Type I and type II alveolar epithelial cells (AECs) are derived from the same progenitor cell, but little is known about the factors that regulate their differentiation into separate phenotypes. An alteration in lung expansion alters the proportion type II AECs in the fetal lung, indicating that this may be a regulatory factor. Our aim was to quantify the changes in the proportion of type I and type II AECs caused by increased fetal lung expansion and to provide evidence for transdifferentiation of type II into type I cells. Lung tissue samples were collected from ovine fetuses exposed to increased lung expansion induced by 2, 4, or 10 days of tracheal obstruction (TO). The identities and proportions of AEC types were determined with electron microscopy. The proportion of type II cells was reduced from 28.5 +/- 2.2% in control fetuses to 9.4 +/- 2.3% at 2 days of TO and then to 1.9 +/- 0.8% at 10 days. The proportion of type I AECs was not altered at 2 days of TO (63.1 +/- 2.3%) compared with that of control cells (64.8 +/- 0.5%) but was markedly elevated (to 89.4 +/- 0.9%) at 10 days of TO. The proportion of an intermediate AEC type, which displayed characteristics of both type I and type II cells, increased from 5.7 +/- 1.3% in control fetuses to 23.8 +/- 5.1% by 2 days of TO and was similar to control values at 10 days of TO (7.7 +/- 0.9%). Our data show that increases in fetal lung expansion cause time-dependent changes in the proportion of AEC types, including a transient increase in an intermediate cell type. These data provide the first evidence to support the hypothesis that increases in fetal lung expansion induce differentiation of type II into type I AECs via an intermediate cell type.

Basal lung expansion is an important determinant of alveolar epithelial cell (AEC) phenotype in the fetus. Because basal lung expansion increases toward term and is reduced after birth, we hypothesized that these changes would be associated with altered proportions of AECs. AEC proportions were calculated with electron microscopy in fetal and postnatal sheep. Type I AECs increased from 4.8 +/- 1.3% at 91 days to 63.0 +/- 3.6% at 111 days of gestation, remained at this level until term, and decreased to 44.8 +/- 1.8% after birth. Type II AECs increased from 4.3 +/- 1.5% at 111 days to 29.6 +/- 4.1% at 128 days of gestation, remained at this level until term, and then increased to 52.9 +/- 1.5% after birth. Surfactant protein (SP)-A, -B and -C mRNA levels increased with increasing gestational age before birth, but the changes in SP expression after birth were inconsistent. Thus before birth type I AECs predominate, whereas after birth type II AECs predominate, possibly due to the reduction in basal lung expansion associated with the entry of air into the lungs.

The factors that control the differentiation of alveolar epithelial cells (AECs) into type-I and type-II cells in vivo are largely unknown. As sustained increases in fetal lung expansion induce type-II AECs to differentiate into type-I cells, our aim was to determine whether reduced fetal lung expansion can induce type-I AECs to trans-differentiate into type-II AECs. Chronically catheterised fetal sheep were divided into two age-matched control groups and three experimental groups (n = 5 for each). The experimental groups were exposed to either: (1) 10 days of increased lung expansion induced by tracheal obstruction (TO), (2) 10 days of TO followed by 5 days of reduced lung expansion induced by lung liquid drainage (LLD), or (3) 10 days of TO followed by 10 days of LLD. evidence to indicate that prolonged increases in cell stretch stimulate differentiation of type-II into type-I AECs, via an intermediate cell type (Flecknoe et al. 2000). We have recently shown that the proportion of type-II AECs decreases from ~30 % to ≤ 2 % during a 10 day period of tracheal obstruction (TO) in fetal sheep, whereas the proportion of type-II AECs increases from ~60 % tõ 90 % (Flecknoe et al. 2000).

Preterm infants are at high risk of developing ventilator-induced lung injury (VILI), which contributes to bronchopulmonary dysplasia. To investigate causes of VILI, we have developed an animal model of in utero ventilation (IUV). Our aim was to characterize the effects of IUV on the very immature lung, in the absence of nonventilatory factors that could contribute to lung pathology. Fetal sheep were ventilated in utero at 110 d gestation for 1, 6, or 12 h (two groups; n ϭ 5 each). Lung tissue was collected at 12 h after initiating IUV in the 1, 6, and one 12 h IUV groups. Lung liquid was replaced in the second 12 h IUV group and tissues collected at 117 d. Operated, nonventilated 110 and 117 d fetuses were controls. IUV reduced secondary septal crest densities, simplified distal airsacs, caused abnormal collagen and elastin deposition, and stimulated myofibroblast differentiation and cellular proliferation. IUV causes VILI in very immature lungs in the absence of other complicating factors and reproduces bronchopulmonary dysplasia -like changes in lung morphology. IUV offers a novel method for dissociating VILI from other iatrogenic factors that could contribute to altered lung development caused by VILI. (Pediatr Res 64: 387-392, 2008) V ery preterm infants (Ͻ30 wk gestation) commonly require resuscitation and assisted ventilation that are closely associated with lung injury (1). Many of these infants (ϳ30%) subsequently develop bronchopulmonary dysplasia (BPD), which is characterized by pathologic changes in lung structure, including larger more simplified alveoli, abnormal capillary growth and elastin deposition (2,3), nonuniform inflation, fibrosis, and edema (4,5).The causes of neonatal lung injury are complex and not fully understood. Animal models used previously to investigate the causes include prolonged ventilation of prematurely delivered baboons (6) and lambs (5). However, it was necessary to ventilate the animals for hours or days to maintain their viability while allowing sufficient time for pathologic changes to develop. During this time, other interventions can be required to ensure survival, including temperature control, higher inspired oxygen levels and ventilation pressures as well as the administration of surfactant, i.v. fluids, nutrition and drugs. As a result, it is difficult to separate the factors causing ventilator-induced lung injury (VILI) from the additional treatments required for survival. To avoid these problems, we have developed a model of VILI, which involves ventilation of very immature fetal lambs in utero.Previously in utero ventilation (IUV) was used to study birth-related changes in cardiopulmonary physiology in late gestation, but not VILI (7-9). IUV has numerous advantages as a model for VILI as the fetus remains on placental support and is not dependent upon ventilation for gas exchange. Thus, individual ventilation parameters can be examined independently and the fetus can remain alive in utero for extended periods following IUV without the need for additio...

Marsupials are born with structurally immature lungs and rely, to varying degrees, on cutaneous gas exchange. With a gestation of 13 d and a birth weight of 13 mg, the fat-tailed dunnart (Sminthopsis crassicaudata) is one of the smallest and most immature marsupial newborns. We determined that the skin is almost solely responsible for gas exchange in the early neonatal period. Indeed, fewer than 35% of newborn dunnarts were observed to make any respiratory effort on the day of birth, with pulmonary ventilation alone not meeting the demand for oxygen until approximately 35 d postpartum. Despite the lack of pulmonary ventilation, the phrenic nerve had made contact with the diaphragm, and the respiratory epithelium was sufficiently developed to support gas exchange on the day of birth. Both type I and type II (surfactant-producing) alveolar epithelial cells were present, with fewer than 7% of the cells resembling undifferentiated alveolar epithelial precursor cells. The type I epithelial cells did, however, display thickened cytoplasmic extensions, leading to a high diffusion distance for oxygen. In addition, the architecture of the lung was immature, resembling the early canalicular stage, with alveolarization not commencing until 45 d postpartum. The pulmonary vasculature was also immature, with a centrally positioned single-capillary layer not evident until 100 d postbirth. These structural limitations may impede efficient pulmonary gas exchange, forcing the neonatal fat-tailed dunnart to rely predominately on its skin, a phenomenon supported by a low metabolic rate and small size.

The cAMP response element binding protein 1 (Creb1) transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1 −/− fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1−/− fetal mice during development. Lungs of Creb1−/− fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1−/− lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1−/− lung on a Crem −/− genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1 −/− lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara), and neuroendocrine cells showed delayed onset of expression in the Creb1−/− lung. Finally, gene expression analyses of the E17.5 Creb1 −/− lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

Glucocorticoids play a vital role in fetal respiratory development and act via the intracellular glucocorticoid receptor (GR) to regulate transcription of key target genes. GR-null mice die at birth due to respiratory dysfunction associated with hypercellularity and atelectasis. To identify events associated with this lung phenotype we examined perinatal cellular proliferation rates and apoptotic indices. We demonstrate that compared to wild-type controls, day 18.5 postcoitum (p.c.) GR-null mouse lungs display significantly increased cell proliferation rates (1.8-fold P < 0.05) and no change in apoptosis. To examine underlying molecular mechanisms, we compared whole genome expression profiles by microarray analysis at 18.5 days p.c. Pathways relating to cell proliferation, division and cell cycle were significantly down-regulated while pathways relating to carbohydrate metabolism, kinase activities and immune responses were significantly up-regulated. Differential levels of gene expression were verified by quantitative-RT-PCR and/or Northern analysis. Key regulators of proliferation differentially expressed in the lung of 18.5 p.c. GR-null lungs included p21CIP1 (decreased 2.9-fold, P < 0.05), a negative regulator of the cell cycle, and Mdk (increased 6.0-fold, P < 0.05), a lung growth factor. The more under-expressed genes in 18.5 p.c. GR-null lungs included Chi3l3 (11-fold, P < 0.05), a macrophage inflammatory response gene and Ela1 (9.4-fold, P < 0.05), an extracellular matrix remodeling enzyme. Our results demonstrate that GR affects the transcriptional status of a number of regulatory processes during late fetal lung development. Amongst these processes is cell proliferation whereby GR induces expression of cell cycle repressors while suppressing induction of a well characterized cell cycle stimulator.

We hypothesized that short periods of mechanical ventilation of very immature lungs can induce persistent bronchiolar remodeling that may adversely affect later lung function. Our objectives were to characterize the effects of brief, positive-pressure ventilation per se on the small airways in very immature, surfactant-deficient lungs and to determine whether the effects persist after the cessation of ventilation. Fetal sheep (0.75 of term) were mechanically ventilated in utero with room air (peak inspiratory pressure 40 cmH2O, positive end-expiratory pressure 4 cmH2O, 65 breaths/min) for 6 or 12 h, after which tissues were collected; another group was studied 7 days after 12-h ventilation. Age-matched unventilated fetuses were controls. The mean basement membrane perimeter of airways analyzed was 548.6 Ϯ 8.5 m and was not different between groups. Immediately after ventilation, 21% of airways had epithelial injury; in airways with intact epithelium, there was more airway smooth muscle (ASM) and less collagen, and the epithelium contained more mucin-containing and apoptotic cells and fewer proliferating cells. Seven days after ventilation, epithelial injury was absent but the epithelium was thicker, with greater cell turnover; there were increased amounts of bronchiolar collagen and ASM and fewer alveolar attachments. The increase in ASM was likely due to cellular hypertrophy rather than hyperplasia. We conclude that brief mechanical ventilation of the very immature lung induces remodeling of the bronchiolar epithelium and walls that lasts for at least 7 days; such changes could contribute to later airway dysfunction. epithelium; airway smooth muscle; collagen; proliferation; apoptosis ADVANCES IN PERINATAL CARE have led to improved survival following very preterm birth, with infants born as early as 23-24 wk of gestation now being capable of survival and leading essentially normal lives. However, numerous studies investigating the long-term outcomes of very preterm birth have shown that children (14,17,25,27) and young adults (15) who were born preterm (Ͻ32 wk of gestation) are at an increased risk of having impaired lung function, including reductions in forced expiratory volume in 1 s (FEV 1 ) and forced expiratory flow rates (FEF 25-75% ). Together, these follow-up studies suggest that the small conducting airways may be permanently affected by very preterm birth, especially if these infants subsequently developed bronchopulmonary dysplasia (BPD).Histological studies of the lungs of infants who were mechanically ventilated following preterm birth have shown evidence of damage to the large and small airways. In the larger airways, a loss of epithelium from the trachea and main bronchi (28) and an increased area of submucosal glands (18, 30) have been reported. Studies that have investigated the smaller airways have shown a greater amount of airway smooth muscle (ASM) (18,19,39,43), increased numbers of goblet cells and altered epithelial cells (18,43). Similar findings have been observed in animal models of v...