Impaired Elastin Deposition in Fstl1?/? Lung Allograft under the Renal Capsule

Lung alveolar development in late gestation is a process important to postnatal survival. Follistatin-like 1 (Fstl1) is a matricellular protein of the Bmp antagonist class, which is involved in the differentiation/maturation of alveolar epithelial cells during saccular stage of lung development. This study investigates the role of Fstl1 on elastin deposition in mesenchyme and subsequent secondary septation in the late gestation stage of terminal saccular formation. To this aim, we modified the renal capsule allograft model for lung organ culture by grafting diced E15.5 distal lung underneath the renal capsule of syngeneic host and cultured up to 7 days. The saccular development of the diced lung allografts, as indicated by the morphology, epithelial and vascular developments, occurred in a manner similar to that in utero. Fstl1 deficiency caused atelectatic phenotype companied by impaired epithelial differentiation in D3 Fstl1^−/− lung allografts, which is similar to that of E18.5 Fstl1^−/− lungs, supporting the role of Fstl1 during saccular stage. Inhibition of Bmp signaling by intraperitoneal injection of dorsomorphin in the host mice rescued the pulmonary atelectasis of D3 Fstl1^−/− allografts. Furthermore, a marked reduction in elastin expression and deposition was observed in walls of air sacs of E18.5 Fstl1^−/− lungs and at the tips of the developing alveolar septae of D7 Fstl1^−/− allografts. Thus, in addition to its role on alveolar epithelium, Fstl1 is crucial for elastin expression and deposition in mesenchyme during lung alveologenesis. Our data demonstrates that the modified renal capsule allograft model for lung organ culture is a robust and efficient technique to increase our understanding of saccular stage of lung development.     

Deletion of STK40 Protein in Mice Causes Respiratory Failure and Death at Birth

STK40 is a putative serine/threonine kinase and was shown to induce extraembryonic endoderm differentiation from mouse embryonic stem cells. However, little is known about its physiological function in vivo. Here, we generate Stk40 knock-out mice and demonstrate that loss of the Stk40 gene causes neonatal lethality at birth. Further examination reveals that the respiratory distress and atelectasis occur in the homozygous mutants. The maturation of lung and alveolar epithelium is delayed in the mutant, as indicated by narrowed air spaces, thickened interstitial septa, and increased glycogen content in the lungs of Stk40^−/− mice. The reduction in levels of T1-α, SP-B, and SP-C indicates delayed maturation of both type I and type II respiratory epithelial cells in Stk40^−/− lungs. Moreover, Stk40 is found to be most highly expressed in lungs of both fetal and adult mice among all organs tested. Mechanistically, a genome-wide RNA microarray analysis reveals significantly altered expression of multiple genes known to participate in lung development. The expression of some genes involved in lipid metabolism, immune response, and glycogen metabolism is also disrupted in the lung of Stk40^−/− mice. Protein affinity purification identifies RCN2, an activator of ERK/MAPK signaling, as an STK40-associated protein. Consistently, Stk40 deficiency attenuates the ERK/MAPK activation, and inhibition of ERK/MAPK activities reduces surfactant protein gene expression in lung epithelial cells. Collectively, this study uncovers an important role of STK40 for lung maturation and neonatal survival. STK40 may associate with RCN2 to activate ERK/MAPK signaling and control the expression of multiple key regulators of lung development.     

Intermediate filaments in the developing type II cell of fetal monkey lung

Anticytokeratin monoclonal antibody was used to study epithelial cell development in fetal monkey lungs taken from animals of different ages. It is well established that the overall maturity of fetal lung depends greatly on the maturation of type II epithelial cells in the alveolus. In this study, we have correlated the cytokeratin phenotype of mammalian epithelial cells with pneumocyte maturation. We show that differentiation and maturation of the type II cell is related to intermediate filament expression. Twenty-four fetal monkeys (Macaca nemestrina) were delivered by cesarean section at a gestational age of 135-140 days (term = 168 days) and divided into two groups. One group of animals was sacrificed during the first 3 hr of life, and the other group was maintained in incubators for 92-120 hr. Anticytokeratin monoclonal antibody recognizes only alveolar type I and type II epithelial cells. In the first 3 hr of life, the cytokeratin was localized only at the alveolar surface and at the cytoplasmic periphery of the type II cells of these premature animals. However, at the age of 92-120 hr, the epithelia in the lungs reacted more intensely than they did during the first 3 hr. Electron microscopy revealed and confirmed that the type II cells were matured and abundant intermediate filaments appeared in the cytoplasm. The filaments appeared to form either aggregates or parallel filament bundles and few were closely associated with the lamellar bodies. In the immature type II cells at 0-3 hr of life, few intermediate filaments could be localized in the cytoplasm, and no parallel filament bundle was observed, though many appeared in the 92-120 hr lungs. This suggests that the intermediate filaments have a functional significance in the development and maturation of the type II cell. The location and stability of keratin filaments in type II cells may confer the structural strength necessary for cells covering a free surface in the alveoli during lung maturation.     

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.     

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.     

Mechanical stretch promotes alveolar epithelial type II cell differentiation.

Functional maturation of pulmonary alveolar epithelial cells is crucial for extrauterine survival. Mechanical distension and mesenchymal-epithelial interactions play important roles in this process. We hypothesized that mechanical stretch simulating fetal breathing movements is an important regulator of pulmonary epithelial cell differentiation. Using a Flexercell Strain Unit, we analyzed effects of stretch on primary cultures of type II cells and cocultures of epithelial and mesenchymal cells isolated from fetal rat lungs during late development. Cyclic stretch of isolated type II cells increased surfactant protein (SP) C mRNA expression by 150 +/- 30% over controls (P < 0.02) on gestational day 18 and by 130 +/- 30% on day 19 (P < 0.03). Stretch of cocultures with fibroblasts increased SP-C expression on days 18 and 19 by 170 +/- 40 and 270 +/- 40%, respectively, compared with unstretched cocultures. On day 19, stretch of isolated type II cells increased SP-B mRNA expression by 50% (P < 0.003). Unlike SP-C, addition of fibroblasts did not produce significant additional effects on SP-B mRNA levels. Under these conditions, we observed only modest increases in cellular immunoreactive SP-B, but secreted saturated phosphatidylcholine rose by 40% (P < 0.002). These results indicate that cyclic stretch promotes developmentally timed differentiation of fetal type II cells, as a direct effect on epithelial cell function and via mesenchymal-epithelial interactions. Expression of the SP-C gene appears to be highly responsive to mechanical stimulation.     

Dual functions for LTBP in lung development: LTBP‐4 independently modulates elastogenesis and TGF‐β activity

The latent TGF‐β binding proteins (LTBP) ‐1, ‐3, and ‐4 are extracellular proteins that assist in the secretion and localization of latent TGF‐β. The null mutation of LTBP‐4S in mice causes defects in the differentiation of terminal air‐sacs, fragmented elastin, and colon carcinomas. We investigated lung development from embryonic day 14.5 (E14.5) to day 7 after birth (P7) in order to determine when the defects in elastin organization initiate and to further examine the relation of TGF‐β signaling levels and air‐sac septation in Ltbp4S?/? lungs. We found that defects in elastogenesis are visible as early as E14.5 and are maintained in the alveolar walls, in blood vessel media, and subjacent airway epithelium. The air‐sac septation defect was associated with excessive TGF‐β signaling and was reversed by lowering TGF‐β2 levels. Thus, the phenotype is not directly reflective of a change in TGF‐β1, the only TGF‐β isoform known to complex with LTBP‐4. Reversal of the air‐sac septation defect was not associated with normalization of the elastogenesis indicating two separate functions of LTBP‐4 as a regulator of elastic fiber assembly and TGF‐β levels in lungs. J. Cell. Physiol. 219: 14–22, 2009. © 2008 Wiley‐Liss, Inc.     

Mesenchymal control of branching pattern in the fetal mouse lung

The effect of mesenchyme on specialization of respiratory epithelium in the fetal mouse was tested in organ cultures. Heterologous combinations were made between respiratory and non-respiratory lung epithelia and the corresponding mesenchymes. Isolated terminal respiratory buds of fetal mouse lungs were recombined with mesenchyme from chick lung parabronchi, mouse trachea or from the avascular, non-respiratory air sacs of chick lungs. Isolated non-branching chick air sacs were combined with mouse terminal bud mesenchyme or mesenchyme from the respiratory branches of chick lungs. Air sac epithelia branched in a pattern characteristic of the chick lung when combined with chick respiratory mesenchyme and in a pattern characteristic of mouse lung when combined with mouse terminal bud mesenchyme. Mouse terminal bud epithelia did not branch with either mouse tracheal mesenchyme or chick air sac mesenchyme but branched in a chick pattern with chick parabronchial mesenchyme. Electron microscopic examination of the cultures showed that all chick air sac epithelial cultures failed to produce surfactant (lamellar bodies) even when they branched. Control cultures of mouse terminal buds contained large numbers of lamellar bodies; mesenchyme which suppressed branching reduced the number of lamellar bodies to only a few in a small proportion of the cells. Culture medium supplemented with growth factors and hormones increased the number of lamellar bodies in heterologous mouse combinations but did not bring the number to control levels. Supplemented medium had no effect on lamellar body production by chick air sac epithelium. The results indicate that branching pattern is determined by the mesenchyme surrounding the epithelial primordium. However, the capacity to synthesize surfactant is determined by the source of the epithelium; mesenchyme may control the degree of expression but not the absolute presence or absence of the differentiated condition.     

Ontogeny of pulmonary alveolar epithelial markers of differentiation

We studied differentiation of the pulmonary epithelium in the periphery of fetal rat lung in vivo and in vitro by comparing the ontogeny of cell-surface glycoconjugates with that of surfactant phospholipids. Apical surface binding of the lectin Maclura pomifera agglutinin (MPA) and expression of a 200-kDa MPA-binding glycoprotein (MPA-gp200) was evident at 20 days gestation in type 2 cells, but did not correlate with ultrastructural features of type 2 cell differentiation. Epithelial cells isolated from peripheral lung of 18-day gestation fetal rats displayed hormone-sensitive surfactant synthesis prior to the hormone-insensitive expression of MPA-gp200. Expression of MPA-gp200 occurred in association with the appearance of many new apical surface proteins suggesting a hormone-independent process of polar membrane differentiation. Thus membrane and secretory differentiation are discordant and can be dissociated. In vivo binding of Ricinus communis 1 agglutinin (RCA1), an apical marker of the differentiated alveolar type 1 cell occurred in undifferentiated peripheral lung epithelial cells as early as 18 days gestation, disappeared from differentiating type 2 cells and appeared in differentiated type 1 cells. Both undifferentiated fetal epithelial cells at 18 days gestation and fully differentiated type 1 cells express multiple glycoproteins with terminal beta-linked galactose residues which bind RCA1. Some of these RCA1-binding glycoproteins appear to be similar. These observations suggest that alveolar epithelial type 1 cells may derive directly from undifferentiated peripheral lung epithelial cells as well as from fully differentiated type 2 cells. In addition, terminal differentiation of fetal lung peripheral epithelium into type 1 and type 2 cells may involve repression as well as induction of differentiation-related genes.     

Development of human fetal lung in organ culture compared with in utero ontogeny

Summary 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.     

Disruption of Sorting Nexin 5 Causes Respiratory Failure Associated with Undifferentiated Alveolar Epithelial Type I Cells in Mice

Sorting nexin 5 (Snx5) has been posited to regulate the degradation of epidermal growth factor receptor and the retrograde trafficking of cation-independent mannose 6-phosphate receptor/insulin-like growth factor II receptor. Snx5 has also been suggested to interact with Mind bomb-1, an E3 ubiquitin ligase that regulates the activation of Notch signaling. However, the in vivo functions of Snx5 are largely unknown. Here, we report that disruption of the Snx5 gene in mice (Snx5^-/- mice) resulted in partial perinatal lethality; 40% of Snx5^-/- mice died shortly after birth due to cyanosis, reduced air space in the lungs, and respiratory failure. Histological analysis revealed that Snx5^-/- mice exhibited thickened alveolar walls associated with undifferentiated alveolar epithelial type I cells. In contrast, alveolar epithelial type II cells were intact, exhibiting normal surfactant synthesis and secretion. Although the expression levels of surfactant proteins and saturated phosphatidylcholine in the lungs of Snx5^-/- mice were comparable to those of Snx5^+/+ mice, the expression levels of T1α, Aqp5, and Rage, markers for distal alveolar epithelial type I cells, were significantly decreased in Snx5 ^-/- mice. These results demonstrate that Snx5 is necessary for the differentiation of alveolar epithelial type I cells, which may underlie the adaptation to air breathing at birth.     

Ex vivo expanded human cord blood-derived hematopoietic progenitor cells induce lung growth and alveolarization in injured newborn lungs

Background

We investigated the capacity of expanded cord blood-derived CD34+ hematopoietic progenitor cells to undergo respiratory epithelial differentiation ex vivo, and to engraft and attenuate alveolar disruption in injured newborn murine lungs in vivo.

Methods

Respiratory epithelial differentiation was studied in CD34+ cells expanded in the presence of growth factors and cytokines (“basic” medium), in one group supplemented with dexamethasone (“DEX”). Expanded or freshly isolated CD34+ cells were inoculated intranasally in newborn mice with apoptosis-induced lung injury. Pulmonary engraftment, lung growth and alveolarization were studied at 8 weeks post-inoculation.

Results

SP-C mRNA expression was seen in 2/7 CD34+ cell isolates expanded in basic media and in 6/7 isolates expanded in DEX, associated with cytoplasmic SP-C immunoreactivity and ultrastructural features suggestive of type II cell-like differentiation. Administration of expanding CD34+ cells was associated with increased lung growth and, in animals treated with DEX-exposed cells, enhanced alveolar septation. Freshly isolated CD34+ cells had no effect of lung growth or remodeling. Lungs of animals treated with expanded CD34+ cells contained intraalveolar aggregates of replicating alu-FISH-positive mononuclear cells, whereas epithelial engraftment was extremely rare.

Conclusion

Expanded cord blood CD34+ cells can induce lung growth and alveolarization in injured newborn lungs. These growth-promoting effects may be linked to paracrine or immunomodulatory effects of persistent cord blood-derived mononuclear cells, as expanded cells showed limited respiratory epithelial transdifferentiation.     

Fetal and postnatal lung defects reveal a novel and required role for Fgf8 in lung development

The fibroblast growth factor, FGF8, has been shown to be essential for vertebrate cardiovascular, craniofacial, brain and limb development. Here we report that Fgf8 function is required for normal progression through the late fetal stages of lung development that culminate in alveolar formation. Budding, lobation and branching morphogenesis are unaffected in early stage Fgf8 hypomorphic and conditional mutant lungs. Excess proliferation during fetal development disrupts distal airspace formation, mesenchymal and vascular remodeling, and Type I epithelial cell differentiation resulting in postnatal respiratory failure and death. Our findings reveal a previously unknown, critical role for Fgf8 function in fetal lung development and suggest that this factor may also contribute to postnatal alveologenesis. Given the high number of premature infants with alveolar dysgenesis and lung dysplasia, and the accumulating evidence that short-term benefits of available therapies may be outweighed by long-term detrimental effects on postnatal alveologenesis, the therapeutic implications of identifying a factor or pathway that can be targeted to stimulate normal alveolar development are profound.