ATII Cells Research Articles

Receptors for Advanced Glycation End‐Products (RAGE) are Transcriptionally Inhibited by FoxA2 in Diverse Pulmonary Cell Types

The receptor for advanced glycation end‐products (RAGE) has been developmentally identified on the membranes of ATI cells and has been implicated in the spreading and adherence that characterize the transitioning of ATII cells to ATI cells. We have previously demonstrated that TTF‐1 and Egr‐1, transcriptional regulators that function during lung development, regulate RAGE transcription. However, the precise pattern of transcription factors that limit RAGE expression to pulmonary‐specific cell types remained unclear. In the current study, transcriptional regulation of RAGE was assessed by transfection of ATII‐like alveolar epithelium (A‐549), bronchiolar epithelium (Beas2B), and macrophages (RAW 264.7) with a luciferase reporter containing 0.9‐kb of the mouse RAGE promoter. In addition to the reporter construct, each cell type was transfected with FoxA2, a winged double helix DNA binding protein that influences respiratory epithelial cell differentiation. In each case, FoxA2 repressed RAGE transcription in vitro. Mutagenesis of a proximal promoter region revealed that FoxA2 directly interacts with the coding sequence for RAGE. These data show that FoxA2 may negatively regulate RAGE expression by respiratory epithelium during pulmonary development. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).

Potassium channels as operators of alveolar epithelial repair

A common feature of inflammatory lung diseases such as acute lung injury and acute respiratory distress syndrome (ARDS) is extensive damage and remodeling of alveolar epithelium. A better understanding of determinants of alveolar regeneration is thus necessary to develop strategies able to restore alveolar integrity. Recent studies have shown that K+ channels (KCh) are key components of cell proliferation and migration processes, necessary for tissues repair. We therefore postulated that KCh play a role in respiratory epithelial repair.Using a wound‐healing assay, we first showed that glibenclamide and clofilium, inhibitors of KATP and KvLQT1 channels, decrease repair rates of A549 and primary alveolar ATII cell monolayers and their inhibitory effects are additive. The role of KvLQT1 and KATP was then confirmed by a molecular approach using specific siRNAs. On the contrary, KvLQT1 and KATP activators significantly stimulate the wound repair. We also evaluated the influence of KCh on cell growth and we showed that clofilium dose‐dependently inhibits alveolar cell growth and induces an accumulation of cells arrested in G0/G1 phase.In conclusion, we demonstrated that KCh, particularly KvLQT1 and KATP, are involved in epithelial repair processes and should be identify as novel targets to promote alveolar repair necessary to ARDS resolution.Project funded by Canadian Institute of Health Research.

Contribution of IL-33–activated type II innate lymphoid cells to pulmonary eosinophilia in intestinal nematode-infected mice

When animals are infected with helminthic parasites, resistant hosts show type II helper T immune responses to expel worms. Recently, natural helper (NH) cells or nuocytes, newly identified type II innate lymphoid cells, are shown to express ST2 (IL-33 receptor) and produce IL-5 and IL-13 when stimulated with IL-33. Here we show the relevant roles of endogenous IL-33 for Strongyloides venezuelensis infection-induced lung eosinophilic inflammation by using Il33(-/-) mice. Alveolar epithelial type II cells (ATII) express IL-33 in their nucleus. Infection with S. venezuelensis or intranasal administration of chitin increases in the number of ATII cells and the level of IL-33. S. venezuelensis infection induces pulmonary accumulation of NH cells, which, after being stimulated with IL-33, proliferate and produce IL-5 and IL-13. Furthermore, S. venezuelensis infected Rag2(-/-) mice increase the number of ATII cells, NH cells, and eosinophils and the expression of IL-33 in their lungs. Finally, IL-33-stimulated NH cells induce lung eosinophilic inflammation and might aid to expel infected worms in the lungs.

Endothelial-monocyte activating polypeptide II disrupts alveolar epithelial type II to type I cell transdifferentiation

BackgroundDistal alveolar morphogenesis is marked by differentiation of alveolar type (AT)-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP) II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII→ATI trans-differentiation has not been explored.MethodIn a controlled nonvascular environment, an in vitro model of ATII→ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation.ResultsHere, we show that EMAP II significantly blocked ATII→ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA.ConclusionOur findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia.

Gene expression profiles of alveolar type II cells of chronic obstructive pulmonary disease: a case–control study

ObjectivesThe aim of this study was to identify the gene expression pattern specific in alveolar epithelial type II cells (ATII cells) isolated from patients with chronic obstructive pulmonary disease (COPD).DesignCase...

Responsive culture platform to examine the influence of microenvironmental geometry on cell function in 3D.

We describe the development of a well-based cell culture platform that enables experimenters to control the geometry and connectivity of cellular microenvironments spatiotemporally. The base material is a hydrogel comprised of photolabile and enzyme-labile crosslinks and pendant cell adhesion sequences, enabling spatially-specific, in situ patterning with light and cell-dictated microenvironment remodeling through enzyme secretion. Arrays of culture wells of varying shape and size were patterned into the hydrogel surface using photolithography, where well depth was correlated with irradiation dose. The geometry of these devices can be subsequently modified through sequential patterning, while simultaneously monitoring changes in cell geometry and connectivity. Towards establishing the utility of these devices for dynamic evaluation of the influence of physical cues on tissue morphogenesis, the effect of well shape on lung epithelial cell differentiation (i.e., primary mouse alveolar type II cells, ATII cells) was assessed. Shapes inspired by alveoli were degraded into hydrogel surfaces. ATII cells were seeded within the well-based arrays and encapsulated by the addition of a top hydrogel layer. Cell differentiation in response to these geometries was characterized over 7 days of culture with immunocytochemistry (surfactant protein C, ATII; T1α protein, alveolar type I (ATI) differentiated epithelial cells) and confocal image analysis. Individual cell clusters were further connected by eroding channels between wells during culture via controlled two-photon irradiation. Collectively, these studies demonstrate the development and utility of responsive hydrogel culture devices to study how a range of microenvironment geometries of evolving shape and connectivity might influence or direct cell function.

TGF-beta 1 induced fibroblast proliferation is mediated by the FGF-2/ERK pathway

Pulmonary fibrosis, defined as the accumulation of connective tissue in the lungs, is a severe and often fatal form of interstitial lung disease. Transforming growth factor-beta (TGF-beta) is a powerful activator of connective tissue synthesis and fibroblast proliferation in the lung, and a critical paracrine signal for the development of pulmonary fibrosis. To investigate signaling pathways downstream of TGF- beta that contribute to lung fibrosis, TGF- beta stimulation of fibroblasts was replicated by treating NIH3T3 fibroblasts with conditioned medium (CM) from TGF- beta -treated type II alveolar epithelial cells (ATII cells). The data showed that fibroblast growth factor 2 (FGF-2) signaling is responsible for TGF-beta 1 CM-induced fibroblast proliferation, while it does not affect TGF-beta 1 CM-induced fibrotic differentiation. Moreover, fibroblast proliferation and differentiation induced by TGF- beta CM was totally abrogated by pretreatment of NIH3T3 cells with the specific ERK1/2 inhibitor, PD98059. These findings indicate that FGF-2 secreted by alveolar epithelial cells in response to TGF- beta 1 induces fibroblast proliferation and fibrotic activation through the ERK kinase pathway.

Immunohistochemical detection and regulation of α5nicotinic acetylcholine receptor (nAChR) subunits by FoxA2 during mouse lung organogenesis

Backgroundα5 nicotinic acetylcholine receptor (nAChR) subunits structurally stabilize functional nAChRs in many non-neuronal tissue types. The expression of α5 nAChR subunits and cell-specific markers were assessed during lung morphogenesis by co-localizing immunohistochemistry from embryonic day (E) 13.5 to post natal day (PN) 20. Transcriptional control of α5 nAChR expression by FoxA2 and GATA-6 was determined by reporter gene assays.ResultsSteady expression of α5 nAChR subunits was observed in distal lung epithelial cells during development while proximal lung expression significantly alternates between abundant prenatal expression, absence at PN4 and PN10, and a return to intense expression at PN20. α5 expression was most abundant on luminal edges of alveolar type (AT) I and ATII cells, non-ciliated Clara cells, and ciliated cells in the proximal lung at various periods of lung formation. Expression of α5 nAChR subunits correlated with cell differentiation and reporter gene assays suggest expression of α5 is regulated in part by FoxA2, with possible cooperation by GATA-6.ConclusionsOur data reveal a highly regulated temporal-spatial pattern of α5 nAChR subunit expression during important periods of lung morphogenesis. Due to specific regulation by FoxA2 and distinct identification of α5 in alveolar epithelium and Clara cells, future studies may identify possible mechanisms of cell differentiation and lung homeostasis mediated at least in part by α5-containing nAChRs.

Regulation of Alveolar Epithelial Na+ Channels by ERK1/2 in Chlorine-Breathing Mice

The mechanisms by which the exposure of mice to Cl(2) decreases vectorial Na(+) transport and fluid clearance across their distal lung spaces have not been elucidated. We examined the biophysical, biochemical, and physiological changes of rodent lung epithelial Na(+) channels (ENaCs) after exposure to Cl(2), and identified the mechanisms involved. We measured amiloride-sensitive short-circuit currents (I(amil)) across isolated alveolar Type II (ATII) cell monolayers and ENaC single-channel properties by patching ATII and ATI cells in situ. α-ENaC, γ-ENaC, total and phosphorylated extracellular signal-related kinase (ERK)1/2, and advanced products of lipid peroxidation in ATII cells were measured by Western blot analysis. Concentrations of reactive intermediates were assessed by electron spin resonance (ESR). Amiloride-sensitive Na(+) channels with conductances of 4.5 and 18 pS were evident in ATI and ATII cells in situ of air-breathing mice. At 1 hour and 24 hours after exposure to Cl(2), the open probabilities of these two channels decreased. This effect was prevented by incubating lung slices with inhibitors of ERK1/2 or of proteasomes and lysosomes. The exposure of ATII cell monolayers to Cl(2) increased concentrations of reactive intermediates, leading to ERK1/2 phosphorylation and decreased I(amil) and α-ENaC concentrations at 1 hour and 24 hours after exposure. The administration of antioxidants to ATII cells before and after exposure to Cl(2) decreased concentrations of reactive intermediates and ERK1/2 activation, which mitigated the decrease in I(amil) and ENaC concentrations. The reactive intermediates formed during and after exposure to Cl(2) activated ERK1/2 in ATII cells in vitro and in vivo, leading to decreased ENaC concentrations and activity.

Rescue of murine silica-induced lung injury and fibrosis by human embryonic stem cells

Alveolar type II pneumocytes (ATII cells) are considered putative alveolar stem cells. Since no treatment is available to repair damaged epithelium and prevent lung fibrosis, novel approaches to induce regeneration of injured alveolar epithelium are desired. The objective of this study was to assess both the capacity of human embryonic stem cells (HUES-3) to differentiate in vitro into ATII cells and the ability of committed HUES-3 cells (HUES-3-ATII cells) to recover in vivo a pulmonary fibrosis model obtained by silica-induced damage. In vitro differentiated HUES-3-ATII cells displayed an alveolar phenotype characterised by multi-lamellar body and tight junction formation, by the expression of specific markers such as surfactant protein (SP)-B, SP-C and zonula occludens (ZO)-1 and the activity of cystic fibrosis transmembrane conductance regulator-mediated chloride ion transport. After transplantation of HUES-3-ATII cells into silica-damaged mice, histological and biomolecular analyses revealed a significant reduction of inflammation and fibrosis markers along with lung function improvement, weight recovery and increased survival. The persistence of human SP-C, human nuclear antigen and human DNA in the engrafted lungs indicates that differentiated cells remained engrafted up to 10 weeks. In conclusion, cell therapy using HUES-3 cells may be considered a promising approach to lung injury repair.

Indispensable role of factor for adipocyte differentiation 104 (fad104) in lung maturation

Factor for adipocyte differentiation 104 (fad104) is a regulator of adipogenesis and osteogenesis. Our previous study showed that fad104-deficient mice died immediately after birth, suggesting fad104 to be essential for neonatal survival. However, the cause of this rapid death is unclear. Here, we demonstrate the role of fad104 in neonatal survival. Phenotypic and morphological analyses showed that fad104-deficient mice died due to cyanosis-associated lung dysplasia including atelectasis. Furthermore, immunohistochemistry revealed that FAD104 was strongly expressed in ATII cells in the developing lung. Most importantly, the ATII cells in lungs were immature, and impaired the expression of surfactant-associated proteins. Collectively, these results indicate that fad104 has an indispensable role in lung maturation, especially the maturation and differentiation of ATII cells.

Up-Regulation of Receptors for Advanced Glycation End-Products by Alveolar Epithelium Influences Cytodifferentiation and Causes Severe Lung Hypoplasia

Receptors for advanced glycation end-products (RAGE) are cell-surface receptors expressed by pulmonary tissue that influence alveolar type (AT) II-ATI transition required for normal alveolar formation. However, the precise contribution of RAGE in interactions between pulmonary epithelium and splanchnic mesenchyme during lung organogenesis remains uncertain. To test the hypothesis that RAGE misexpression adversely affects lung morphogenesis, conditional transgenic mice were generated that overexpress RAGE. Mice that overexpress RAGE throughout embryogenesis experienced 100% mortality and significant lung hypoplasia coincident with large, vacuous areas in the periphery when compared with normal airway and alveolar architecture observed in control mouse lungs. Flow cytometry and immunohistochemistry employing cell-specific markers for distal (forkhead box protein A2) and respiratory (thyroid transcription factor-1) epithelium, ATII cells (pro-surfactant protein-C), and ATI cells (T1-α) demonstrated anomalies in key epithelial cell populations resulting from RAGE up-regulation. These results reveal that precise regulation of RAGE expression is required during lung formation. Furthermore, abundant RAGE results in profound alterations in epithelial cell differentiation that culminate in severe respiratory distress and perinatal lethality.

β2-Adrenergics in Hypoxia Desensitize Receptors but Blunt Inhibition of Reabsorption in Rat Lungs

Alveolar edema and decreased inspired Po(2) decrease the oxygen supply to alveolar epithelia, impairing β(2)-adrenergic receptor (β2AR) signaling and alveolar reabsorption. β2AR agonists potently stimulate alveolar reabsorption. Thus, hypoxia impairs a major defense mechanism that provides protection from alveolar edema. Because in vivo data on the combined effects of prolonged hypoxia and β2AR agonist treatment on β2AR signaling are sparse, we tested whether in vivo hypoxia augments the inactivation of β2AR during prolonged stimulation. Rats were exposed to normoxia (N) and hypoxia (8% O(2); H), and were also treated with terbutaline (T; 2.5 mg/kg, intraperitoneal, twice daily) or saline (S) for 4 days. β2AR signaling was studied in alveolar epithelial (ATII) cells and in whole-lung tissue from treated rats. The terbutaline-stimulated formation of cyclic adenosine monophosphate was decreased by approximately 40% in whole lung and in ATII cells of NT, HS, and HT. The effects were not additive. The β2AR number was increased in HS, but decreased in NT and HT. Treatment increased the G-protein-coupled receptor kinase 2 protein in the plasma membranes of ATII cells, but did not affect G proteins. In vivo hypoxia significantly decreased total and amiloride-sensitive alveolar fluid reabsorption, which was prevented by acute alveolar treatment and 4 days of systemic terbutaline treatment. The αENaC (subunit of epithelial Na channels) protein in plasma membranes was increased in HT, without effects on mRNA. These results indicate that prolonged alveolar hypoxia and treatment with terbutaline impaired β2AR signaling in alveolar epithelia and in whole lungs, and this signaling was not further impaired by hypoxia. Despite impaired β2AR signaling, treatment with terbutaline for 4 days prevented the inhibition of alveolar reabsorption caused by in vivo hypoxia.

Effect of therapeutic hypercapnia on type I alveolar cells in transplanted lung in rats

Objective To investigate the effects of therapeutic hypercapnia on type Ⅱ alveolar cells (ATⅡ ) in the transplanted lung in rats.Methods Twenty-eight pathogen free adult male Wistar rats weighing 180-220 g were randomly divided into 2 groups (n= 14 each) : control group (group C) and therapeutic hypercapnia group (group T). The animals were anesthetized with intraperitoneal 3% pentobarbital 30 mg/kg, tracheostomized and mechanically ventilated with 50% O2-50% N2 (VT 10 ml/kg, RR 60 bpm, I∶ E1∶1). Left lung transplantation was performed. In group T starting from the beginning of reperiusion of the transplanted lung, the animals were ventilated with a mixture of 50% O2-N2 and C02(in appropriate concentrations) to keep PaCO2 between 80-100 mm Hg. After 90 min of reperfusion of the transplanted lung, blood samples were collected from pulmonary vein of the transplanted lung and blood gas analysis was performed. Oxygenation index was calculated.AT II cells were isolated from the transplanted lung and purified and examined with electronic microscope. The apoptosis rate of AT Ⅱ cells was detected by flow cytometry. Results Oxygenation index was significantly higher, the apoptotic rate of ATⅡ cells lower, the damage to ATⅡ cells was less in group T than in group C.Conclusion Therapeutic hypercapnia can protect the AT Ⅱ cells in the transplanted lung and improve the function of the trans planted lung. Key words: Hypercapnia; Lung transplantation; Pulmonary alveoli; Epithelial cells

Sepsis impairs alveolar epithelial function by downregulating Na-K-ATPase pump

Widespread vascular endothelial injury is the major mechanism for multiorgan dysfunction in sepsis. Following this process, the permeability of the alveolar capillaries is augmented with subsequent increase in water content and acute respiratory distress syndrome (ARDS). Nevertheless, the role of alveolar epithelium is less known. Therefore, we examined alveolar fluid clearance (AFC) using isolated perfused rat lung model in septic rats without ARDS. Sepsis was induced by ligating and puncturing the cecum with a 21-gauge needle. AFC was examined 24 and 48 h later. The expression of Na-K-ATPase proteins was examined in type II alveolar epithelial cells (ATII) and basolateral membrane (BLM). The rate of AFC in control rats was 0.51 ± 0.02 ml/h (means ± SE) and decreased to 0.3 ± 0.02 and 0.33 ± 0.03 ml/h in 24 and 48 h after sepsis induction, respectively (P < 0.0001). Amiloride, significantly decreased AFC in sepsis; conversely, isoproterenol reversed the inhibitory effect of sepsis. The alveolar-capillary barrier in septic rats was intact; therefore the finding of increased extravascular lung water in early sepsis could be attributed to accumulation of protein-poor fluid. The expression of epithelial sodium channel and Na-K-ATPase proteins in whole ATII cells was not different in both cecal ligation and puncture and control groups; however, the abundance of Na-K-ATPase proteins was significantly decreased in BLMs of ATII cells in sepsis. Early decrease in AFC in remote sepsis is probably related to endocytosis of the Na-K-ATPase proteins from the cell plasma membrane into intracellular pools, with resultant inhibition of active sodium transport in ATII cells.

Persistent over‐expression of RAGE in adult mouse lung causes airspace enlargement coincident with emphysema

Receptors for advanced glycation end-products (RAGE) are multiligand cell-surface receptors detected abundantly in pulmonary tissue. RAGE signaling assists in the differentiation of alveolar type (AT) I cells during development and can enhance epithelial adhesion to the basement membrane. RAGE is elevated in COPD which is characterized by enlarged airspaces and inflammation and previous work revealed increased RAGE in both cells and lungs exposed to tobacco smoke. However, the precise contribution of RAGE to emphysema is yet unknown. We generated transgenic mice that conditionally over-express RAGE in ATII cells. RAGE was induced from weaning (20 days of age) until sacrifice date at age 50, 80, and 110 days (representing 30, 60, and 90 days of induced RAGE). H&E staining and mean linear intercepts (Lm) revealed incremental dilation of alveolar spaces as RAGE over-expression persisted. Immunohistochemistry using antibodies for ATI (T1alpha) and ATII (proSP-C) cells revealed a decrease in ATI contribution to respiratory surface area and a marked decrease in ATII cell quantity in the RAGE transgenic mice. Staining for FoxJ1 revealed a detectible decrease in ciliated proximal lung epithelium when compared to controls. These data support the concept that RAGE may function in the progression of diseases such as emphysema. Supported by the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU MEG (PRR)

Inhibition of ion transport across ATII cells by chlorine

Lung fluid homeostasis is regulated by ion transport across respiratory and alveolar epithelia. We sought to determine the mechanisms by which chlorine gas (Cl2) exposure damages ion transport of alveolar type II cells resulting in pulmonary edema. Rat ATII cells were cultured under a liquid-liquid interface and an air liquid interface to produce high resistance monolayers. The purity of ATII cells were more than 90% as proved by anti-SP-C staining. Confluent monolayers were covered with 50 μL of lung epithelila lining fluid and exposed to Cl2 (200 ppm for 30 min) which resulted in significant inhibition of total (Isc) and amiloride sensitive (ΔIscam) currents both at 1h and 24 h post exposure. Western blotting studies showed a 20% decrease of total αENaC 24 h post exposure. These changes were at least in part due to activation of mitogen activated kinase ERK1/2 by reactive intermediates: western blotting studies revealed significant increases in the phosphorylated ERK1/2/total ERK1/2 ratio at 1h post exposure. Pre-incubation of ATII cells with either antioxidants or the ERK1/2 inhibitor (U01260) prevented this activation and reversed the decrease of Isc and ΔIscam. In addition, post exposure (1, 8, 21 h) administration of a mixture of low molecular weight scavengers prevented the decrease of ΔIscam 24 h post exposure, attenuated apoptosis. Supported by NIEHS grants 5U01ES015676 and 1U54NS063739.

Gene expression changes with differentiation of cord blood stem cells to respiratory epithelial cells: a preliminary observation

IntroductionOwing to wide availability, low cost and avoidance of ethical concerns, umbilical cord blood (UCB) provides an attractive source of stem cells for investigational and therapeutic uses. In this study, we sought to characterize the gene expression changes as stem cells from UCB differentiate toward alveolar type II pneumocytes (ATII).MethodsControl and experimental cells were cultured in maintenance medium (mesenchymal stem cell growth medium) or differentiation medium (small airway growth medium (SAGM)), respectively, for 8 days. Total RNA was isolated from control and experimental groups for gene expression profiling and real-time polymerase chain reaction assay.ResultsAnalysis of only mixed cell lines (n = 2) with parameters including a P value of 0.01 and an intergroup gap of 2.0 yielded a set of 373 differentially expressed genes. Prominently upregulated genes included several genes associated with ATII cells and also lung cancers: ALDH3A1, VDR and CHKA. Several upregulated genes have been shown to be integral or related to ATII functioning: SGK1, HSD17B11 and LEPR. Finally, several upregulated genes appear to play a role in lung cancers, including FDXR and GP96. Downregulated genes appear to be associated with bone, muscle and central nervous system tissues as well as other widespread tissues.ConclusionsTo the best of our knowledge, this accounting of the gene expression changes associated with the differentiation of a human UCB-derived stem cell toward an ATII cell represents the first such effort. Dissecting which components of SAGM affect specific gene regulation events is warranted.

Embryonic over‐expression of RAGE in mouse lung causes an imbalance between apoptosis and proliferation leading to severe lung hypoplasia

Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors prominently detected in lung epithelium. Previous experiments demonstrate that over-expression of RAGE in murine alveolar type (AT) II cells during development causes lung hypoplasia and neonatal lethality. In order to characterize lung hypoplasticity, proliferation and apoptosis were evaluated in RAGE over-expressing mice. Histology spanning developmental milestones revealed marked loss of lung tissue beginning in the canalicular stage of lung development and continuing throughout the saccular period (embryonic day (E) 15.5–18.5). In situ cell death detection using TUNEL assay revealed a significant elevation in apoptotic cells in RAGE over-expressing pulmonary tissue at E17.5 compared to age-matched controls. The balance between cell death and proliferation was also characterized by PCNA staining. These findings were further supported by electron micrographic analysis of E18.5 lungs that demonstrated extracellular matrix degradation and ATI and ATII cell death in RAGE over-expressed mice. The data suggest that elevated RAGE expression alters the normal balance between proliferation and apoptosis in the developing lung leading to significant loss in respiratory surface area. Supported by the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).

Effect of exogenous surfactants on viability and DNA synthesis in A549, immortalized mouse type II and isolated rat alveolar type II cells

BackgroundIn mechanically ventilated preterm infants with respiratory distress syndrome (RDS), exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has not been studied directly on alveolar type II cells (ATII cells), a key cell type responsible for alveolar function and repair.ObjectiveThe aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis.MethodsCurosurf® and Alveofact® were applied to two ATII cell lines (human A549 and mouse iMATII cells) and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation.ResultsCurosurf® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models.ConclusionThis study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis.