Profiling target genes of FGF18 in the postnatal mouse lung: possible relevance for alveolar development.

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.

Gene expression profiling in lung fibroblasts reveals new players in alveolarization.

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)-gamma 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.

Surfactant maturation is not delayed in human fetuses with diaphragmatic hernia.

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.

Differential expression of matrix metalloproteinases and inhibitors in developing rat lung mesenchymal and epithelial cells.

Lung development requires extracellular matrix remodeling. This involves matrix metalloproteinases (MMPs) and their endogenous inhibitors [tissue inhibitors of metalloproteinases (TIMPs)]. Because these have been generally studied only in whole lung, we focused specifically on mesenchymal and epithelial cells freshly isolated at various developmental stages. In fibroblasts, the most striking developmental change was a peak (fourfold the prenatal level) of membrane type 1 (MT1)-MMP transcript during alveolarization, consistent with the known crucial role of MT1-MMP in this process. TIMP-1 and -2 mRNAs transiently increased on postnatal d (pn) 3. In alveolar epithelial cells (AECs), MMP-2 expression was maximal on fetal d (f) 19 when alveolar type II cells (ATII) differentiate and on pn5; by contrast, MT1-MMP expression changed little and TIMP-1 expression decreased with advancing gestation. In cells expressing in vitro the ATI phenotype, TIMP-1 and -2 activities were nine- and fivefold those in cells expressing ATII features, respectively, whereas ATII presented higher MMP-2 activity and were the only cell type to express MMP-9. This indicates higher remodeling potential for ATII. Pulmonary mesenchymal and epithelial cells have therefore quite distinct MMP/TIMP expression patterns. Changes in cell compartments should be specifically documented in developing lung diseases such as bronchopulmonary dysplasia in which changes in MMP activities have been reported.

Decreased lung fibroblast growth factor 18 and elastin in human congenital diaphragmatic hernia and animal models.

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.

Control mechanisms of lung alveolar development and their disorders in bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease that occurs in very premature infants and is characterized by impaired alveologenesis. This ultimate phase of lung development is mostly postnatal and allows growth of gas-exchange surface area to meet the needs of the organism. Alveologenesis is a highly integrated process that implies cooperative interactions between interstitial, epithelial, and vascular compartments of the lung. Understanding of its underlying mechanisms has considerably progressed recently with identification of structural, signaling, or remodeling molecules that are crucial in the process. Thus, the pivotal role of elastin deposition in lung walls has been demonstrated, and many key control-molecules have been identified, including various transcription factors, growth factors such as platelet-derived growth factor, fibroblast growth factors, and vascular endothelial growth factor, matrix-remodeling enzymes, and retinoids. BPD-associated changes in lung expression/content have been evidenced for most of these molecules, especially for signaling pathways, through both clinical investigations in premature infants and the use of animal models, including the premature baboon or lamb, neonatal exposure to hyperoxia in rodents, and maternal-fetal infection. These findings open therapeutic perspectives to correct imbalanced signaling. Unraveling the intimate molecular mechanisms of alveolar building appears as a prerequisite to define new strategies for the prevention and care of BPD.

Developmental changes in endothelial vasoactive and angiogenic growth factors in the human perinatal lung.

Little is known of the mechanisms underlying the marked fall in pulmonary vascular resistance that occurs at birth, but changes in the expression of endothelial vasoactive and angiogenic factors during lung development might play a key role. Nitric oxide, endothelin-1, and vascular endothelial growth factor have critical effects on vascular tone and cell growth. Here, we investigated the protein expression of endothelial nitric oxide synthase, endothelin-1 and its receptors, and vascular endothelial growth factor in pulmonary necropsy samples from 14 fetuses of different gestational ages and from 5 infants. Expression of endothelin-1 and its receptor endothelin-A was strong and stable. In contrast, expression of the endothelin-B receptor was weak in early gestation, then increased markedly in mid-gestation and remained high thereafter. The expression of endothelial nitric oxide synthase and vascular endothelial growth factor fell markedly after mid-gestation and remained low thereafter. These data point to a discrepancy between maturational and functional changes in human pulmonary vascular structures. The weak perinatal expression of endothelial nitric oxide could suggest that other potent vasodilatory mediators are responsible for the marked vasodilation observed at birth.

FGF-18 is upregulated in the postnatal rat lung and enhances elastogenesis in myofibroblasts.

The fibroblast growth factors (FGFs) are key players in fetal lung development, but little is known about their status in postnatal lung. Here, we investigated the expression pattern of FGF-18 transcripts through the perinatal period and evidenced a sevenfold increase after birth that paralleled changes in elastin expression. In vitro, recombinant human (rh)FGF-18 had a mitogenic activity on day 21 fetal rat lung fibroblasts and stimulated its own expression in the latter, whereas FGF-2 inhibited it. At 50 or 100 ng/ml, rhFGF-18 increased the expression of alpha-smooth muscle actin (alpha-SMA; 2.5-fold), a characteristic marker of myofibroblasts, of tropoelastin (6.5-fold), of lysyl oxidase (2-fold), and of fibulins 1 and 5 (8- and 2.2-fold) in confluent fibroblasts isolated from fetal day 21 lung; similar results were obtained with fibroblasts from day 3 postnatal lungs. Elastin protein expression was also slightly increased in fetal fibroblasts. Lung analysis on day 4 in rat pups that had received rhFGF-18 (3 microg) on days 0 and 1 showed a 1.7-fold increase of tropoelastin transcripts, whereas alpha-SMA transcripts were unchanged. In contrast, rhFGF-2 markedly decreased expression of elastin in vitro and in vivo and of fibulin 5 in vitro. In addition, vitamin A, which is known to enhance alveolar development, elevated FGF-18 and elastin expressions in day 2 lungs, thus advancing the biological increase. We postulate that FGF-18 is involved in postnatal lung development through stimulating myofibroblast proliferation and differentiation.

Surfactant phospholipids and proteins are increased in fetal sheep with pulmonary hypertension secondary to fetal systemic arteriovenous fistula.

To determine whether prenatal surfactant storage was altered in a model of systemic arteriovenous fistula (SAVF) with pulmonary hypertension, a fistula was created between the internal jugular vein and the carotid artery in 120-day fetal lambs, and surfactant material was explored at 134 days. Total phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) were increased in whole lung tissue. Phospholipid analysis of isolated lamellar body fraction evidenced a specific increase of surfactant pool size: TPL and DSPC in this fraction were enhanced 1.9 and 2.9 times, respectively, when referred to DNA. Although the steady-state level of transcripts of surfactant protein (SP)-A and SP-B was not found to be changed at the time of death, semiquantitative Western blot analysis revealed elevated SP-A and SP-B protein contents three- and twofold, respectively. These findings indicate markedly enhanced accumulation of surfactant material in the presence of surgically induced prenatal pulmonary hypertension. Although total lung cell number was increased by 26%, SP-B immunolabeling indicated that increased surfactant amount did not result from an increased alveolar type II cell proportion, but rather from an increased rate of storage. Whether similar changes in surfactant are encountered in human neonates with persistent pulmonary hypertension is worthy of investigation.

Effects of mild vitamin a deficiency on lung maturation in newborn rats: a morphometric and morphologic study.

OBJECTIVE: To evaluate the effects of a 60% vitamin A deficiency (VAD) on the two postnatal stages of lung development: alveolarization and microvascular maturation. Lungs from deficient rats were compared to age-matched controls. STUDY DESIGN: Starting at 3 weeks before mating, female rats were maintained under a diet lacking vitamin A. Due to the slow depletion of the vitamin A liver stores the pregnant rats carried to term and delivered pups under mild VAD conditions. Mothers and offspring were then kept under the same diet what resulted in a mean reduction of vitamin A plasma concentration of about 60% vs. controls during the whole experimental period. Pups were sacrificed on days 4, 10 and 21 and their lungs fixed and analyzed by means of a combined morphologic and morphometric investigation at light and electron microscopic levels. RESULTS: During the whole experiment, body weights of VAD animals were lower than controls with a significant decrease on day 10. On days 4, 10 and 21 the pulmonary structure was in a comparable gross morphologic state in both groups. Despite this morphologic normality, quantitative alterations in some functional parameters could be detected. On day 4, lung volume and the volume and surface area of air spaces were decreased, while the arithmetic mean barrier thickness and type 2 pneumocyte volume were increased in the VAD group. On day 21, some changes were again manifest mainly consisting in an augmentation of the vascularization and a decrease in interstitial volume in deficient animals. CONCLUSIONS: Mild VAD causes no gross disturbances in the postnatal phases of lung development in rats. However, a body weight-related transient retardation of lung maturation was detectable in the first postnatal week. At 3 weeks, the VAD lungs showed a more mature vascular system substantiated by an increase in volume of both capillary volume and the large non-parenchymal vessels. In view of these quantitative alterations, we suspect that mild VAD deregulates the normal phases of body and lung growth, but does not induce serious functional impairments.

Effects of vascular endothelial growth factor on isolated fetal alveolar type II cells.

Previous investigations gained from in vivo or lung explant studies suggested that VEGF is an autocrine proliferation and maturation factor for developing alveolar type II cells. The objective of this work was to determine whether VEGF exerted its growth and maturation effects directly on isolated type II cells. These were isolated from 19-day fetal rat lung and cultured in defined medium. The presence of VEGF receptor-2 was assessed in cultured cells at the pre- and posttranslational levels. Recombinant VEGF(165), formerly found to be active on lung explants, failed to enhance type II cell proliferation estimated by thymidine and 5-bromo-2'-deoxy-uridine incorporation. It increased choline incorporation in saturated phosphatidylcholine by 27% but did not increase phospholipid surfactant pool size. VEGF (100 ng/ml) left unchanged the transcript level of surfactant proteins (SP)-A, SP-C, and SP-D but increased SP-B transcripts to four times the control steady-state level. VEGF slightly retarded, but did not prevent, the in vitro transdifferentiation of type II into type I cells, as assessed by immunolabeling of the type I cell marker T1alpha. We conclude that, with the exception of SP-B expression, which appears to be controlled directly, the previously observed effects of this VEGF isoform on type II cells are likely to be exerted indirectly through reciprocal paracrine interactions involving other lung cell types.

Maturational factors modulate transcription factors CCAAT/enhancer-binding proteins alpha, beta, delta, and peroxisome proliferator-activated receptor-gamma in fetal rat lung epithelial cells.

Previous investigations have evidenced the importance of CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor (PPAR)gamma for lung development, especially for alveolar type II cells (ATII). This prompted us to explore whether ATII maturation-promoting mediators controlled their expression in isolated ATII. In whole rat lung, C/EBPalpha, beta, delta, and PPARgamma mRNAs increased 3-5 times between gestational day 18 and term (Day 22), dropped around birth, then reincreased. C/EBPbeta and delta, but not PPARgamma, displayed similar profile in isolated ATII; C/EBPalpha transcript disappeared and the protein became hardly detectable in isolated cells. In cultured ATII, dexamethasone increased C/EBPbeta and PPARgamma mRNAs 2-4 times, and cyclic AMP increased C/EBPbeta and delta mRNAs approximately 1.5 times. Whereas retinoic acid increased C/EBPbeta and PPARgamma mRNAs 1.5 times in ATII in vitro, vitamin-A deficiency strongly decreased fetal lung C/EBPalpha, beta, and PPARgamma transcripts in vivo. C/EBPbeta, delta, and PPARgamma mRNAs were also increased in vitro by epidermal growth factor and keratinocyte growth factor, whereas they were unchanged by the maturation inhibitor transforming growth factor-beta. C/EBPalpha expression was not reinduced by any mediator. Changes in transcripts were reflected in protein levels analyzed through Western blotting. These results argue for a role of these factors in ATII functional maturation, and indicate a multifactorial control of their ontogeny.

Dexamethasone potentiates keratinocyte growth factor-stimulated SP-A and SP-B gene expression in alveolar epithelial cells.

Keratinocyte growth factor (KGF, or fibroblast growth factor 7) was previously reported to enhance the synthesis of surfactant in alveolar type II cells. We investigated the possible interactions between KGF and a glucocorticoid, dexamethasone (Dex), on surfactant protein (SP) gene expression. In cultured fetal rat type II cells, KGF and Dex induced greater-than-additive stimulating effects on SP-A and SP-B expressions that were enhanced three-fold and 30-fold, respectively, but had only additive effects on SP-C expression. Using murine lung epithelial (MLE) cells, KGF increased SP-A, SP-B (up to two-fold), and SP-C (up to three-fold) mRNA levels in a dose-dependent way. Dex 10(-9) to 10(-7) M increased SP-A and SP-B mRNA 1.5-fold and SP-C mRNA two-fold. Consistent with type II cell findings, simultaneous treatment by KGF and Dex induced a synergistic increase of SP-A and SP-B transcripts (three-fold and 4.5-fold, respectively), but not of SP-C transcripts. SP-A protein was present in MLE-15 and was increased about three-fold by KGF plus Dex. Expression study of a reporter gene placed under either the SP-A or the SP-B gene regulatory sequences and transfected in MLE-15 cells indicated that the Dex-KGF synergy was achieved mainly through a transcriptional effect for SP-A, and both transcriptional and nontranscriptional effects for SP-B. For the latter, increased mRNA stability was evidenced with the aid of actinomycin D. The Dex-KGF synergy may have potential interest for diseases associated with surfactant deficiency.

Expression of surfactant proteins and thyroid transcription factor 1 in an ovine model of congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: The question of delayed lung maturation in congenital diaphragmatic hernia (CDH) is pending. Data about surfactant proteins (SPs) are sparse in human fetuses and discrepant in the ovine CDH model. The purpose of this study was to investigate, in the ovine surgically created CDH model, the expression of SPs and of thyroid transcription factor 1 (TTF-1), a key regulator of lung development that also controls the expression of surfactant proteins. METHODS: Diaphragmatic hernia (DH) was created surgically in lamb fetuses on day 85 of gestation. On day 139, 5 DH and 6 control fetuses were retrieved by cesarean section. The mRNA levels for SPs and TTF-1 were determined by Northern blot analysis; SP-A and SP-B protein levels were assessed by Western blot analysis. RESULTS: In DH lungs, SP-A, SP-B, and SP-C messenger RNAs were diminished by 82%, 67%, and 32%, respectively, compared with control level. SP-A and SP-B protein amounts were decreased consistently. TTF-1 expression was not altered in the surgical model. CONCLUSIONS: SP's deficiency appears to be a common feature of the various CDH models. By contrast with the nitrofen model, TTF-1 expression was not altered in the surgical model indicating different underlying molecular mechanisms in both models.

High concentrations of keratinocyte growth factor in airways of premature infants predicted absence of bronchopulmonary dysplasia.

Premature lungs are highly susceptible to lung injuries, leading to bronchopulmonary dysplasia (BPD). Keratinocyte growth factor (KGF) is produced by the developing lung and may reduce the risk of BPD by preventing injury to the lung epithelium and enhancing its repair. To determine whether KGF concentrations in the airways during the initial phase of hyaline membrane disease are correlated with subsequent development of BPD defined as the need for supplemental oxygen at a postconceptional age of 36 weeks, we obtained tracheal aspirates within 3 hours of birth (Day 0) from 91 intubated neonates with a gestational age of 30 weeks or less. Repeat samples were obtained from 42 neonates within 5 days after birth. KGF in aspirate supernatants was measured by enzyme-linked immunosorbent assay. On Day 0, KGF was detected in all but six neonates. A significant increase in KGF concentration was found from the first to the second samples. The highest KGF concentration within 5 days after birth (KGF(max)) was significantly higher in survivors without BPD than in those with BPD. A KGF(max) value higher than 110 pg/ml had a positive predictive value of 95% for absence of BPD. KGF may hold promise for the treatment of very premature neonates.