Synergism of cortisol and thyrotropin-releasing hormone in lung maturation in fetal sheep

The effects of fetal infusions of cortisol and thyrotropin-releasing hormone (TRH) singly and together on pressure-volume relationships and saturated phosphatidylcholine (SPC) concentrations in the lungs were studied in 28 fetal sheep delivered at 128 days of gestation. Four groups each of 7 fetuses were infused with either saline (for 156 h), TRH (25 micrograms/h in 60-s pulses for 156 h), TRH (for 156 h) combined with cortisol (1 mg/h for 84 h), or cortisol (for 84 h). Cortisol had no effect on SPC concentrations, whereas both TRH and cortisol plus TRH increased the concentration of SPC in lavage fluid but not lung tissue. Neither cortisol nor TRH significantly affected lung distensibility [V40; 0.64 +/- 0.04 and 0.57 +/- 0.10 (SE) ml/g, respectively, vs. 0.41 +/- 0.03 ml/g in controls] or stability (V5; 0.24 +/- 0.01 and 0.35 +/- 0.07 ml/g vs. 0.24 +/- 0.03 ml/g), whereas treatment with a combination of the two hormones was associated with a fourfold increase in V40 (1.70 +/- 0.16 ml/g) and V5 (1.03 +/- 0.15 ml/g). Since raised concentrations of cortisol, triiodothyronine, and estradiol-17 beta (treatment with cortisol) had no effect on V40 and V5, whereas similar hormonal changes associated with elevated prolactin levels (treatment with cortisol plus TRH) had marked effects, we conclude that prolactin plays an essential part in the synergism of cortisol and TRH.     

Synergistic effect of cortisol and thyrotrophin releasing hormone on lung maturation in the fetal sheep entails connective tissue maturation.

Pressure-volume relationships and collagen and elastin contents were measured in the lungs of fetal sheep infused either with saline (n = 4), thyrotrophin-releasing hormone (TRH; n = 6), cortisol (n = 9) or TRH plus cortisol (n = 10) at 128 days of gestation (term = 149 days) for 7 days. Lung distensibility (V40 = 1.8 +/- 0.1 ml/g wet wt; mean +/- SD) and stability (V5 = 0.6 +/- 0.1) increased along with collagen (C) (10.1 +/- 2.7 micrograms/mg) and elastin (E) contents (128 +/- 35 ng/mg) in the animals infused with TRH plus cortisol and were significantly higher (p < 0.05) than those observed in TRH (V40 0.62 +/- 0.07; V5 0.32 +/- 0.04; C 3.53 +/- 1.3; E 38.2 +/- 8.3), cortisol (V4 0.66 +/- 0.6; V5 0.27 +/- 0.03; C 4.27 +/- 0.8; E 41.02 +/- 12.7) or saline infused fetuses (V40 0.40 +/- 0.1; V5 0.20 +/- 0.06; C 3.28 +/- 0.9; E 31.5 +/- 9.2). Plasma concentrations of prolactin (PRL), triiodothyronine (T3) and cortisol (F) were also higher in the group of fetuses infused with both hormones in comparison with the other groups. In fetuses treated with TRH plus cortisol, PRL (32 +/- 8.3 ng/ml) and T3 (308.3 +/- 36 micrograms/dl) were significantly higher than in those infused with cortisol alone (PRL 3.7 +/- 2.3; T3 128 +/- 30) or with saline (PRL 4.2 +/- 1.6; T3 < 5 micrograms/dl). In the group treated with TRH alone, PRL also increased significantly (37 +/- 6.4), but T3 increased only slightly (18 +/- 3.4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic hormonal effects on lung maturation in fetal sheep

Cortisol has minimal effects on lung maturation in fetal sheep before 130 days gestation. To test whether there is enhancement of cortisol action by other hormones, cortisol (F), triiodothyronine (T3), epinephrine (E), prolactin (PRL), and epidermal growth factor (EGF), alone or in combination, were infused into fetal sheep for 84 h between 124 and 128 days gestation. A mixture of F + T3 + PRL, but not any combination of two hormones, increased both distensibility [1.71 +/- 0.12 (SE) ml of air/g wet wt at 40 cmH2O, V40] and stability (1.16 +/- 0.09 ml of air per g wet wt at 5 cmH2O, V5) to near full-term values, above values resulting from treatment with F alone (0.91 +/- 0.12 and 0.43 +/- 0.09 ml/g, P less than 0.01). Only F had an effect when given alone, V40 increasing (P less than 0.05). Treatment with F + T3 (0.81 +/- 0.18 ml/g) and F + E (0.77 +/- 0.07 ml/g) increased V5 above values obtained with F alone (P less than 0.05). Alveolar saturated phosphatidylcholine (SPC) was higher after treatment with F + T3 (161 +/- 52 micrograms/g), F + T3 + PRL (156 +/- 53 micrograms/g, P less than 0.05), and F + E (113 +/- 40 micrograms/g, P = 0.07) than after F (12 +/- 3 micrograms/g). We conclude that F, T3, and PRL have a synergistic effect on the development of distensibility and stability of the ovine fetal lung.     

IL-1beta stimulates alveolar fluid absorption in fetal guinea pig lungs via the hypothalamus-pituitary-adrenal gland axis

We tested the hypothesis that interleukin (IL)-1beta-induced cortisol synthesis stimulates alveolar fluid clearance in preterm fetuses. IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. Fetuses were obtained by abdominal hysterotomy at 61 and 68 days gestation and instilled with isosmolar 5% albumin in the lungs, and alveolar fluid movement was measured over 1 h from the change in alveolar protein concentration. Alveolar fluid clearance was induced at 61 days gestation and stimulated at 68 days gestation by IL-1beta, which both were attenuated by cortisol synthesis inhibition. Plasma ACTH and cortisol concentrations were increased by IL-1beta at both gestational ages, and metyrapone reduced cortisol concentrations. IL-1beta was mostly low or undetectable in both fetal and maternal blood. Prenatal alveolar fluid clearance, when present as well as IL-1beta induced, was always propranolol and amiloride sensitive, suggesting that beta-adrenoceptor stimulation and amiloride-sensitive Na+ channels were critical for fluid absorption. IL-1beta increased lung beta-adrenoceptor density at gestation day 61, and cortisol synthesis inhibition attenuated the increased beta-adrenoceptor density. Epithelial Na+ channel and Na+-K+-ATPase subunit expressions were both increased by IL-1beta and attenuated by cortisol synthesis inhibition. These results may explain why babies delivered preterm after intrauterine inflammation have a reduced risk of developing severe respiratory distress.     

Endogenous opioids modulate fetal rabbit lung maturation

To test the hypothesis that endogenous opioids modulate fetal lung development, separate groups of pregnant rabbits received daily injections of saline, morphine (1 mg/kg body wt), or the opioid antagonist naloxone (0.4 and 5.0 mg) for 10 days during their last trimester of pregnancy. The corresponding groups of fetuses were then delivered prematurely on day 28 of gestation (term approximately 31 days) and evaluated with respect to differences in body weight, lung weight, and the ratios of wet to dry lung weight and lung dry weight to body weight, the static inflation and deflation air and saline pressure-volume (P-V) characteristics of the lungs, and lung morphology. Mean values for body weight, lung weight, and the ratios of lung wet to dry weight and lung dry weight to body weight were not significantly different among the saline control (C), morphine (M)-, and naloxone (NLX)-treated fetuses. On the other hand, the fetal air P-V curves varied significantly (P less than 0.001), wherein the M-treated group depicted increased lung distensibility and alveolar stability on lung deflation, whereas the opposite was obtained in the NLX-treated fetuses. Moreover, morphometric analyses demonstrated that the mean alveolar air space-to-tissue ratio in lungs from M-treated fetuses were significantly greater than that observed either in C or in NLX-treated fetuses (P less than 0.05); however, the air space-to-tissue ratio did not significantly vary between the C and NLX-treated animals. These observations provide new evidence that endogenous opioids enhance fetal lung maturation.     

The effects of hydrocortisone on lung structure in fetal lambs

The effect of cortisol infusion on fetal lung development was studied in lambs. Changes were compared with those of control groups of saline-infused fetuses of the same age (day 132) and normal late gestation fetuses (142 +/- 4.6 days). Cortisol was infused into five fetal lambs at 129 days of gestation at a rate of 17.0 mg/day. Four fetuses were delivered by hysterotomy at the onset of labour-like uterine activity (58 +/- 3 h). In cortisol-infused fetuses the concentration of cortisol in fetal plasma and tracheal fluid rose to levels similar to those in normal fetuses during the last week of gestation. Progesterone concentration in maternal plasma declined at about 48 h after the start of treatment. Cortisol-infused lambs showed increases in fixed lung volume, specific lung volume, absolute volume of both parenchyma and non-parenchyma and the proportion of the parenchyma which was potential airspace and a decrease in the proportion of parenchyma. For cortisol-infused lambs Type II cell size and the abundance of lamellar bodies, and the volume fraction of cell occupied by the nucleus were similar to the 142 day group, whereas Golgi apparatus and RER were closer to age matched saline-infused (day 132) controls. Glycogen content was midway between the two control groups. We conclude that infusion of cortisol for about 60 h at physiological levels, beginning at 0.85 of gestation, accelerates many, but not all aspects of pulmonary parenchymal maturation, expressed in terms either of morphogenesis of the gas exchange area or differentiation of Type II alveolar cells.     

Spatial distribution of collagen and elastin fibers in the lungs

Surface tension forces acting on the thin-wall alveolar septa and the collagen-elastin fiber network are major factors in lung parenchymal micromechanics. Quantitative serial section analysis and morphometric evaluations of planar sections were used to determine the spatial location of collagen and elastin fibers in Sprague-Dawley rat and normal human lung samples. A large concentration of connective tissue fibers was located in the alveolar duct wall in both species. For rats, the tissue densities of collagen and elastin fibers located within 10 microns of an alveolar duct were 13 and 9%, respectively. In human lung samples, the tissue densities of collagen and elastin fibers within 20 microns of an alveolar duct were 18 and 16%, respectively. In both species, bands of elastin fibers formed a continuous ring around each alveolar mouth. In human lungs, elastin fibers were found to penetrate significantly deeper into alveolar septal walls than they did in rat lungs. The concentration of connective tissue elements in the alveolar duct walls of both species is consistent with their proposed roles as the principal load-bearing elements of the lung parenchyma.     

Pulmonary maturation in the hypophysectomised ovine fetus. Differential responses to adrenocorticotrophin and cortisol

Pulmonary maturation in six ovine fetuses hypophysectomised by a cryosurgical method at 0.7-0.8 of pregnancy and delivered by hysterotomy at 152.2 +/- 2.9 (SD) days was compared with that in seven control fetuses delivered at 144.5 +/- 3.5 days. Both the wet and the dry weight of the lungs was less in the hypophysectomised fetuses but total DNA did not differ. Lung volumes at 40 cm of H2O and at 5 cm of H2O on deflation in hypophysectomised fetuses were less than one-third that of controls. Saturated phosphatidylcholine, as an estimate of surfactant, was lower in both lung tissue and lavage fluid. A further group of hypophysectomised fetuses was infused intravenously either with cortisol at 1 mg/h for 72 h (n = 6), or with ACTH1-24 at 5 microgram/h for 84 h (n = 6) before delivery at 155.0 +/- 2.1 days and 154.2 +/- 3.9 days respectively. None of the indices of pulmonary maturation in the cortisol-treated fetuses differed from those in untreated hypophysectomised fetuses whereas values for lung volumes at 40 and 5 cm of H2O in ACTH-treated fetuses were more than twice those of untreated hypophysectomised fetuses and did not differ significantly from controls. In addition, the amount of saturated phosphatidylcholine in lavage fluid was greater in ACTH-treated fetuses (0.13 +/- 0.10 mg/g) than in untreated hypophysectomised fetuses (0.04 +/- 0.48 mg/g). Lung volume at 40 cm of H2O in four fetuses that were thyroidectomised at the time of hypophysectomy responded to ACTH as in hypophysectomised fetuses with intact thyroids but other indices were unaffected. We conclude that hypophysectomy retards pulmonary maturation in fetal sheep. Since ACTH restores distensibility and increases alveolar surfactant in the absence of other pituitary hormones it is likely that ACTH has a major role in lung maturation. The lack of response to cortisol suggests that the effect of ACTH is not mediated only by circulating cortisol.     

Histochemical analyses of altered fetal lung development following single vs multiple courses of antenatal steroids.

A single course of antenatal steroids is widely used during preterm labor to promote fetal lung maturation. However, little is known regarding efficacy and safety of multiple courses of antenatal steroids. In animal models and clinical trials, treatment with glucocorticoids can inhibit growth. The present study of single- vs multiple-course steroids in pregnant ewes analyzes the effects of steroids vs placebo on fetal lung histopathology. Single-course groups received dexamethasone (Dex) 6 mg or normal saline every 12 hr for 48 hr at 104-106 days of gestation (term = 150 days). Multiple-course groups received the first course at 76-78 days; this was repeated weekly for 5 weeks. At 108 days, lungs were analyzed using immunohistochemistry for alpha-smooth muscle actin, a myofibroblast marker and proliferating cell nuclear antigen. Cell injury/death was evaluated using TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) analysis. Although fetal growth was restricted by either single or multiple courses of Dex, alveolar development was accelerated as measured by mean linear intercepts. Alveolar walls were thinner, developing septa were longer, and septal myofibroblasts were increased for both Dex groups compared with controls. Cell proliferation increased following multiple steroid courses, especially in the distal parenchyma, with a corresponding decrease in apoptosis. These observations suggest that Dex promotes alveolarization, whether given in single or multiple courses.     

The retino-hypothalamic tract is involved in prolactin regulation in fetal sheep.

To investigate the role of the retino-hypothalamic tract on fetal prolactin regulation, we examined the effect of ocular enucleation on fetal plasma prolactin. Eleven fetuses of Suffolk ewes were chronically catheterized during fall, and six of them were subjected to bilateral ocular enucleation. All ewes were kept at 12h:12h light:dark cycle (lights on at 0800 and off at 2000). The experiments were performed 5-9 days after surgery (GA control fetuses 125 +/- 1.5, optical enucleation 121.3 +/- 1.5 days). Blood samples were taken from fetuses hourly around the clock, and plasma prolactin and cortisol were measured by radioimmunoassay (RIA). Luteinizing hormone (LH) and Growth hormone (GH) were measured in pooled plasma samples from control and enucleated fetuses by RIA. Average plasma prolactin was 5-fold lower in enucleated than in control fetuses (9.6 +/- 0.5 and 54.2 +/- 3.3 ng/ml, SEM; P < 0.005). Both control and enucleated fetuses presented circadian rhythm of prolactin with acrophase between 1400 and 1830 h. An enucleated fetus was tested for response of prolactin to TRH. Prolactin increased as described in the literature. There was no change in plasma concentration of cortisol, LH or GH after ocular enucleation. Our data indicate that the optical pathway participates in prolactin regulation in the fetal sheep.     

Activated MCTC mast cells infiltrate diseased lung areas in cystic fibrosis and idiopathic pulmonary fibrosis

Background

Although mast cells are regarded as important regulators of inflammation and tissue remodelling, their role in cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) has remained less studied. This study investigates the densities and phenotypes of mast cell populations in multiple lung compartments from patients with CF, IPF and never smoking controls.

Methods

Small airways, pulmonary vessels, and lung parenchyma were subjected to detailed immunohistochemical analyses using lungs from patients with CF (20 lung regions; 5 patients), IPF (21 regions; 7 patients) and controls (16 regions; 8 subjects). In each compartment the densities and distribution of MC_T and MC_TC mast cell populations were studied as well as the mast cell expression of IL-6 and TGF-β.

Results

In the alveolar parenchyma in lungs from patients with CF, MC_TC numbers increased in areas showing cellular inflammation or fibrosis compared to controls. Apart from an altered balance between MC_TC and MC_T cells, mast cell in CF lungs showed elevated expression of IL-6. In CF, a decrease in total mast cell numbers was observed in small airways and pulmonary vessels. In patients with IPF, a significantly elevated MC_TC density was present in fibrotic areas of the alveolar parenchyma with increased mast cell expression of TGF-β. The total mast cell density was unchanged in small airways and decreased in pulmonary vessels in IPF. Both the density, as well as the percentage, of MC_TC correlated positively with the degree of fibrosis. The increased density of MC_TC, as well as MC_TC expression of TGF-β, correlated negatively with patient lung function.

Conclusions

The present study reveals that altered mast cell populations, with increased numbers of MC_TC in diseased alveolar parenchyma, represents a significant component of the histopathology in CF and IPF. The mast cell alterations correlated to the degree of tissue remodelling and to lung function parameters. Further investigations of mast cells in these diseases may open for new therapeutic strategies.     

Corticosteroid and thyrotropin-releasing hormone effects on preterm sheep lung function

Four groups of twin sheep fetuses were catheterized at 121 days of gestational age and intravenously infused with saline, 0.75 mg.kg-1.h-1 cortisol for 60 h, five intermittent bolus injections of 5 micrograms/kg thyrotropin-releasing hormone (TRH) at 12-h intervals, or both hormones before delivery at 128 days. At birth, the lambs were randomized to receive surfactant or no treatment. Surfactant treatment improved lung function of all the groups. Corticosteroids alone and in combination with TRH improved compliance and gas exchange as well as pressure-volume curves. Corticosteroids alone dramatically decreased the recovery of intravenously administered radiolabeled albumin in the lung tissue and air space and improved the pulmonary response to surfactant treatment. There were no additional effects of TRH when given with corticosteroids on lung function or albumin leak. There were no changes in alveolar surfactant-saturated phosphatidylcholine pool sizes after any hormone treatment. The single significant effect of combined corticosteroid and TRH treatment was a fivefold increase in surfactant protein A in alveolar lavage fluid relative to all other groups.     

Increased number and altered phenotype of lymphatic vessels in peripheral lung compartments of patients with COPD

Background

De novo lymphatic vessel formation has recently been observed in lungs of patients with moderate chronic obstructive pulmonary disease (COPD). However, the distribution of lymphatic vessel changes among the anatomical compartments of diseased lungs is unknown. Furthermore, information regarding the nature of lymphatic vessel alterations across different stages of COPD is missing. This study performs a detailed morphometric characterization of lymphatic vessels in major peripheral lung compartments of patients with different severities of COPD and investigates the lymphatic expression of molecules involved in immune cell trafficking.

Methods

Peripheral lung resection samples obtained from patients with mild (GOLD stage I), moderate-severe (GOLD stage II-III), and very severe (GOLD stage IV) COPD were investigated for podoplanin-immunopositive lymphatic vessels in distinct peripheral lung compartments: bronchioles, pulmonary blood vessels and alveolar walls. Control subjects with normal lung function were divided into never smokers and smokers. Lymphatics were analysed by multiple morphological parameters, as well as for their expression of CCL21 and the chemokine scavenger receptor D6.

Results

The number of lymphatics increased by 133% in the alveolar parenchyma in patients with advanced COPD compared with never-smoking controls (p < 0.05). In patchy fibrotic lesions the number of alveolar lymphatics increased 20-fold from non-fibrotic parenchyma in the same COPD patients. The absolute number of lymphatics per bronchiole and artery was increased in advanced COPD, but numbers were not different after normalization to tissue area. Increased numbers of CCL21- and D6-positive lymphatics were observed in the alveolar parenchyma in advanced COPD compared with controls (p < 0.01). Lymphatic vessels also displayed increased mean levels of immunoreactivity for CCL21 in the wall of bronchioles (p < 0.01) and bronchiole-associated arteries (p < 0.05), as well as the alveolar parenchyma (p < 0.001) in patients with advanced COPD compared with never-smoking controls. A similar increase in lymphatic D6 immunoreactivity was observed in bronchioles (p < 0.05) and alveolar parenchyma (p < 0.01).

Conclusions

This study shows that severe stages of COPD is associated with increased numbers of alveolar lymphatic vessels and a change in lymphatic vessel phenotype in major peripheral lung compartments. This novel histopathological feature is suggested to have important implications for distal lung immune cell traffic in advanced COPD.     

Parenchymal mechanics at the alveolar level

Three current questions about alveolar mechanics are discussed: how is the liquid lining of the alveolus maintained, how does surface tension affect alveolar geometry and lung recoil, and how does the parenchyma respond to nonuniform deformations? Modeling the parenchyma as an interconnected network of alveolar walls is emphasized and modifications that are needed to make the models quantitatively successful are suggested.--Wilson, T. A. Parenchymal mechanics at the alveolar level.     

Inflammation and lung maturation from stretch injury in preterm fetal sheep

Mechanical ventilation is a risk factor for the development of bronchopulmonary dysplasia in premature infants. Fifteen minutes of high tidal volume (V(T)) ventilation induces inflammatory cytokine expression in small airways and lung parenchyma within 3 h. Our objective was to describe the temporal progression of cytokine and maturation responses to lung injury in fetal sheep exposed to a defined 15-min stretch injury. After maternal anesthesia and hysterotomy, 129-day gestation fetal lambs (n = 7-8/group) had the head and chest exteriorized. Each fetus was intubated, and airway fluid was gently removed. While placental support was maintained, the fetus received ventilation with an escalating V(T) to 15 ml/kg without positive end-expiratory pressure (PEEP) for 15 min using heated, humidified 100% nitrogen. The fetus was then returned to the uterus for 1, 6, or 24 h. Control lambs received a PEEP of 2 cmH(2)O for 15 min. Tissue samples from the lung and systemic organs were evaluated. Stretch injury increased the early response gene Egr-1 and increased expression of pro- and anti-inflammatory cytokines within 1 h. The injury induced granulocyte/macrophage colony-stimulating factor mRNA and matured monocytes to alveolar macrophages by 24 h. The mRNA for the surfactant proteins A, B, and C increased in the lungs by 24 h. The airway epithelium demonstrated dynamic changes in heat shock protein 70 (HSP70) over time. Serum cortisol levels did not increase, and induction of systemic inflammation was minimal. We conclude that a brief period of high V(T) ventilation causes a proinflammatory cascade, a maturation of lung monocytic cells, and an induction of surfactant protein mRNA.     

A simple method for the differential characterization of alveoli and alveolar ducts in injured lungs

RATIONALE AND HYPOTHESIS: Previous studies evaluating the histoarchitecture of distal airspaces have been shown to be limited by the difficulty in adequately differentiating alveoli and alveolar ducts. This limitation has been specially noticed in studies addressing lung recruitment and in situations of diffuse alveolar damage (DAD), where generic nominations for distal airspaces had to be created, such as "peripheral airspaces" (PAS) and "large-volume gas-exchanging airspaces" (LVGEA). Elastic stains have been largely used to describe normal lung structures. Weigert's resorcin-fuchsin staining (WRF) demarcates the thickened free portions of the ductal septum facilitating its recognition. We hypothesized that this staining could help in differentiating alveoli from alveolar ducts in distorted lung parenchyma. MATERIAL AND METHODS: Samples of control lungs and of DAD lungs induced by mechanical ventilation (VILI) were stained with hematoxylin-eosin (HE) and with WRF. Using morphometry we assessed the volume proportion of alveoli, alveolar ducts and LVGEA in control and VILI lungs. RESULTS: WRF stained VILI lungs showed a significant decrease in the volume proportion of LVGEA and alveoli and a significant increase in the volume proportion of alveolar ducts when compared to HE stained samples. CONCLUSION: We conclude that WRF staining is useful to distinguish alveolar ducts from alveoli in a DAD model, and suggest that it should be routinely used when morphometric studies of lung parenchyma are performed.     

Androgen receptors in fetal rabbit lung and the effect of fetal sex on the levels of circulating hormones and pulmonary hormone receptors

High affinity (KD = 0.2 nM), low capacity (3.6-5.0 fmol/mg protein), androgen-specific binding proteins with characteristics typical of androgen receptors were identified in the lungs of rabbit fetuses between the 26 and 29th day of gestation and in the lungs of adult rabbits. While androgen receptor concentrations increased significantly from late gestation to adulthood (P less than 0.01), no sex-related differences were observed in either the binding affinities or concentrations of the receptors at any age tested. Similarly, no sex-related differences were found in the levels of progesterone, cortisol and cortisone in the fetal circulation, or in the levels of progesterone receptors, glucocorticoid receptors and beta-adrenergic receptors in the fetal lung at 26 days of gestation. It is concluded that the fetal lung interacts directly with circulating androgens via specific androgen receptors and that the suggested male disadvantage with respect to lung maturation in the perinatal period does not appear to be associated with sex-related differences in the levels of pulmonary androgen, glucocorticoid, progesterone or beta-adrenergic receptors.     

Effects of oligohydramnios on lung growth and maturation in the fetal rat

Oligohydramnios (OH) retards fetal lung growth by producing less lung distension than normal. To examine effects of decreased distension on fetal lung development, we produced OH in rats by puncture of uterus and fetal membranes at 16 days of gestation; fetuses were delivered at 21 or 22 days of gestation. Controls were position-matched littermates in the opposite uterine horn. OH lungs had lower weights and less DNA, protein, and water, but no differences in saturated phosphatidylcholine, surfactant proteins (SP)-A and -B, and mRNA for SP-A, -B, -C, and -D. To evaluate effects on epithelial differentiation, we used RTI(40) and RTII(70), proteins specific in lung to luminal surfaces of alveolar type I and II cells, respectively. At 22 days of gestation, OH lungs had less RTI(40) mRNA (P < 0.05) and protein (P < 0.001), but RTII(70) did not differ from controls. With OH, type I cells (in proportion to type II cells) covered less distal air space perimeter (P < 0.01). We conclude that OH, which retards lung growth, has little effect on surfactant and impedes formation of type I cells relative to type II cells.     

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.