A novel function for the protein tyrosine phosphatase Shp2 during lung branching morphogenesis.

Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must be tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. Herein, we demonstrate for the first time an important function for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. We show that Shp2 is specifically expressed in embryonic lung epithelial buds, and that loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, we provide direct evidence that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. We demonstrate that Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung was associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation was not affected. Our results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis.

Bronchial ligation enhances murine fetal lung development in whole-organ culture.

Evidence exists from both congenital anomalies and animal models that normal fetal lung development is dependent on maintenance of fluid pressure within the developing "airways." Fetal tracheostomy, allowing free egress of airway fluids, results in lung hypoplasia, indicating that some airway distending pressure is required for normal lung development to occur. In contrast, fetal tracheal ligation, which increases fetal airway pressure, reverses lung hypoplasia in animal models. The authors' experiments test the hypothesis that large airway obstruction accelerates the development of murine lungs in vitro in whole-organ culture. Fetuses from time-dated pregnant CD-1 mice at day 14 of gestation were removed (term, 20 days), and the lungs were excised. The left bronchus of each lung was ligated (n = 26), after which the left lung was isolated and cultured at 37 degrees C (95% air, 5% CO2) in BGJb media supplemented with vitamin C and antibiotics. Some fetal lungs were cultured under similar conditions without bronchial ligation (n = 11). After 7 days in culture, the lungs were taken for various analyses. The lungs were fixed in either formaldehyde and processed for paraffin embedding for light microscopic evaluation and morphometric data collection, or were freshly minced and aliquots taken for total protein and DNA content. Several more ligated and unligated lungs were processed for ultrastructural analysis. Morphometric analysis on transverse sections of lungs showed significant differences in the lung tissue size, thickness, epithelial cell height, luminal areas, perimeters, and total number of airspaces (airway + primordial alveolar airspaces). It was evident that bronchial ligation promoted lung development. The ligated lungs displayed thinning of the primordial alveolar walls with cuboidal epithelial cells. The total number of airspaces per field was lower for better developed ligated lungs because of the increased area of airspaces compared with that of the unligated lungs. The dorsoventral tissue thickness (in micrometers) of the ligated lungs was significantly greater than that of the unligated lungs (124.1 +/- 7.0 v 89.6 +/- 8.0); the average outer perimeter of the primordial alveolar airspaces was greater for ligated lungs (404.56 +/- 19.0 microns v 256.85 +/- 17.0 microns). Similarly, the luminal diameter of the spaces of ligated lungs was almost double that of the unligated lungs (38.0 +/- 2.0 microns v 20.3 +/- 2.0 microns), as was the luminal surface area. The morphometric data, which suggest enhanced maturation of the ligated lungs, are supported by results of ultrastructural studies. Ligated lungs had significantly more lamellar bodies. Although total protein and DNA content were greater among the ligated lungs, the protein/DNA ratios did not differ among the groups. The intraluminal pressure (airway pressure) of ligated lungs was 2.9 mm Hg and 3.1 mm Hg at 2 and 4 days in organ culture; the respective pressures for unligated lungs were 1.0 mm Hg and 0.8 mm Hg. These data support the hypothesis that mechanical distending pressure resulting from airway obstruction not only improves pulmonary architecture but also accelerates lung development in vitro. Although these effects have been seen in in vivo models, this is the first proposed in vitro organ culture model. This model may prove to be a powerful tool for the study of molecular mechanisms of mammalian lung development with respect to mechanical and chemical (cytokines, hormones) stimuli.

Regenerative healing of incisional wounds in murine fetal lungs maintained in organ culture.

Although fetal dermal repair is known to be fundamentally different from adult healing, the response to wounding in other organs is less well characterized. Scarless repair in mid-gestation dermis with a transition to adult-type healing at term has been shown in fetal organ culture. A lung explant culture system was used to investigate whether wound repair in the fetal lung shows characteristics similar to those found in fetal dermis. Lungs from 14-day and 18-day Cd-1 murine fetuses and 2-day-old newborns, (term = 20 days, n = 24) were wounded by linear incision and incubated at 37 degrees C, in a 21% O2, 5% CO2 environment, in BGJb supplemented with vitamin C and antibiotics. Medium was changed daily. Samples were fixed at 7 days and embedded in paraffin. Sections were stained with hematoxalyn-eosin and Masson Trichrome. Additional 14-day and 18-day samples were frozen in freon and immunohistochemical staining for TGF-beta performed. Other frozen tissues from each time point were homogenized and used to assay for endogenous TGF-beta levels by Western blot analysis. Histology showed reconstitution of tissue architecture across the wound in 14-day and 18-day specimens. In representative histological sections, intact bronchial architecture developed across the previous wound site. No cellular inflammatory response was observed, and collagen deposition was undetectable at the site of the wound by Trichrome staining. By 22 days the lung explants showed a much less ordered repair, including disorganized collagen deposition.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro induction of type II pneumocyte-related differentiation in a clonal fetal bronchiolo-alveolar epithelial cell line (M3E3/C3).

The aim of the present study is to investigate the differentiation of a cloned fetal Syrian hamster lung epithelial cell line, M3E3/C3, to assume morphological and biochemical features of Type II pneumocytes (phospholipid synthesis). The use of a soft agar overlay and a differentiation medium, based on RPMI 1640 combined with hormone supplements, increased the cellular content of phosphatidylcholine (PC) from 48.6% in the conventional culture without any of these factors (referred to as 'control') to 64.7% (p < 0.02). The other cell membrane-associated components, phosphatidylethanolamine (p < 0.05), sphingomyelin (p < 0.001), phosphatidylserine (n. s.), phosphatidic acid (p < 0.02) and phosphatidylinositol (p < 0.02) decreased. The content of phosphatidylglycerol showed no essential change (from 11.2% to 8.4%) and the content of disaturated phospholipids decreased from 32.0 to 23.4 micrograms/10(6) cells (p < 0.002). The phospholipid pattern of these differentiated cells is in rough accordance with that of primary isolated Type II pneumocytes. They incorporated 3H-choline over a period of four hours at a higher rate in the Type II pneumocyte-specific phospholipids, PC and dipalmitoyl-phosphatidylcholine (DPPC), than the undifferentiated control. The radiolabelling of PC and DPPC in the differentiated cells, after 3 hours of incubation with 3H-choline, was about 3.2-fold and 2.2-fold, respectively, higher than that in the control cells (p < 0.001). Intracytoplasmatic phospholipid granules were evident in the differentiated cells by light and fluorescence microscopy (modified Papanicolaou stain, Phosphin 3 R fluorescence). Furthermore, the differentiated cells had a high activity of alkaline phosphatase, whereas the control cells showed only little activity of this enzyme. Ultrastructurally, many concentric multilayered osmiophilic bodies, well developed Golgi apparatuses and many cytoplasmic protrusions comparable to microvilli, were detectable in the cuboidal shaped differentiated cells. The control cells remained wide and flattened on the plastic surface and produced a fibrillar extracellular matrix. In the simultaneously studied fetal lung fibroblasts none of these specific features were noted. These results indicate a specific differentiation capacity of the clonal fetal cell line, M3E3/C3, by closely resembling Type II pneumocytes.

Furosemide differentially relaxes airway and vascular smooth muscle in fetal, newborn, and adult guinea pigs.

Furosemide, an inhibitor of Cl-dependent Na+,K+ cotransport, is the most frequently used diuretic in newborns. Recently, furosemide was also demonstrated to decrease bronchial hyper-responsiveness in adults, although little is known about the direct effect of furosemide on smooth muscle of immature animals. This in vitro study was designed to determine the action of furosemide on airway and vascular smooth muscle during ontogeny. Extrathoracic trachea (ET), main stem bronchi, main pulmonary artery, and thoracic aorta ring segments from fetal, newborn, and adult Hartley albino guinea pigs were suspended in HEPES solution for measurement of isometric tension. Furosemide (30 or 300 microM) was administered after preconstriction with an ED35-70 concentration of histamine or acetylcholine for airway and ED40-100 concentration of norepinephrine for vessels. Furosemide (30 microM) caused significant relaxation of airway smooth muscle at all ages. After histamine-induced preconstriction, fetal airway segments exhibited greatest relaxation (183 +/- 28%), with newborn airway demonstrating 123 +/- 15% relaxation and modest relaxation seen in adults (40 +/- 4%). This pattern was similar for both ET and bronchus and appeared greater for histamine compared with ACh preconstriction. Epithelial removal slightly enhanced relaxation. Furosemide also relaxed pulmonary artery segments, but at a 10-fold higher concentration. In striking contrast to the pattern seen in airway, adult pulmonary artery relaxed more than newborn and newborn, more than fetus. Cyclooxygenase blockade and endothelium removal did not change pulmonary artery relaxation. Furosemide did not significantly relax aorta after NE preconstriction. Taken together, these results suggest that furosemide may be more effective in relaxing airway compared with vascular smooth muscle, and the ontogeny of these responses indicates a greater efficacy and selectivity in airways of immature animals.

Secretory differentiation and cell type identification of a human fetal bronchial epithelial cell line (HFBE).

A human fetal bronchial epithelial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions. Despite a somewhat fibroblastic shape the cells maintained immunoreactivity to cytokeratin, carcinoembryonic antigen and epithelial membrane antigen. When grown on a collagen gel in a growth-hormone-supplemented medium, their spindle shape became more conspicuous. With an additional supplement of vitamin A (6 micrograms/ml), most of the cells underwent differentiation by producing many bright inclusion bodies which proved to be strongly positive with periodic acid-Schiff and weakly positive with alcian blue staining. Electron microscopy revealed a well-developed rough endoplasmic reticulum, an enlarged Golgi apparatus and many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE cells cultured on collagen gel with vitamin A secreted hyaluronic acid and neutral glycoproteins containing mainly N-linked glycoproteins whose glycans were of a complex type. A monoclonal antibody (SEC-41) generated against the neutral glycoproteins detected a glycoprotein of approximately 52 kDa in the spent culture medium of differentiated HFBE cells. This antibody also reacted with the intracytoplasmic secretory granules in these cells. When tested on frozen sections of lung tissue, the immunohistochemical reactivity of the SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung, and respiratory epithelial cells in the fetal lung. Moreover, this antibody could detect secretory glycoprotein in broncho-alveolar lavages from two patients. This paper clearly demonstrates that cells derived from human fetal bronchial epithelium can be cultivated in an undifferentiated precursor state and, under appropriate culture conditions, can be stimulated to undergo differentiation into a Clara cell type.

Induction and characterization of secretory differentiation in human fetal bronchial epithelial cell line (HFBE) cultured on collagen gel in growth hormone and vitamin A-supplemented medium.

A type of secretory differentiation was induced and characterized in a human fetal bronchial epithelial cell line (HFBE), which was grown on a collagen substratum in a basal differentiative medium (BDM) containing growth hormones and with supplementation of various concentrations of vitamin A (VA). HFBE cells grown on a collagen gel in BDM with or without VA assumed a spindle shape with thick cytoplasm arranged in strands running parallel to each other. Under a phase-contrast microscope, cells cultured in the absence of VA possessed a small number of bright inclusion bodies, which proved to be positive to PAS and almost negative to alcian-blue (AB) staining. Electron microscopy revealed well-developed rough endoplasmic reticulum (rER), enlarged Golgi apparatus and a small number of high-density granules resembling serous or Clara cell granules. HFBE cells treated with VA at levels higher than 6 mu/ml showed a remarkable increase of the secretory granules and contained amorphous material in the rER. Addition of a low concentration of VA (6 ng/ml) only stimulated the growth of HFBE cells. In contrast, higher concentrations of VA significantly inhibited the growth and 3H-thymidine incorporation into DNA in a dose-dependent manner. HFBE cells cultured on collagen gel with VA secreted products with 2 different molecular weights into the medium. A high molecular weight-product, consisting of void volume fractions from a Bio-gel A 15-m column, was identified as hyaluronic acid based on the results obtained from the DEAE-ion exchange chromatography and specific enzymatic digestion. A low molecular weight-product fractionated on the A 15-m was tentatively identified as mainly neutral glycoproteins containing N-linked glycans. While the secretion of hyaluronic acid was inhibited by VA in a dose-dependent manner, the secretion of the neural glycoproteins was most enhanced by VA in the range from the physiological concentration of 600 ng/ml to 6 micrograms/ml. These biochemical data on the secretory products, together with the morphological findings, demonstrate that the HFBE cell line serves as a new model for investigating the cellular differentiation of human lung epithelium.

Secretory differentiation of human fetal bronchial epithelial cells in culture. A study by histochemistry and electron microscopy.

Human fetal bronchial epithelial (HFBE) cells at 6-8 passages were cultivated on a collagen gel for 10 days. A basal differentiative medium (BDM), consisting of RPMI 1640 supplemented with hormones and growth factors, was employed. Histochemistry, scanning electron microscopy and transmission electron microscopy revealed that HFBE cells developed secretory granules when cultivated on collagen gel in BDM. They were electron-dense and stained positive for PAS but negative for alcian blue. On additional treatment with 8 micrograms/ml vitamin A (VA), the number of secretory granules was increased. Moreover, the HFBE cells lost their surface microvilli, and dilation of rough endoplasmic reticulum was more marked than in culture without VA.