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SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 2 (

International audience; Rationale: Promoting endogenous pulmonary regeneration is crucial after damage to restore normal lungs and prevent the onset of chronic adult lung diseases.Objectives: To investigate whether the cell-cycle inhibitor p16INK4a limits lung regeneration after newborn bronchopulmonary dysplasia (BPD), a condition characterized by the arrest of alveolar development, leading to adult sequelae.Methods: We exposed p16INK4a-/- and p16INK4a ATTAC (apoptosis through targeted activation of caspase 8) transgenic mice to postnatal hyperoxia, followed by pneumonectomy of the p16INK4a-/- mice. We measured p16INK4a in blood mononuclear cells of preterm newborns, 7- to 15-year-old survivors of BPD, and the lungs of patients with BPD.Measurements and Main Results: p16INK4a concentrations increased in lung fibroblasts after hyperoxia-induced BPD in mice and persisted into adulthood. p16INK4a deficiency did not protect against hyperoxic lesions in newborn pups but promoted restoration of the lung architecture by adulthood. Curative clearance of p16INK4a-positive cells once hyperoxic lung lesions were established restored normal lungs by adulthood. p16INK4a deficiency increased neutral lipid synthesis and promoted lipofibroblast and alveolar type 2 (AT2) cell development within the stem-cell niche. Besides, lipofibroblasts support self-renewal of AT2 cells into alveolospheres. Induction with a PPARγ (peroxisome proliferator-activated receptor γ) agonist after hyperoxia also increased lipofibroblast and AT2 cell numbers and restored alveolar architecture in hyperoxia-exposed mice. After pneumonectomy, p16INK4a deficiency again led to an increase in lipofibroblast and AT2 cell numbers in the contralateral lung. Finally, we observed p16INK4a mRNA overexpression in the blood and lungs of preterm newborns, which persisted in the blood of older survivors of BPD.Conclusions: These data demonstrate the potential of targeting p16INK4a and promoting lipofibroblast development to stimulate alveolar regeneration from childhood to adulthood.

Background: Fetal determinants of airway function, such as in utero exposure to maternal cigarette smoke (CS), may create a predisposition to adult airflow obstruction and chronic obstructive pulmonary disease (COPD) in adulthood. It has been suggested that active smoking in adolescence and preexisting airflow obstruction have synergistic deleterious effects. Objective: We used a mouse model to investigate whether there is a synergistic effect of exposure to CS in utero and during adolescence on lung function. Methods: Female C57Bl/6J mice were exposed to CS or to filtered room air during pregnancy. Exposure to CS began 2 weeks before mating and continued until delivery. After birth, the pups were not exposed to CS until day 21 (D21). Between D21 and D49, corresponding to “adolescence,” litters were randomized for an additional 4 weeks of exposure to CS. Lung morphometry, lung mechanics, and the expression of genes involved in senescence were evaluated in different subsets of mice on D21 and D49. Results: In utero exposure to CS induced significant lung function impairment by D21. CS exposure between D21 and D49 induced significant functional impairment only in mice exposed to CS prenatally. On D49, no difference was observed between subgroups in terms of lung p53, p16, p21, and Bax mRNA levels. Conclusions: Our findings suggest that prenatal and adolescent CS exposure have a synergistic effect on lung function in mice. The combined effect did not appear to be a consequence of early pulmonary senescence. Citation: Drummond D, Baravalle-Einaudi M, Lezmi G, Vibhushan S, Franco-Montoya ML, Hadchouel A, Boczkowski J, Delacourt C. 2017. Combined effects of in utero and adolescent tobacco smoke exposure on lung function in C57Bl/6J mice. Environ Health Perspect 125:392–399; http://dx.doi.org/10.1289/EHP54

Introduction: Lung regeneration is essential for damage recovering after lung injury at different age of life. Whether lipofibroblasts (LIF) are able to enhance endogenous alveolar regeneration once alveoli are damaged remains to be determined. Aim: To determine in a mouse model of bronchopulmonary dysplasia if a lipid switch of fibroblasts to LIF induce a curative lung regeneration. Methods: Wt C57BL/6J mice were exposed to hyperoxia (85% O2) or room air from days 3 to 14 of post-natal life. Rosiglitazone (RGZ), a PPARγ agonist which activates fibroblasts differentiation into LIF, was administered intraperitoneally at a dose of 5µg/g of mice daily, 5 days a week, starting after hyperoxia exposure, until adulthood (D60). Tissues were immunostained with anti-ADRP and anti-proSpC antibodies for LIF and AT2 detection respectively. Number of alveoli were estimated by the mean linear intercept and secondary septation by septal crest number (Weigert). Results: Compared to mice exposed to room air, mice exposed to hyperoxia presented a significant decreased number of LIF in lung associated with an arrested alveolar development revealed by a reduced number of alveoli and a concomitant disruption of secondary septation. Compared to hyperoxic, non-treated mice, RGZ administration increased the number of LIF and AT2 cells in the lung, associated with an increase in the number of alveoli and septal crests at adulthood. Conclusion: Switching fibroblasts towards LIF through PPARγ agonist administration induces lung regeneration after hypoalveolization due to post-natal hyperoxia. Administration of PPARγ agonists can be a new pharmacologic strategy to promote lung recovery after injury.

Introduction La bronchopneumopathie chronique obstructive represente la 4e cause de mortalite dans le monde et ne dispose a ce jour d’aucun traitement curatif. L’emphyseme, qui en est une des composantes, resulte de la destruction progressive des septums inter-alveolaires, conduisant a une insuffisance respiratoire. Parmi les marqueurs de senescence, des etudes montrent une surexpression du suppresseur de tumeurs p16 Ink4a dans les poumons de patients ayant un emphyseme compare aux fumeurs ou non-fumeurs sains. D’autres etudes ont montre l’influence de p16 Ink4a sur l’adipogenese. L’objectif est de determiner si la deletion de p16 Ink4a previent le developpement d’un emphyseme pulmonaire induit par une instillation d’elastase a un temps precoce ou tardif ainsi que les mecanismes impliques, en particulier l’induction de la lipogenese et l’activation des lipofibroblastes. Methodes Des souris adultes p16 Ink4a ko et sauvages (WT) ont recu une instillation intratracheale de 2UI d’elastase porcine pancreatique. Les souris ont ete sacrifiees 21 jours ou 90 jours apres l’instillation pour quantification morphometrique de l’emphyseme, evaluation de la septation secondaire et analyse de l’expression des genes de l’adipogenese et des marqueurs de pneumocytes. Evaluation dans un second temps des remaniements de la matrice extracellulaire. Resultats A 21 jours, la deletion de p16 Ink4a n’influence pas le degre d’emphyseme mais entraine une proliferation des pneumocytes de types 2, atteste par qPCR ou immunohistochimie ainsi qu’une activation de la lipogenese par rapport aux souris WT. A 90 jours, les souris p16 Ink4a presentaient un emphyseme significativement moins severe, des marqueurs de proliferation de pneumocytes 2 mais pas d’activation de l’adipogenese. A ce temps tardif, apparaissaient chez les souris p16 Ink4a des remaniements fibrotiques. Conclusion La deletion de p16 Ink4a ne protege pas de l’emphyseme induit par l’elastase dans un premier temps mais est associee a une amelioration des lesions a un temps tardif. Cet effet sous-tend un processus de regeneration alveolaire avec un remaniement secondaire la matrice extracellulaire.

Introduction: Air pollution (AP) represents the largest environmental risk for health. Such considerations however rely on the static quantification of only a few individual components of AP, without considering their interactions, chemical reactivity and synergistic toxicity. We therefore developed an innovative approach to analyze the health effects of “real life” model atmospheres at the preclinical level. Here, we report the first results of the feasibility study. Methods: A realistic atmosphere, representative of a 2017 pollution event in Paris, was generated (CESAM atmospheric simulation chamber - cesam.cnrs.fr). The chamber was connected to stalling cabinets where mice were exposed during 6 or 48 hours. Lungs, spleen, adipose tissue, heart, mesenteric ganglions were then harvested to address the expression of detoxification and antioxidant genes, as well as total cell count (TCC) and inflammatory cytokines expression in BAL fluid Results: The atmosphere contained 57.3 µg/m3 particulate matter, 114±11 ppb NO2 and 242±101 ppb O3. No mortality and no weight loss was observed. Increased expression of Ahr, Cyp1a1, Hmox1 and NQO1 was detected in the lungs, and increased TCC and KC concentrations in the BAL. Interestingly, we found extra pulmonary modifications; increased expression of Ahr, Cyp1a1, Hmox1 and NQO1 in the spleen, Hmox1, Cyp1a1 and Cyp1b1 in ganglions, and Hmox1 in the adipose tissue and heart. Limited modifications were detected after 6h. Conclusion: These preliminary results demonstrate the feasibility of our innovative experimental approach, which represents a versatile tool to get a better understanding of AP impacts on health.

Anthropic nanoparticles (NP) are increasingly produced and emitted, with accompanying concerns for human health. Currently there is no global understanding as to the exact mechanistics of NP toxicity, as the traditional nanotoxicological approaches only provide a restricted overview. To address this issue, we performed an in-depth transcriptomic analysis of human macrophages exposed to a panel of welding-related metal oxide NP that we previously identified in welders lungs (Fe2O3, Fe3O4, MnFe2O4 and CrOOH NP). Utilizing the specified analysis criteria (|fold change| ≥1.5, p ≤ 0.001), a total of 2164 genes were identified to be differentially expressed after THP-1 macrophage exposure to the different NP. Performing Gene Ontology enrichment analysis, for cellular content, biological processes and Swiss-Prot/Protein Information Resource keywords the data show for the first time a profound modification of gene differential expression in response to the different NP, among which MnFe2O4 NP were the most potent to induce THP-1 macrophage activation. The transcriptomic analysis utilized in the study, provides novel insights into mechanisms that could contribute to NP-induced adverse effects and support the need for widened approaches to supplement existing knowledge of the processes underlying NP toxicity which would have not been possible using traditional nanotoxicological studies.

Introduction: Bronchopulmonary dysplasia (BPD), characterized by airspace enlargement, develops in premature infants. BPD leads to persistent lung consequences in adulthood, being considered as an early determinant of adult lung diseases. P16Ink4 is a key factor implicated in aging-related lung diseases and its levels are increased in cord blood cells of preterm infants. Whether p16 is involved in BPD and its persistent consequences at the adult age remains to be determined. Aims: To determine in a mouse model of BPD: 1) if p16 deletion protects against lung alterations in new born and adult mice, and 2) if these effects are mediated by cellular senescence. Methods: Mouse pups were exposed to hyperoxia (85% O2) or room air from day 3 to 14 of life. Morphometry (mean linear intercept), elastin content (Weigert), collagen deposition (sirius red), cell senescence (βgalactosidase activity and p21) and cell proliferation (Ki67) were evaluated. Results: At day 14, hyperoxia induced airspace enlargement and cellular senescence, without modification of collagen deposition, nor cell proliferation. Hyperoxic p16-/- mice were not protected against alveolar disruption, but presented an increased alveolar wall thickness and cell proliferation. At day 60, hyperoxic mice showed persistence of airspace enlargement and senescence, without modification of collagen deposition, nor cellular proliferation. Hyperoxic p16-/- mice have reduced airspace enlargement, but increased collagen deposition, without modulation of senescence markers. Conclusions: P16 did not modulated hyperoxia-induced alveolar disruption at young age but could play a crucial role in BPD consequences in adults. This effect may be independent of cellular senescence.

International audience; Due to the growing commercial applications of manufactured nanoparticles (NPs), toxicological studies on NPs, especially during the critical window of development, are of major importance. The aim of the study was to assess the impact of respiratory exposure to metallic and metal oxide NPs during pregnancy on lung development of the offspring and to determine the key parameters involved in lung alterations. Pregnant mice were exposed to weekly doses of 100 mu g (total dose 300 mu g) of titanium dioxide (TiO2), cerium oxide (CeO2), silver (Ag) NPs or saline solution by nonsurgical intratracheal instillation. The offspring lungs were analyzed at different stages of lung development: fetal stage (gestational day 17.5), pulmonary alveolarization (post-delivery day 14.5) and lung maturity (post-delivery day 49.5). Regardless of the type of NP, maternal exposure during gestation induced long-lasting impairment of lung development of the offspring. This effect was accompanied by: i) decreased placental efficiency together with the presence of NPs in placenta, ii) no increase of inflammatory mediators present in amniotic fluid, placenta or offspring lungs and iii) decreased pulmonary expression of vascular endothelial growth factor-alpha (VEGF-alpha) and matrix metalloproteinase 9 (MMP-9) at the fetal stage, and fibroblast growth factor-18 (FGF-18) at the alveolarization stage. Respiratory exposure to metallic NPs during pregnancy induces stereotyped impairment of lung development with a lasting effect in adult mice, independently of the chemical nature of the NP.

Bronchopulmonary dysplasia (BPD) is the main respiratory sequela of extreme prematurity. Its pathophysiology is complex, involving interactions between host and environment, likely to be significantly influenced by genetic factors. Thus, the clinical presentation and histological lesions have evolved over time, along with the reduction in neonatal injuries, and the care of more immature children. Impaired alveolar growth, however, is a lesion consistently observed in BPD, such that it is a key feature in BPD, and is even the dominant characteristic of the so-called "new" forms of BPD. This review describes the key molecular pathways that are believed to be involved in the genesis of BPD. Much of our understanding is based on animal models, but this is increasingly being enriched by genetic approaches, and long-term respiratory functional studies.

Little is known about the molecular basis of lung alveolarization. We used a microarray profiling strategy to identify novel genes that may regulate the secondary septation process. Rat lung fibroblasts were extemporaneously isolated on postnatal days 2, 7, and 21, i.e., before, during, and after septation, respectively. Total RNA was extracted, and cRNAs were hybridized to Affymetrix rat genome 230 2.0 microarrays. Expression levels of a selection of genes were confirmed by real-time PCR. In addition to genes already known to be upregulated during alveolarization including drebrin, midkine, Fgfr3, and Fgfr4, the study allowed us to identify two remarkable groups of genes with opposite profiles, i.e., gathering genes either transiently up- or downregulated on day 7. The former group includes the transcription factors retinoic acid receptor ( RXR)-γ and homeobox ( Hox) a2, a4, and a5 and genes involved in Wnt signaling ( Wnt5a, Fzd1, and Ndp); the latter group includes the extracellular matrix components Comp and Opn and the signal molecule Slfn4. Profiling in whole lung from fetal life to adulthood confirmed that changes were specific for alveolarization. Two treatments that arrest septation, hyperoxia and dexamethasone, inhibited the expression of genes that are upregulated during alveolarization and conversely enhanced that of genes weakly expressed during alveolarization and upregulated thereafter. The possible roles of these genes in secondary septation are discussed. Gene expression profiling analysis on freshly isolated cells represents a powerful approach to provide new information about differential regulation of genes during alveolarization and pathways potentially involved in the pathogenesis of bronchopulmonary dysplasia.

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.

Introduction La dysplasie bronchopulmonaire (DBP) est une pathologie du nouveau-ne premature favorisee par une oxygenotherapie precoce et caracterisee par un developpement pulmonaire anormal avec elargissement des espaces aeriens. L’hypoalveolisation et le defaut de septation des saccules primitifs correspondent a un defaut de production d’elastine par les fibroblastes. Les pneumocytes 2 pourraient aussi jouer un role en se differenciant en pneumocytes 1 si ceux-ci sont alteres. Cependant, la physiopathologie de cette maladie reste mal connue. La senescence, dont p16Ink4a est un acteur cle, est un arret permanent de la proliferation limitant le renouvellement des tissus et pourrait etre impliquee dans ce processus par le biais d’un defaut de differenciation et/ou reparation. L’objectif de ce travail est de determiner le role causal de p16 et de la senescence cellulaire sur la DBP dans un modele murin. Methodes Des souriceaux sauvages et p16-/-, ont ete exposes a une FiO 2 a 85 %, dans une enceinte hermetique, pendant la periode d’alveolisation, de j3 apres la naissance a j14. La morphometrie (intercepte lineaire moyen, surface alveolaire), la quantite d’elastine et sa distribution (coloration de Weigert) ont ete evaluees. La senescence a ete quantifiee par mesure de l’activite beta-galactosidase sur poumon frais et par l’expression en qPCR sur poumon total de p21. La division cellulaire a ete mesuree par immunohistochimie (Ki67). L’expression de l’elastine et de marqueurs inflammatoire (IL6, IL1β) a ete evaluee par qPCR sur poumon total. Resultats Apres exposition a l’hyperoxie, les souriceaux presentaient (1) une diminution de la surface alveolaire, une augmentation de l’intercepte lineaire moyen, une diminution de la quantite d’elastine et du nombre de cretes d’elastine, semblables aux lesions de poumons dysplasiques, (2) des marqueurs de senescence augmentes (expression accrue de la beta-galactosidase et de l’ARNm de p21), (3) une inflammation (IL6 et Il1β en ARNm). Il n’existait pas de difference entre les souriceaux sauvages et p16-/- pour les parametres sus-cites. En revanche, les souriceaux p16-/- en hyperoxie, compares aux sauvages, presentaient une division cellulaire accrue (Ki67), au sein du parenchyme pulmonaire. Conclusion Le modele d’hyperoxie se caracterise par une hypoalveolisation et une senescence majoree, non modulees par p16. En revanche, la deletion de p16, via une division cellulaire accrue, pourrait permettre une meilleure reparation a l’âge adulte.

Intra-uterine growth restriction (IUGR) dramatically increases the risk of bronchopulmonary dysplasia in preterm babies, a disease characterized by arrested alveolarization and abnormal microvascular angiogenesis. We have previously described a rodent low protein diet (LPD) model of IUGR inducing impaired alveolarization, but failed to demonstrate any modification of the classical factors involved in lung development. We performed a genome-wide microarray analysis in 120 rat pups with LPD-induced IUGR and their controls, at three key time points of the alveolarization process: postnatal day 4 (P4): start of alveolarization; P10: peak of the alveolarization process and P21: end of the alveolarization process. Results were analysed using Arraymining, DAVID and KEGG software and validated by qRT-PCR and western blots. Considering a cut-off of 2:1 as significant, 67 transcripts at P4, 102 transcripts at P10 and 451 transcripts at P21 were up-regulated, and 89 transcripts at P4, 25 transcripts at P10 and 585 transcripts at P21 were down-regulated. Automatic functional classification identified three main modified pathways, 'cell adhesion molecules', 'cardiac muscle contraction' and 'peroxisome proliferator-activated receptor' (PPAR). Protein analysis confirmed involvement of the PPAR pathway, with an increase of FABP4, an activator of this pathway, at P4 and an increase of adiponectin at P21. Other data also suggest involvement of the PPAR pathway in impaired alveolarization. Our results show that deregulation of the PPAR pathway may be an important component of the mechanism inducing impaired alveolarization observed in IUGR. The complete dataset is available as GEO profiles on the Gene Expression Omnibus (GEO) database ( www.ncbi.nih.gov/geo/, GEO Accession No. GSE56956).

Alveolar macrophages (AM) express gelatinase B, a member of the matrix metalloproteinase family involved in the degradation and remodeling of extracellular matrix components. We evaluated the expression of gelatinase B in the course of idiopathic pulmonary fibrosis (IPF) by studying alveolar macrophages in culture AM and bronchoalveolar lavage fluid from 12 untreated patients with IPF, 11 patients with IPF under treatment with steroid and immunosuppressive agents, and 10 control subjects. By using zymography and quantitative image analysis, latent gelatinase B, as well an 88-kD active form, were investigated in culture medium (24 h) of AMs and were found to be significantly increased (P < 0.01) in untreated patients exhibiting severe IPF when compared with control subjects (4.1 +/- 1.7 versus 0.3 +/- 0.2 10(5) arbitrary units [AU]/10(4) AM for the 92-kD form). Concomitant studies of gelatinase B levels associated with cultured AM extracts or freshly harvested AM showed similar results, both at the mRNA and protein levels, respectively. Immunocytochemical studies on freshly harvested AM demonstrated that the enzyme was located mainly at the cell, suggesting some involvement of gelatinase B in AM migration. In contrast, gelatinase B activity secreted by AM tended to be normal in patients with IPF under steroid and immunosuppressive treatment. Simultaneously, level of the gelatinase B activity in epithelial lining fluid was increased in untreated IPF patients, whereas it was normal in treated patients. These results suggest that AM of patients with IPF are primed for gelatinase B expression and that steroid and immunosuppressive treatment induces negative modulation of the gelatinase B overexpression. We conclude that gelatinase B may play a role in lung remodeling in IPF.

Type II pneumocytes are key cells of the alveolar epithelium. They lie on the alveolar basement membrane, which influences their phenotype and functions. We hypothesized that type II pneumocytes degrade basement membrane components by producing gelatinases, members of the matrix metalloproteinase family. To investigate this hypothesis, we used primary cultures of rat type II pneumocytes and cultures of the human A549 cell line. We found by zymography that 70-kDa gelatinase was present in media conditioned by these cells. This 70-kDa gelatinase was identified as gelatinase A by a Western blot, and the presence of its mRNA was demonstrated by reverse transcription-polymerase chain reaction. A 95-kDa gelatinase could be induced under certain conditions. Production of gelatinases may take place during the turnover of basement membranes, in physiological and in pathophysiological processes. This was suggested by the increase in production of both gelatinases that we observed after in vitro exposure to LPS or interleukin-1. The presence of tissue inhibitors of matrix metalloproteinase-1 and -2 was also demonstrated, suggesting that degradation of extracellular matrix by type II pneumocytes is tightly regulated.

International audience; Intrauterine growth restriction (IUGR) in preterm infants increases the risk of bronchopulmonary dysplasia, characterized by arrested alveolarization. We evaluated the impact of two different rat models (nitric oxide synthase inhibition or protein deprivation) of IUGR on alveolarization, before, during, and at the end of this postnatal process. We studied IUGR rat pups of dams fed either a low protein (LPD) or a normal diet throughout gestation and pups of dams treated by continuous infusion of Nω-nitro-L-arginine methyl ester (L-NAME) or its diluent on the last four days of gestation. Morphometric parameters, alveolar surface (Svap), mean linear intercept (MLI) and radial alveolar count (RAC) and transcriptomic analysis were determined with special focus on genes involved in alveolarization. IUGR pups regained normal weight at day 21 in the two treated groups. In the LPD group, Svap, MLI and RAC were not different from those of controls at day 4, but were significantly decreased at day 21, indicating alveolarization arrest. In the L-NAME group, Svap and RAC were significantly decreased and MLI was increased at day 4 with complete correction at day 21. In the L-NAME model, several factors involved in alveolarization, VEGF, VEGF-R1 and -R2, MMP14, MMP16, FGFR3 and 4, FGF18 and 7, were significantly decreased at day 4 and/or day 10, while the various factors studied were not modified in the LPD group. These results demonstrate that only maternal protein deprivation leads to sustained impairment of alveolarization in rat pups, whereas L-NAME impairs lung development before alveolarization. Known growth factors involved in lung development do not seem to be involved in LPD-induced alveolarization disorders, raising the question of a possible programming of altered alveolarization.

International audience; RATIONALE: Bronchopulmonary dysplasia is the most common chronic respiratory disease in premature infants. Genetic factors might contribute to bronchopulmonary dysplasia susceptibility. OBJECTIVES: To identify genetic variants involved in bronchopulmonary dysplasia through a genome-wide association study. METHODS: We prospectively evaluated 418 premature neonates (gestational age

Better understanding alveolarization mechanisms could help improve prevention and treatment of diseases characterized by reduced alveolar number. Although signaling through fibroblast growth factor (FGF) receptors is essential for alveolarization, involved ligands are unidentified. FGF18, the expression of which peaks coincidentally with alveolar septation, is likely to be involved. Herein, a mouse model with inducible, lung-targeted FGF18 transgene was used to advance the onset of FGF18 expression peak, and genome-wide expression changes were determined by comparison with littermate controls. Quantitative RT-PCR was used to confirm expression changes of selected up- and downregulated genes and to determine their expression profiles in the course of lung postnatal development. This allowed identifying so-far unknown target genes of the factor, among which a number are known to be involved in alveolarization. The major target was adrenomedullin, a promoter of lung angiogenesis and alveolar development, whose transcript was increased 6.9-fold. Other genes involved in angiogenesis presented marked expression increases, including Wnt2 and cullin2. Although it appeared to favor cell migration notably through enhanced expression of Snai1/2, FGF18 also induced various changes consistent with prevention of epithelial-mesenchymal transition. Together with antifibrotic effects driven by induction of E prostanoid receptor 2 and repression of numerous myofibroblast markers, this could prevent alveolar septation-driving mechanisms from becoming excessive and deleterious. Last, FGF18 up- or downregulated genes of extracellular matrix components and epithelial cell markers previously shown to be up- or downregulated during alveolarization. These findings therefore argue for an involvement of FGF18 in the control of various developmental events during the alveolar stage.

Although chorioamnionitis and glucocorticoids (GC) are both known to have potential adverse effects on alveolar development, the use of GC is generalized because of their demonstrated benefits in premature newborns. The objective of this study was to analyze the cumulative effects of GC and chorioamnionitis on lung development and infectious process. In a model of ESCHERICHIA COLI chorioamnionitis controlled by antibiotics, pregnant rabbits were randomized among five groups: (1) E. COLI infection alone, (2) infection plus one betamethasone injection (0.1 mg.kg(-1)), (3) infection plus two betamethasone injections, (4) betamethasone alone, (5) control. Lung morphometric analysis, bronchoalveolar lavage, and bacteriologic tissue cultures were performed after spontaneous delivery. In the context of chorioamnionitis, one betamethasone treatment significantly decreased birth weight and lung volume versus controls (30 ± 1.40 versus 52.40 ± 2.54 g, and 1.92 ± 0.67 versus 2.15 ± 0.74 cm(3), respectively, p < 0.05). Two betamethasone treatments significantly decreased specific alveolar area (279.8 ± 46 cm(2)/100 g versus 510.90 ± 54.1 cm(2)/100 g), specific interstitium volume (0.98 ± 0.09 cm(3)/100 g versus 1.78 ± 0.16 cm(3)/100 g), and specific elastin fiber length (57.4 ± 10.5 versus 183.6 ± 8.1 cm/100 g). These results suggest that glucocorticoid treatment might represent an additional risk factor for lung development in the instance of prenatal infection.

During the perinatal period, lungs undergo changes to adapt to air breathing. The genes involved in these changes are developmentally regulated by various signaling pathways, including the cyclic nucleotide cAMP. As PDE4s are critical enzymes for regulation of cAMP levels, the objective of this study was to investigate PDE4's ontogeny in developing rat lung during the perinatal period. Pulmonary PDE4 activity, PDE4A-D, PDE4B, and PDE4D variant expression levels, PDE4B and PDE4D protein levels, and PDE4D localization in distal lung were determined. PDE4 activity increased towards term, dropped at birth, and increased thereafter to reach a plateau at the end of the second week of life. PDE4B2 and PDE4D long forms demonstrated a pattern of expression that increased markedly at birth. After birth, PDE4D was expressed in alveolar epithelial and mesenchymal cells. The study, therefore, evidenced striking variations in expression patterns among the PDE4 family that differed from changes in global PDE4 activity.

International audience; Lung hypoplasia (LH) is a life-threatening congenital abnormality with various causes. It involves vascular bed underdevelopment with abnormal arterial muscularization leading to pulmonary hypertension. Because underlying molecular changes are imperfectly known and sometimes controversial, we determined key factors of angiogenesis along intrauterine development, focusing at the angiopoietin (ANG)/Tie-2 system. Lung specimens from medical terminations of pregnancy (9-37 wk) were used, including LH due to congenital diaphragmatic hernia (CDH) or other causes, and nonpulmonary disease samples were used as controls. ELISA determination indicated little ANG-1 change during pregnancy and no effect of LH, whereas Tie-2 declined similarly between 9 and 37 wk in LH and controls. By contrast, ANG-2 markedly increased in LH from 24 wk, whereas it remained stable in controls. Because VEGF increased also, this was interpreted as an attempt to overcome vascular underdevelopment. Hypothesizing that its inefficiency might be due to impaired downstream mechanism, endothelial nitric oxide synthase (eNOS) was determined by semiquantitative Western blot and found to be reduced by approximately 75%, mostly in the instance of CDH. In conclusion, angiogenesis remains defective in hypoplastic lungs despite reactive enhancement of VEGF and ANG-2 production, which could be due, at least in part, to insufficient eNOS expression.

Acute lung injury and compromised alveolar development characterize bronchopulmonary dysplasia (BPD) of the premature neonate. High levels of keratinocyte growth factor (KGF), a cell-cell mediator with pleiotrophic lung effects, are associated with low BPD risk. KGF decreases mortality in hyperoxia-exposed newborn rodents, a classic model of injury-induced impaired alveolarization, although the pulmonary mechanisms of this protection are poorly defined. These were explored through in vitro and in vivo approaches in the rat. Hyperoxia decreased by 30% the rate of wound closure of a monolayer of fetal alveolar epithelial cells, due to cell death, which was overcome by recombinant human KGF (100 ng/ml). In rat pups exposed to >95% O2 from birth, increased viability induced by intraperitoneal injection of KGF (2 μg/g body wt) every other day was associated with prevention of neutrophil influx in bronchoalveolar lavage (BAL), prevention of decreases in whole lung DNA content and cell proliferation rate, partial prevention of apoptosis increase, and a markedly increased proportion of surfactant protein B-immunoreactive cells in lung parenchyma. Increased lung antioxidant capacity is likely to be due in part to enhanced CAAT/enhancer binding protein α expression. By contrast, KGF neither corrected changes induced by hyperoxia in parameters of lung morphometry that clearly indicated impaired alveolarization nor had any significant effect on tissue or BAL surfactant phospholipids. These findings evidence KGF alveolar epithelial cell protection, enhancing effects on alveolar repair capacity, and anti-inflammatory effects in the injured neonatal lung that may account, at least in part, for its ability to reduce mortality. They argue in favor of a therapeutic potential of KGF in the injured neonatal lung.

We examined the effects of platelet-activating factor (PAF) on lung epithelial permeability by measuring the clearance of intratracheally administered 99m-technetium-labeled diethylene triamine penta-acetic acid (99mTc-DTPA) in guinea-pigs which were anaesthetised, paralysed and mechanically ventilated. The clearance of the radiolabeled tracer molecule 99mTc-DTPA from airways to the blood was expressed as changes in counts/min corrected for background. For each guinea-pig, 99mTc-DTPA clearance was assessed before and after i.v. PAF administration, when tracheal pressure had returned to near control values. Doses of 10, 50 and 100 ng/kg of PAF caused dose-dependent increases in 99mTc-DTPA clearance of 7 +/- 3%, 38 +/- 7% and 65 +/- 11% respectively. The respective effects of 0.5 mg/kg of the beta 2-adrenergic agonist salbutamol and 0.3 mg/kg of the alpha 1-adrenergic agonist methoxamine on the increase in lung epithelial permeability induced by 50 ng/kg PAF were also studied. Salbutamol significantly reduced the acute bronchoconstrictor effects of PAF, but did not affect the increase in lung epithelial permeability, which was 58 +/- 10%. Conversely, methoxamine significantly enhanced the bronchoconstrictor effects of PAF but inhibited the lung epithelial permeability increase, which was only 10 +/- 13%. In the absence of PAF, salbutamol significantly increased this permeability by 49 +/- 11%, whereas methoxamine alone slightly reduced, it by -11 +/- 4%. These results demonstrate that PAF increases lung epithelial permeability and suggest that vascular surface area recruitment may explain this increase.

International audience; BACKGOUND: Alveolarization requires coordinated extracellular matrix remodeling, a process in which matrix metalloproteinases (MMPs) play an important role. We postulated that polymorphisms in MMP genes might affect MMP function in preterm lungs and thus influence the risk of bronchopulmonary dysplasia (BPD). METHODS AND FINDINGS: Two hundred and eighty-four consecutive neonates with a gestational age of

International audience; RATIONALE: Lung hypoplasia in congenital diaphragmatic hernia (CDH) seems to involve impaired alveolar septation. We hypothesized that disturbed deposition of elastin and expression of fibroblast growth factor 18 (FGF18), an elastogenesis stimulus, occurs in CDH. OBJECTIVES: To document FGF18 and elastin in human CDH and ovine surgical and rat nitrofen models and to use models to evaluate the benefit of treatments. METHODS: Human CDH and control lungs were collected post mortem. Diaphragmatic hernia was created in sheep at 85 days; fetal lungs were collected at 139 days (term = 145 days). Pregnant rats received nitrofen at 12 days; fetal lungs were collected at 21 days (term = 22 days). Some of the sheep fetuses with hernia underwent tracheal occlusion (TO); some of the nitrofen-treated pregnant rats received vitamin A. Both treatments are known to promote lung growth. MEASUREMENTS AND MAIN RESULTS: Coincidental with the onset of secondary septation, FGF18 protein increased threefold in control human lungs, which failed to occur in CDH. FGF18 labeling was found in interstitial cells of septa. Elastin staining demonstrated poor septation and markedly decreased elastin density in CDH lungs. Consistently, lung FGF18 transcripts were diminished 60 and 83% by CDH in sheep and rats, respectively, and elastin density and expression were diminished. TO and vitamin A restored FGF18 and elastin expression in sheep and rats, respectively. TO restored elastin density. CONCLUSIONS: Impaired septation in CDH is associated with decreased FGF18 expression and elastic fiber deposition. Simultaneous correction of FGF18 and elastin defects by TO and vitamin A suggests that defective elastogenesis may result, at least partly, from FGF18 deficiency.

International audience; BACKGROUND: Pulmonary hypoplasia and persistent pulmonary hypertension account for significant mortality and morbidity in neonates with congenital diaphragmatic hernia (CDH). Global lung immaturity and studies in animal models suggest the presence of surfactant deficiency that may further complicate the pathophysiology of CDH. However, data about surfactant status in human fetuses with CDH at birth are contradictory. The lack of a chronological study of surfactant content in late pregnancy has been a significant limitation. The appropriateness of administering surfactant supplements to neonates with CDH is therefore a debated question. METHODS AND FINDINGS: We investigated surfactant content in human fetuses with CDH compared to age-matched fetuses with nonpulmonary diseases used as controls. Concentrations of disaturated phosphatidylcholine and surfactant proteins were found to be similar at a given stage of pregnancy, with both components showing a similar pattern of increase with progressing pregnancy in fetuses with CDH and in control fetuses. Thyroid transcription factor 1, a critical regulator of surfactant protein transcription, similarly displayed no difference in abundance. Finally, we examined the expression of three glucocorticoid-regulated diffusible mediators involved in lung epithelial maturation, namely: keratinocyte growth factor (KGF), leptin, and neuregulin 1 beta 1 (NRG1-beta1). KGF expression decreased slightly with time in control fetuses, but remained unchanged in fetuses with CDH. Leptin and NRG1-beta1 similarly increased in late pregnancy in control and CDH lungs. These maturation factors were also determined in the sheep fetus with surgical diaphragmatic hernia, in which surfactant deficiency has been reported previously. In contrast to the findings in humans, surgical diaphragmatic hernia in the sheep fetus was associated with decreased KGF and neuregulin expression. Fetoscopic endoluminal tracheal occlusion performed in the sheep model to correct lung hypoplasia increased leptin expression, partially restored KGF expression, and fully restored neuregulin expression. CONCLUSIONS: Our results indicate that CDH does not impair surfactant storage in human fetuses. CDH lungs exhibited no trend toward a decrease in contents, or a delay in developmental changes for any of the studied surfactant components and surfactant maturation factors. Surfactant amounts are likely to be appropriate to lung size. These findings therefore do not support the use of surfactant therapy for infants with CDH. Moreover, they raise the question of the relevance of CDH animal models to explore lung biochemical maturity.

Local overexpression of genes that promote lung defense or repair may be helpful in protecting the immature neonatal lung from injuries, but whether the vectors used to administer these genes affect physiological postnatal lung growth has not been investigated. We explored the effect on alveolarization of E1-deleted Adnull vector (Ad5-LMP-null) given intratracheally to 3-day-old rats. Three Adnull doses were evaluated 10(8), 5 x 10(8), and 10(9) TCID(50). Lung morphometry on day 21 showed significant growth disorders with the two higher doses. With 5 x 10(8) TCID(50), absolute lung volume increased significantly (+16%), as did absolute (+20%) and specific (+32%) alveolar airspace volumes, whereas alveolar surface density decreased by 13% (p < 0.009 for all parameters). Lung inflammation was mild, nonsignificant, and occurred mainly with the highest Adnull dose, indicating that it was unlikely to contribute to our results. Adnull instillation induced a significant#10; decrease in terminal bronchiolar cell proliferation as evaluated by proliferating cell nuclear antigen immunostaining (p = 0.02), as well as a 23% decrease in absolute parenchyma elastic fiber length (p = 0.02). Furthermore, lung tropoelastin mRNA content decreased by 25% (p < 0.02). In conclusion, E1-deleted adenoviral vectors can induce lung growth disorders when instilled into the airways of neonatal rats. Interactions with lung matrix turnover may be the main explanation to these deleterious effects.

BACKGROUND: Prolonged neonatal exposure to hyperoxia is associated with high mortality, leukocyte influx in airspaces, and impaired alveolarization. Inhibitors of type 4 phosphodiesterases are potent anti-inflammatory drugs now proposed for lung disorders. The current study was undertaken to determine the effects of the prototypal phosphodiesterase-4 inhibitor rolipram on alveolar development and on hyperoxia-induced lung injury. METHODOLOGY/FINDINGS: Rat pups were placed under hyperoxia (FiO2>95%) or room air from birth, and received rolipram or its diluent daily until sacrifice. Mortality rate, weight gain and parameters of lung morphometry were recorded on day 10. Differential cell count and cytokine levels in bronchoalveolar lavage and cytokine mRNA levels in whole lung were recorded on day 6. Rolipram diminished weight gain either under air or hyperoxia. Hyperoxia induced huge mortality rate reaching 70% at day 10, which was prevented by rolipram. Leukocyte influx in bronchoalveolar lavage under hyperoxia was significantly diminished by rolipram. Hyperoxia increased transcript and protein levels of IL-6, MCP1, and osteopontin; rolipram inhibited the increase of these proteins. Alveolarization was impaired by hyperoxia and was not restored by rolipram. Under room air, rolipram-treated pups had significant decrease of Radial Alveolar Count. CONCLUSIONS: Although inhibition of phosphodiesterases 4 prevented mortality and lung inflammation induced by hyperoxia, it had no effect on alveolarization impairment, which might be accounted for by the aggressiveness of the model. The less complex structure of immature lungs of rolipram-treated pups as compared with diluent-treated pups under room air may be explained by the profound effect of PDE4 inhibition on weight gain that interfered with normal alveolarization.