Decreased VEGF concentration in lung tissue and vascular injury during ARDS.

Endothelial injury is an important prognostic factor in acute respiratory distress syndrome (ARDS). Decreased production of vascular endothelial growth factor (VEGF) in ARDS may favour vascular lesions, since VEGF promotes endothelial survival by inhibiting apoptosis. This study sought to document low VEGF levels in lung tissue from ARDS patients, to determine whether the cause was injury to alveolar type II cells (the main pulmonary source of VEGF) and to evaluate the vascular consequences. Lung specimens were obtained by open biopsy or autopsy from 29 patients with severe ARDS (two survivors) and five controls. As compared with controls, homogenates of lung tissue from ARDS patients contained less VEGF (median (interquartile range) ARDS 8.2 (4.7-12.2) versus controls 28.4 (9.9-47.1) ng x g(-1) protein). Increased immunostaining with surfactant protein B was seen in ARDS lungs. Extensive cellular apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling staining), including endothelial and alveolar type II cells, was demonstrated, and vascular bed density (CD31 immunostaining) decreased in ARDS lungs as compared with controls. VEGF levels were negatively correlated to apoptotic endothelial cell counts. In conclusion, decreased vascular endothelial growth factor levels in lung tissue may participate in the decrease in lung perfusion in acute respiratory distress syndrome.

A model of pulmonary adenocarcinoma in transgenic mice expressing the simian virus 40 T antigen driven by the rat Calbindin-D9K (CaBP9K) promoter.

Lung cancer is the most frequent cause of cancer deaths. Its origin and development remain poorly understood, partly because of the lack of pertinent animal models. This study produced transgenic mice expressing the simian virus (SV) 40 T antigen (Tag) driven by a 1011 base-pair DNA fragment of the rat Calbindin-D9K (CaBP9K) promoter. All transgenic animals developed multifocal pulmonary tumours with pathological and ultrastructural features consistent with adenocarcinomas. Using immunohistochemistry, northern blot or western blot, tumours were found to express the transcription factor TTF-1, as well as specific markers of the peripheral airway Clara cells (CC10) and alveolar type II cells (surfactant proteins A, B, C, and D). This model, with its similarities to human adenocarcinoma, should be useful not only for addressing the mechanisms underlying the development and progression of lung cancer, but also for testing new therapeutic approaches.

The ciliated hepatic foregut cyst, an unusual bronchiolar foregut malformation: a histological, histochemical, and immunohistochemical study of 7 cases.

The ciliated hepatic foregut cyst is an unusual solitary cystic lesion of the liver. In a series of 7 cases of hepatic ciliated cysts, we performed a histological, histochemical, and immunohistochemical study to better define the histogenesis of this rare entity. The patients were 4 women and 3 men, aged 39 to 75 years. Four patients presented with abdominal pain. In 3 cases the cyst was discovered incidentally on ultrasonography. The cysts measured from 1 to 4 cm in diameter. Microscopically, the lining of the columnar epithelium was composed of ciliated cells and mucin secreting goblet cells. The wall was composed of bands of smooth-muscle fibers surrounded by an outer fibrous capsule. The goblet cells stained with PAS, alcian blue, and high-iron diamine. The immunohistochemical study showed that endocrine cells were present within the cyst epithelium, positive for chromogranin, synaptophysin, bombesin, and calcitonin, and negative for serotonin, somatostatin, glucagon, insulin, gastrin, and pancreatic polypeptide. In all the cases, immunoreactivity of some cells for CC10 strongly suggested the presence of Clara cells. Our study shows that the epithelium lining ciliated hepatic foregut cysts has histological, histochemical, and immunohistochemical features similar to those observed in the bronchiolar epithelium. This lesion is a developmental ventral foregut abnormality that could arise from a bronchiolar bud of the tracheobronchial diverticulum.

Ultrastructural evaluation of lung maturation in a sheep model of diaphragmatic hernia and tracheal occlusion.

In fetuses with diaphragmatic hernia (DH) lung development is impaired, and pulmonary hypoplasia is one of the main factors responsible for the poor outcome of the disease. A possible treatment consists of occluding trachea during lung development to retain pulmonary fluid and to force the lung to expand. Although it appeared promising at first, this technique has recently been reported to decrease type II cell number and to induce surfactant deficiency. The aim of this study was to investigate lung maturation further through ultrastructural examination in a fetal lamb model of DH created at 85 d, followed or not by endoscopic balloon tracheal occlusion (TO) at 120 d of gestation. The proportion of alveolar epithelial type I and type II cells was altered by both treatments: the type I/type II cell ratio, which was about 2 in control lungs, was decreased 4.5-fold in DH lungs but was increased 4.5-fold in DH+TO lungs. The proportion of undifferentiated cells was increased in DH lungs. Indeterminate cells sharing features of type II and type I cells that were not observed in controls were seldom seen in DH lungs and were numerous in DH+TO lungs. The number of lamellar bodies per type II cell was decreased in both DH and DH+TO groups. In DH lungs, wall structure presented an immature appearance, with cellular connective tissue and poor secondary septation of saccules. In DH+TO lungs, primary septa appeared more mature, with reduced connective tissue, but secondary septa were still buds, although elastin was present at their tips. A single capillary layer was found in all three groups (control, DH, and DH+TO) with no sign of septal capillary pairing. This first investigation in DH and DH+TO lungs through transmission electron microscopy thus enabled us to show that compression and forced expansion of the lung are both responsible for alterations in type II cell differentiation and septal development.

Keratinocyte growth factor enhances maturation of fetal rat lung type II cells.

Keratinocyte growth factor (KGF) or fibroblast growth factor (FGF)-7, a peptide produced by stromal cells and in particular by lung mesenchyme, has recently been shown to influence early lung morphogenesis and to be a mitogen for fetal and adult alveolar type II cells. Although contradictory findings have been reported regarding its effects on surfactant protein expression, its effects on surfactant phospholipids have not been studied. We investigated the effects of KGF on the synthesis of surfactant components by cultured fetal rat type II cells isolated during the late gestational period, when surfactant accumulates in preparation for extrauterine life. We show that KGF is a potent stimulus of surfactant phospholipid synthesis, particularly for the major component of surfactant, disaturated phosphatidylcholine (DSPC). KGF increased choline incorporation into DSPC in a dose-dependent manner up to 25 ng/ml (1.3 x 10(-9) M), and this effect was greater for surfactant than for nonsurfactant DSPC. KGF was several times more potent in this respect than acidic FGF at the same molar concentration. KGF, similar to epidermal growth factor, also stimulated acetate incorporation and increased the surfactant phospholipid and DSPC content of cultured cells twofold. These effects correlated with increased choline phosphate cytidylyltransferase activity and increased fatty acid synthase activity and gene expression. KGF also induced a dose-dependent stimulation of surfactant protein-A, -B, and -C gene expression, leading to a 2- to 3-fold increase in their messenger RNAs. KGF therefore stimulates the synthesis of all surfactant components in developing type II cells at the time of surfactant accumulation. Its secretion by lung fibroblasts may thus be an important factor in promoting the maturation of fetal lung epithelium and the synthesis of sufficient surfactant. The results suggest that KGF could provide a new therapeutic agent for the management of the immature or injured lung.

Lung growth and maturation after tracheal occlusion in diaphragmatic hernia.

Tracheal occlusion (TO) was performed at 120 d of gestation by noninvasive endoscopic technique using a releasable latex balloon, in fetal lambs with diaphragmatic hernia (DH) established at 85 d. The lungs were studied at 139 d in five fetuses with DH + TO, five fetuses with DH only, and six control fetuses. Fluid retention consecutive to TO allowed fetal lungs to grow. Histological pulmonary structure was more mature in DH + TO than in DH alone. The growth-inducing effect of TO was however incomplete, with an increased protein/DNA ratio. Tissue phospholipids were increased, but this was not reflected in the surfactant compartment. The major surfactant component, disaturated phosphatidylcholine, was reduced to 58% of its control value in DH, and further reduced to 17.5% of its control value in DH + TO. The proportion of surfactant protein B immunoreactive cells, assumed to represent the proportion of type II cells, was increased in DH (27% of all parenchymal cells), and reduced in DH + TO (7.8%) as compared with control fetuses (15%). In conclusion, although noninvasive tracheal occlusion in utero is feasible and may partly compensate the adverse effects of DH on lung organogenesis, it reduces the number of type II cells and induces a dramatic surfactant deficit. Using this technique in human fetuses requires careful consideration until further evaluation of lung functional characteristics has been achieved in this experimental model.

Isolation and characterization of surfactant-like particles in rat and human colon.

The source of a phospholipid-rich layer recovered from the surface of the mammalian colon has been obscure. This report describes the isolation of a low-density membrane from the surface of rat and human colons (d = 1.07-1.08 g/ml), with a low cholesterol-to-phospholipid ratio and phosphatidylcholine as its major phospholipid. Electron microscopy shows unilamellar and partially coiled membranes. Compared with microvillous membranes isolated from underlying mucosa, this extracellular membrane is enriched for tissue-unspecific alkaline phosphatase and surfactant protein A. It does not contain small intestinal marker proteins (intestinal alkaline phosphatase and sucrase-isomaltase). The human membrane contains only traces of the colonic microvillous membrane marker, carcinoembryonic antigen. Antiserum against the rat colonic membrane does not recognize colonic microvillous membrane or small intestinal surfactant-like particle proteins. Antiserum against human colonic membrane identifies one protein in the surfactant-like particle from the adjacent small intestine and two proteins in the colonic microvillous membrane. These data show that the colonocyte microvillous membrane is covered by another membrane with a different protein composition. Enrichment for surfactant protein A suggests that this colonic membrane is another example of a surfactant-like particle sharing proteins with pulmonary surfactant.

Production of transforming growth factor (TGF) beta by fetal lung cells.

TGF beta is supposed to play an important role in the process of epithelial maturation in the developing fetal lung. Using an immunofluorescence approach, we showed that fetal rat lung fibroblasts elaborate the three TGF beta isoforms known in mammals (TGF beta 1, beta 2 and beta 3) whereas epithelial cells appear to synthesize only TGF beta 1 and beta 3. Isolated fibroblasts secrete the three isoforms. Biological assay of TGF beta activity in fibroblast-conditioned media did not reveal significant changes according to the stage when fibroblasts were isolated.

Fetal rat lung type II cell differentiation in serum-free isolated cell culture: modulation and inhibition.

Undifferentiated fetal rat lung epithelial cells were isolated on gestational days 15 or 17 (term 22 days) and cultured in a defined medium. On plastic, most of the cells developed structurally abnormal lamellar bodies. On a basement membrane matrix (BMM), they sequentially accumulated glycogen and formed typical lamellar bodies. Biochemical analysis of the latter indicated that they had a phospholipid composition typical of surfactant for cells on BMM but not on plastic and that surfactant protein A appeared on BMM only. Progressing maturation from day 1 to day 6 in culture was demonstrated for 17-day cells on BMM by a sevenfold increase of labeled precursor incorporation into surfactant phospholipids. Exposure to medium conditioned by 21-day fetal fibroblasts enhanced incorporation already after a 1-day culture. The antisteroid RU 486 had no effect on differentiation, whereas transforming growth factor-beta, a factor produced by lung mesenchyme at early fetal stages, inhibited it markedly. Alveolar epithelial type II cells appear to be committed early, but their maturational process would be prevented until a definite gestational stage.