Differentiation-arrested rat fetal lung in primary monolayer cell culture

Summary Differentiation-arrested lung cell cultures were developed from fetal rats of various gestational ages. In contrast to previously published observations with cultures in a pO₂ of ∼142 mm Hg, cultures developed in a pO₂ of ∼30 mm Hg, close to the normal fetal arterial pO₂, have improved plating efficiency and a slightly increased growth rate. They did not, however, show gestation-dependent increases of choline incorporation into phospholipids, nor did immature lung cell cultures respond to dexamethasone or triiodothyronine, singly or in combination, by increased choline incorporation into saturated lecithin. The incorporation of choline and glycerol into lipids suggested a mature rate of lipid synthesis by immature cultures at a pO₂ ∼30 mm Hg, despite preservation of an immature morphology. Electron microscope observations revealed no gross differences between immature cultures developed at either pO₂. The cellular mechanisms underlying these differences are unclear but suggest that oxygen tension may significantly influence results obtained with in vitro studies of lipid synthesis by immature lung. This work was supported by grants from the Medical Research Council of Canada, the Ontario Thoracic Society, and the Physicians' Services Incorporated Foundation.     

The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity

The cellular mechanism by which glucocorticoids stimulate phosphatidylcholine biosynthesis has been studied in the fetal rat lung in vivo and in cultured fetal rat lung cells of varying levels of complexity. Administration of dexamethasone to pregnant rats at 18 days gestation resulted in a significant increase in saturated phosphatidylcholine content in fetal lung 24 h after injection. Dexamethasone administration increased the activity of fetal lung choline-phosphate cytidylyltransferase by 34%. It had no effect on the activities of fetal lung choline kinase and choline phosphotransferase. Exposure of fetal lung type II cells in organotypic cultures (which contain both type II cells and fibroblasts) to cortisol resulted in a 1.6-fold increase in the incorporation of [Me-3H]choline into saturated phosphatidylcholine. The activities of the enzymes in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 105% increase in choline-phosphate cytidylyltransferase activity. Treatment of monolayer cultures of fetal type II cells with cortisol-conditioned medium from fetal lung fibroblasts resulted in a 1.5-fold increase in saturated phosphatidylcholine production. This effect correlated with a doubling of choline-phosphate cytidylyltransferase activity. Additional evidence that this stimulatory action is mediated by fibroblast-pneumonocyte factor, produced by fetal lung fibroblasts in response to cortisol, was obtained. The factor was partially purified from cortisol-conditioned medium of fetal lung fibroblasts by gel filtration and affinity chromatography. Based on biological activity, a 3000-fold purification was obtained. Stimulation of saturated phosphatidylcholine synthesis in type II cells by fibroblast-pneumonocyte factor was maximal within 60 min of incubation. Pulse-chase experiments indicated that the stimulatory effect was correlated with an increased conversion of choline phosphate into CDP choline. Moreover, the enhanced phosphatidylcholine formation by fetal type II cells in response to fibroblast-pneumonocyte factor was accompanied by decreased levels of cellular choline phosphate. These findings further support the concept that glucocorticoid action on surfactant-associated phosphatidylcholine synthesis occurs ultimately at the level of the alveolar type II cell and involves fibroblast-pneumonocyte factor which stimulates the activity of choline-phosphate cytidylyltransferase.     

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.     

Effect of cortisol on the synthesis of lamellar body glycerophospholipids in fetal rabbit lung tissue in vitro

The effect of cortisol on the rate of choline incorporation into tissue phosphatidylcholine was investigated in lung explants from fetal rabbits of 19-28 days gestational age. The explants were incubated in medium with or without fetal calf serum for up to 7 days. When lung tissues were incubated in serum-free medium, a stimulatory effect of cortisol on tissue phosphatidylcholine synthesis was found in explants from 21-, 24-, 26- and 28-day fetal rabbits; a stimulatory effect of cortisol was observed in 19-day fetal lung explants only if fetal calf serum was present in the culture medium. To assess directly the effect of cortisol on the synthesis of lamellar body phosphatidylcholine, choline incorporation into phosphatidylcholine associated with a purified lamellar body fraction isolated from lung explants of 21- and 28-day fetal rabbits was also investigated. Cortisol caused a marked stimulation of synthesis and accumulation of lamellar body phosphatidylcholine in lung explants from both 21- and 28-day fetal rabbits. The magnitude of the stimulatory effect of cortisol on the rate of synthesis of lamellar body phosphatidylcholine was always greater than the effect of cortisol on the rate of choline incorporation into lipids of tissue homogenates. The relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol were also significantly altered in lung explants from 21- and 28-day fetal rabbits by cortisol treatment. Lamellar bodies that were formed initially in the fetal lung explants were enriched in phosphatidylcholine and phosphatidylinositol and had a relatively low phosphatidylglycerol content. With cortisol treatment there was a decrease in the relative rate of synthesis of lamellar body phosphatidylinositol and an increase in the relative rate of synthesis of phosphatidylglycerol. The stimulatory effect of cortisol on the synthesis of lamellar body phosphatidylcholine was observed at an earlier time-point of incubation than was the effect of cortisol on the relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol. The temporal sequence of the cortisol-induced changes in the synthesis of lamellar body glycerophospholipids, therefore, reflects that which occurs with maturation in vivo.     

Alveolar pre-type II cells from the fetal rabbit lung: effect of confluence on the production of disaturated phosphatidylcholine

Pre-type II alveolar cells isolated from the fetal rabbit lung on the 24th gestational day have been maintained in vitro for 14 days in a chemically defined medium supplemented with hormone-stripped serum. These cells replicate in culture. Measurement of the incorporation of [14C]choline into cellular disaturated phospholipid indicated that those cells grown in vitro under standard conditions for 8 days (pre-confluent) incorporate the radioactive precursor at a similar rate to cells maintained for 14 days (post-confluent). Both dexamethasone and serum-free medium conditioned by monolayer cultures of fetal rabbit lung fibroblasts stimulated [14C]choline incorporation into disaturated phosphatidylcholine (PC) by the pre- and post-confluent cultures after 24 or 48 h of exposure: the conditioned medium was more effective than the steroid. These treatments had little effect on choline incorporation into disaturated phosphatidylcholine of preconfluent cells during the first 12 h. A marked response occurred by 24 h after which the labelling of disaturated phosphatidylcholine plateaued. In contrast, with post-confluent cells labelling of disaturated PC increased in a more linear fashion and only plateaued after 72 h. Determination of the ratio of incorporation of [14C]choline into disaturated versus unsaturated phospholipid indicated that serum-free medium conditioned by monolayer cultures of fetal lung fibroblasts specifically increased the level of radioactive precursor in the disaturated phospholipid in both the pre- and post-confluent cell monolayers.     

The effects of insulin and hyperglycemia on surfactant phospholipid synthesis in organotypic cultures of type II pneumocytes

Organotypic cultures of fetal type II epithelial cells were incubated in media containing insulin at concentrations ranging from 10 to 400 microunits/ml. Exposure to insulin resulted in increased glucose uptake from the media and in the rate of glucose conversion to CO2. Furthermore, both glucose uptake and CO2 production were dependent on the glucose concentration in the media. Surfactant and residual phosphatidylcholine fractions were isolated from the organotypic cultures by sucrose density centrifugation. The presence of low doses of insulin (10-25 microunits/ml) caused a significant increase in the incorporation of glucose into both surfactant and residual phosphatidylcholine. Insulin at levels of 100 microunits/ml or higher resulted in a significant decrease in glucose incorporation into both phosphatidylcholine fractions. Increasing the media glucose concentration from 5.6 to 20 mM caused a 2- to 2.5-fold increase in glucose utilization for surfactant and residual phospholipid synthesis, but did not produce any significant changes in choline incorporation into either surfactant or residual phosphatidylcholine. The addition of 400 microunits/ml of insulin to media containing 20 mM glucose, however, resulted in a 20% decrease in choline incorporation into surfactant phosphatidylcholine but had no effect on choline incorporation into residual phosphatidylcholine. These results suggest that insulin is an important hormone regulating fetal lung maturation and that hyperinsulinemia may be responsible for the delayed lung development in infants of diabetic mothers.     

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.     

A continuous-flow culture system for organ culture of large explants of adult tissue: effect of oxygen tension on mouse molar periodontium

A modified continuous-flow culture system (CFCS) was developed to maintain large explants of periodontium from adult mouse in organ culture. The culture medium was stored in a reservoir outside of the incubator, pumped via polyvinyl tubing into small glass culture chambers that were placed in the oxygenator and then collected in a waste flask. Medium was analyzed for pO2, pCO2 and pH during the culture period. Three-molar and single-molar explants of periodontium were maintained for 48 hr in the CFCS at two different pO2 ranges: 100 to 120 mm Hg and 400 to 420 mm Hg. [3H]Proline was added 24 hr prior to sacrifice. Light-microscope morphological and radioautographic observations suggested that cell viability and incorporation of [3H]proline, probably into newly synthesized protein, increased with an increase in pO2 and was related to a pO2 gradient extending from the periphery to the center of the explants.     

Glucocorticoids affect the synthesis of pulmonary fibroblast-pneumonocyte factor at a pretranslational level

Glucocorticoids accelerate fetal lung maturation by acting on the fetal lung fibroblast to induce the synthesis of fibroblast-pneumonocyte factor which in turn stimulates pulmonary surfactant synthesis by the alveolar type II cell. We have studied the site of glucocorticoid regulation of fibroblast-pneumonocyte factor synthesis in primary cultures of fetal rat lung fibroblasts. Conditioned media from fetal rat lung fibroblasts exposed to cortisol stimulate [Me-3H]choline incorporation into saturated phosphatidylcholine by primary cultures of fetal rat lung alveolar type II cells. This effect is blocked by the presence of actinomycin D during the first, but not the second, 24 h of incubation of the fibroblasts with cortisol. Cycloheximide blocks this effect if present during either the first or second 24 h of incubation. We fractionated mRNA from fetal rat lung fibroblasts incubated in the presence or absence of dexamethasone and observed that cell-free translation products from a fraction of approximately 500 bases possess biological activity in the bioassay. Such activity is only present in cell-free translation products of mRNA isolated from fibroblasts treated with dexamethasone. These results suggest that glucocorticoids act at a pretranslational level to induce production of fibroblast-pneumonocyte factor and that the primary translation products are biologically active.     

A comparison of the specificity of phosphatidylcholine synthesis by human fetal lung maintained in either organ or organotypic culture.

Human fetal lung (14-18 weeks gestation) was maintained in either organ or organotypic culture. By 4 days in organ culture or 14 days in organotypic culture, epithelial cells within both culture systems exhibited well-developed apical microvilli and possessed numerous intracellular lamellar bodies characteristic of surfactant phospholipid stores. However, analysis of the pattern of synthesis of individual molecular species of phosphatidylcholine by [14C]choline incorporation and reversed-phase h.p.l.c. showed that this apparent maturation was not paralleled by an increased synthesis of the dipalmitoyl species in either culture system. By contrast, the fractional synthesis of dipalmitoyl phosphatidylcholine, expressed as a percentage of total [14C]choline incorporation, decreased with time in both organ and organotypic culture. Moreover, these fractions were not significantly different from those measured in parallel monolayer cultures of mixed human fetal lung cells that displayed mainly fibroblast morphology. These results suggest that the synthesis pattern of phosphatidylcholine species by lung cells in culture is determined principally by their incubation conditions and not by their state of apparent maturation.     

Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation

An oxystat system was employed in conjunction with a single-photon counting apparatus for simultaneous monitoring of oxygen uptake, oxidative decomposition of membrane lipids, and occurrence of electronically excited species during microsomal lipid peroxidation. During NADPH/ADP-iron-promoted lipid peroxidation at a steady state oxygen partial pressure (pO2) of 30 mm Hg, complex time relationships among oxygen uptake, malondialdehyde (MDA) formation, and low-level chemiluminescence were observed. While the first two parameters occurred nearly simultaneously, low-level chemiluminescence occurred with a significant delay. A decrease of the steady state pO2 to 3 mm Hg led to significant increases of the lag phases of all three parameters and a further enhancement of the time displacement of low-level chemiluminescence in relation to oxygen uptake and MDA formation. At a pO2 of 0.5 mm Hg, the lowest pO2 maintained during this study, no low-level chemiluminescence was observed while oxygen uptake and MDA formation were still detected. In contrast, during NADPH/CCl4-promoted lipid peroxidation at a pO2 of 0.5 mm Hg a sudden drastic rise of low-level chemiluminescence accompanying oxygen uptake and MDA formation was observed. At pO2 between 0.5 and 3 mm Hg all three parameters occurred nearly concomitantly during the entire incubation. At pO2 levels above 3 mm Hg all three parameters showed principally the same behavior. However, the respective maxima of low-level chemiluminescence were reached with some delay. The present observations support the assumption that the decomposition of membrane lipid peroxyl radicals to MDA and the formation of electronically excited species proceed via different pathways. The time displacement between oxygen uptake and MDA formation, on the one hand, and low-level chemiluminescence, on the other hand, depends on the type of initiating radical system and on the steady state pO2 level. It is suggested that the differences are due to distinct subsets (chemical or spatial) of secondary peroxyl radicals in the membrane.     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Development of fibroblast-type-II cell communications in fetal rabbit lung organ culture.

The development of the fetal lung is regulated by fibroblast-type-II cell communications which involve fibroblast pneumonocyte factor (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming growth-factor beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day 19 of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the fibroblast. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.     

Cell cultures of fetal rat brain : Growth and marker enzyme development

Growth and enzyme development in cell cultures of fetal rat brain were influenced by type of growth medium, cell density, and age of fetal tissue source. Cells grew better in one medium (DMEM), but the other (F12G) enhanced development of choline acetyltransferase activity. One type of growth medium (DMEM) lost efficacy 2 weeks after preparation of complete medium. Cell division rate was density dependent, and choline acetyltransferase development was related to time in culture and cell concentration. Some results suggested division of choline acetyltransferase producing cells. Differences in age of tissue source resulted primarily in differences in growth: cultures of 21 day fetal cells developed more protein per 10⁶ cells inoculated than cultures of cells from younger animals; there was little difference in enzyme activity per culture. Conditions may be controlled such that fetal rat brain cells will grow and express differentiated functions in culture in a predictable manner.     

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells.

Administration of dexamethasone to pregnant rats at 19 days gestation increased phosphatidylcholine synthesis (45%) from radioactive choline in type II cells. This enhanced synthesis of phosphatidylcholine was accompanied by an increased conversion of choline phosphate into CDP-choline. Similar results were obtained by incubating organotypic cultures of 19-day-fetal rat lung with cortisol. The increased conversion of choline phosphate into CDP-choline correlated with an enhanced choline-phosphate cytidylyltransferase activity (31% after dexamethasone treatment; 47% after cortisol exposure) in the cell homogenates. A similar increase (26% after dexamethasone treatment; 39% after cortisol exposure) was found in the microsomal-associated enzyme. No differences in cytosolic enzyme activity were observed. The specific activity of the microsomal enzyme was 3-4 times that of the cytosolic enzyme. Most of the enzyme activity was located in the microsomal fraction (58-65%). The treatments had no effect on the total amount of enzyme recovered from the cell homogenates. These results, taken collectively, are interpreted to indicate that the active form of cytidylyltransferase in type II cells is the membrane-bound enzyme and that cytidylyltransferase activation in type II cells from fetal rat lung after maternal glucocorticoid administration occurs by binding of inactive cytosolic enzyme to endoplasmic reticulum.     

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.     

Testosterone regulation of sex differences in fetal lung development.

Fetal lung development, in particular surfactant synthesis, exhibits a sexual dimorphism. Dihydrotestosterone (DHT) has been shown to delay fetal pulmonary surfactant production, but the potential role for testosterone is unknown. Both testosterone and DHT are potent masculinizing hormones, yet in some instances, an end organ specificity for DHT is present. We hypothesized that the delay in fetal lung surfactant production is dependent upon DHT such that inhibition of the synthesis of DHT from the precursor hormone testosterone would eliminate the sex difference by allowing the male fetus to produce surfactant at the female level. We tested this hypothesis using 17 beta-N,N-diethylcarbamoyl-4-aza-4-methyl-5-alpha-androstane-3-one (4-MA), a potent inhibitor of the enzyme 5 alpha-reductase, which converts testosterone into DHT. First, studies were performed in vivo. 4-MA (20 mg/kg/day) or an equivalent volume of vehicle was injected into pregnant rabbits from Day 12 through Day 26 of gestation. On Day 26, the fetuses were delivered, the lungs were lavaged, and fetal sex was noted. Treatment with 4-MA resulted in a lack of any male-female difference in the anogenital distance and no DHT was detected in the serum of any treated fetus. Phosphatidylcholine (PC), saturated phosphatidylcholine (SPC), and sphingomyelin (S) were measured in the lung lavage, and were expressed as the ratios of PC to sphingomyelin (PC:S) and SPC to sphingomyelin (SPC:S). Sex differences in the PC to sphingomyelin ratio of 4-MA-treated fetuses (female PC:S ratio, 1.43 +/- 0.14; male PC:S ratio, 1.00 +/- 0.13 [mean +/- SE]; P = 0.04) and in the SPC:S ratio of the 4-MA-treated group (female SPC:S ratio, 0.68 +/- 0.10; male SPC:S ratio, 0.35 +/- 0.10; P = 0.03) were present after treatment with 4-MA. The effect of testosterone and of 4-MA on fibroblast pneumonocyte factor (FPF) production was studied in vitro. Fetal rat lung fibroblasts were cultured to confluence with either no added androgen, DHT, testosterone, or testosterone plus 4-MA, and conditioned media for FPF were prepared. Conditioned media were added to fetal Type II cell cultures and FPF activity was measured as the degree of stimulation of the incorporation of [3H] choline into SPC. The conversion of radiolabeled testosterone to DHT by the fibroblasts was inhibited by 4-MA (10(-5) M). Conditioned media from untreated female fibroblasts stimulated with cortisol exhibited significant FPF activity ([3H]choline incorporation into SPC, 140 +/- 17% of control).(ABSTRACT TRUNCATED AT 400 WORDS)     

VEGF induces airway epithelial cell proliferation in human fetal lung in vitro

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen involved in normal and abnormal angiogenesis. VEGF mRNA and protein are abundant in distal epithelium of midtrimester human fetal lung. In the present study, we identified immunoreactivity for KDR, a major VEGF-specific receptor, in distal lung epithelial cells of human fetal lung tissue, suggesting a possible autocrine or paracrine regulatory role for VEGF in pulmonary epithelial cell growth and differentiation. Addition of exogenous VEGF to human fetal lung explants resulted in increased epithelium volume density and lumen volume density in the tissues, both morphometric parameters of tissue differentiation. Cellular proliferation demonstrated by bromodeoxyuridine uptake was prominent in distal airway epithelial cells and increased in the VEGF-treated explants. VEGF-treated explants also demonstrated increased surfactant protein (SP) A mRNA, SP-C mRNA, and SP-A protein levels compared with controls. However, SP-B mRNA levels were unaffected by VEGF treatment. [(3)H]choline incorporation into total phosphatidylcholine was increased by VEGF treatment, but incorporation into disaturated phosphatidylcholine was not affected by exogenous VEGF. Based on these observations, we conclude that VEGF may be an important autocrine growth factor for distal airway epithelial cells in the developing human lung.     

Accelerated fetal lung maturation by estrogen is associated with an epithelial-fibroblast interaction

Summary The role of epithelial-mesenchymal interactions in the stimulation of lung development by estrogen is now investigated using organ cultures of lung from male and female fetal rats taken from Days 17 to 21 of gestation. Estradiol at 1 μg/ml was found to reduce cell proliferation in explants taken during a rapid growth phase (Day 18) and to stimulate surfactant synthesis in both males and females only in Day 20 explants when cell division is much slower. At this time more epithelial cells from estrogen-treated explants contained lamellar bodies, which were also secreted to fill the air sacs. These cultures also showed a significant increase in the frequency of cell-to-cell contacts between epithelial cells and fibroblasts. Uptake of tritiated estradiol by explants increased from Day 18 onward, and by autoradiography, labeling was located predominantly over fibroblasts. Using pure cultures of fetal and adult cells, uptake of labeled estradiol was significantly higher in fibroblasts than in corresponding epithelial cells, and estradiol did not directly enhance palmitate incorporation into epithelial cells. The results suggest that the earlier maturation and increased surfactant synthesis in female fetal lung is related at least in part to enhanced binding of estrogen by the fibroblast with subsequent transfer of a maturation factor to the fetal epithelium. This research project was supported by grants from the Medical Research Council of Canada and the Council for Tobacco Research, U.S.A., Inc.     

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.