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Protein deprivation from the neonatal period impairs lung development in the rat.

The effects of early protein-deficiency on lung development were studied in the rat. Newborn male rats were nursed in groups of eight. Control dams and pups received a 15% protein diet during the whole experiment. Test mothers only received 12 and 8% proteins from the 7th and the 14th day of lactation. Test rats fed a diet of 8% protein were weaned at 21 days and maintained on the low protein diet for 1 month. This caused a marked decrease in body weight gain, but lung/body weight ratio was not affected. Lung DNA and protein content, lung protein concentration, but not lung DNA concentration were decreased, suggesting the presence of fewer and smaller cells than in control lungs. Volume-pressure curves were performed on excised lungs. With air-filling, the curve obtained in rats fed a diet with 8% proteins was shifted downward and to the right, even when expressed as percent of maximal volume. Its exponential analysis assessed a decrease in lung compliance. In contrast, with saline-filling, the recoil pressure was decreased in rats fed a diet with 8% proteins. Both with air and saline-filling, the pressure at lung rupture was increased in malnourished rats. It is concluded that protein-deficiency from the neonatal period increases surface forces, decreases tissue elastic forces, and augments resistance of the lung to rupture.

Animals↗

Guinea pig lung development: antioxidant enzymes and premature survival in high O2.

Whereas guinea pigs have advanced prenatal morphological lung development, their surfactant development is not "precocious" compared with other small laboratory animals. To investigate whether maturation of the antioxidant enzyme (AOE) system coincides more closely with surfactant development or with morphological maturation, we assayed fetal guinea pig lungs at gestational days 49-69 for superoxide dismutase, catalase, and glutathione peroxidase activities. We found that elevations in pulmonary AOE occurred in parallel with increases in surfactant during the final 10-15% of gestation. Since newborn guinea pigs behave more like adult animals in their relative intolerance to hyperoxia, we explored whether prematurely delivered guinea pigs would tolerate high O2 exposure better than full-term newborns. We found that prematures have markedly improved hyperoxic tolerance compared with newborns (time at which 50% of animals died in greater than 95% O2, 6.4 days vs. 4.5 days, respectively, P less than 0.05); and (unlike newborns) premature pups are capable of mounting an elevated AOE response to hyperoxic challenge. Thus premature guinea pigs behave more like full-term newborns of other species in respect to hyperoxic tolerance, an additional precocious feature of guinea pig development.

Animals↗

Lung development in diamniotic twins discordant for complete urinary tract obstruction.

OBJECTIVES: To investigate the effect of anhydramnios on the lung development of 1 twin in the presence of a normal amniotic fluid volume in its diamniotic co-twin. METHODS: Three sets of diamniotic twins, discordant for complete urinary tract obstruction and anhydramnios, were followed prospectively with regular ultrasound scans and after delivery. RESULTS: All 3 twins with complete urinary tract obstruction and anhydramnios died within 2 days after birth, with confirmed severe pulmonary hypoplasia. In every case the twin with a normal amount of surrounding amniotic fluid had a normal postnatal outcome. CONCLUSIONS: The observation that a normal amniotic fluid volume in one sac does not protect the anhydramniotic twin from pulmonary hypoplasia has important implications for the aetiology of the condition and for the possibility of therapeutic septostomy. These results are discussed in relation to previous human and animal studies.

Adult↗

Lymphangiogenesis in the developing lung promoted by VEGF-A.

Understanding the basic processes of late-stage pulmonary vascular development is essential as this period corresponds to the stage when preterm infants have increased chance of survival. During this period, refinement of the gas exchange unit leads to close apposition of the capillary vasculature and airway epithelium through thinning of the mesenchyme, formation of alveolar septae and functional adaptation of endothelial cells into vessels including pulmonary lymphatics. The pulmonary lymphatic network promotes efficient gas exchange through maintaining interstitial fluid balance. Through conditional transgene regulation, we found that a modest, pathologically relevant increase in vascular endothelial growth factor A (VEGF-A) in distal lung during only the perinatal period adversely affected final refinement of the gas exchange unit. VEGF-A induction disrupted the established vascular network, increased endothelial cell number, altered endothelial ultrastructure and reduced mesenchymal thinning. In addition, VEGF-A induction caused a 3-fold increase in small vessels identified as lymphatics in distal lung. mRNA levels of lymphangiogenic factors VEGF-D/-C were unchanged, while levels of the cognate receptor VEGFR-3 increased. The responses to VEGF-A induction in the perinatal period differ from those during early lung development when endothelial migration, but not proliferation altered initial vascular patterning (Akeson, A.L., Greenberg, J.M., Cameron, J.E., Thompson, F.Y., Brooks, S.K., Wiginton, D., Whitsett, J.A., 2003. Temporal and spatial regulation of VEGF-A controls vascular patterning in the embryonic lung. Dev. Biol. 264, 443-455). The late-stage response resembles that of adult lung to VEGF-inducing stimuli including injury and disease. These data suggest that VEGF-A influences the balance between development of blood and lymphatic vasculature during lung organogenesis.

Animals↗

l7Rn6 encodes a novel protein required for clara cell function in mouse lung development.

The highly secretory Clara cells play a pivotal role in protecting the lung against inflammation and oxidative stress. This study reports the positional cloning of a novel protein required for Clara cell physiology in mouse lung development. The perinatal lethal N-ethyl-N-nitrosourea-induced l7Rn6(4234SB) allele contained a nonsense mutation in the previously hypothetical gene NM_026304 on chromosome 7. Whereas l7Rn6 mRNA levels were indistinguishable from wild type, l7Rn6(4234SB) homozygotes exhibited decreased expression of the truncated protein, suggesting protein instability. During late gestation, l7Rn6 was widely expressed in the cytoplasm of lung epithelial cells, whereas perinatal expression was restricted to the bronchiolar epithelium. Homozygosity for the l7Rn6(4234SB) allele did not affect early steps in lung patterning, growth, or cellular differentiation. Rather, mutant lungs demonstrated severe emphysematous enlargement of the distal respiratory sacs at birth. Clara cell pathophysiology was evident from decreased cytoplasmic CCSP and SP-B protein levels, enlargement and disorganization of the Golgi complex, and formation of aberrant vesicular structures. Additional support for a role in the secretory pathway derived from l7Rn6 localization to the endoplasmic reticulum. Thus, l7Rn6 represents a novel protein required for organization and/or function of the secretory apparatus in Clara cells in mouse lung.

Amino Acid Sequence↗

[Vimentin expression during the differentiation of smooth myocytes of the bronchial wall in developing lungs].

Dynamics of vimentine synthesis and intensity of DNA synthesis in cells of myoblastic differon and epitheliocytes were analysed in developing lungs by means of immunocytochemistry. Monoclonal antibodies (MCA) reaction to vimentine with different cell structures alters in the course of prenatal ontogenesis. Granular type of the reaction, turning into the diffuse one was described on the early stages of differentiation. Expression of vimentine grows smaller in the course of differentiation in all the cell groups. The reaction was not in fact observed in definitive smooth myocytes. The process of the reduction of the MCA reaction intensity goes along with the DNA synthesis decrease in the cells described.

Aging↗

Effect of tyrosine kinase inhibition on surfactant protein A gene expression during human lung development.

Epidermal growth factor (EGF) stimulates surfactant protein (SP) A synthesis in human fetal lung explants. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase with subsequent activation of second messengers. We hypothesized that inhibition of EGF-receptor tyrosine kinase activity would block SP-A expression in spontaneously differentiating cultured human fetal lung tissue. Midtrimester fetal lung explants were exposed for 4 days to genistein (a broad-range inhibitor of tyrosine kinases) and tyrphostin AG-1478 (a specific inhibitor of EGF-receptor tyrosine kinase). Genistein significantly decreased SP-A and SP-A mRNA levels without affecting either tissue viability or the morphological differentiation of alveolar type II cells. Tyrphostin AG-1478 also decreased SP-A content and SP-A mRNA levels in cultured fetal lung explants. Treatment with EGF could not overcome the inhibitory effects of either genistein or tyrphostin on SP-A; however, only tyrphostin inhibited EGF-receptor tyrosine phosphorylation. We conclude that specific inhibition of EGF-receptor tyrosine kinase with tyrphostin AG-1478 blocks the expression of SP-A during spontaneous differentiation of cultured human fetal lung tissue. Furthermore, exposure to genistein also decreases SP-A expression and blocks the effects of EGF in human fetal lung tissue without inhibiting EGF-receptor tyrosine phosphorylation. These findings support the importance of tyrosine kinase-dependent signal transduction pathways in the regulation of SP-A during human fetal lung development.

Embryonic and Fetal Development↗

Hypophysectomy and porcine fetal lung development.

The effect of hypophysectomy on the development of the lung parenchyma and maturation of pulmonary alveolar type II cells was examined in the fetal pig. In fetuses from four different gilts, hypophysectomy was performed on gestational day 69 or 70. Littermates from the same gilts served as controls. Fetuses were delivered by caesarean section at term (113 +/- 1 days of gestation), and the lungs were fixed by intratracheal instillation. Plasma cortisol and thyroxine concentrations in the umbilical artery were markedly lower for hypophysectomized fetuses compared with control fetuses. Body weight was similar for both groups of fetuses. Total lung volume was 53% smaller in hypophysectomized fetuses compared with control fetuses. However, alveolar septal tissue and capillary luminal volumes were similar in both groups. Total lung alveolar surface area was twice as great in control animals compared with hypophysectomized animals. The volumes of epithelium, interstitium, and endothelium of centriacinar alveolar septa per unit surface area of epithelial basal lamina were 3.7, 4.8, and 2.4 times greater in hypophysectomized fetuses compared with control fetuses. Alveolar type II cell composition also differed significantly between groups. The volume fraction of glycogen in type II cells was 51% for hypophysectomized fetuses and 12% for control fetuses, while lamellar body volume fraction was 8% in hypophysectomized fetuses and 23% in control fetuses. The frequency of alveolar type II cell contact with mesenchymal interstitial cells via foot processes was 5 times greater in the lungs of control animals compared with hypophysectomized animals. These findings demonstrate significant effects of hypophysectomy on the morphogenetic and cytodifferentiation activities of all major tissue compartments of the pulmonary gas exchange area during the final trimester of fetal development.

Animals↗

Restricted fetal growth and lung development: a morphometric analysis of pulmonary structure.

Intrauterine growth restriction (IUGR) in humans increases the risk of lung disease and impaired function suggesting that adverse intra-uterine conditions can alter lung development. We hypothesized that placental restriction (PR) of fetal growth would alter lung structure in late gestation. PR involved removal of implantation sites in pre-pregnant ewes. Normal (n = 7) and PR (n = 11) fetuses were delivered at day 140 gestation. Lungs were fixed by tracheal infusion, processed and analyzed by morphometry. PR reduced ponderal index (PI) of lambs by 13%, increased lung volume:body weight (BW) (19%), and decreased the proportion of lung volume that comprised parenchyma from 86.5(2.6)% to 76.7(2.1)% with no change in absolute volume of non-parenchyma. Within the parenchyma, PR increased the proportion comprising airspace from 42.0(2.2)% to 55.5(1.7)% with smaller (-13%) more dense (18%) airsacs/alveoli present. The overall effect was a reduction in total gas-exchange surface density (-10%). Lung wet-weight and volume, parenchymal volume, gas-exchange tissue, and airspace volumes and gas-exchange surface area correlated positively with BW and crown-rump length (CRL) for all animals. The relative lung weight and volume correlated negatively with BW, CRL, and lung weight:BW with PI. Lung weight, lung volume, parenchymal volume, airspace perimeter, percent of parenchymal gas-exchange tissue, gas-exchange surface density, and area correlated positively with PI. The results indicate increased sparing of lung growth but with increasing structural changes, predominantly within lung parenchyma, with increasing growth restriction. Structural alterations associated with PR and poor fetal growth may be important in the pathogenesis of impaired lung function associated with IUGR.

Animals↗

Effects of anti-mouse EGF antiserum on prenatal lung development in fetal mice.

To clarify the precise role of epidermal growth factor (EGF) on fetal lung development, rabbit anti-mouse EGF (anti-mEGF) antiserum was administered to pregnant mice from days 10 to 17 during late gestation. Control mice were administered either normal rabbit serum (NRS) or physiological saline (PS). Serum EGF was not detected in fetuses from anti-mEGF antiserum treated mothers, but the level in NRS treated control animals was 4.73 +/- 0.66 ng/mL. One day prior to birth, the fetuses were removed and their body and lung weights were measured. There was no difference between body weights and lung weights of anti-mEGF antiserum treated animals and NRS-treated control animals. On light microscopic morphometry, there was no obvious difference between pulmonary architecture of anti-mEGF antiserum treated animals and NRS treated control animals. On transmission electron microscopy, osmiophillic lamellar inclusion bodies were less prominent in the type II epithelial cells in anti-mEGF antiserum treated animals. Electron microscopic morphometric study revealed that the osmiophillic lamellar inclusion bodies in type II epithelial cells of anti-mEGF antiserum treated animals were fewer in number and had decreased area fraction. These findings support the previous finding that EGF promotes epithelial cell differentiation of the fetal lung without affecting body weight and lung weight.

Animals↗

Genes involved in the adrenal pathway of glucocorticoid synthesis are transiently expressed in the developing lung.

We have studied the expression of genes involved in glucocorticoid synthesis in the developing lungs of male and female mouse fetuses on gestation days (GD) 15-18 (surge of surfactant, GD 17; term, GD 19). High levels of steroidogenic acute regulatory protein, cytochrome P450 cholesterol side chain cleavage, 3beta-hydroxysteroid dehydrogenase type 1, 21-hydroxylase, and 11 beta-hydroxylase mRNAs were observed in three of the six litters studied on GD 15 and in none of the 14 litters analyzed between GD 16 and 18. Of these three litters, two showed high expression levels for these five genes in lung tissues from female fetuses only, whereas in the remaining litter, only tissues from male fetuses presented high expression of these genes. In contrast, 11 beta-hydroxysteroid dehydrogenase type 1 mRNA level was very low on GD 15 and presented a gradual increase between GD 15 and 18 with no sex difference. Our data indicate that, like the mature adrenal, the fetal lung expresses all genes required in glucocorticoid synthesis from cholesterol. In addition, our results demonstrate that transient expression of these genes on GD 15 in the fetal lung occurs for both male and female fetuses, 2 d before the surge of surfactant synthesis, which is stimulated by glucocorticoids.

3-Hydroxysteroid Dehydrogenases↗

Neuregulin-1 induces branching morphogenesis in the developing lung through a P13K signal pathway.

Neuregulin-1 (NRG-1) induces signal transduction through the activation of its receptor, a heterodimer of human epidermal growth factor receptors 2 and 3 (HER2/HER3). Signal transduction through this receptor/ligand system plays a critical role in the developing heart, mammary gland, and nervous systems. Previous studies showed that NRG-1-induced HER2 activation resulted in pulmonary epithelial cell proliferation in the human fetal lung. The authors hypothesized that NRG-1 further contributes to lung development and maturation by inducing branching morphogenesis. In the present study, the authors show that NRG-1, HER2, and HER3, but not HER4, are expressed in the developing mouse lung. Addition of NRG-1 to fetal lung explants increased lung branching morphogenesis by 32% (P < .05). This increase in branching was blocked by 2C4, an antibody directed against HER2 that inhibits its dimerization and subsequent NRG-1-induced signal transduction. To gain an understanding of the intracellular signaling pathways involved in NRG-1-induced branching morphogenesis, the authors specifically blocked the phosphatidylinositol-3 kinase (PI3K) and mitogen activation protein kinase (MAPK) pathways. Inhibition of PI3K signaling significantly decreased NRG-1-induced branching morphogenesis (P < .05). Inhibition of NRG-1-induced MAPK activation had no effect on explant branching morphogenesis. These data suggest that NRG-1, binding to the HER2/HER3 heterodimer receptor complex, induces pulmonary branching morphogenesis through HER2 activation of the PI3K pathway.

Animals↗

Growth factors in lung development and disease: friends or foe?

Growth factors mediate tissue interactions and regulate a variety of cellular functions that are critical for normal lung development and homeostasis. Besides their involvement in lung pattern formation, growth and cell differentiation during organogenesis, these factors have been also implicated in modulating injury-repair responses of the adult lung. Altered expression of growth factors, such as transforming growth factor beta1, vascular endothelial growth factor and epidermal growth factor, and/or their receptors, has been found in a number of pathological lung conditions. In this paper, we discuss the dual role of these molecules in mediating beneficial feedback responses or responses that can further damage lung integrity; we shall also discuss the basis for their prospective use as therapeutic agents.

Animals↗

Localization of epidermal growth factor receptor in alveolar epithelium during human fetal lung development in vitro.

Epidermal growth factor (EGF) enhances alveolar type II cell differentiation. In human fetal lung explants, EGF stimulates surfactant protein A (SP-A) synthesis. This effect may occur through a direct interaction of the ligand on EGF receptors located within distal pulmonary epithelium during alveolar type II cell differentiation. To determine if EGF receptor is present in alveolar epithelium, immunostaining for EGF receptor and in situ hybridization for EGF receptor mRNA were performed in human fetal lung explants undergoing alveolar type II cell differentiation in vitro. After 4 days in culture, EGF receptor immunostaining was present in alveolar epithelium from human fetal lung explants compared to minimal immunostaining in undifferentiated human fetal lung epithelium prior to culture. In situ hybridization revealed increased EGF receptor mRNA in differentiated type II cells from cultured explants, with minimal EGF receptor mRNA detected in undifferentiated epithelium from tissue prior to culture. Immunogold staining revealed EGF receptors on the cytoplasmic membranes of epithelial cells lining the prealveolar ducts in human fetal lung explants after 2 days in culture. Alveolar type II cell differentiation in vitro was confirmed ultrastructurally by the presence of lamellar bodies and biochemically by an increase in SP-A content. Thus, EGF receptor is found in alveolar epithelium during differentiation, which suggests an important role for EGF during human fetal lung development.

Embryonic and Fetal Development↗

Creation and repair of diaphragmatic hernia in the fetal lamb: lung development and morphology.

Left-sided diaphragmatic hernias were created in 26 lambs at about 78 days' gestation and repaired at 106 to 124 days' gestation. Nine of these lambs were delivered at term and lived much longer than the nonrepaired lambs delivered at term. The normal development of the fetal lamb lung is compared and contrasted with the development of the lung after creation of a diaphragmatic hernia and also with the changes in morphology resulting from in-utero repair. Creation of a diaphragmatic hernia resulted in marked delay in the development of alveoli and at term the lung had small, thick-walled terminal air-spaces with few capillaries and no true alveoli when compared with the thin-walled alveoli in normal lungs. Another striking feature was an apparent increase in the frequency of type II alveolar cells in diaphragmatic hernia lungs. In-utero repair of the diaphragmatic hernia resulted in a more normal appearance with true alveoli developing by term, although capillaries appeared to be less numerous and type II cells more numerous than in normal lungs. Surprisingly, there appears to be little difference between the left and right lungs in lambs with diaphragmatic hernia.

Animals↗

[Morphological study of the basement membrane of the developing lung in rats].

In order to know the significance of the basement membrane in the developing lung, an immunohistochemical study and an electron microscopical study were conducted. Wistar-Imamichi rats, which were 15, 18 and 20 day-old embryos and 5 day-old newborns, were used. The ABC method (labeled antibody method) and immunofluorescence method (FITC-labeled antibody method) were introduced, using antisera against laminin, type IV collagen and fibronectin which are the major components of the basement membrane. The basement membrane of 15 day-old embryos (pseudo-glandular period) showed very wide fine structures: the lamina lucida and lamina densa could not be defined. The width of the basement membrane became narrow in 18 and 20 day-old embryonic lungs, and the fine structural appearance of lamina lucida and lamina densa became visible after birth. There was no difference between laminin and type IV collagen immunostainability. The width of the type IV collagen and laminin immunoreactions gradually became narrow with the fine structural maturation of the basement membrane. Fibronectin immunostaining with the FITC-labeled method presented a diffuse nodular pattern, while that with the peroxidase labeled antibody method displayed a notched linear pattern. In 15 day-old embryos, fibronectin was mainly localized in the basement membrane zone of pseudo-glandular tubules and in the mesenchymal tissue. This immunoreaction in the mesenchymal tissue gradually disappeared.

Animals↗

Altered lung development after prenatal nicotine exposure in young lambs.

There is compelling evidence that prenatal nicotine exposure permanently alters lung development and airway function. The aim of this study was to determine how prenatal nicotine exposure alters proximal and distal airway function. Thirteen lambs were continuously exposed during the last fetal trimester to low-dose nicotine (LN) and 12 to a moderate dose (MN) (maternal s.c. dose: 0.5 and 1.5 mg/kg/d, respectively). Ten lambs served as controls (C). Proximal airway function was measured by lung mechanics. A multiple-breath N2 washout technique was used to measure lung volume (functional residual capacity) and efficiency of gas mixing in distal airways, i.e. terminal respiratory units (moment ratio and nitrogen clearance). In comparison with C, both LN and MN had significantly reduced specific airway conductance to the same extent at a median study age of 12, 25, and 51 d, indicating signs of proximal airway obstruction. Distal airway function showed significant improvement in LN. Ventilation and functional residual capacity were unaffected. In summary, prenatal nicotine exposure induced airway obstruction in proximal airways and improved gas mixing in distal airways, possibly reflecting restriction in proximal airway growth and accelerated maturation of the acinar part of the lung, respectively. We speculate that prenatal nicotine exposure has a disparate impact on airway development and function. The effect on the distal airways seemed to be inversely related to dose, which was not the case in the large airways. The altered airway function persisted during the study period, indicating that the effects of prenatal nicotine exposure might be permanent.

Animals↗

Identification of laminin domains involved in branching morphogenesis: effects of anti-laminin monoclonal antibodies on mouse embryonic lung development.

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal anti-laminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50-100 micrograms/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.

Animals↗