-1 immunostaining. The involvement of VEGF and FGF18 in alveolarization, (16, 28) may suggest a possible protective effect of D-NO–induced changes on these factors; however, such an effect was not observed. In particular, it would have been expected that the increase in VEGR2 mRNA expression was associated with an increase in PECAM-1/capillary density, which was not observed. This apparent discrepancy may be explained by an increase in the density of receptors per cell without an increase in the number of endothelial cells. Such discordance between VEGFR expression and capillary density has already been described (39), albeit in a different model.

The lack of physiologic effect of D-NO may be due to the broad and severe pathophysiologic consequences of hyperoxia, raising the possibility that the enhanced expression of growth factors/receptors was insufficient to reach a protective level. In contrast, later administration of recombinant VEGF in a different model has been shown to restore alveolarization (9). The severity of our model of lung injury may be linked to the oxidative stress induced by hyperoxia, and significant concerns could be raised about possible increases in this stress in the presence of NO, as reactive nitrogen species have been implicated in lung epithelium lesions (40). In our study, however, neutrophil counts were not higher in the "hyperoxia with D-NO" group as compared with the "hyperoxia-diluent" group, an increase which would probably have occurred if reactive nitrogen species had been implicated.

Alternatively, changes in other factors or pathways induced by hyperoxia, but not attenuated by D-NO treatment, may have contributed to impaired alveolar development. For example, D-NO therapy did not reduce neutrophil influx, nor restore FGFR3 expression in hyperoxia. In addition, the effects of D-NO on generation of reactive oxygen species was not assessed. In contrast with gene knock-out models, which may induce an impairment of alveolarization with a single defect, thus highlighting the importance of a single factor, oxygen toxicity involves a multifactorial process in which a decrease or increase in a single factor or a few factors is not sufficient to induce impairment or correction of alveolarization. Among these factors, the specific toxicity of oxygen may be determinant.

In summary, our results in the rat confirm the previously observed effects of NO on elastin deposition in baboon lung (10), show that the effects of NO on VEGF/VEGFR2 observed in other organs (7) are also observed in the lung, and demonstrate, for the first time, stimulant effects on FGF18 and FGFR4. Although D-NO failed to prevent hyperoxia-induced lung morphologic changes and to significantly increase survival under the present experimental conditions, this study nevertheless demonstrates several molecular mechanisms that are likely to have contributed to the protective effects exerted by NO in previous investigations.


   Footnotes
 
E.L. and O.B. have contributed equally to this work.

E.L. was supported by a grant from the Société Francçaise de Néonatologie.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0254OC on February 16, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 8, 2005

Accepted in final form February 1, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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