Phase Contrast Imaging Reveals Low Lung Volumes and Surface Areas in the Developing Marsupial

Marsupials are born with immature lungs when compared to eutherian mammals and rely, to various extents, on cutaneous gas exchange in order to meet metabolic requirements. Indeed, the fat-tailed dunnart is born with lungs in the canalicular stage of development and relies almost entirely on the skin for gas exchange at birth; consequently undergoing the majority of lung development in air. Plane radiographs and computed tomography data sets were acquired using phase contrast imaging with a synchrotron radiation source for two marsupial species, the fat-tailed dunnart and the larger tammar wallaby, during the first weeks of postnatal life. Phase contrast imaging revealed that only two lung sacs contain air after the first hour of life in the fat-tailed dunnart. While the lung of the tammar wallaby was comparatively more developed, both species demonstrated massive increases in air sac number and architectural complexity during the postnatal period. In addition, both the tammar wallaby and fat-tailed dunnart had lower lung volumes and parenchymal surface areas than were expected from morphometrically determined allometric equations relating these variables to body mass during the neonatal period. However, lung volume is predicted to scale with mass as expected after the neonatal marsupial reaches a body mass of ∼1 g and no longer relies on the skin for gas exchange. Decreased lung volume in the marsupial neonate further supports the maxim that cutaneous gas exchange occurs in the marsupial neonate because the respiratory apparatus is not yet capable of meeting the gas exchange requirements of the newborn.     

Epithelial-mesenchymal co-culture model for studying alveolar morphogenesis

Division of large, immature alveolar structures into smaller, more numerous alveoli increases the surface area available for gas exchange. Alveolar division requires precise epithelial-mesenchymal interactions. However, few experimental models exist for studying how these cell-cell interactions produce changes in 3-dimensional structure. Here we report an epithelial-mesenchymal cell co-culture model where 3-dimensional peaks form with similar cellular orientation as alveolar structures in vivo. Co-culturing fetal mouse lung mesenchyme with A549 epithelial cells produced tall peaks of cells covered by epithelia with cores of mesenchymal cells. These structures did not form when using adult lung fibroblasts. Peak formation did not require localized areas of cell proliferation or apoptosis. Mesenchymal cells co-cultured with epithelia adopted an elongated cell morphology closely resembling myofibroblasts within alveolar septa in vivo. Because inflammation inhibits alveolar formation, we tested the effects of E. coli lipopolysaccharide on 3-dimensional peak formation. Confocal and time-lapse imaging demonstrated that lipopolysaccharide reduced mesenchymal cell migration, resulting in fewer, shorter peaks with mesenchymal cells present predominantly at the base. This epithelial-mesenchymal co-culture model may therefore prove useful in future studies of mechanisms regulating alveolar morphogenesis.     

Epithelial-mesenchymal co-culture model for studying alveolar morphogenesis

Division of large, immature alveolar structures into smaller, more numerous alveoli increases the surface area available for gas exchange. Alveolar division requires precise epithelial-mesenchymal interactions. However, few experimental models exist for studying how these cell-cell interactions produce changes in 3-dimensional structure. Here we report an epithelial-mesenchymal cell co-culture model where 3-dimensional peaks form with similar cellular orientation as alveolar structures in vivo. Co-culturing fetal mouse lung mesenchyme with A549 epithelial cells produced tall peaks of cells covered by epithelia with cores of mesenchymal cells. These structures did not form when using adult lung fibroblasts. Peak formation did not require localized areas of cell proliferation or apoptosis. Mesenchymal cells co-cultured with epithelia adopted an elongated cell morphology closely resembling myofibroblasts within alveolar septa in vivo. Because inflammation inhibits alveolar formation, we tested the effects of E. coli lipopolysaccharide on 3-dimensional peak formation. Confocal and time-lapse imaging demonstrated that lipopolysaccharide reduced mesenchymal cell migration, resulting in fewer, shorter peaks with mesenchymal cells present predominantly at the base. This epithelial-mesenchymal co-culture model may therefore prove useful in future studies of mechanisms regulating alveolar morphogenesis.     

Retarded development of noenatal rat lung by maternal malnutrition

Inadequate dietary intake during late pregnancy may have significant effects on the developing fetal lung which undergoes rapid cellular multiplication and differentiation shortly before birth. The morphology, glycogen distribution and acid phosphatase activity in normal and starved neonatal rats have been studied sequentially, by using histochemical and cytochemical methods. It has been shown that the normal pattern of lung growth and enzymatic development is retarded in neonates of malnourished mothers. A slowed rate of cellular division and differentiation in the critical prenatal period resulted in a more immature air-blood barrier at birth, with glycogen retention by some epithelial cells. Delayed Type 2 cell maturation with diminished acid phosphatase activity suggests a decrease in surfactant production in the malnourished newborn. In addition, fewer alveolar macrophages with reduced acid phosphatase activity were observed in the perinatal period of starved rats; this finding might have implications for the handling of inhaled bacteria shortly after birth. These results indicate that nutritional status of the mother has a marked effect on fetal lung growth and development by inhibiting cellular proliferation, differentiation and enzyme development by epithelial and macrophagic cells.     

Oxidative stress and NF-κB signaling are involved in LPS induced pulmonary dysplasia in chick embryos

Inflammation or dysbacteriosis-derived lipopolysaccharides (LPS) adversely influence the embryonic development of respiratory system. However, the precise pathological mechanisms still remain to be elucidated. In this study, we demonstrated that LPS exposure caused lung maldevelopment in chick embryos, including higher embryo mortality, increased thickness of alveolar gas exchange zone, and accumulation of PAS⁺ immature pulmonary cells, accompanied with reduced expression of alveolar epithelial cell markers and lamellar body count. Upon LPS exposure, pulmonary cell proliferation was significantly altered and cell apoptosis was inhibited as well, indicating a delayed progress of pulmonary development. LPS treatment also resulted in reduced CAV-1 expression and up-regulation of Collagen I, suggesting increased lung fibrosis, which was verified by Masson staining. Moreover, LPS induced enhanced Nrf2 expression in E18 lungs, and the increased reactive oxygen species (ROS) production was confirmed in MLE-12 cells in vitro. Antioxidant vitamin C restored the LPS induced down-regulation of ABCA3, SP-C and GATA-6 in MLE-12 cells. Furthermore, LPS induced activation of NF-κB signaling in MLE-12 cells, and the LPS-induced decrease in SP-C expression was partially abrogated by blocking NF-κB signaling with Bay-11–7082. Bay-11–7082 also inhibited LPS-induced increases of ROS and Nrf2 expression. Taken together, we have demonstrated that oxidative stress and NF-κB signaling are involved in LPS induced disruption of pulmonary cell development in chick embryos.     

Ⅱ型肺泡上皮细胞与特发性肺纤维化的关系研究进展

特发性肺纤维化（IPF）是一种严重影响肺通气与换气功能的下呼吸道慢性疾病,其发病机理目前尚不明确,表现为异常的间质炎症和纤维化,以及肺泡结构的破坏。而Ⅱ型肺泡上皮细胞（ATⅡ）作为维持肺结构和功能的关键细胞,在肺部纤维化的发生和发展中极其重要。在IPF中,各种原因所致的ATⅡ的受损和衰老凋亡,可能是纤维化发生的是始动因素。而在这之后,关于临时基质的形成、成纤维细胞的聚集、激活以及间质-上皮转化的过程,异常的ATⅡ也参与其中,并发挥着重要的作用。     

Paracrine cellular and extracellular matrix interactions with mesenchymal progenitors during pulmonary alveolar septation

Alveolar development in humans primarily occurs postnatally and requires a carefully orchestrated expansion of distal epithelial and mesenchymal progenitor populations and coordinated differentiation, to create a highly segmented gas‐exchange surface. The regulation of alveolarization normally assimilates cues from paracrine cell–cell, cell–extracellular matrix, and mechanical interactions which are superimposed on cells and the extracellular matrix through phasic respiratory movement. In bronchopulmonary dysplasia, the entire process is precociously initiated when cellular and extracellular components are adapted to the saccular stage where movement and circulation are much more limited. This review focuses on mesenchymal cells (fibroblasts, endothelial cells, and pericytes), and epithelial cells are primarily discussed as sources of growth factor ligands or recipients of ligands produced by mesenchymal cells. Some interstitial fibroblasts differentiate to contractile myofibroblasts, containing a smooth muscle‐actin rich cytoskeleton, which connects with tensile and elastic elements in the extracellular matrix, and together comprise a load‐bearing network that diffuses mechanical forces during respiration. Other interstitial fibroblasts assimilate neutral lipid droplets, which regulate the differentiation of distal epithelial progenitors and surfactant production by alveolar type 2 cells. Pericytes organize and reinforce the capillary network as it expands to match the coverage of type 1 epithelial cells. Hyperoxia and the mechanical load imposed by positive pressure mechanical ventilation disrupt these paracrine interactions, leaving thickened alveolar walls, airways and arterioles, thereby diminishing gas‐exchange surface area. Better understanding of these mechanisms of alveolar septation will lead to more effective treatments to preserve and perhaps augment the surface usual sequence of events that drive alveolarization. Birth Defects Research (Part A) 100:227–239, 2014. © 2014 Wiley Periodicals, Inc.     

Reduced activity of the epithelial sodium channel in malaria-induced pulmonary oedema in mice

Lung complications during malaria infection can range from coughs and impairments in gas transfer to the development of acute respiratory distress syndrome (ARDS). Infecting C57BL/6 mice with Plasmodium berghei K173 strain (PbK) resulted in pulmonary oedema, capillaries congested with leukocytes and infected red blood cells (iRBCs), and leukocyte infiltration into the lungs. This new model of malaria-associated lung pathology, without any accompanying cerebral complications, allows the investigation of mechanisms leading to the lung disease. The activity of the amiloride-sensitive epithelial sodium channel (ENaC) in alveolar epithelial cells is decreased by several respiratory tract pathogens and this is suggested to contribute to pulmonary oedema. We show that PbK, a pathogen that remains in the circulation, also decreased the activity and expression of ENaC, suggesting that infectious agents can have indirect effects on ENaC activity in lung epithelial cells. The reduced ENaC activity may contribute to the pulmonary oedema induced by PbK malaria.     

T1alpha,a lung type I cell differentiation gene,is required for normal lung cell proliferation and alveolus formation at birth

T1alpha, a differentiation gene of lung alveolar epithelial type I cells, is developmentally regulated and encodes an apical membrane protein of unknown function. Morphological differentiation of type I cells to form the air-blood barrier starts in the last few days of gestation and continues postnatally. Although T1alpha is expressed in the foregut endoderm before the lung buds, T1alpha mRNA and protein levels increase substantially in late fetuses when expression is restricted to alveolar type I cells. We generated T1alpha null mutant mice to study the role of T1alpha in lung development and differentiation and to gain insight into its potential function. Homozygous null mice die at birth of respiratory failure, and their lungs cannot be inflated to normal volumes. Distal lung morphology is altered. In the absence of T1alpha protein, type I cell differentiation is blocked, as indicated by smaller airspaces, many fewer attenuated type I cells, and reduced levels of aquaporin-5 mRNA and protein, a type I cell water channel. Abundant secreted surfactant in the narrowed airspaces, normal levels of surfactant protein mRNAs, and normal patterns and numbers of cells expressing surfactant protein-B suggest that differentiation of type II cells, also alveolar epithelial cells, is normal. Anomalous proliferation of the mesenchyme and epithelium at birth with unchanged numbers of apoptotic cells suggests that loss of T1alpha and/or abnormal morphogenesis of type I cells alter the proliferation rate of distal lung cells, probably by disruption of epithelial-mesenchymal signaling.     

Congenital bronchiolo-alveolar airway malformation in a cynomolgus macaque (Macaca fascicularis)

Pulmonary congenital anomalies in animals are rare. Previously reported malformations include accessory lung formation, pulmonary hypoplasia, pulmonary agenesis, and various forms of hamartoma. Congenital bronchiolo-alveolar airway malformation, a new entity, is described in a 1-day-old male cynomolgus macaque. This neonate experienced breathing difficulties shortly after birth and died while therapy was being administered. Grossly, the right lung was markedly increased in size, firm, and pink. Histopathologically, sections of right lung showed irregular bronchiole-like and alveolus-like structures. There was marked widening of alveolar septae by loosely arranged mesenchymal cells and many centrally located capillaries. Alveoli were lined by cuboidal epithelial cells. There were scattered islands of immature cartilage. A grossly enlarged lung containing bronchiole-like and alveolus-like structures, immature cartilage islands, and many capillaries within alveolar septae on histopathologic examination, is inconsistent with previously described congenital pulmonary anomalies in animals and humans.     

Alveolar Type II Epithelial Cell Dysfunction in Rat Experimental Hepatopulmonary Syndrome (HPS)

The hepatopulmonary syndrome (HPS) develops when pulmonary vasodilatation leads to abnormal gas exchange. However, in human HPS, restrictive ventilatory defects are also observed supporting that the alveolar epithelial compartment may also be affected. Alveolar type II epithelial cells (AT2) play a critical role in maintaining the alveolar compartment by producing four surfactant proteins (SPs, SP-A, SP-B, SP-C and SP-D) which also facilitate alveolar repair following injury. However, no studies have evaluated the alveolar epithelial compartment in experimental HPS. In this study, we evaluated the alveolar epithelial compartment and particularly AT2 cells in experimental HPS induced by common bile duct ligation (CBDL). We found a significant reduction in pulmonary SP production associated with increased apoptosis in AT2 cells after CBDL relative to controls. Lung morphology showed decreased mean alveolar chord length and lung volumes in CBDL animals that were not seen in control models supporting a selective reduction of alveolar airspace. Furthermore, we found that administration of TNF-α, the bile acid, chenodeoxycholic acid, and FXR nuclear receptor activation (GW4064) induced apoptosis and impaired SP-B and SP-C production in alveolar epithelial cells in vitro. These results imply that AT2 cell dysfunction occurs in experimental HPS and is associated with alterations in the alveolar epithelial compartment. Our findings support a novel contributing mechanism in experimental HPS that may be relevant to humans and a potential therapeutic target.     

Assessment of inhibited alveolar‐capillary membrane structural development and function in bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of extreme prematurity and is defined clinically by dependence on supplemental oxygen due to impaired gas exchange. Optimal gas exchange is dependent on the development of a sufficient surface area for diffusion. In the mammalian lung, rapid acquisition of distal lung surface area is accomplished in neonatal and early adult life by means of vascularization and secondary septation of distal lung airspaces. Extreme preterm birth interrupts secondary septation and pulmonary capillary development and ultimately reduces the efficiency of the alveolar‐capillary membrane. Although pulmonary health in BPD infants rapidly improves over the first few years, persistent alveolar‐capillary membrane dysfunction continues into adolescence and adulthood. Preventative therapies have been largely ineffective, and therapies aimed at promoting normal development of the air‐blood barrier in infants with established BPD remain largely unexplored. The purpose of this review will be: (1) to summarize the histological evidence of aberrant alveolar‐capillary membrane development associated with extreme preterm birth and BPD, (2) to review the clinical evidence assessing the long‐term impact of BPD on alveolar‐capillary membrane function, and (3) to discuss the need to develop and incorporate direct measurements of functional gas exchange into clinically relevant animal models of inhibited alveolar development. Birth Defects Research (Part A) 100:168–179, 2014. © 2014 Wiley Periodicals, Inc.     

Loss of Hox5 function results in myofibroblast mislocalization and distal lung matrix defects during postnatal development

Alveologenesis is the final stage of lung development and is responsible for the formation of the principle gas exchange units called alveoli. The lung mesenchyme, in particular the alveolar myofibroblasts, are drivers of alveolar development, however,few key regulators that govern the proper distribution and behavior of these cells in the distal lung during alveologenesis have been identified. While Hox5 triple mutants(Hox5 aabbcc) exhibit neonatal lethality, four-allele, compound mutant mice(Hox5 AabbCc) are born in Mendelian ratios and are phenotypically normal at birth. However, they exhibit defects in alveologenesis characterized by a BPD-like phenotype by early postnatal stages that becomes more pronounced at adult stages. Invasive pulmonary functional analyses demonstrate significant increases in total lung volume and compliance and a decrease in elastance in Hox5 compound mutants. SMA+ myofibroblasts in the distal lung are distributed abnormally during peak stages of alveologenesis and aggregate, resulting in the formation of a disrupted elastin network. Examination of other key components of the distal lung ECM, as well as other epithelial cells and lipofibroblasts reveal no differences in distribution. Collectively, these data indicate that Hox5 genes play a critical role in alveolar development by governing the proper cellular behavior of myofibroblasts during alveologenesis.     

Beta-catenin regulates differentiation of respiratory epithelial cells in vivo

An activated form of beta-catenin [Catnb(Delta(ex3))] was expressed in respiratory epithelial cells of the developing lung. Although morphogenesis was not altered at birth, air space enlargement and epithelial cell dysplasia were observed in the early postnatal period and persisted into adulthood. The Catnb(Delta(ex3)) protein caused squamous, cuboidal, and goblet cell dysplasia in intrapulmonary conducting airways. Atypical epithelial cells that stained for surfactant pro protein C (pro-SP-C) and had morphological characteristics of alveolar type II cells were observed in bronchioles of the transgenic mice. Catnb(Delta(ex3)) inhibited expression of Foxa2 and caused goblet cell hyperplasia associated with increased staining for mucins and the MUC5A/C protein. In vitro, both wild type and activated beta-catenin negatively regulated the expression of the Foxa2 promoter. Catnb(Delta(ex3)) also caused pulmonary tumors in adult mice. Activation of beta-catenin caused ectopic differentiation of alveolar type II-like cells in conducting airways, goblet cell hyperplasia, and air space enlargement, demonstrating a critical role for the Wnt/beta-catenin signal transduction pathway in the differentiation of the respiratory epithelium in the postnatal lung.     

T1α, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth

T1α, a differentiation gene of lung alveolar epithelial type I cells, is developmentally regulated and encodes an apical membrane protein of unknown function. Morphological differentiation of type I cells to form the air-blood barrier starts in the last few days of gestation and continues postnatally. Although T1α is expressed in the foregut endoderm before the lung buds, T1α mRNA and protein levels increase substantially in late fetuses when expression is restricted to alveolar type I cells. We generated T1α null mutant mice to study the role of T1α in lung development and differentiation and to gain insight into its potential function. Homozygous null mice die at birth of respiratory failure, and their lungs cannot be inflated to normal volumes. Distal lung morphology is altered. In the absence of T1α protein, type I cell differentiation is blocked, as indicated by smaller airspaces, many fewer attenuated type I cells, and reduced levels of aquaporin-5 mRNA and protein, a type I cell water channel. Abundant secreted surfactant in the narrowed airspaces, normal levels of surfactant protein mRNAs, and normal patterns and numbers of cells expressing surfactant protein-B suggest that differentiation of type II cells, also alveolar epithelial cells, is normal. Anomalous proliferation of the mesenchyme and epithelium at birth with unchanged numbers of apoptotic cells suggests that loss of T1α and/or abnormal morphogenesis of type I cells alter the proliferation rate of distal lung cells, probably by disruption of epithelial-mesenchymal signaling.     

Influence of maternal diabetes on basement membranes, type 2 cells, and capillaries in the developing rat lung

To determine the effect of maternal diabetes on rat lung development, we studied the ultrastructure of the alveolar wall from the ninteenth day of gestation (term = 22 days) through the eighth postnatal day in fetal and neonatal rats of mothers with streptozotocin-induced diabetes. In normal fetal lung development, epithelial basement membranes develop large discontinuities beneath type 2 cells, through which cytoplasmic foot processes extend into the interstitium. Maternal diabetes delays the appearances of these epithelial basement membrane discontinuities and reduces the number of type 2 cell processes that penetrate it. These alterations in epithelial basement membrane are reversed after birth. There is no ultrastructural evidence of a delay in type 2 cell maturation as assessed by lamellar body volume density morphometry. Endothelial basement membranes, which are not present around the growing pulmonary capillary bed in the pseudoglandular lung, are seen late in normal gestation, primarily around capillaries forming the mature air-blood barrier. This development of endothelial basement membrane may be delayed in the fetuses of diabetic mothers and reflects a significant delay in the expansion of the pulmonary capillary network in these animals as assessed by morphometric volume density measurements. This effect on capillary growth is not reversed in the newborn animals through 8 days after birth. The summation of these effects indicates a generalized slowing of fetal lung development by maternal diabetes, some of which effects persist after birth and may continue to influence lung development during the period of postnatal alveolar septal growth.     

Cone visual pigments in two marsupial species: the fat-tailed dunnart (Sminthopsis crassicaudata) and the honey possum (Tarsipes rostratus)

Uniquely for non-primate mammals, three classes of cone photoreceptors have been previously identified by microspectrophotometry in two marsupial species: the polyprotodont fat-tailed dunnart (Sminthopsis crassicaudata) and the diprotodont honey possum (Tarsipes rostratus). This report focuses on the genetic basis for these three pigments. Two cone pigments were amplified from retinal cDNA of both species and identified by phylogenetics as members of the short wavelength-sensitive 1 (SWS1) and long wavelength-sensitive (LWS) opsin classes. In vitro expression of the two sequences from the fat-tailed dunnart confirmed the peak absorbances at 363 nm in the UV for the SWS1 pigment and 533 nm for the LWS pigment. No additional expressed cone opsin sequences that could account for the middle wavelength cones could be amplified. However, amplification from the fat-tailed dunnart genomic DNA with RH1 (rod) opsin primer pairs identified two genes with identical coding regions but sequence differences in introns 2 and 3. Uniquely therefore for a mammal, the fat-tailed dunnart has two copies of an RH1 opsin gene. This raises the possibility that the middle wavelength cones express a rod rather than a cone pigment.