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We determined the effects of sustained and cyclical prenatal mechanical strain on the hypoplastic lung of the ovine model of congenital diaphragmatic hernia. Over a period of 4 weeks in late gestation, repeated cyclical tracheal occlusion for 23 hours with 1-hour release stimulated minimal growth, but promoted maturation with the development of a saccular lung. In contrast, a cycle consisting of 47 hours with 1-hour release induced optimal lung growth and morphologic maturation of the hypoplastic lung parenchyma. Sustained occlusion resulted in exaggerated lung growth, exceeding that of unaffected controls, and abnormal alveolar development. The extent of induction of lung growth by mechanical strain was inversely proportional to the number of alveolar type II cells remaining in the lung epithelium. These studies show that, although mechanical strain is capable of inducing lung growth and differentiation, cyclical strain is a prerequisite for normal development and that mechanically induced growth occurs at the expense of the alveolar type II cell. We conclude that cyclical strain may allow optimal alveolar development while maintaining a population of alveolar type II cells and may thus facilitate an improvement in postnatal lung function in infants with congenital diaphragmatic hernia.

Specialisation of the respiratory portion of human fetal lung commences around 20-24 weeks gestation. In contrast, human fetal lung in vitro has the capacity to self-differentiate from 12 weeks gestation when grown in media devoid of growth factors or hormones, suggesting activation of autocrine or paracrine factors in vitro, or removal of the fetus from in utero inhibitory mechanisms. Prostaglandins play a key role during in vitro human fetal lung development and are synthesised by prostaglandin H synthase-1 (PGHS-1) and inactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH) with formation of inactive 13,14-dihydro-15-keto-prostaglandins. We have used quantitative immunohistochemistry to determine expression and localisation of PGHS-1, PGDH, PGE2 and 13,14-dihydro-15-keto-PGE2 (PGEM) in human fetal lung with in situ hybridisation to localise PGHS-1 and PGDH mRNA. For the catabolic enzyme PGDH, amounts of mRNA, protein and enzyme product PGEM are increased within epithelium of distal as compared to more proximal airways. For PGHS-1, comparable amounts of mRNA, protein and enzyme product PGE2 are found in proximal and distal lung epithelium. Catabolism by PGDH is a sensitive mechanism for regulating bioavailability of prostaglandins and we propose that active catabolism of prostaglandins within human fetal lung epithelium is an inhibitory mechanism retarding epithelial differentiation in utero.

Thyroid hormones are involved in the regulation of fetal lung development, and maturation is accelerated in animal models by antepartum exposure to raised concentrations of the receptor-active thyroid hormone triiodothyronine and glucocorticoids. It is essential that the nature of the regulation of the spatial and temporal metabolism of iodothyronines in the human fetus and infant is known before effective therapies can be developed to modify human lung maturation. Thyroid hormone bioavailability to the human fetus is regulated in part by enzymatic deiodination and reversible sulfation of iodothyronines, with contributions from other factors such as fetomaternal and fetoamniotic hormone transfers, fetal thyroid gland production, and the activities of plasma membrane transporters mediating uptake of iodothyronines from plasma into tissues., (Copyright 2001 S. Karger AG, Basel.)

Reversible sulphation, catalysed by sulphotransferases and sulphatases, of biologically active compounds such as androgens and oestrogens is a sensitive mechanism for regulating their bioavailability, and we have previously hypothesised that this process plays a significant role in the regulation of human fetal lung development. Sulphation is also a major detoxification reaction, contributing significantly to the body's chemical defence mechanism. We have used qualitative and semiquantitative immunological studies to determine the temporal expression and localisation of phenol and hydroxysteroid sulphotransferases during human lung development. Our results show that in the early fetal lung, phenol sulphotransferase expression is at its highest, and is most widely distributed throughout the developing respiratory epithelium. With later development, expression levels decrease and become predominantly restricted to the more proximal airways. In contrast, hydroxysteroid sulphotransferase is present only at very low levels in the early-gestation lung but expression increases rapidly through gestation to reach an apparent peak by 1 year postnatal age. The proximal-to-distal gradients of phenol and hydroxysteroid sulphotransferase expression were similar in mature respiratory epithelium, with immunoreactivity in ciliated cells, non-ciliated secretory cells and basal cells, but with no apparent expression in mucus-secreting cells. These studies provide supporting evidence for the hypothesis that hydroxysteroid sulphotransferase, an androgen-inactivating enzyme, contributes to the role of androgens in retarding the maturation of human lung in utero.

Human fetal lung at 16-19 weeks gestation has a partially differentiated epithelium, and in organ culture, distal airsacs dilate and the epithelium autodifferentiates to type I and II pneumatocytes, processes regulated by endogenous prostaglandin PGE2. Human fetal trachea, at the same gestation, has a terminally differentiated mucociliary epithelium but after 4-6 d in organ culture, develops squamous metaplasia. Tracheal cultures restricted to 3 d have normal phase-contrast and light microscopy appearances and immunohistochemical reactivities (epithelium: cytokeratin 7,8,18; glutathione S-transferase pi-isozyme; epithelial membrane antigen and mesenchyme; desmin; vimentin). In human fetal trachea organ cultures, the predominant prostaglandins released are 6-keto-PGF1 alpha, PGF2 alpha, and PGE2, a pattern similar to that previously described for human adult trachea and lung. In fetal lung cultures, 13,14-dihydro-15-keto-PGF2 alpha is the major prostaglandin released with lesser amounts of 13,14-dihydro-15-keto-PFG2 alpha,PGF2 alpha,PGE2, and 6-keto-PGF1 alpha. Human fetal lung in vitro has the competence to self-differentiate, as early as 12 weeks gestation and presence of high levels in fetal lung of the inactive metabolite 13,14-dihydro-15-keto-PGE2 relative to PGE2 suggests that active prostaglandin catabolism may be one of the mechanisms to retard this stage of maturation in vivo by limiting PGE2 availability. Surprisingly, the profile of prostaglandins released from fetal lung organ culture does not change to that of a mature lung with terminal differentiation of the epithelium, and this may indicate differences in the expression of key prostaglandin-metabolizing enzymes in developing human fetal lung in culture and with in utero ontogeny.

PGE2 and PGF2 alpha are released into the media of human fetal lung organ cultures in decreasing amounts with time. This decline in PGs is not due to culture failure or loss of synthetic capacity, which can be stimulated by fetal bovine serum, nor is it due to increased catabolism of PGE2 to 13,14-dihydro-15-keto-PGE2 (PGEM) or of PGF2 alpha to 13,14-dihydro-15-keto-PGF2 alpha (PGFM). Immunohistochemically reactive PGs are not retained within lung cells. Antisera against methyl-moximated derivatives of PGEM or PGFM and preceded by derivatization on tissue sections of PGs by methyl-moximation not only demonstrate the localization of PGEM and PGFM in epithelial cells and blood vessels, but also show an overall decline in immunoreactivity with time. In addition electron microscopy of uncultured fetal lung removed directly after termination reveals various degrees of mitochondrial damage and in some cases plasma membrane blebs which resolve during the period in culture and as fetal lung self-differentiates. It is proposed that oxidative and mechanical stresses, occurring during termination of pregnancy or tissue preparation, result in cell damage and increased lung prostaglandin production, which, although decreasing during culture as cells recover, is sufficient to trigger terminal self-differentiation.

In utero, at around 23 wk gestation, the progenitor epithelium of distal airway differentiates into type I and type II pneumatocytes. Human fetal lung organ cultures, as early as 12 wk gestation, have the competence to self-differentiate. Distal airway epithelial immunoreactivity to cytokeratins CK 7, 8, and 18 decreases with differentiation both in utero and in organ culture, whereas reactivity to epithelial membrane antigen remains constant in both. As distal airways dilate, the mean percentage airspace of fetal lungs in organ culture increases to 58%, equivalent to lung of gestation 26.0 +/- 7.3 wk. In organ culture, capillary blood vessels, visualized by vimentin immunoreactivity, remodel and more closely approximate the epithelium but without direct invasion. In utero, at 23 wk gestation, elastin appears as condensation around airways and forms a basis for secondary crests which, by 29 wk gestation, evolve into alveolar septae. In organ culture, no elastin is deposited, no secondary or alveolar crests form, and the lung retains a simple saccular structure. Differentiation of the terminal airway epithelium and mesodermal maturational events to facilitate gas exchange, such as capillary invasion or secondary-alveolar crest formation, are almost synchronous in human lung in utero but clearly dissociate in organ culture.

Immunohistochemical studies in human fetal lung have shown that epithelial and endothelial cells are both strongly and equally reactive for PGE2. In contrast, epithelial PGF2 alpha reactivity varied between fetuses, in some as intense as endothelial staining and in others very much less. As lung organ cultures differentiated, the intensity of PGE2 staining declined in airways and blood vessels, although it was still weakly positive at 10 days. In contrast, epithelial cells rapidly became negative for PGF2 alpha, whereas PGF2 alpha positivity was retained in blood vessels, albeit less obviously. PGF2 alpha and PGE2 were released into the media of organ cultures in decreasing amounts as cultures progressed. Amounts of released PGF2 alpha were greater by 2- to 10-fold than PGE2. Our findings suggest that the endogenous production of prostaglandins by human fetal lung in organ culture has a key role in the self-differentiation process that occurs in the absence of sera or added growth factors or hormones.

The sulfation of steroid hormones and xenobiotics by human fetal lung cytosol was examined. 1-Naphthol and estrone were extensively sulfated, whereas paracetamol and dehydroepiandrosterone were not good substrates for the pulmonary enzyme. Investigation of the thermostability and inhibition by 2,6-dichloro-4-nitrophenol (DCNP) of the 1-naphthol and estrone sulfotransferase (ST) activities revealed that the estrone ST activity was more thermolabile and more readily inhibited by DCNP than was the 1-naphthol ST activity. Anion exchange chromatography by FPLC resulted in the resolution of two 1-naphthol ST activities, with the estrone ST activity co-eluting with the more basic 1-naphthol ST activity. When human fetal lung cytosol was subjected to gel filtration FPLC, both the 1-naphthol and estrone ST activities had the same native molecular weight of 63,000 Da. this is the first demonstration of estrogen ST activity in human fetal lung. These results suggest that there are at least two forms of sulfotransferase in human fetal lung and that this tissue is capable of sulfating both xenobiotics and endogenous compounds.

Addition of PGE2, but not PGF2 alpha, to fetal lung organ cultures accelerates the process of self-differentiation with increased dilatation of terminal airsacs and differentiation of the epithelial lining. Indomethacin reduces the endogenous production by organ cultures of PGE2, PGF2 alpha, 13,14-dihydro-15-keto-PGE2, and 13,14-dihydro-15-keto-PGF2 alpha and retards the process of self-differentiation. Prolonged exposure of cultures to indomethacin results in cell necrosis. Indomethacin inhibition of self-differentiation can be reversed and accelerated by the addition of PGE2. Addition of PGF2 alpha in the presence of indomethacin prevents indomethacin-associated cell necrosis but does not accelerate dilatation or differentiation beyond that of cultures in sera-free media without additions. We propose that the endogenous production of PGE2 is a key process in the mechanism of self-differentiation of human fetal lung in organ culture.

The developmental expression of the alpha-, mu- and pi-class glutathione S-transferases has been defined in human lung and kidney using radioimmunoassay, immunohistochemistry and column chromatography. Expression of alpha-class enzymes increased significantly after about 40 weeks gestation in kidney but not lung, while expression of mu isoenzymes was continuous throughout development in both tissues. Expression of the pi isoenzyme fell during in utero ontogeny in lung, the pattern of down-regulation being similar to that previously observed in liver. There was no change in the expression of this isoenzyme in kidney. Comparison of the expression of the glutathione S-transferases in developing lung, kidney and liver shows some common patterns of expression suggesting these genes are under similar regulatory control.

The putative involvement of reactive oxygen species in the etiology of lung damage in infants receiving mechanical ventilation has been examined by comparing the levels of peroxidation and expression of the antioxidant enzymes, CuZn and Mn superoxide dismutase, in lungs from control and affected infants as well as from fetuses and infants who died postnatally after term delivery. Mean levels (+/- SD) of lung peroxidation, determined with a thiobarbituric acid method, were similar in affected and control premature neonates and in fetal subjects (1.87 +/- 1.26, 1.92 +/- 2.07, and 1.19 +/- 1.36 nmol/mg protein, respectively). Expression of CuZn and Mn superoxide dismutases was also similar in these subjects and in the patients who died postnatally. Thus activity measurements and immunoblotting studies showed continuous expression of these enzymes throughout development with no apparent change in protein levels or size. Immunohistochemical examination of lung tissue showed expression of CuZn and Mn superoxide dismutases in epithelial, smooth muscle, endothelial, and some mesenchyme components. In patients with bronchopulmonary dysplasia, alveolar walls were thickened by an excess of fibrous tissue and terminal air spaces were lined mainly by type II pneumatocytes. All structures, including abnormal fibrous components, were positive for both CuZn and Mn superoxide dismutase. Our data show that, unlike some experimental animals, expression of at least these antioxidant enzymes in human infants born prematurely is similar to that in adults, and indicate that such infants are better adapted for life in an oxygen-containing environment than previously suspected.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyclonal antisera to the alpha and pi isoenzymes of glutathione S-transferase have been used in immunohistochemical studies of developing human lung. In utero expression of the pi set was down-regulated in distal airway cells and the first appearance of pi-negative cells coincided with phenotypic differentiation. In contrast, in the early phase of fetal lung organ culture pi isoenzyme was detected in all differentiated epithelial cells and only as culture progressed did focal negativity develop. The alpha set showed no developmental changes in utero or in organ culture.

The developmental expression of Cu,Zn superoxide dismutase in human lung and erythrocytes has been studied using activity measurements, immunoblotting and immunohistochemistry. Enzyme activity in erythrocytes increased significantly during gestation but no developmental trend was seen in lung. Immunoblotting identified a single enzyme form that was present in a variety of tissues and immunohistochemistry showed the enzyme to have widespread distribution in lung tissue. These data indicate that Cu,Zn superoxide dismutase is consistently expressed during human development and that, unlike in other species, no late-fetal surge in expression occurs.

The development of glutathione S-transferase and glutathione peroxidase activities has been studied in human lung cytosols. Whilst no clear change in glutathione peroxidase activity was identified, expression of the acidic glutathione S-transferase isoenzyme decreased markedly after 15 weeks of gestation so that at birth the level of activity of this isoenzyme was only about 20% of that in samples obtained during the first trimester. Basic glutathione S-transferase isoenzymes were weakly expressed during development and usually comprised less than 10% of cytosolic activity. Ion-exchange studies identified several basic isoenzymes that may correspond to the alpha, beta, gamma, delta and epsilon set previously identified in liver. Weak expression of apparently near-neutral isoenzymes was also detected; they were detected in only a few cytosols.