Nitric Oxide 38 (2014) 8–16

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Early inhaled nitric oxide at high dose enhances rat lung development after birth S. Duong-Quy a,1, T. Hua-Huy a,1, H. Pham b, X. Tang a,c, J.C. Mercier d, O. Baud b,e, A.T. Dinh-Xuan a,⇑ a

Assistance Publique-Hôpitaux de Paris, Université Paris Descartes, Hôpital Cochin, Service de Physiologie, 75014 Paris, France INSERM UMR 676, Université Paris-Diderot, Hôpital Robert Debré, APHP, 75019 Paris, France c Department of Respiratory Disease, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China d Assistance Publique-Hôpitaux de Paris, Hôpital Robert Debré, Service des Urgences Pédiatriques, 75019 Paris, France e Assistance Publique-Hôpitaux de Paris, Hôpital Robert Debré, Réanimation et pédiatrie néonatales, 75019 Paris, France b

a r t i c l e

i n f o

Article history: Received 16 January 2014 Revised 9 February 2014 Available online 22 February 2014 Keywords: Lung development Angiogenesis Alveolarization Inhaled NO VEGF/VEGFR-2

a b s t r a c t Rational: Inhaled nitric oxide (NO) is frequently administered to full term and preterm newborns in various clinical settings in order to alleviate pulmonary hypertension whilst improving oxygenation. However, the physiological effect of NO on early postnatal lung development has not yet been clearly described. We therefore investigated whether NO administered by inhalation affects lung development at early postnatal life. Methods: Pregnant rats were placed in a chamber containing 5 ppm (iNO-5 ppm group) and 20 ppm NO (iNO-20 ppm group), started from the last day of their pregnancy in order to keep rat pups under ambient NO from birth to 7 days postnatal. Control animals were kept at room air and all rat pups were sacrificed at postnatal day 7 and day 14. Results: Lung-to-body weight and wet-to-dry lung weight ratios did not significantly differ among 3 groups at postnatal day 7 and day 14. Vascular volume densities (Vv) in both NO groups (5 and 20 ppm) were higher than controls (P < 0.05; P < 0.001). Pulmonary vessel number was significantly increased in iNO-20 ppm group. Radial alveolar counts (RAC) and mean linear intercepts (MLI) markedly increased (consistent with increased alveolarization) in iNO-20 ppm group. This was associated with upregulation of VEGF/VEGFR-2, MT1-MMP/MMP2 and HO-1 protein expression in iNO-20 ppm group. Conclusions: We concluded that inhaled NO at 20 ppm enhanced lung development possibly through increased expression of HO-1, VEGF/VEGFR-2, and MMP2 at early stage of postnatal rat life. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Inhaled nitric oxide (NO) is a potential therapy in very ill preterm and term newborns. Recent studies showed that early use of inhaled NO in preterm newborn might decrease the risk of brain injury [1], improve neurological development outcome, and prevent incidence of bronchopulmonary dysplasia [2]. It is known that preterm newborns with severe respiratory failure are at high risk of mortality due to intracranial hemorrhage and lung injury. Inhaled NO is an important adjunctive treatment to current therapy of persistent pulmonary hypertension (PPH) in term newborns with hypoxemic respiratory failure. As inhaled NO is a selective pulmonary vasodilator [3]; it can selectively decrease pulmonary vascular resistance ⇑ Corresponding author. Address: Service de Physiologie-Explorations Fonctionnelles, Hôpital Cochin, 27 rue du faubourg Saint-Jacques, 75014 Paris, France. Fax: +33 158412345. E-mail address: [email protected] (A.T. Dinh-Xuan). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.niox.2014.02.004 1089-8603/Ó 2014 Elsevier Inc. All rights reserved.

and ventilation – perfusion mismatching, leading to oxygenation improvement [4]. Further, in severe respiratory failure newborns, inhaled NO also reduces the requirement for extracorporeal membrane oxygenation and improves short term pulmonary outcomes [5]. Although the rational of dose and duration of inhaled NO in very ill preterm and term newborn has been demonstrated by previous studies [6,7], the effects of inhaled NO on normal lung development have not been completely understood. It is recognized that NO has a potential role in airway branching morphogenesis and vascular development. NO is produced endogenously from the amino acid L-arginine by enzymatic action of nitric oxide synthases (NOS) which is expressed in endothelial and epithelial cells [8]. A number of studies have shown that eNOS-deficient mice exhibit severe abnormalities in lung development with a high mortality due to fatal respiratory distress [9,10]. However, the precise mechanism by which pulmonary vascular bed develops during fetal and early postnatal life under effects of inhaled NO has not been completely understood. Recently, the role of endogenous NO on

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lung vascular growth has been suggested by its effect on upregulation of vascular endothelial growth factor (VEGF), a key factor for angiogenesis during embryonic and fetal development [11]. NO modulates a number of VEGF-regulating factors such as hypoxiainducible factors-1alpha (HIF-1a) and heme oxygenase-1 (HO-1) which induce VEGF synthesis during lung development [12]. VEGF binds to its receptor VEGFR-1 (flt-1) and VEGFR-2 (KDR in human and flk-1 in mice), which then interact and modify the biological effects of VEGF [13]. While VEGFR-1 acts as a silent receptor for VEGF due to its relatively weak kinase activity, VEGFR-2 plays an important role for lung development via phosphatidylinositol 3-kinase/Akt pathway [14]. Beside the angiogenesis and vasculogenesis mediated by VEGF/VEGFR-2 pathway, lung development before and after birth is marked by an alveolarization. In early postnatal life, the factors controlling alveolar stage are abundant and very complex. Among of these, the matrix metalloproteinase (MMPs), especially matrix metalloproteinase 2 (MMP2 or gelatinase A) and its activator MT1-MMP, play an important role in alveolarization [15]. The activity of MMPs is mediated by hypoxia or hyperoxia status and oxidative stress [16]. However, until now, the effect of exogenous NO (inhaled NO) on normal lung development has not been clearly demonstrated. Therefore, we hypothesize that inhaled NO at early postnatal life affects lung growth via VEGF/VEGFR-2 pathway. Then, we postulate that the effect of inhaled NO on postnatal lung growth will depend on the dose and the duration of inhalation. To test this hypothesis, we first determined whether inhaled NO during an early postnatal life changes alveolar and vascular growth in normal neonatal rat lungs. Then, we described the effect of inhaled NO at low and high dose on VEGF/VEGFR-2 pathway and its regulating factors in animal model. Materials and methods Animals and experimental protocols This study was approved by the National Institute of Health and Medical Research and complied with the instructions of the Institutional Animal Care and Use Committees INSERM 676-Paris. Pregnant Sprague–Dawley rats (SDR) were purchased from Charles River Laboratory (L’Arbresle, France). Animals were maintained at 20–24 °C in autoclaved cages and exposed to alternative day–night cycles every 12 h (lights on at 8:00 am) throughout the study period with free access to standard food and water. Rat pups were delivered naturally at termed gestation. Pregnant SDR were randomized into three groups at the last day of their gestation: SDR exposed to room air (control group), SDR exposed to low-dose NO at 5 ppm (iNO-5 ppm group), and SDR exposed to high-dose NO at 20 ppm (iNO-20 ppm group). Twentyfour hours before giving birth, pregnant SDR from inhaled NO groups were placed in a transparent Plexiglas chambers, connected to NO source (BioSpherix, Redfield-IL, USA) in order that neonatal rats could expose to inhaled NO since their first inspiration. NO and NO2 levels were continuously monitor with specific apparatus (Datex-Ohmeda, Inc., Madison, WI53707-7550, USA). The concentration of NO2 was kept less than 1 ppm. Inhaled NO was performed only for 7 days. Rat pups were killed at postnatal day 7 and day 14 to obtain lung tissue for histology, morphometric, and protein analysis. All postnatal day 14 rat pups were maintained at room air condition from day 7. At least 6 animals were used for each group unless otherwise noted. Chemical reagents and antibodies All chemical reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise noted. For all washing steps

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in immunohistochemical (IHC) staining and Western blot, phosphate buffered saline (PBS, pH 7.4) was used with Tween 20 (0.05%), noted as PBS-T. Primary antibodies for CD31 (PECAM-1), VEGF and VEGFR-2 (Flk-1), 3-nitrotyrosine, b-actin, and HRPconjugated secondary antibodies were purchased from Santa Cruz Biotechnology Incorporation. Vectastain Elite ABC Kit for secondary biotinylated antibodies for IHC and haematoxylin was purchased from Vector Laboratories Inc. (Burlingame, CA, USA). Quantikine VEGF Immunoassay kit was supplied by R&D Systems Europe Ltd (Abingdon, UK). Rat hemeoxygenase-1 (HO-1) ELISA Kit was purchased from Stressgen (Ann Arbor, MI, USA). Antibodies used in Western blot were diluted in non-fat milk (5%), PBS-T, and those for immunohistochemistry in PBS. Lung tissue preparations Rat lungs were first flushed in situ with PBS via right ventricular puncture and pulmonary artery to get rid of blood from pulmonary circulation. Rat lungs were removed from thoracic cavity. Right lungs were dissected, snap-frozen in liquid nitrogen, and stored at 80 °C until use for Western blot or ELISA. Left lungs were washed in physiological solution (NaCl 0.9%, pH 7.4), infused with 4% paraformaldehyde (PFA in PBS, pH 7.4) at 20 cm water pressure via an intratracheal catheter for 1 h [15]. Main left bronchus was ligated under pressure, and left lungs immersed into the same fixative solution for 24 h at room temperature. Lung tissues were then dehydrated and embedded in paraffin by automatic procedure (Shandon Citadel 2000 Tissue Processor, Rankin Biomedical Corp., MI, USA). Wet-to-dry lung weight Other groups of newborn rats (n = 4 in each group) were used for determining wet-to-dry lung weight. Rat lungs were removed from thoracic cavity. After trachea and stem bronchi removal, the whole lung was weighed to obtain wet weight. Lungs were then dried to constant weight in a microwave oven with a low power of 200 W for 60 min as previously described with a few modifications [17]. Lung alveolarization assessment Lung blocks were cut into 5-lm sections and serially mounted onto SuperFrost Plus slides (Braunschweig, Germany). All lungs were sectioned by the same manner, with symmetrical slices from hilum to pleural surface. Haematoxylin and eosin staining was carried out on all sections to evaluate the alveolarization. Alveolarization was assessed by performing radial alveolar counts (RAC) and median linear intercepts (MLI). Images of each section were captured with a magnified digital camera through a Leica microscope (Leica Microsystems, Wetzlar, Germany). The RAC, described by Emery and Mithal [18], represents the alveolar number across terminal respiratory units. To assess the RAC, respiratory bronchioles were identified as bronchioles lined by epithelium in one part of the wall. Then, a perpendicular line was drawn from the centre of respiratory bronchiole to the outer edge of acinus, as defined by a connective tissue septum or pleura. The number of septa intersected by this line was counted. For MLI assessment, the technique had been previously described by Dunnill and Thurlbeck [19,20]. The MLI represents the average size of alveoli or distance between airspace walls. The same images as above mentioned were used. To measure the intercepts, a transparent sheet with 10 horizontal and 11 vertical lines was laid over the images. The intercepts of alveolar walls with these lines were counted. Intercepts of bronchioli, blood vessels or septa were counted for one half since they are more or less part

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of the structure of surrounding alveolar spaces. Images with bronchi, large bronchioli or blood vessels were excluded from the measurements. The MLI was measured by dividing the total length of lines drawn across the lung section by the number of intercepts encountered. At least ten counts were performed for each animal. Immunohistochemistry for pulmonary vessel number and vascular density volume Angiogenesis has been evaluated by measuring pulmonary vessel number and vascular density volume, using von Willebrand Factor (vWF; Dako) and CD31 for endothelial specific markers. The paraffin-embedded slides were deparaffinized and rehydrated by serial immersions in xylene, ethanol, and water. Endogenous peroxidase activity was reduced by immersion in 0.3% hydrogen peroxide in methanol for 30 min. Sections were digested with protease (Pronase from Streptomyces griseus, Fluka, Steinheim, Japan) for 30 min at room temperature and washed with PBS at 4 °C to block protease activity. The sections were then covered in blocking serum 10% (Vector Laboratories) for 20 min and incubated with primary antibody for 2 h (1:200). After incubation, the sections were washed with PBS and incubated with biotinylated secondary antibody for 30 min. It was then incubated with HRP–streptavidin complex and washed with PBS. The desired stain intensity was obtained after incubating the sections with HRP substrate mixture. The sections were dehydrated by sequential immersion in ethanol and xylene after counter-stained with hematoxylin. Pulmonary vessel number was determined by counting microvessels (20–80 lm) stained with vWF per high-power field (10 magnification). The fields were chosen randomly and all vessels adjacent to large airways were excluded. Pulmonary vessel density (Vv) was measured by using a grid of 100 points superimposed on color photographs (40 magnification) from ten random noncontiguous fields per animal. The Vv was calculated as the ratio of the number of points falling on CD31 stained sites to points on lung parenchyma, excluding large vessels and airways. Immunohistochemistry for VEGF and VEGFR-2 For semi-quantitative of VEGF and VEGFR-2, lung sections for each group were deparaffinized, rehydrated, and digested with protease as described above. After washing, sections were covered in blocking serum 10% for 60 min and then incubated with primary antibody diluted at 1:50 in PBS for 60 min. After incubation, the sections were washed with PBS and incubated with goat anti-rabbit secondary antibody diluted at 1:200 in PBS for 60 min. The sections were washed again with PBS and incubated with BiotinStreptavidin enzymes using Vectastain Elite ABC Kit, washed in PBS, and developed with diaminobenzidine (DAB) tetrahydrochloride peroxidase substrate kit (Vector Laboratories). The sections were then dehydrated by sequential immersion in ethanol and xylene after counter stained with haematoxylin. For VEGF and VEGFR-2 expression, the staining points were counted by intensity score and normalized by surface unit. Ten counts were performed for each animal. Immunohistochemistry for 3-nitrotyrosine (3-NT) To assess the possibility of NO induced-lung inflammation and nitrosative stress, IHC staining for 3-NT was performed on rat lung sections at 7 days as previously described for VEGF and VEGFR-2 with a mouse monoclonal IgG2b raised against 3-nitrotyrosine (sc-32731, diluted at 1:100 and incubated for 2 h at room temperature) as primary antibody and a biotinylated goat anti-mouse IgG as secondary antibody (BA-9200, diluted at 1:200 and incubated for 1 h at room temperature). Negative control was done by replac-

ing primary antibody with non-immune mouse immunoglobulin (I-2000, Vector Laboratories Inc.) as manufacturers’ instructions. For 3-nitrotyrosine expression, lung sections were evaluated in peribronchovascular interstitial spaces (40-fold magnification), noted by its intensity from 0 to 3 (0: none, 1: mild, 2: moderate, and 3: strong) on 10–15 counts per animal. Western blot analysis for VEGF, VEGFR-2, HIF-1, MMP2, and MT1MMP Frozen lung samples were homogenized in ice-cold buffer containing Tris (25 mM), EGTA (1 mM), PMSF (1 mM), 2-mercaptoethanol (0.1%), and protease inhibitor cocktail tablet (Roche Applied Science, Mannheim, Germany). The homogenates were centrifuged at 21,000g at 4 °C for 20 min to remove tissue debris. Protein contents of homogenate lysates of lung tissues were measured by using a BCA Protein assay kit (Pierce Biotechnology, Rockford, IL, USA). For VEGF and VEGFR-2, equal amounts of lysates (30 g of total protein content per sample), were subjected to electrophoresis on SDS–PAGE gels (12% for VEGF and 7.5% for VEGFR2), and then transferred to PVDF membranes (Immobilon-P, Millipore, MA, USA). Membranes were blocked in 5% non-fat milk, 0.1% Twen-20 in PBS for 1 h at room temperature and then hybridized overnight at 4 °C with rabbit anti-VEGF or rabbit anti-Flk-1 as primary antibodies diluted at 1:500. After being washed in PBS-T, membranes were incubated with HRP-linked goat anti-rabbit, diluted at 1:10,000. Protein bands were developed on the film by using Enhanced Chemi-Luminescence (ECL) Plus reagent, according to the manufacturer’s instructions (Amersham, Biosciences, Orsay, France). The protein density was quantified by using Image Software System (Genius 2, Syngene, Cambridge, UK). The equal amounts of proteins loaded per well were verified by stripping the blots with Tris–HCl (pH 6.7) 62.5 mM, SDS 2%, and b-mercapto-ethanol 100 mM for 30 min at 55 °C. The membranes were reprobed with a mouse polyclonal anti-b-actin antibody (1:1000; sc-9104, Santa Cruz Biotechnology) and then incubated with HRP-linked goat anti-rabbit, diluted at 1:10,000. Results were presented as ratios of VEGF or VEGFR-2/b-actin expression. For HIF-1a, MMP2, and MT1-MMP, Western blot analysis was performed as described above with different parameters: amount of lysates (80, 20, 10 lg protein/sample; respectively), SDS–PAGE gels (8%, 10%, 10%; respectively), dilution of primary antibody (1:500, 1:500, 1:1000; respectively), and dilution of secondary antibody (1:10,000, 1:10,000, 1:20,000; respectively). Determination of VEGF and HO-1 by ELISA The concentrations of VEGF and HO-1 in lung homogenates were determined by enzyme-linked immunosorbent assay using the Quantikine VEGF Immunoassay kit and the Rat HO-1 ELISA Kit, according to the manufacturer’s instructions. Briefly, a mouse monoclonal antibody specific for rat VEGF or HO-1 was pre-coated on the wells of the provided immunoassay plate. The protein in lung homogenate lysates was captured by the immobilized antibody and detected with a specific rabbit polyclonal antibody. The polyclonal antibody was subsequently bound by an anti-rabbit IgG antibody conjugated to horseradish peroxidase. Tetramethylbenzidine (TMB) was used as peroxidase substrate, giving a blue color in proportion to the amount of captured VEGF or HO-1. The color development was stopped with an acid stop solution (H2SO4 0.2 M) which converted the endpoint color to yellow. The intensity of the color was measured in a microplate reader at 450 nm. VEGF and HO-1 concentrations from the sample were quantified by interpolating absorbance readings from a standard curve generated with the calibrated VEGF or HO-1 protein standard provided.

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Statistical analysis Statistical comparisons were performed with t-Student test or Fisher’s exact test between two groups or with analysis of variance (ANOVA) among 3 groups or more by using the SPSS 16.0 software (Chicago, IL, USA). Data were presented as means ± standard error of the mean (SEM). Differences were considered significant at P < 0.05.

Results

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ence among the 3 groups (17 ± 2 in control group, 18 ± 2 in iNO5 ppm group, and 19 ± 3 in iNO-20 ppm group; P > 0.05). The results of lung vascular volume density (Vv) stained with CD31 showed that, at postnatal day 7, lung Vv in iNO-5 ppm and iNO-20 ppm groups were significantly higher than those in control group (P < 0.05, P < 0.001, respectively). Especially, lung Vv in iNO20 ppm rat pups were significantly more abundant than those in iNO-5 ppm group (P < 0.05). But, at postnatal day 14, there was no significant difference among the 3 groups (Fig. 1A). Effects of inhaled NO on alveolarization

Effect of inhaled NO on lung weight, body weight, lung-to-body weight ratio, and wet-to-dry lung weight ratio Six animals for each group were included in the present study. At postnatal day 7, lung weights in iNO-5 ppm and iNO-20 ppm rat pups were significantly increased in comparison with those in control rat pups (P < 0.05, P < 0.001, respectively; Table 1). Among inhaled NO-exposed rat pups, lung weights in iNO-20 ppm group were significantly higher than those in iNO-5 ppm group (P < 0.001). At the same time-point, body weights in iNO-20 ppm rat pups were significantly higher than those in control and iNO5 ppm groups (P < 0.005, P < 0.01, respectively). Lung-to-body weight ratios were not significantly different among the 3 groups at postnatal day 7. There was a trend toward an increased wetto-dry lung weight ratio (P = 0.08) in iNO-20 ppm rat pups as compared to that from controls group (Table 1). However, this ratio was not significantly different as evaluated by one-way ANOVA method among the 3 studied groups (P = 0.2). At postnatal day 14, after weaning from inhaled NO and recovering in room air, there was no significant difference among the 3 groups for lung weight, lung-to-body weight ratio, and wet-to-dry lung weight ratio. However, body weights were significantly higher in iNO-20 ppm rat pups than those in control and iNO-5 ppm groups at postnatal day 14 (P < 0.005, P < 0.001, respectively) (Table 1). Effects of inhaled NO on pulmonary vessel density Pulmonary vessel number was counted on microvessels stained with vWF. The result showed that at postnatal day 7, the vessel numbers in iNO-20 ppm group were significantly higher than those in control and iNO-5 ppm groups (16 ± 2 versus 10 ± 1 and 12 ± 1; P < 0.01 and P < 0.05; respectively). There was no significant difference between iNO-5 ppm and control rat pups (12 ± 1 versus 10 ± 1, P > 0.5). At postnatal day 14, there was no significant differ-

In comparison with control rat pups, lung structure was changed with inhaled NO at high dose. At postnatal day 7, RAC was significantly higher in iNO-20 ppm rat pups than in control and iNO5 ppm groups (8.7 ± 1.5 versus 7.2 ± 1.0 and 7.4 ± 1.1, P < 0.05, P < 0.05; respectively). In iNO-5 ppm group, RAC was slightly higher than in control group but the difference was not statistically significant (0.05 < P < 0.2). At postnatal day 14, there was no significant difference among the 3 groups (9.7 ± 1.4 with control group, 9.8 ± 1.5 with iNO-5 ppm group, and 10.0 ± 1.5 with iNO20 ppm group; P > 0.05). For MLI assessment, we found that at postnatal day 7, the median diameter of alveoli in iNO-20 ppm rat pups was significantly lower than that in control and iNO-5 ppm rat pups (54.9 ± 0.8 lm versus 60.0 ± 1.0 lm, and 58.5 ± 0.3 lm; P < 0.001 and P < 0.001; respectively). There was no significant difference for MLI between iNO-5 ppm and control groups. At postnatal day 14, MLI were not significantly different among the 3 groups (54.7 ± 0.8 lm in control group, 53.6 ± 0.9 lm in iNO-5 ppm group, and 53.1 ± 0.7 lm in iNO-20 ppm group, P > 0.05). Effect of inhaled NO on lung inflammation Immunohistochemical staining on rat lung sections showed 3nitrotyrosine expression was predominantly present on endothelial layers of arteries and arterioles. 3-NT expression was less important in bronchi, bronchioles, and was seldom observed in interstitial tissue and alveoli (Fig. 1B). Semi-quantitative assessment found no significant difference (P > 0.05) among the 3 groups at postnatal day 7 (2.18 ± 0.093 with controls (n = 5), 2.20 ± 0.092 with iNO-5 ppm group (n = 4), and 2.25 ± 0.056 with iNO-20 ppm group (n = 6)).

Table 1 Lung weight, body weight, lung-to-body weight ratio, and wet-to-dry lung weight ratio in air-exposed (control) and iNO-exposed rat pups. Control

iNO-5 ppm

iNO-20 ppm

Postnatal day 7 Lung wt, g Body wt, g Lung wt:Body wt Wet-to-dry lung wt

0.29 ± 0.02 15.72 ± 0.32 0.018 ± 0.001 3.13 ± 0.18

0.31 ± 0.01⁄ 16.34 ± 0.28 0.019 ± 0.001 3.34 ± 0.19

0.34 ± 0.02⁄⁄⁄,à 17.35 ± 0.36⁄⁄,/ 0.020 ± 0.001 3.61 ± 0.17£

Postnatal day 14 Lung wt, g Body wt, g Lung wt:Body wt Wet-to-dry lung wt

0.48 ± 0.02 36.74 ± 0.73 0.013 ± 0.001 4.17 ± 0.14

0.47 ± 0.02 35.25 ± 1.15 0.014 ± 0.001 4.14 ± 0.19

0.50 ± 0.01 40.56 ± 0.40⁄⁄,à 0.013 ± 0.00 3.99 ± 0.12

Values are means ± SEM; n = 6 for each group. g: gram; ⁄P < 0.05, ⁄⁄P < 0.005, P < 0.001 versus controls; /P < 0.01 and àP < 0.001 versus iNO-5 ppm group; £ P = 0.08 versus controls. ⁄⁄⁄

Fig. 1A. Effects of inhaled NO on pulmonary vascular volume density (Vv). At postnatal day 7, lung Vv in iNO-5 ppm and iNO-20 ppm groups was significantly higher than that in control group (P < 0.05, P < 0.001). Lung Vv in iNO-20 ppm group was significantly higher than that in iNO-5 ppm group (P < 0.05). At postnatal day 14, there was no significant difference among the 3 groups.

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Fig. 1B. Expression of 3-nitrotyrosine evaluated by immunostaining in lung sections at postnatal day 7 as described in Methods. A, B: negative control (primary antibody was replaced by mouse IgG serum with the same dilution). C, D: control rats elevated in room air. E, F: iNO-5 ppm rats. G, H: iNO-20 ppm rats. A, C, E, G: photos with 20 magnification. B, D, F, H: photos with 40 magnification. At postnatal day 7, there was no significant difference among the 3 groups (P > 0.05).

VEGF and VEGFR-2 (Flk-1) staining and protein expressions At postnatal day 7, VEGF immunostaining in pulmonary vessels and in bronchial airways from iNO-20 ppm rat pups was significantly stronger than that from control and iNO-5 ppm groups (P < 0.05 and P < 0.05, respectively; Figs. 2A and 2B). There was no significant difference between iNO-5 ppm and control groups. At postnatal day 14, the expression of VEGF detected by immunostaining in pulmonary vessels and bronchial airways was not significantly different among the 3 groups (Figs. 2A and 2B). At postnatal day 7, VEGFR-2 immunostaining in pulmonary vessels was significantly increased in iNO-5 ppm and iNO-20 ppm groups as compared to controls (P < 0.05 and P < 0.05, respectively; Fig. 3A). In bronchial airways, VEGFR-2 immunostaining in iNO20 ppm rat pups was significantly higher than that in control and iNO-5 ppm group (P < 0.05 and P < 0.05; respectively; Fig. 3B). At postnatal day 14, there was no significant difference among the 3 groups for VEGFR-2 immunostaining in pulmonary vessels and bronchial airways (Figs. 3A and 3B).

Fig. 2A. Expression of VEGF assessed by immunostaining in endothelial cells. At postnatal day 7, expression of VEGF was significantly higher in iNO-20 ppm group than in control and iNO-5 ppm groups (P < 0.05, P < 0.05, respectively). At postnatal day 14, there was no significant difference among the 3 groups.

Fig. 2B. Expression of VEGF assessed by immunostaining in epithelial cells. At postnatal day 7, expression of VEGF was significantly higher in iNO-20 ppm group than in control and iNO-5 ppm groups (P < 0.05, P < 0.05, respectively). At postnatal day 14, there was no significant difference among the 3 groups.

Fig. 3A. Expression of VEGFR-2 assessed by immunostaining in endothelial cells. At postnatal day 7, expression of VEGFR-2 was significantly higher in iNO-5 ppm and iNO-20 ppm groups than controls (P < 0.05). At postnatal day 14, there was no significant difference among the 3 groups.

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Western blot analysis at postnatal day 7 showed that VEGF protein density in iNO-20 ppm group was significantly higher than that in control group but not than that in iNO-5 ppm group (P < 0.05 and P = 0.23, respectively; Fig. 4A). VEGFR-2 protein expression at postnatal day 7 in both iNO-5 ppm and iNO20 ppm groups was significantly higher than that in control group (P < 0.05 and P < 0.05, respectively; Fig. 4B). At postnatal day 14, VEGF and VEGFR-2 protein expression was not significantly different among the 3 groups (Figs. 4A and 4B). VEGF protein concentration measured by ELISA at postnatal day 7 in iNO-20 ppm group was significantly higher than that in control and iNO-5 ppm groups (Table 2). At postnatal day 14, there was no significant difference among the 3 groups (70.1 ± 8.3 pg/ mL with controls, 80.5 ± 4.2 pg/mL with iNO-5 ppm group, and 92.2 ± 4.7 pg/mL with iNO-20 ppm group; P > 0.05).

VEGFR2 β-actin

Expression of HIF-1 and concentration of HO-1 At postnatal day 7, HIF-1 protein expression measured by Western blot was not significantly different among the 3 groups (Table 2). HO-1 protein concentration, measured by ELISA, was sig-

Fig. 4B. VEGFR-2 protein expression in the developing lung. At postnatal day 7, VEGFR-2 protein density was significantly higher in iNO-5 ppm and iNO-20 ppm groups than controls (P < 0.05). At postnatal day 14, there was no significant difference among the 3 groups.

nificantly higher in iNO-20 ppm group as compared to control group (Table 2). In contrast, there was no significant difference among the 3 groups at postnatal day 14 for HIF-1 protein expression and HO-1 protein concentration (data not shown). Expression of MMP2 and MT1-MMP

Fig. 3B. Expression of VEGFR-2 assessed by immunostaining in epithelial cells. At postnatal day 7, expression of VEGFR-2 was significantly higher in iNO-20 ppm group than in control and iNO-5 ppm groups (P < 0.05). At postnatal day 14, there was no significant difference among the 3 groups.

MMP2 protein expression at day 7 was significantly enhanced in iNO-20 ppm group as compared to that in control and iNO-5 ppm groups (P < 0.01 and P < 0.05, respectively; Fig. 5A). The ratios of MMP2 active/inactive form were significantly higher in iNO20 ppm group as compared to control and iNO-5 ppm groups (0.60 ± 0.08 versus 0.26 ± 0.05 and 0.30 ± 0.09; P < 0.01 and P < 0.05; respectively). At the same time-point, MT1-MMP protein expression measured by Western blot was significantly increased in iNO-20 ppm group as compared to control and iNO-5 ppm groups (Fig. 5B). Protein expression was no longer significantly different among the 3 groups at postnatal day 14 (Figs. 5A and 5B). Discussion Although inhaled NO is used for more than a decade to treat very ill newborns with hypoxemic respiratory failure [21,22], the question as to whether it may affect normal lung development after birth has not been fully investigated. This question is worth to be addressed because of the possibility of the use of inhaled NO, notwithstanding its inconsistent beneficial effects, in preterm infants in whom lung structure continues to develop after birth [21,22]. Moreover, the effect of inhaled NO on lung development in early postnatal life and its dose-related effects are still controversial [16].

VEGF β-actin

Fig. 4A. VEGF protein expression in the developing lung. At postnatal day 7, VEGF protein density was significantly higher in iNO-20 ppm group than controls (P < 0.05). At postnatal day 14, there was no significant difference among the 3 groups.

Table 2 VEGF, HO-1, and HIF-1 proteins measured by ELISA and Western blot in air-exposed (control) and iNO-exposed postnatal day 7 rat pups.

VEGF (pg/mL) HO-1 (ng/mL) HIF-1 (arbitrary units for WB)

Control

iNO-5 ppm

iNO-20 ppm

84.92 ± 10.27 3.18 ± 0.54 2802 ± 126

115.21 ± 9.45 6.20 ± 1.37 2444 ± 370

144.06 ± 6.43⁄⁄⁄,– 7.27 ± 1.19⁄ 1889 ± 323

Values are means ± SEM; n = 5 for each group. ⁄P < 0.05, ⁄⁄⁄P < 0.001 versus controls; P < 0.05 versus iNO-5 ppm group.

–

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MMP2 β-actin

Fig. 5A. MMP2 protein expression in the developing lung. At postnatal day 7, MT1MMP protein density was significantly higher in iNO-20 ppm group than in control and iNO-5 ppm groups (P < 0.01, P < 0.05, respectively). At postnatal day 14, there was no significant difference among the 3 groups.

MT1-MMP β-actin

Fig. 5B. MT1-MMP protein expression in the developing lung. At postnatal day 7, MT1-MMP protein density was significantly higher in iNO-20 ppm group than in control and iNO-5 ppm groups (P < 0.01, P < 0.05, respectively). At postnatal day 14, there was no significant difference among the 3 groups.

The results of our study showed that (1) low dose (5 ppm) of inhaled NO enhanced vascular volume density and increased VEGFR2 expression; (2) high dose (20 ppm) of inhaled NO enhanced angiogenesis and alveolarization, whilst increasing VEGF/VEGFR-2 protein expression and MMPs protein activity, and these effects disappeared after inhaled NO discontinuation; (3) overexpression of VEGF/VEGFR-2 seen with inhaled NO was associated with increased HO-1 expression. The effects of inhaled NO on lung development can be assessed using experimental models of preterm or term newborn animals submitted to abnormal conditions like hypoxia or hyperoxia during the antenatal and postnatal periods [23]. In these conditions, inhaled NO led to pulmonary vasodilatation, oxygenation improvement, and lung structural recovery [24], the latter effect still being a debated question. In our study, all rat pups had 24 h before delivery inhaled NO that was maintained 7 days after birth in order to allow us to assess the effects of inhaled NO on lung growth at the first breath of postnatal life. Furthermore, two different doses of inhaled NO, a low dose of 5 ppm and a higher dose of 20 ppm,

were chosen based on previous animal studies [24–27]. In rats, alveolarization begins in utero and alveolar stage is completed during the postnatal period during which the alveolar numbers markedly increase during the first weeks after birth [28]. During the same period, the pulmonary circulation develops concomitantly with distal lung air space growth to optimize gas-blood exchange [29]. In the present study, at postnatal day 7, although high dose (20 ppm) of inhaled NO enhanced angiogenesis and alveolarization and significantly increased lung weight and body weight, it did not affect lung-to-body weight ratios in all 3 study groups (Table 1). In our study, dry-to-wet lung weight ratios did not significantly differ among the 3 groups (controls, iNO-5 ppm, and iNO-20 ppm) at postnatal day 7 and day 14, suggesting that increased lung weight and body weight during inhaled NO with high dose was more related to lung growth than lung edema. Inhaled NO has a theoretical pulmonary vasodilator effect that might acutely increase blood volume and lung weight. The pulmonary effect of inhaled NO however is best seen in animals with pulmonary hypertension (the higher the pulmonary vascular resistance the greater pulmonary vasodilatation obtained with inhaled NO). In animals with normal pulmonary arterial pressure the vasodilatory effect of inhaled NO is less pronounced. In our study, it was not possible to eliminate the potential vasodilatory effect of inhaled NO in rat pups breathing NO, and we suspect that such vasodilatory effect might partially account for increased lung weights in iNO-5 ppm and iNO20 ppm groups as compared to control animals. There are theoretical grounds to hypothesize that inhaled NO might be harmful in inflammatory lungs by the production of peroxynitrite derived from NO and superoxide anion. To address this issue, we have measured 3-nitrotyrosine expression, a marker of peroxynitrite harmful effects, in lung tissue of rat pups. As there was no significant difference among the 3 experimental groups (Fig. 1B), we suggest that inhaled NO is unlikely to contribute to nitrosative stress in this study. In neonatal mice, Le Cras et al. showed that VEGF overexpression might cause lung edema due to pulmonary hemorrhage and inflammation [30]. Kunig et al. also stated that in hyperoxia rat pups, treatment with VEGF overexpression transiently increased lung edema at early stage of lung development after birth [31]. This discrepancy could be explained by the moderate degree of upregulation of VEGF/VEGFR2 pathway induced by inhaled NO, as compared to VEGF gene-targeted method in other studies. In our study, the results showed that either low or high dose of inhaled NO enhanced angiogenesis at early postnatal life (day 7), an effect that did not last up to one week after weaning from inhaled NO (day 14). Low doses (5 ppm) of inhaled NO slightly increased vascular volume density but not micro-vessel numbers. The effects of inhaled NO on angiogenesis after birth have been observed by recent studies. Lin et al. found that inhaled NO increased vessel volume density in neonatal rats exposed to hyperoxia (>95% oxygen at Denver’s altitude) breathing NO as compared with control animals (kept in room air) [24]. In our study, increase of angiogenesis at postnatal day 7 in rat pups breathing NO was associated with upregulation of VEGFR-2 (with 5 ppm) and VEGF/ VEGFR-2 (with 20 ppm) (Figs. 2A–4B). The possible mechanism for the effect of inhaled NO on angiogenesis might be mediated via VEGF/VEGFR-2 pathway promoting vascularization. Low doses (5 ppm) of inhaled NO increased angiogenesis at early postnatal life by means of VEGFR-2 upregulation. High doses (20 ppm) of inhaled NO increased angiogenesis through upregulation of VEGF and VEGFR-2 activity. Similarly, the stimulating role of inhaled NO on upregulation of VEGF and VEGFR-2 has been confirmed by in vitro and in vivo studies [10,32]. Increased VEGF synthesis is observed in vascular smooth muscle cells transfected with plasmid containing either endothelial

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NOS (eNOS) or inducible NOS (iNOS) genes, an effect that was dependent on the amount of endogenous NO [33,34]. Increased VEGF production has also been found in numerous cell types treated with exogenous NO donors. In our study, inhaled NO at 20 ppm significantly increased VEGF protein determined by Western blot and ELISA. Although the mechanisms by which VEGF synthesis is mediated by endogenous NO have been shown recently [9,12], the role of exogenous NO (inhaled NO) on upregulation of VEGF/ VEGFR pathway is still debated. First, most studies indicated that activation of sGC by NO did not modulate VEGF synthesis [35]. Second, recent studies suggested that NO might enhance VEGF synthesis in normoxic conditions via HIF-1 transcription factor by blocking HIF-1a degradation through inhibition of proline hydroxylation or by increasing HIF1a stabilizing through nitrosylation of E3 ubiquitin ligase [35– 38]. Additionally, NO-induced VEGF synthesis is mediated by HO1 [37]. HO-1 is a stress-inducible enzyme that is synthesized in response to numerous stimuli, including NO and reactive oxygen species (ROS) [39]. Dulak et al. indicated that HO-1 product is involved in the increase of VEGF synthesis in both normoxic and hypoxic conditions [38]. Consistently, the results of our study showed that HIF-1 protein level was not increased in inhaled NO rat pups whereas HO-1 protein concentration in these groups was higher than control group (Table 2). In the present study, although vascular density was increased in iNO-5 ppm-exposed rat pups, lung alveolar septation did not differ from control group, an effect that is consistent with results obtained with iNO-20 ppm at postnatal day 7. One week after weaning from inhaled NO, lung growth became similar in all 3 study groups. These findings suggest that low dose (5 ppm) of inhaled NO slightly and transiently enhanced angiogenesis (vascular vessel density) but did not affect alveolar septation. High dose (20 ppm) of inhaled NO promoted pulmonary vascularization (vascular density and vessel number) and alveolarization (RAC and MLI). In rats, the first week of postnatal life is the most critical period for lung development. Therefore, the interaction between pulmonary vascular development and alveolar septum growth at this stage is essential for normal alveolarization [38]. Consequently, stress to the lung during this period of development might disrupt the normal interaction between angiogenesis and alveolarization as what is seen in bronchopulmonary dysplasia [40]. In our study, although vascular density was increased in inhaled NO groups as compared with control group, alveolarization enhancement was only seen with high dose-inhaled NO. We postulate that there are other factors that might be implicated in the cross talk between angiogenesis and alveolarization. Recent studies suggest that branching of epithelium and formation of alveoli are two crucial processes resulting from the combination of cellular differentiation and dynamic remodeling of the extracellular matrix associated with matrix metalloproteinases (MMPs) activity during lung development [41]. MMPs are considered as key molecules of embryonic and postnatal lung development processes [37,41,42–45]. MT1-MMP (MMP-14) plays a major role in alveolar septation that is necessary to increase pulmonary surface in order to optimize blood gas exchange at postnatal life [41]. MMP-2 (gelatinase A), activated by MT1-MMP via pro-MMP-2, has also a preponderant role in the mechanisms of alveolarization [42]. In support of this contention are the facts that both MMP-2 null mice and MT1-MMP null mice have abnormal alveolar air space (emphysema-like) [43]. MT1-MMP is present in epithelial cells throughout lung growth reaching the peak of gene expression during the first week of postnatal life or during alveolar stage. Similarly, location of MMP-2 in epithelial and endothelial cells is also demonstrated during lung development [44]. We found that 20 ppm of inhaled NO enhanced MT1-MMP and MMP2 protein expression, but these effects had been abolished

15

after weaning from inhaled NO and recovery of the animals breathing room air (Figs. 5A and 5B). MT1-MMP and MMP-2 protein expression might result from direct effect of inhaled NO on MMPs activity. We postulate that increase of MMPs protein expression with inhaled NO (20 ppm) might play a critical role in alveolarization and pulmonary angiogenesis, as suggested by several animal studies investigating the effect of MT1-MMP on endothelial cell migration and capillary tube formation [43,46]. MT1-MMP can therefore be viewed as active downstream effectors of NO-induced angiogenesis [47]. Although this study provides new evidence on the effect of inhaled NO on normal rat lung development at early postnatal life, a number of limitations still remain. First, the structural changes related to elastic recoil, lung compliance, and surfactant component had not been evaluated in our study. These changes could clarify the effect of inhaled NO on postnatal lung function. Second, we were not able to explain why with a low dose, inhaled NO could enhance vascular density by individual increase of VEGFR-2 but without VEGF protein expression. Third, in our study, the mechanisms by which normal established lung growth after weaning from inhaled NO were not clarified. Fourth, it is still debated as to whether NO directly affect vascular cells or affect bone marrow cells which could be the origin of lung progenitor cells, and the design of our study was not set to specifically address this important issue. Conclusions The results of our study showed that inhaled NO at high dose enhanced lung growth possibly through increased HO-1, VEGF/ VEGFR-2, and MT1-MMP/MMP2 expression and/or activity at early stage of termed postnatal rat life. The effects of inhaled NO on angiogenesis and alveolarization were transient and disappeared after discontinuation of NO exposure. These findings support the current knowledge about the use of inhaled NO to improve lung development in very ill preterm and in term newborn, and provide circumstantial evidence for a pivotal role of the balance of VEGF/VEGFR-2 pathway in morphological lung control after birth. Further studies using different doses of inhaled NO and time points during postnatal life are warranted to better understand the effects of inhaled NO on lung growth after birth. References [1] J.P. Kinsella, G.R. Cutter, W.F. Walsh, D.R. Gerstmann, C.L. Bose, C. Hart, K.C. Sekar, R.L. Auten, V.K. Bhutani, J.S. Gerdes, T.N. George, W.M. Southgate, H. Carriedo, R.J. Couser, M.C. Mammel, D.C. Hall, M. Pappagallo, S. Sardesai, J.D. Strain, M. Baier, S.H. Abman, Early inhaled nitric oxide therapy in premature newborns with respiratory failure, N. Engl. J. Med. 355 (2006) 354–364. [2] R.J. Martin, M.C. Walsh, Inhaled nitric oxide for preterm infants: who benefits?, N Engl. J. Med. 353 (2005) 82–84. [3] J. Pepke-Zaba, T.W. Higenbottam, A.T. Dinh-Xuan, D. Stone, J. Wallwork, Inhaled nitric oxide as a cause of selective pulmonary vasodilation in pulmonary hypertension, Lancet 338 (1991) 1173–1174. [4] S.H. Abman, J.L. Griebel, D.K. Parker, J.M. Schmidt, D. Swanton, J.P. Kinsella, Acute effects of inhaled nitric oxide in severe hypoxemic respiratory failure in pediatrics, J. Pediatr. 124 (1994) 881–888. [5] R.H. Clark, J.L. Huckaby, T.J. Kueser, M.W. Walker, W.M. Southgate, J.A. Perez, B.J. Roy, M. Keszler, Clinical inhaled nitric oxide research group, low-dose nitric oxide therapy for persistent pulmonary hypertension: 1-year follow-up, J. Perinatol. 23 (2003) 300–303. [6] Neonatal Inhaled Nitric Oxide Study Group, Inhaled nitric oxide in full-term and nearly full-term infants with hypoxic respiratory failure, N. Engl. J. Med. 336 (1997) 597–604. [7] J.P. Kinsella, S.R. Neish, E. Shaffer, S.H. Abman, Low-dose inhalational nitric oxide in persistent pulmonary hypertension of the newborn, Lancet 340 (1992) 819–820. [8] H. Li, T. Wallerath, U. Förstermann, Physiological mechanisms regulating the expression of endothelial-type NO synthase, Nitr Oxide 7 (2002) 132–147. [9] R.N. Han, D.J. Stewart, Defective lung vascular development in endothelial nitric oxide synthase-deficient mice, Trends. Cardiovasc. Med. 16 (2006) 29– 34.

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