Epidermal growth factor in the rat lung

Summary Epidermal Growth Factor (EGF) in pharmacological doses is able to induce precoccious lung maturation in rabbits and sheeps. As EGF is probably acting in a para- or autocrine way, we have searched for EGF in the lungs.We report EGF immunoreactivity to be present in the type II pneumocytes of the rat from a couple of days prior to birth and throughout life. Further, we report EGF immunoreactivity to be present in cells in the bronchi and the bronchioles from day 20–21 of gestation and throughout life. G-200 gelchromatography of lung extracts indicates that the EGF-reactive material is a high molecular weight form of EGF.Since previous studies have shown that EGF in pharmacological doses is able to promote lung maturation, our results may imply a physiological role for EGF in the lungs.     

Immunohistochemical localization of epidermal growth factor receptor (EGF-R) in normal and diseased newborn lung tissues

The distribution of EGF receptors (EGF-R) was examined in normal, hyaline membrane diseased and pneumonic newborn lung tissues by immunohistochemical methods under the light microscope. The PAP technique with polyclonal antibodies was performed to demonstrate the EGF receptor localisation in these tissues. Strong EGF-R reactivity was observed on bronchiolar epithelium and type I and type II alveolar cells in normal newborn lung tissues; whereas, poor reactivity was observed in alveolar macrophages. On the other hand, strong immunoreactivity was detected in type I alveolar cells and alveolar macrophages in hyaline membrane disease, but no reactivity was present in type II alveolar cells. The strongest immunoreactivity was observed in alveolar macrophages of newborn pneumonic lung tissues. In conclusion, the most meaningful form of reactivity was observed in normal newborn lung tissues of airway track and respiration area. This result is related with the maturation of the lungs after birth.     

大鼠肺内表皮生长因子(EGF)之分布及缺氧对其含量的影响

本文用免疫体金银法对大白鼠肺内表皮生长因子(Epidermal growth factor,EGF)进行了定位研究,并观察缺氧时肺内 EGF 含量的变化。结果显示正常大鼠肺泡细胞、支气管粘膜及肺内血管壁上均含有 EGF 免疫阳性反应物质;缺氧时,肺内 EGF 含量增加。这提示 EGF 不仅对肺具有一定的生理学影响,而且可能参与缺氧时肺的某些病理学过程。     

The receptor for epidermal growth factor is present in human fetal kidney, liver and lung

In animals a pharmacological doses of the growth-promoting peptide epidermal growth factor (EGF) has an effect on the growth and/or maturation of several organs such as the lung, the kidney, the liver and the gastrointestinal tract. Since EGF elicits its function via binding to specific cellular receptors the presence of these receptors predicts a possible physiological role for EGF and EGF agonists. We have studied the presence of the EGF-receptor on human fetal membrane preparations from the kidney, the liver, the lung and the placenta (gestational age 13-20 weeks). The 4 membrane preparations all bind labeled EGF thus allowing us to calculate the apparent affinity constant and the number of receptors present per mg of membrane protein. The apparent affinity constant (gestational age 13-20 weeks) varies between 0.5 and 3.5 X 10(9) mol-1, median 1.3 X 10(9) mol-1 (n = 40). No difference is observed for the 4 tissues examined, and no difference is found as a function of the gestational age. The number of receptors present per mg of membrane protein (gestational age 16-20 weeks) are (range and (median) 90-220 (130) fmol, n = 10 for the kidney, 80-480 (250) fmol, n = 9 for the liver, 90-690 (300) fmol, n = 10 for the lung, and 2100-4200 (3400) fmol, n = 7 for the placenta. Results for a fetus of gestational age 13 weeks show high values for kidney receptors (240 fmol) and lung receptors (800 fmol) and low values for the placenta receptors (410 fmol).     

Systemic Biochemical Effects of Inhaled NO in Rats: Increased Expressions of NOS III, Nitrotyrosine-, and Phosphotyrosine-Immunoreactive Proteins in Liver and Kidney Tissues

Inhaled nitric oxide (iNO) has been shown to reduce pulmonary hypertension associated with several disease states. The effects of iNO are thought to be restricted to the pulmonary vasculature because of its rapid inactivation by hemoglobin. Recent data have suggested, however, that iNO can form nitrosothiols, which can be carried throughout the circulation, thus increasing the half life and bioactivity on NO. Other studies have shown that iNO can affect intestinal ischemia and renal hemodynamics. In this study, rats were exposed to 49 +/- 4 ppm or 107 +/- 13 ppm NO for 4 h and the lung, spleen, liver, and kidney tissues were removed and measured for NOS II and NOS III protein, nitrotyrosine (NT), and phosphotyrosine (PT) immunoreactivity. Following 107 ppm iNO, increases in NOS III protein expression, NT, and PT were observed in the liver and kidney, but not in the lung or spleen. No such increases were noted after the lower dose of iNO. These results paralleled those shown for isobutyl nitrite that we reported earlier and indicated that iNO can cause changes in protein chemistry in organs and tissues beyond the lungs. Since iNO produced little systemic hemodynamic effects, it is unlikely that the observed biochemical alterations were derived secondarily from physiological changes.     

Epidermal growth factor influences the developmental clock regulating maturation of the fetal lung fibroblast

Growth factors may play a significant role in regulating the orderly progression of organ growth and differentiation during fetal development. We hypothesized that epidermal growth factor (EGF) would help regulate the development of surfactant synthesis in the fetal lung by influencing fibroblast-epithelial cell interactions. The effect of EGF (10 ng per ml) on the ability of the fetal lung fibroblast to produce fibroblast pneumonocyte factor (FPF) was studied in sex-specific fibroblasts cultured from day 16, day 17 or day 18 fetal mouse lungs. FPF which is normally not produced by day 16 fibroblasts, is found only in female fibroblasts on day 17, and then in both males and females on day 18. EGF advanced this pattern such that female fibroblasts produced activity on day 16 and fibroblasts from both sexes produced FPF activity on day 17 and day 18. Fibroblasts from an androgen receptor-deficient mouse model confirmed that the effect of EGF was sex-specific and related to the state of development of the fetal lung. We conclude that EGF advances the fetal lung fibroblast through specific stages of development. It appears, therefore, to help control the timing of the clock regulating fetal lung maturation.     

Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants.

We have recently reported (J. Filmus, M. N. Pollak, R. Cailleau, and R. N. Buick, Biochem. Biophys. Res. Commun. 128:898-905, 1985) that MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited in vitro pharmacological doses of EGF. We have derived several MDA-468 clonal variants which are resistant to EGF-induced growth inhibition. These clones had a number of EGF receptors, similar to normal human fibroblasts, and had lost the EGF receptor gene amplification. Karyotype analysis showed that MDA-468 cells had an abnormally banded region (ABR) in chromosome 7p which was not present in the variants. It was shown by in situ hybridization that the amplified EGF receptor sequences were located in that chromosome, 7pABR. Five of the six variants studied were able to generate tumors in nude mice, but their growth rate was significantly lower than that of tumors derived from the parental cell line. The variant that was unable to produce tumors was found to be uniquely dependent on EGF for growth in soft agar.     

肺部菌群与呼吸道疾病的相互关系

由于呼吸道黏膜免疫系统具有很好的防御保护作用和强大的清除病原体的能力,过去学术界曾经一度认为健康机体的肺是无菌的。随着不依赖于体外培养的第二代测序技术的发展,关于肺部共生微生物的结构组成及其免疫调节功能的研究越来越受重视。肺部菌群的结构组成与出生方式、饮食结构、生活环境和抗生素使用等多种因素有关,生命早期的肺部菌群的形成和发育会影响全生命周期的呼吸道疾病的发生和发展。肺部菌群通过与宿主免疫系统相互作用调节肺部免疫稳态,还可以与肠道菌群、呼吸道病毒相互作用影响呼吸道感染。因此,干预生命早期肺部菌群的结构组成可以成为预防和控制呼吸道疾病的有效策略和新靶点。     

Intermediate filaments in the developing type II cell of fetal monkey lung

Anticytokeratin monoclonal antibody was used to study epithelial cell development in fetal monkey lungs taken from animals of different ages. It is well established that the overall maturity of fetal lung depends greatly on the maturation of type II epithelial cells in the alveolus. In this study, we have correlated the cytokeratin phenotype of mammalian epithelial cells with pneumocyte maturation. We show that differentiation and maturation of the type II cell is related to intermediate filament expression. Twenty-four fetal monkeys (Macaca nemestrina) were delivered by cesarean section at a gestational age of 135-140 days (term = 168 days) and divided into two groups. One group of animals was sacrificed during the first 3 hr of life, and the other group was maintained in incubators for 92-120 hr. Anticytokeratin monoclonal antibody recognizes only alveolar type I and type II epithelial cells. In the first 3 hr of life, the cytokeratin was localized only at the alveolar surface and at the cytoplasmic periphery of the type II cells of these premature animals. However, at the age of 92-120 hr, the epithelia in the lungs reacted more intensely than they did during the first 3 hr. Electron microscopy revealed and confirmed that the type II cells were matured and abundant intermediate filaments appeared in the cytoplasm. The filaments appeared to form either aggregates or parallel filament bundles and few were closely associated with the lamellar bodies. In the immature type II cells at 0-3 hr of life, few intermediate filaments could be localized in the cytoplasm, and no parallel filament bundle was observed, though many appeared in the 92-120 hr lungs. This suggests that the intermediate filaments have a functional significance in the development and maturation of the type II cell. The location and stability of keratin filaments in type II cells may confer the structural strength necessary for cells covering a free surface in the alveoli during lung maturation.     

Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth

We examined whether lung growth after pneumonectomy (PNX) invokes normal signaling pathways of postnatal development. We qualitatively and quantitatively assessed the immunoexpression of epidermal growth factor (EGF), its receptor (EGFR), surfactant proteins (SP) [SP-A and -D and surfactant proproteins (proSP)-B and -C] and proliferating cell nuclear antigen (PCNA) in immature and mature dog lung. We also assayed these proteins in lungs of immature dogs 3 wk or 10 mo after they underwent right PNX compared with simultaneous matched sham controls. During maturation, alveolar cell proliferation is regionally regulated in parallel with EGF and EGFR levels and inversely correlated with SP-A and proSP-C levels. In contrast, post-PNX lung growth is not associated with EGF or EGFR upregulation but with markedly increased SP-A level and moderately increased SP-D level; proSP-B and proSP-C levels did not change. We conclude that 1) signaling of EGF axis and differential regulation of SPs persist during postnatal lung development, 2) post-PNX lung growth is not a simple recapitulation of maturational responses, and 3) SP-A and SP-D may modulate post-PNX lung growth.     

Immunohistochemical localization of epidermal growth factor in the second-trimester human fetus

Epidermal growth factor (EGF) is considered to be important in mammalian neonatal growth and development. In order to clarify its developmental role, we have investigated, by immunohistochemistry, the localization of EGF and the time of its first appearance in various organs from a series of 25 midtrimester human fetuses with a gestational age ranging from 13 to 22 weeks. The first detectable EGF immunoreactivity occurred in week 15–16 fetuses in the placenta, the skin, the distal tubules of the kidney, the surface epithelium of the stomach, and the tips of the small intestinal villi, as well as in a few Paneth cells. Glandular structures, such as the glands of the cardia and the pyloric part of the stomach. Brunner's glands of the duodenum, the pancreas, and the submucous glands of the trachea, showed positive EGF immunoreactivity later (week 17). Thus, apart from the kidney, staining of the surface epithelia seems to precede staining of the EGF-producing glandular structures and EGF is not present in the glands before these have already differentiated.     

Epidermal growth factor increases lung liquid clearance in rat lungs

Epidermal growthfactor (EGF) has been reported to stimulate the proliferation ofepithelial cells and increase Na⁺flux andNa⁺-K⁺-ATPasefunction in alveolar epithelial cell monolayers. Increases inNa⁺-K⁺-ATPasein alveolar type II cells (AT2) have been associated with increasedactive Na⁺ transport and lungedema clearance across the rat alveolar epithelium in a model ofproliferative lung injury. Thus we tested whether administration ofaerosolized EGF to rat lungs would increase activeNa⁺ transport and lung liquidclearance. Sixteen adult Sprague-Dawley male rats were randomized tothree groups. To a group of six rats, an aerosol generated from 20 µgof EGF in saline was delivered to the lungs, to a second group of fiverats only aerosolized saline was delivered, and a third group of fiverats without treatment served as the control. Forty-eight hourspostaerosolization of rat lungs with EGF there was an ~40% increasein active Na⁺ transport and lungliquid clearance compared with control rats, in the absence of changesin²²Na⁺,[³H]mannitol, andalbumin permeabilities. TheNa⁺-K⁺-ATPaseactivity in AT2 cells harvested from these lungs was increased in ratsthat received aerosolized EGF compared with AT2 cells from both controlrats and rats receiving aerosolized saline. These results support thehypothesis that in vivo delivery of EGF aerosols upregulates alveolarepithelialNa⁺-K⁺-ATPaseand increases lung liquid clearance in rats.

     

Lack of change in the composition of fetal lamb lung surfactant during gestation

Fetal surfactant from lamb lung fluids collected daily from day 114 to day 146 of gestation, was isolated by centrifugation (pellet material) and further purified by sucrose density gradient centrifugation. The concentration of the pellet material from lung fluid (crude surfactant) increased from day 125 till day 135 and fluctuated strongly from that period onwards, whereas lung fluid secretion increased linearly until a few days before parturition. The pellet phospholipid composition changed with gestational age, suggesting biochemical maturation of the surfactant-producing system. The purified surfactant fraction, of which approximately 85% was phosphatidylcholine, did not change however from day 122 onwards except for a small increase in the percentage of phosphatidylglycerol. Alveolar wash surfactant or the lamellar body material, isolated from fetal lungs at different gestational ages had the same composition as surfactant from lung fluids. Only the composition of lamellar bodies of '125 day' lungs differed slightly from that of the lung fluid surfactant. The similar characteristics of all purified surfactant fractions throughout gestation indicate that, in the fetal lamb, lung maturation is associated with an increase in surfactant production no significant changes in phospholipid composition.     

Neuron specific enolase: a marker for the early development of nerves and endocrine cells in the human lung

Neuron specific enolase (NSE), an isoenzyme of the glycolytic enzyme enolase, has been established by immunocytochemical means as a marker of morphological and functional maturation in central neurons and appears late in development. However, little is known about the presence of NSE in developing peripheral neurons and endocrine cells and its relationship to the development of classical neurotransmitters and peptides. We therefore investigated the appearance of NSE immunoreactivity in nerves and mucosal endocrine cells of the human respiratory tract in foetal, neonatal and adult life. NSE was found to be present in neuroblasts, nerve fibres and endocrine cells from the earliest period of gestation examined (8 weeks), before the appearance of acetylcholinesterase activity (10-12 weeks), dopamine-beta-hydroxylase (20 weeks), vasoactive intestinal polypeptide (20 weeks) or calcitonin (20 weeks). Bombesin-like immunoreactivity was found in a small proportion of mucosal endocrine cells as early as eight weeks in the foetal respiratory tract. These findings indicate that unlike central neurons and their processes, peripheral neurons of the lung contain NSE immunoreactivity well before full maturation and establishment of synaptic contact with end organs.     

Dexamethasone increases adult rat lung surfactant lipids

Prenatal administration of glucocorticoids stimulates epithelial cell maturation and induces a precocious development of pulmonary surfactant. The response of the adult lung to steroid administration is less well understood. We administered dexamethasone (2 mg X kg-1 X day-1) to adult male rats for 1 wk by daily subcutaneous injection. After pentobarbital anesthesia we lavaged the lungs and also isolated lamellar bodies from the tissue. Lipid analyses of the extracellular and intracellular surfactant compartments showed two- to fourfold greater amounts of total phospholipids and disaturated phosphatidylcholine compared with control. These changes were not found in kidney nor liver and were not present in plasma membrane, mitochondrial, or microsomal fractions from lungs. Morphometric analyses of the type II cells showed that anatomic measures of the lamellar body pool did not increase. We conclude that glucocorticoids have a significant effect to increase lung surfactant lipid pools of adult rat lungs by changing the phospholipid content of lamellar bodies, without changing lamellar body volume.     

Stage‐dependent effects of epidermal growth factor on Ca2+ efflux in mouse oocytes

Epidermal growth factor (EGF) has received much attention recently for its positive effects on mammalian oocyte maturation and embryo development and its potential importance in cytoplasmic maturation of oocytes. Calcium (Ca²⁺) homeostasis in germinal vesicle stage oocytes has also been suggested to play a role in cytoplasmic maturation. This study examined the effects of EGF on Ca²⁺ mobilization as measured by its efflux from mouse oocytes at three time periods throughout maturation (0–4 hr, 4–8 hr, and 12 hr). Immature cumulus oocyte complexes (COCs) removed from the ovary for less than 4 hr exhibit oscillations in Ca²⁺ efflux that initiated 5–30 min following EGF stimulation. This response was not observed in COCs matured for 4–8 hr or 12 hr or in unstimulated 0–4 hr COCs. Denuded oocytes and cumulus cells did not show the same response to EGF (8.2 nM and 16.4 nM). Immunohistochemistry for detection of the EGF receptor along with EGF internalization studies showed that receptors are present both on cumulus cells and the oocyte but EGF appears to be internalized mainly by the cumulus cells. These data demonstrate that EGF induces oscillations in Ca²⁺ efflux in COCs 0–4 hr old and this response is mediated by the cumulus cells. Mol. Reprod. Dev. 53:244–253, 1999. © 1999 Wiley‐Liss, Inc.     

Selected contribution: mechanisms that may stimulate the resolution of alveolar edema in the transplanted human lung.

Pulmonary edema is common in organ donors and lung transplant recipients. Therefore, we assessed the responsiveness of human donor lungs to pharmacological agents that stimulate clearance of alveolar edema. Organ donors whose lungs were rejected for transplantation were studied. After resection, transport (4 degrees C), and rewarming (37 degrees C) of lungs, alveolar fluid clearance was measured with (n = 8 donors) or without (n = 23 donors) beta-adrenergic stimulation. Terbutaline-stimulated clearance (10(-4) M) was higher than unstimulated clearance (7.1 +/- 1.3 vs. 4.8 +/- 2.4%/h, P < 0.01). Second, we determined whether medications given to the organ donor were associated with the extent of pulmonary edema or the rate of alveolar fluid clearance in the harvested lung. Preharvest administration of dopamine in low to moderate doses was associated with faster alveolar fluid clearance (r = 0.62, P < 0.01). Preharvest administration of diuretics was associated with lower extravascular lung water-to-dry weight ratios. This study provides the first evidence that a beta(2)-adrenergic agonist stimulates alveolar fluid clearance in the human donor lung. Aerosolized beta(2)-adrenergic agonists may have therapeutic value for hastening the resolution of alveolar edema during the management of donors before resection of lungs for transplantation or in the posttransplant setting.