Restoring effects of vitamin A on surfactant synthesis in nitrofen-induced congenital diaphragmatic hernia in rats.

Congenital diaphragmatic hernia (CDH) is a major cause of refractory respiratory failure in the newborn. Besides pulmonary hypoplasia, the pathophysiology of CDH also includes surfactant deficiency. Vitamin A (vit A) is important for various aspects of lung development. We hypothesized that antenatal treatment with vit A would stimulate lung surfactant synthesis in experimental CDH induced in rats by maternal ingestion of the herbicide nitrofen (2,4-dichloro-phenyl-p-nitrophenyl-ether) on Day 12. Fetuses were assigned to six experimental groups: (1) controls from rats that received olive oil, the vehicle; (2) fetuses from rats that received olive oil on Day 12 and vit A orally (15,000 IU) on Day 14; (3) nitrofen (N)-exposed fetuses without diaphragmatic hernia (N/no DH); (4) N/no DH from rats given vit A on Day 14; (5 ) nitrofen-exposed fetuses with DH (N/+DH); (6) N/+DH from rats given vit A on Day 14. Fetuses were delivered by C-section at Day 21. Lung DNA content was lowered in the nitrofen group as compared with the controls group, but increased by subsequent vit A treatment. Lung surfactant disaturated phosphatidylcholine was reduced in the N/+DH group and restored to control level by vit A. The expression level of surfactant proteins (SP) -A and -C was decreased in vit A-treated control rats and in nitrofen-exposed fetuses with or without DH. Vit A restored SP-A and -C mRNA expression to control levels in N/+DH. SP-B expression was lowered in N/no DH and increased by vit A in this group. The proportion of type II cells assessed by SP-B immunolabeling was lowered in N/+DH and restored by vit A treatment. We conclude that antenatal treatment with vit A restores lung maturation in nitrofen-induced hypoplastic lungs with CDH. These findings point out vit A as a potential therapeutical agent for correcting surfactant deficiency in CDH.

Surfactant proteins in the digestive tract, mesentery, and other organs: evolutionary significance.

For years, the so-called surfactant proteins (SPs) that were discovered in the phospholipid-rich material designated pulmonary surfactant, were considered to be lung-specific. The fact that surfactant-like materials composed of phospholipids are secreted by a number of other organs recently prompted several groups to search for SP expression in these organs also. The hydrophilic proteins SP-A and SP-D and their transcripts have been found in a number of tissues, including gastric and intestinal mucosae, mesothelial tissues (mesentery, peritoneum, and pleura), synovial cells, Eustachian tube and sinus, and possibly in salivary glands, pancreas, and urinary tract. By contrast, the hydrophobic proteins SP-B and SP-C actually appear to be expressed in lung epithelium only. SP-A and SP-D belong to the innate defence system against pathogens and play a role as opsonins for facilitating phagocytosis. Their expression appears as a general feature of organs exposed to pathogens because they present an interface with the external milieu. Although this function has thus far been investigated in the lung only through the gene-targeting approach, increased expression of SP-A in the infected middle ear and of SP-D in the Helicobacter-infected antrum argues for such a function also in other organs. In organs that are not exposed to external pathogens, their role is likely to exert anti-inflammatory and immunomodulatory functions, as suggested by increased SP-A immunoreactivity in rheumatoid disease. SP-A and SP-B have been found in association with phospholipids in the lung of all air-breathing vertebrates, including the most primitive forms represented by lungfish, which implies that the surfactant system had a single evolutionary origin. Immunochemical proximity of the proteins among vertebrates indicates considerable conservation during evolution. Moreover, the finding of an SP-A-like protein in intestine and swim bladder of actinopterygian fish implies that the ancestral form of the protein was already present before the emergence of lung structures.

Role of keratinocyte growth factor in the control of surfactant synthesis by fetal lung mesenchyme.

Fetal lung maturation is regulated by mesenchymal-epithelial cell communication, which plays a major role in the control of surfactant synthesis by alveolar type II cells. We have recently shown that keratinocyte growth factor (KGF), also called fibroblast growth factor-7, enhances the maturation of fetal alveolar epithelial type II cells. Here, we investigated, among the factors produced by lung mesenchyme, the part attributable to KGF in the control of surfactant synthesis. Using a KGF-neutralizing antibody, we assessed surfactant phospholipid synthesis by measuring choline incorporation into disaturated phosphatidylcholine of isolated fetal type II cells. We found that KGF accounts for about half of the stimulating activity present in fetal lung fibroblast-conditioned medium (FCM). By contrast, the use of an epidermal growth factor-neutralizing antibody did not alter the FCM-stimulating activity. To further delineate KGF properties as a mesenchymal mediator, we wondered about its possibility to relay glucocorticoid-stimulating activity on the synthesis of the phospholipid moiety of surfactant in fetal lung fibroblasts. A 24-h exposure to dexamethasone led us to detect a 50% increase in the level of KGF messenger RNA (mRNA) in isolated fetal lung fibroblasts. Moreover, anti-KGF antibody totally abolished the further increase of FCM-stimulating activity induced by dexamethasone. Thus, KGF seems to be a major player in mediating glucocorticoid stimulation of fetal lung maturation.

Vitamin A decreases the incidence and severity of nitrofen-induced congenital diaphragmatic hernia in rats.

Congenital diaphragmatic hernia (CDH) is a major cause of refractory respiratory failure in the newborn. Pulmonary hypoplasia often limits survival. Vitamin A (Vit A) is an important signal for lung growth. We hypothesized that antenatal treatment with Vit A would stimulate lung growth and decrease mortality in experimental CDH induced in rats by ingestion of the herbicide nitrofen (2, 4-dichlorophenyl-p-nitrophenyl ether). Nitrofen was administered to pregnant rats on day 12 of gestation (term 22 days). Rats were assigned to five groups: three groups received one dose of oral antenatal Vit A (15,000 IU) before (day 10), concomitant with (day 12), or after (day 14) nitrofen administration; one group received only nitrofen; and a control group received vehicle (olive oil). The incidence of CDH was markedly lower in all groups receiving Vit A (day 10, 44%; day 12, 20%; and day 14, 40%) compared with the nitrofen-treated group (84%; P < 0.05). The 72-h survival was higher in all 3 Vit A-treated groups (day 10, 40%; day 12, 58%; and day 14, 70%) compared with the nitrofen-treated group (16%; P < 0.05). Lung-to-body weight ratio and radial saccular count were significantly increased by Vit A. Antenatal treatment with Vit A lowers the incidence and severity of experimental CDH and increases lung growth and maturation.

Mild vitamin A deficiency delays fetal lung maturation in the rat.

During late pregnancy, the fetal lung stores surfactant in preparation for extrauterine life. Surfactant deficiency, most often due to prematurity, precipitates respiratory distress syndrome (RDS) of the neonate. Although vitamin A (retinol) and retinoic acid have been shown to enhance the synthesis of phospholipid surfactant components, their effect on surfactant-specific proteins is unclear. No attempt has been made to evaluate the consequences of vitamin A restriction on surfactant phospholipid storage or on the expression of the life-essential surfactant protein-B (SP-B). We induced in rats a partial vitamin A deficiency leading to a 30-60% reduction in blood retinol, a status compatible with maintenance of gestation and absence of gross abnormalities in offspring. At term, lung surfactant phospholipids were reduced by 21%, and the major surfactant phospholipid, disaturated phosphatidylcholine (DSPC), was reduced by 27% in vitamin A-deficient (VAD) fetuses. The decrease in surfactant phospholipids and DSPC correlated linearly with plasma retinol, and reached about 50% in fetuses with the lowest retinol concentrations; it was accompanied by reduced expression of the gene for fatty acid synthase, a key enzyme in the synthetic pathway for surfactant-phospholipid lipid precursors. The amounts of SP-A, SP-B, and SP-C messenger RNAs were decreased by 46%, 32%, and 28%, respectively, in VAD fetuses. Consistently, amounts of SP-A and SP-B proteins were diminished as assessed by Western blotting. The proportion of type II cells determined after SP-B labeling was unchanged in VAD as compared with control lungs. Vitamin A deficiency is therefore a cause of lung maturational delay. In view of its rather large incidence in human populations, it may represent an increased risk for RDS and an aggravating factor for prematurity.

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.

The exogenous surfactant Curosurf enhances phosphatidylcholine content in isolated type II cells.

The exogenous surfactant, Curosurf, contains proteins as well as phospholipids. We investigated the possibility that these might affect the reutilization of exogenous phospholipid by type II alveolar cells isolated from rat lung. The time course of incorporation into lamellar bodies of radioactivity from tritiated dipalmitoyl-phosphatidylcholine (DPPC) contained within liposomes was studied. Rates of uptake were compared between liposomes prepared from pure phospholipids and from Curosurf. The incorporation of labelled choline into newly synthesized phosphatidylcholine was also determined in the presence of both preparations. The rate of DPPC incorporation over the first 4 h was the same, but, after 6 and 8 h, the radioactivity associated with lamellar bodies was about 40% higher from Curosurf liposomes. By contrast, both Curosurf and the phospholipid mixture enhanced choline incorporation into phosphatidylcholine to the same extent. We conclude that Curosurf enhances the surfactant-related phosphatidylcholine content of type II cells by two mechanisms, one of which depends on the presence of proteins in exogenous surfactant. The difference in incorporation of radioactivity from liposome-associated labelled dipalmitoyl-phosphatidylcholine cannot be explained just by an increase in reutilization of choline from degraded dipalmitoyl-phosphatidylcholine.

Expression of hydrophilic surfactant proteins by mesentery cells in rat and man.

Human peritoneal dialysis effluent (PDE) contains a phosphatidylcholine-rich compound similar to the surfactant that lines lung alveoli. This material is secreted by mesothelial cells. Lung surfactant is also characterized by four proteins essential to its function. After having long been considered as lung-specific, some of them have been found in gastric and intestinal epithelial cells. To explore further the similarity between lung and peritoneal surfactants, we investigated whether mesothelial cells also produce surfactant proteins. We used rat transparent mesentery, human visceral peritoneum biopsies and PDE. Surfactant proteins were searched for after one- and two-dimensional SDS/PAGE and Western blotting. On a one-dimensional Western blot, bands at 38 and 66 kDa in rat mesentery, and at 38 and 66 kDa in human peritoneal mesothelial cells (in vivo and in vitro) and PDE, corresponded to monomeric and dimeric forms of lung surfactant protein A (SP-A). On two-dimensional Western blots, the 32 and 38 kDa spots in mesentery and PDE localized at the acidic pH appropriate to the SP-A monomer's isoelectric point. SP-D was also identified at the same 43 kDa molecular mass as in lung. SP-B was not detected in mesenteric samples. Expression of SP mRNA species was also assessed by reverse transcriptase-PCR, which was performed with specific primers of surfactant protein cDNA sequences. With primers of SP-A and SP-D, DNA fragments of the same size were amplified in lung and mesentery, indicating the presence of SP-A and SP-D mRNA species. These fragments were labelled by appropriate probes in a Southern blot. No amplification was obtained for SP-B. These results show that mesentery cells produce SP-A and SP-D, although they are of embryonic origin (mesodermal) and are different from those of the lung and digestive tract (endodermal) that secrete these surfactants.

Antibody against pulmonary surfactant protein A recognizes proteins in intestine and swim bladder of the freshwater fish, carp.

An antibody raised against rat pulmonary surfactant protein A (SP-A) bound on Western blots to proteins present in intestinal mucosa and in swim bladder, but not in gills of the carp. The fish protein(s) revealed by the antibody exhibited an electrophoretical behavior similar to that of rat SP-A with a characteristic triplet in the 30- to 35-kDa range. It therefore appears that proteins immunologically very close to mammalian SP-A are present in modern fish which evolved from ancestors that never possessed lungs. The association of SP-A with phospholipid-rich surfactant-like materials appears as a phylogenetically old feature.

Pulmonary surfactant protein A (SP-A) is expressed by epithelial cells of small and large intestine.

Surfactant protein A (SP-A) is the most abundant protein associated with phospholipids in pulmonary surfactant. There are several lines of evidence that pulmonary and gastrointestinal epithelium produce closely related surface-active materials, although the presence of SP-A in gastrointestinal tract has so far not been reported. Indirect immunofluorescence experiments using different antibodies raised against rat pulmonary SP-A showed that some jejunal and colonic but not gastric epithelial cells positively stained for SP-A. Analysis of the proteins in cell lysates from rat small intestine and colon studied by Western blot revealed several immuno-reactive bands, including the characteristic triplet of 26-, 32-, and 38-kDa monomeric proteins, less strongly labeled than in lung cells, and higher molecular mass forms of 66 and 120 kDa also present in lung cells. The 66- and 120-kDa bands displayed the expected isoelectric pH of SP-A after two-dimensional electrophoresis. Alkylation induced conversion of the 120-kDa form (almost completely) and the 66-kDa form (partly) into the 26-38-kDa monomeric species. The presence of SP-A mRNA in rat stomach, small intestine, and colon was then searched for by conventional cDNA/reverse transcriptase-polymerase chain reaction. Products of appropriate size (372 base pairs) identical to that of pulmonary tissue were amplified in small intestine and colon but not in stomach or in other tissues used as controls. Cloning and sequencing of rat colon SP-A cDNA revealed the same sequence as the one reported for rat lung SP-A. Furthermore, analysis of the transcriptional initiation site of SP-A gene in colon by anchored-polymerase chain reaction showed that transcription was initiated at the same site in both colon and lung. These data, which demonstrate that small intestine and colon express SP-A constitutively and that this protein is present in some epithelial cells, extend the concept of intestinal surfactant and underline its close relationships to pulmonary surfactant.

Development of the surfactant system.

Pulmonary surfactant is a lipoprotein complex produced by type II pneumocytes that lines and stabilizes alveoli. Surfactant-specific proteins are early markers of the embryonic lung. Precursors of type II pneumocytes appear to be committed early and are able to pursue their differentiation program in a relatively autonomous way. Despite this potential, surfactant ontogeny and storage actually take place relatively late in the course of fetal development. Surfactant accumulation is, therefore, delayed until a certain stage after which it is stimulated by a number of factors. Recent investigations have pointed out the possible role of paracrine mechanisms in the determinism of this control, especially with regard to retinoids and TGF-beta.

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.

Ontogeny of surfactant proteins and lipid-synthesizing enzymes in cultured fetal lung epithelial cells.

Fetal rat lung epithelial cells were isolated on gestational day 17 (term is 22), separated from fibroblasts, and cultured up to 6 days in a serum-free medium on a basement membrane matrix. Surfactant protein (SP) A, barely detectable by immunostaining at the beginning of the culture, considerably increased in cells and subsequently in the lumen of the epithelial cell clusters. SP-A mRNA, already detectable at culture initiation, progressively increased. By contrast, SP-B and its mRNA appeared after 2-3 days. SP-C mRNA appeared only after 4 days of culture. Cells cultured 6 days had a phospholipid composition similar to that of freshly isolated adult rat type II cells. The enhancement of lipid synthesis between the first and the sixth culture days, reported earlier to occur in these cells, was found to be accompanied by a two- to fivefold increase in amount of mRNAs of lipogenic enzymes and choline phosphate cytidylyltransferase. In conclusion, alveolar epithelial type II cells appear to be capable of full differentiation in vitro, and components of the surfactant system are all regulated developmentally at a pretranslational level.

Retinoids control surfactant phospholipid biosynthesis in fetal rat lung.

Vitamin A (retinol) may play an important role in lung maturation: 1) premature delivery is simultaneously a source of vitamin A deficiency and increased risk of neonatal respiratory distress syndrome and subsequent bronchopulmonary dysplasia (BPD), due to deficit in pulmonary surfactant; 2) neonatal supplementation with retinol reduces the risk of BPD; and 3) fetal rat lung stores retinol in late gestation just before the onset of surfactant synthesis. To test the hypothesis of an implication of retinoids in the control of pulmonary surfactant synthesis, experiments were designed in the pregnant rat, aiming either at enhancing fetal lung vitamin A stores, bringing the active metabolite of vitamin A, retinoic acid (RA), or inhibiting the conversion of retinol to RA with aid of citral. Maternal administration of a single dose of 50,000 IU of retinyl palmitate on day 16 (term 22 days) increased 22 and 29%, respectively, the total phospholipid (TPL) and disaturated fraction of phosphatidylcholine (PC) in extracted fetal surfactant on day 19 but did not change surfactant protein (SP) A concentration. Chronic administration of retinyl palmitate to the mother from day 16 through 20 increased disaturated PC content on day 21 but decreased SP-A concentration. Fetal lung surfactant phospholipids were increased by chronic administration of RA and considerably reduced by citral (-31 and -35% for TPL and PC concns, respectively). RA also enhanced labeled choline incorporation into fetal lung PC on day 20. Given once on day 17, it accelerated the appearance of surfactant precursors on day 18.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Effects of maternal protein-calorie malnutrition on the phospholipid composition of surfactant isolated from fetal and neonatal rat lungs. Compensation by inositol and lipid supplementation.

The effects of a maternal protein-calorie malnutrition during gestation and lactation were analyzed on fetal and postnatal lung growth and maturation, including a surfactant fraction isolated from lung tissue. There was a considerable reduction in body weight and in wet and dry lung weights of malnourished pups. Lung protein and DNA concentrations were similar in both groups except in late gestation (lung hyperplasia) and 2 and 15 d after delivery (hypocellularity). Lung glycogen breakdown was slowed down in malnourished newborns. Surfactant material was decreased the most perinatally and the reduction was more marked than for the nonsurfactant fraction of the lung. Disaturated phosphatidylcholine, the major surface active surfactant component, was decreased the most at birth (1.70 +/- 0.31 nmol/mg wet wt versus 3.68 +/- 0.17 nmol/mg in controls, n = 8) and on d 2 (5.04 +/- 0.53 nmol/mg versus 7.67 +/- 0.44 nmol/mg in controls, n = 8). There was an apparent recovery in the composition of surfactant in malnourished rats 5 d after delivery, due in fact to a decrease in controls, and an actual return to normal levels 15 to 20 d after birth. Postnatal lipid supplementation with Intralipid led to partial recovery on d 10. Inositol supplementation totally reverted the effects of malnutrition on surfactant phospholipids (8.36 +/- 0.94 nmol disaturated phosphatidylcholine/mg wet wt on d 2 versus 7.67 +/- 0.44 nmol/mg in controls and 5.55 +/- 0.62 nmol/mg in untreated malnourished rats, n = 10; 2.43 +/- 0.32 nmol disaturated phosphatidylcholine/mg wet wt on d 10 versus 3.26 +/- 0.32 nmol/mg in controls and 1.18 +/- 0.27 nmol/mg in untreated malnourished rats, n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of effects of epidermal and insulin-like growth factors, gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro.

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10(-9) M. At concentration 10(-8) M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.

Culture of fetal alveolar epithelial type II cells in serum-free medium.

A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle's minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistence of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.