Alveolar Type II Cells Research Articles

Pulmonary surfactant protein SP‐B promotes exocytosis of lamellar bodies in alveolar type II cells

The release of pulmonary surfactant by alveolar type II (ATII) cells is essential for lowering surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to alveolar collapse. Hydrophobic surfactant protein (SP)-B ensures the proper packing of newly synthesized surfactant particles, promotes the formation of the surface active film at the alveolar air-liquid interface and maintains its proper structure along the respiratory dynamics. We report that membrane-associated SP-B efficiently induces secretion of pulmonary surfactant by ATII cells, at the same level as potent secretagogues such as ATP. The presence in the extracellular medium of lipid-protein complexes containing SP-B activates the P2Y2 purinergic signaling pathway that ultimately triggers exocytosis of lamellar bodies by ATII cells. Our data suggest that SP-B prompts Ca2+-dependent surfactant secretion via ATP release from ATII cells. This result implies that SP-B is not only an essential component for the biophysical function of surfactant but is also a central element in the alveolar homeostasis by eliciting autocrine and paracrine cell stimulation.-Martínez-Calle, M., Olmeda, B., Dietl, P., Frick, M., Pérez-Gil, J. Pulmonary surfactant protein SP-B promotes exocytosis of lamellar bodies in alveolar type II cells.

The cytoprotective role of DJ-1 and p45 NFE2 against human primary alveolar type II cell injury and emphysema

Emphysema is characterized by irreversibly enlarged airspaces and destruction of alveolar walls. One of the factors contributing to this disease pathogenesis is an elevation in extracellular matrix (ECM) degradation in the lung. Alveolar type II (ATII) cells produce and secrete pulmonary surfactants and proliferate to restore the epithelium after damage. We isolated ATII cells from control non-smokers, smokers and patients with emphysema to determine the role of NFE2 (nuclear factor, erythroid-derived 2). NFE2 is a heterodimer composed of two subunits, a 45 kDa (p45 NFE2) and 18 kDa (p18 NFE2) polypeptides. Low expression of p45 NFE2 in patients with emphysema correlated with a high ECM degradation. Moreover, we found that NFE2 knockdown increased cell death induced by cigarette smoke extract. We also studied the cross talk between p45 NFE2 and DJ-1. DJ-1 protein is a redox-sensitive chaperone that protects cells from oxidative stress. We detected that cigarette smoke significantly increased p45 NFE2 levels in DJ-1 KO mice compared to wild-type mice. Our results indicate that p45 NFE2 expression is induced by exposure to cigarette smoke, has a cytoprotective activity against cell injury, and its downregulation in human primary ATII cells may contribute to emphysema pathogenesis.

ResolvinD1 stimulates epithelial wound repair and inhibits TGF-β-induced EMT whilst reducing fibroproliferation and collagen production

Acute and chronic inflammatory lung diseases are often associated with epithelial cell injury/loss and fibroproliferative responses. ResolvinD1 (RvD1) is biosynthesized during the resolution phase of inflammatory response and exerts potent anti-inflammatory and promotes resolution of inflammatory lung diseases. The aim of this study was to investigate whether RvD1 exerts protective effects on alveolar epithelial cell function/differentiation and protects against fibroproliferative stimuli. Primary human alveolar type II cells were used to model the effects of RvD1 in vitro upon wound repair, proliferation, apoptosis, transdifferentiation, and epithelial–mesenchymal transition (EMT). Effects of RvD1 upon primary human lung fibroblast proliferation, collagen production, and myofibroblast differentiation were also examined. RvD1 promoted alveolar type II (ATII) cell wound repair and proliferation. RvD1 protected ATII cells against sFas-ligand/TNF-α-induced apoptosis and inhibition on cell proliferation and viability. RvD1 promoted ATII cells transdifferentiation. Moreover, we demonstrate that RvD1 inhibited EMT in response to TGF-β. Furthermore RvD1 inhibited human lung fibroblast proliferation, collagen production, and myofibroblast differentiation induced by both TGF-β and bronchoalveolar lavage fluid from acute respiratory distress syndrome (ARDS) patients. The effects of RvD1 were PI3-kinase dependent and mediated via the resolvin receptor. RvD1 seems to promote alveolar epithelial repair by stimulating ATII cells wound repair, proliferation, reducing apoptosis, and inhibiting TGF-β-induced EMT. While RvD1 reduced fibroproliferation, collagen production, and myofibroblast differentiation. Together, these results suggest a potential new therapeutic strategy for preventing and treating chronic diseases (such as idiopathic pulmonary fibrosis) as well as the fibroproliferative phase of ARDS by targeting RvD1 actions that emphasizes natural resolution signaling pathways.

Isolation and Characterization of Human Alveolar Type II Cells.

Alveolar type II (ATII) cells synthesize, store, and secrete pulmonary surfactant and restore the epithelium after damage to the alveolar epithelium. Isolation of human ATII cells provides a valuable tool to study their function under normal and pathophysiological conditions. Moreover, maintenance of their differentiated phenotype in vitro allows further study of their function. Here we describe a protocol for efficient ATII cell isolation, characterization, and culture.

Intratracheal instillation of alveolar type II cells enhances recovery from acute lung injury in rats

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by excess production of inflammatory factors. Alveolar type II (ATII) cells help repair damaged lung tissue, rapidly proliferating and differentiating into alveolar type I cells after epithelial cell injury. In ALI, the lack of viable ATII favors progression to more severe lung injury. ATII cells regulate the immune response by synthesizing surfactant and other anti-inflammatory proteins and lipids. Cross-talk between ATII and other cells such as macrophages may also be part of the ATII function. The aim of this study was to test the anti-inflammatory and reparative effects of ATII cells in an experimental model of ALI. In this study ATII cells (2.5 × 106 cells/animal) were intratracheally instilled in rats with HCl and lipopolysaccharide (LPS)-induced ALI and in healthy animals to check for side effects. The specific effect of ATII cells was compared with fibroblast transplantation. ATII cell transplantation promoted recovery of lung function, decrease mortality and lung inflammation of the animals with ALI. The primary mechanisms for benefit were paracrine effects of prostaglandin E2 (PGE2) and surfactant protein A (SPA) released from ATII cells that modulate alveolar macrophages to an anti-inflammatory phenotype. To our knowledge, these data are the first to provide evidence that ATII cells secrete PGE2 and SPA, reducing pro-inflammatory macrophage activation and ALI. ATII cells and their secreted molecules have shown an ability to resolve ALI, thereby highlighting a potential novel therapeutic target.

Deletion of SMARCA4 impairs alveolar epithelial type II cells proliferation and aggravates pulmonary fibrosis in mice

Alveolar epithelial cells (AECs) injury and failed reconstitution of the AECs barrier are both integral to alveolar flooding and subsequent pulmonary fibrosis (PF). Nevertheless, the exact mechanisms regulating the regeneration of AECs post-injury still remain unclear. SMARCA4 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF, which is essential for kidney and heart fibrosis. We investigates SMARCA4 function in lung fibrosis by establishing PF mice model with bleomycin firstly and found that the expression of SMARCA4 was mainly enhanced in alveolar type II (ATII) cells. Moreover, we established an alveolar epithelium-specific SMARCA4-deleted SP-C-rtTA/(tetO)7-Cre/SMARCA4f/f mice (SOSM4Δ/Δ) model, as well as a new SMARCA4-deleted alveolar type II (ATII)-like mle-12 cell line. We found that the bleomycin-induced PF was more aggressive in SOSM4Δ/Δ mice. Also, the proliferation of ATII cells was decreased with the loss of SMARCA4 in vivo and in vitro. In addition, we observed increased proliferation of ATII cells accompanied by abnormally high expression of SMARCA4 in human PF lung sections. These data uncovered the indispensable role of SMARCA4 in the proliferation of ATII cells, which might affect the progression of PF.

Differential Expression of VEGF-Axxx Isoforms Is Critical for Development of Pulmonary Fibrosis.

Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)-A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF-A gene produces multiple functional isoforms including VEGF-A165a and VEGF-A165b, a member of the inhibitory family. To date there is no clear information on the role of VEGF-A in IPF. To establish VEGF-A isoform expression and functional effects in IPF. We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin-induced lung fibrosis model we used wild-type MMTV mice and a triple transgenic mouse SPC-rtTA+/-TetoCre+/-LoxP-VEGF-A+/+ to conditionally induce VEGF-A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. IPF and normal lung fibroblasts differentially expressed and responded to VEGF-A165a and VEGF-A165b in terms of proliferation and matrix expression. Increased VEGF-A165b was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII-specific deficiency of VEGF-A or constitutive overexpression of VEGF-A165b inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF-A165b to wild-type mice. These results indicate that changes in the bioavailability of VEGF-A sourced from ATII cells, namely the ratio of VEGF-Axxxa to VEGF-Axxxb, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair.

Abstract 500: Impairment of DNA double strand break repair in human primary alveolar type II cells in emphysema

Abstract Rationale Emphysema is caused by the destruction of alveolar wall septa. The major risk factor for this disease is cigarette smoke and effective therapies are very limited. Alveolar type II (ATII) cells are in the gas exchange portion of the lung. They make and secrete pulmonary surfactant, and proliferate to restore the epithelium after damage to the more sensitive alveolar type I cells. Methods Control ATII cells were isolated from deidentified control non-smoker and smoker organ donors whose lungs were not suitable for transplantation and donated for medical research. Furthermore, as a unique approach, we have developed a new method on how to isolate ATII cells from excess tissue from lung transplants obtained from patients with emphysema using magnetic microbeads. We determined DNA damage, DNA repair, oxidative stress, injury, and inflammation in human primary ATII cells isolated from these individuals in comparison with controls. Results Our data indicates high oxidative stress in human ATII cells induced by cigarette smoke extract in vitro as measured by 4-HNE staining by immunocytofluorescence. We also observed DNA double strand breaks, high DNA damage, and low DNA repair in these cells. Moreover, we found greater proinflammatory response as determined by IL-8 and IL-6 levels by ELISA. Furthermore, ATII cells obtained from patients with emphysema had greater injury compared to cells obtained from control non-smokers and smokers. Conclusion Our results indicate oxidative ATII cell damage induced by cigarette smoke and in emphysema. Observed DNA damage/repair imbalance may contribute to cell death in this disease. The study of ATII cell injury may improve our knowledge on this disease pathogenesis and may lead to novel therapeutic strategies to slow the progression of emphysema. Note: This abstract was not presented at the meeting. Citation Format: Beata Kosmider, Liudmila Vlasenko, Nathaniel Marchetti, Sudhir Bolla, Chenna Mandapati, Nathaniel Xander, Gerard Criner, Karim Bahmed. Impairment of DNA double strand break repair in human primary alveolar type II cells in emphysema [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 500. doi:10.1158/1538-7445.AM2017-500

Abstract 1327: CD147 contributes to extracellular matrix remodeling and degradation

Abstract Chronic obstructive pulmonary disease (COPD) is an incurable, progressive lung disease characterized by shortness and difficulty in breathing. Patients with COPD are at higher risk of developing lung cancer and often are associated with poor outcome of lung cancer diagnosis and treatment. Extracellular matrix (ECM) remodeling and degradation is one of the common hallmarks between cancer and COPD. It contributes to degradation of air sacs (emphysema) or small airways (obstructive bronchiolitis). It is also involved in cancer initiation and progression to assist tumor expansion and cancer cell migration. In this study, we analyzed lung tissue and primary alveolar type II (ATII) cells isolated from patients with emphysema and control organ donors. ATII cells secrete and store pulmonary surfactant and restore the epithelium after damage. We performed RNA sequencing in ATII cells to identify dysregulated proteins, which are involved in ECM degradation in emphysema. To further study the mechanism involved in the ECM degradation in this disease, we analyzed the expression of selected proteins and we focused on the role of extracellular matrix metalloproteinase inducer (CD147). CD147 is a cell membrane protein involved in inducing the production of extracellular matrix metalloproteinases (MMPs). First, we found that cells isolated from patients with emphysema have higher expression of CD147 in ATII cells in comparison with controls as detected by western blotting and RT-PCR. This may explain high ECM degradation in this disease. Second, we observed higher MMPs levels in plasma obtained from individuals with this disease. Third, we found significantly higher expression of cathepsin B in ATII cells isolated from emphysema in comparison with controls. Cathepsin B is an enzyme that facilitates a direct degradation of ECM proteins and activate other proteases capable of degrading ECM. Fourth, our data indicate that CD147 interacts with cathepsin B, which may lead to the activation of MMPs and emphysema progression. In summary, our results show for the first time the important role of CD147 in emphysema development. Moreover, we found that cathepsin B may contribute to CD147-mediated disease progression. Further studies will elucidate its role in lung cancer development in patients with emphysema. Note: This abstract was not presented at the meeting. Citation Format: Tiffany Tan, Beata Kosmider, Nathaniel Marchetti, Sudhir Bolla, Chenna Mandapati, Gerard Criner, Karim Bahmed. CD147 contributes to extracellular matrix remodeling and degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1327. doi:10.1158/1538-7445.AM2017-1327

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

The emission of cerium oxide nanoparticles (CeO2) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO2 induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO2-induced fibrosis. Male Sprague-Dawley rats were exposed to CeO2 (0.15 to 7mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28days after CeO2 (3.5mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO2-exposed rats at 28days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO2 exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO2-exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO2 exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO2 nanoparticle exposure.

Lung injury and inflammation response by chronic intermittent hypoxia in rats

BackgroundChronic intermittent hypoxia is the primary pathophsiological feature of obstructive sleep apnea/hypopnea syndrome. The characteristics of CIH can be imitated by animal experiment models thus to study CIH related systemic organ injuries, including respiratory systems.MethodsSixteen male SD rats were randomly divided into two groups: CIH group (n = 8) and control group (n = 8). The CIH group was exposed to intermittent hypoxia circumstance for 5 weeks (8 h/day) and control group was placed in the same animal chamber exposed to normal air circumstance. Between the two groups, the inflammatory factors of IL-6 and TNF-α within serum and BALF were measured by ELISA; the structure and ultrastructure of lungs were evaluated by HE staining and electronmicroscopy. The nuclear factor-κB (NF-κB) in lung tissue was detected by Western blot.ResultsThe concentration of IL-6 and TNF-α in serum in CIH group increased significantly (138.63 ± 43.82 vs. 41.82 ± 5.24 pg/ml and 126.62 ± 34.81 vs. 73.43 ± 5.72 pg/ml, both P < 0.05). The concentration of IL-6 and TNF-α in BALF in CIH group was higher than that in control group (67.1 ± 24.2 pg/ml vs 39.8 ± 21.5 pg/ml and 36.61 ± 19.17 pg/ml vs 20.31 ± 8.44 pg/ml, respectively, P < 0.05). In the HE staining of lung tissue, results showed that severe inflammatory cell infiltration in alveolar walls and alveolar spaces was found in CIH group and the histological score of CIH group is higher significantly (6.857 ± 0.553 vs. 2.286 ± 0.286 g, P < 0.05); In the electronmicroscopy of TypeIIalveolar cells, results showed that karyotheca and endoplasmic reticulum were damaged obviously in Type II alveolar cells. The NF-κB increased significantly in the CIH group compared with control group (0.43 ± 0.1 vs 0.22 ± 0.05, P < 0.05).ConclusionsAnimal experiment model can be used to imitate the pathophysiologic changes of CIH. There are systematic and local airway inflammation coexisting in CIH rats. CIH leads to inflammatory cell infiltration and organelle damages within lung tissues. We speculated that there was some correlation between inflammation and lung damages in CIH rats.

Stretch-induced apoptosis in rat alveolar epithelial cells is mediated by the intrinsic mitochondrial pathway

ABSTRACTBackground: Alveolar type II (ATII) cells in the lung are exposed to mechanical stretch during breathing and mechanical ventilation. Increased mechanical stretch contributes to lung injury by induction of apoptosis and necrosis in ATII cells. Aim of the Study: In this study, we investigated the intrinsic and the extrinsic apoptosis pathways, and their involvement in our model of stretch-induced apoptosis. Material and Methods: ATII cells were stretched on elastic membranes for 24 h and apoptosis was determined at different time points. Several factors of the intrinsic and the extrinsic pathway were investigated by FACS, ELISA, and Western blot. We also investigated the inhibition of reactive oxygen species (ROS) expression and cytochrome C release and their influence on induction of apoptosis by stretching. Results: In comparison to static cells, ROS generation and cytochrome C release were increased in stretched cells while at the same time, mitochondrial membrane potential was reduced. Both inhibition of ROS generation by tocopherol and inhibition of cytochrome C release by cyclosporine A led to reduction of stretch-induced apoptosis. An increase of caspase 8 and 9 was observed in stretched cells compared to static cells. In contrast, factors of the extrinsic pathway such as TNF-alpha, TRAIL, TNFRI, Fas, FasL, and FADD were not different to static cells at all time points or where not detectable. Conclusion: Together, these findings suggest predominance of the intrinsic pathway in stretch-induced apoptosis.

Crosstalk between Mitochondrial Fission and Oxidative Stress in Paraquat-Induced Apoptosis in Mouse Alveolar Type II Cells.

Paraquat (PQ), as a highly effective and nonselective herbicide, induces cell apoptosis through generation of superoxide anions which forms reactive oxygen species (ROS). Mitochondria, as regulators for cellular redox signaling, have been proved to play an important role in PQ-induced cell apoptosis. This study aimed to evaluate whether and how mitochondrial fission interacts with oxidative stress in PQ-induced apoptosis in mouse alveolar type II (AT-II) cells. Firstly, we demonstrated that PQ promoted apoptosis and release of cytochrome-c (Cyt-c). Furthermore, we showed that PQ broke down mitochondrial network, enhanced the expression of fission-related proteins, increased Drp1 mitochondrial translocation while decreased the expression of fusion-related proteins in AT-II cells. Besides, inhibiting mitochondrial fission using mdivi-1, a selective inhibitor of Drp1, markedly attenuated PQ-induced apoptosis, release of Cyt-c and the generation of ROS. These results indicate that mitochondrial fission involves in PQ-induced apoptosis. Further study demonstrated that antioxidant ascorbic acid inhibited Drp1 mitochondrial translocation, mitochondrial fission and attenuated PQ-induced apoptosis. Overall, our findings suggest that mitochondrial fission interplays with ROS in PQ-induced apoptosis in mouse AT-II cells and mitochondrial fission could serve as a potential therapeutic target in PQ poisoning.

Lethal H1N1 influenza A virus infection alters the murine alveolar type II cell surfactant lipidome.

Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has not been explored. C57BL/6 mice were inoculated intranasally with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days postinfection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by ultra-high-performance liquid chromatography-mass spectrometry. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'-diphosphocholine and 5'-diphosphoethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased amounts of sphingomyelin, cholesterol, and protein. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of acute respiratory distress syndrome in influenza-infected mice.

Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

Epithelial disruption of Gab1 perturbs surfactant homeostasis and predisposes mice to lung injuries.

GRB2-associated-binding protein 1 (Gab1) belongs to Gab adaptor family, which integrates multiple signals in response to the epithelial growth factors. Recent genetic studies identified genetic variants of human Gab1 gene as potential risk factors of asthmatic inflammation. However, the functions of Gab1 in lungs remain largely unknown. Alveolar type-II cells (AT-IIs) are responsible for surfactant homeostasis and essentially regulate lung inflammation following various injuries (3). In this study, in vitro knockdown of Gab1 was shown to decrease the surfactant proteins (SPs) levels in AT-IIs. We further examined in vivo Gab1 functions through alveolar epithelium-specific Gab1 knockout mice (Gab1Δ/Δ). In vivo Gab1 deficiency leads to a decrease in SP synthesis and the appearance of disorganized lamellar bodies. Histological analysis of the lung sections in Gab1Δ/Δ mice shows no apparent pathological alterations or inflammation. However, Gab1Δ/Δ mice demonstrate inflammatory responses during the LPS-induced acute lung injury. Similarly, in mice challenged with bleomycin, fibrotic lesions were found to be aggravated in Gab1Δ/Δ These observations suggest that the abolishment of Gab1 in AT-IIs impairs SP homeostasis, predisposing mice to lung injuries. In addition, we observed that the production of surfactants in AT-IIs overexpressing Gab1 mutants, in which Shp2 phosphatase and PI3K kinase binding sites have been mutated (Gab1ΔShp2, Gab1ΔPI3K), has been considerably attenuated. Together, these findings provide the direct evidence about the roles of docking protein Gab1 in lungs, adding to our understanding of acute and interstitial lung diseases caused by the disruption of alveolar SP homeostasis.

Regulation effect of β-catenin pathway on TGF-β1 induced pulmonary pro-fibrosis

To investigate the regulation effect of β-catenin pathway on transforming growth factor beta1 (TGF-β1) induced pulmonary pro-fibrosis. The rat alveolar typeⅡ cells (RLE-6TN) were divided into four groups: A1.control group; B1.TGF-β1 group was treated with 5 μg/L TGF-β1; C1.pcDNA+ TGF-β1 group was transiently transfected with eukaryotic expression vector pcDNA3.0 (pcDNA) and followed by TGF-β1 treatment (5 μg/L); D1.F-(β-TrCP)-Ecad+ TGF-β1 group was transiently transfected with β-catenin protein knockout vector [F-(β-TrCP)-Ecad] and followed by TGF-β1 treatment (5 μg/L). After 24 hours, cells were observed under the inverted phase contrast microscope, then the expressions of E-cadherin, α-smooth muscle actin (α-SMA) and fibronectin (Fn) in each group were measured by Western blot and the mRNA levels of Snail which was the downstream profibrotic transcription production in cell culture supernatants of each group were detected by real-time fluorescence quantification-polymerase chain reaction (RT-PCR) .The rat alveolar macrophages (CRL-2192) were divided into five groups: A2.control group; B2.Interferon gamma (IFN-γ) group was treated by 20 μg/L IFN-γ; C2.TGF-β1+ IFN-γ group was treated by 20 μg/L IFN-γ with 10 μg/L TGF-β1; D2.F-(β-TrCP)-Ecad+ TGF-β1+ IFN-γ group was transfected with F-(β-TrCP)-Ecad and other dispose was the same as group C2; E2.WTβ-catenin+ TGF-β1+ IFN-γ group was transfected with WTβ-catenin and other dispose was the same as group C2.After 24 hours, protein levels of β-catenin in group A2, B2, C2 were determined by Western blot.Inducible nitric oxide synthase (iNOS) mRNA levels of each group were detected by RT-PCR. The RLE-6TN cells of group B1, C1 showed a change in morphology to spindle-shaped cells, the cells of group D1 maintained a cobblestone morphology. Protein expressions of the fibroblast markers α-SMA and Fn, and mRNA expressions of the downstream profibrotic transcription production Snail of group B1, C1 were significantly higher than group A1, while protein expressions of the epithelial marker E-cadherin were significantly lower.The protein expressions of α-SMA, Fn and mRNA expressions Snail of group D1 were significantly lower than group C1 (0.352±0.076 vs 0.937±0.303, 0.319±0.072 vs 0.903±0.211, 3.675±0.642 vs 9.708±2.031), while the protein expressions of E-cadherin were significantly higher (1.482±0.227 vs 0.604±0.121) (all P<0.05). The steady state protein levels of β-catenin in CRL-2192 cells was low and β-catenin protein expressions of CRL-2192 cells in group A2, B2 and C2 had no significantly statistical differences.The mRNA expressions of iNOS of group B2 cells were significantly higher than group A2, C2, D2, E2 (64.95±4.47 vs 9.87±0.73, 21.32±2.41, 18.35±3.61, 22.87±3.14) (all P<0.01), the expressions of iNOS of group C2, D2, E2 were all higher than group A2 (all P<0.05), but there were no significant differences among group C2, D2 and E2. Inhibition of β-catenin pathway inhibits TGF-β1-induced epithelial-mesenchymal transition (EMT) and has no effect on its anti-inflammation effect.Therefore, β-catenin pathway regulates the pulmonary pro-fibrosis effect of TGF-β1.

Perinatal nicotine exposure induces myogenic differentiation, but not epithelial-mesenchymal transition in rat offspring lung.

Perinatal nicotine exposure alters offspring lung structure and function; however, the underlying mechanisms remain incompletely understood. Whether epithelial-mesenchymal transition (EMT), a known contributor to pulmonary pathology, occurs following moderate perinatal nicotine exposure is not known. Pregnant, pair-fed Sprague Dawley rat dams received either placebo (diluent) or nicotine [1 mg/kg, subcutaneously] once daily from embryonic day (e) 6 to postnatal day (PND) 21. Generation 1 (F1) and 3 (F3) offspring lungs were isolated at PND 21, and using Western analysis, q-RT-PCR and immunohistochemistry examined for evidence of EMT. To gain further supportive evidence for nicotine-induced EMT, embryonic day 19 primary rat lung alveolar type II (ATII) cells were cultured and treated with nicotine for 24 hr. Protein levels of α-smooth muscle actin, fibronectin, and calponin (myogenic differentiation markers) increased significantly. However, surfactant proteins B and C, and cholinephosphate cytidylyltransferase-α (epithelial cell markers), as well as the typical markers of EMT, E-cadherin, N-cadherin, and fibroblast specific protein (FSP)-1, in both F1 and F3 generation lungs, showed no significant change between the nicotine exposed and control dams. Immunostaining of lung sections and data from in vitro treated ATII cells strongly supported the Western data. Enhanced myogenic molecular profile, without evidence of EMT, as evidenced by the absence of the loss of E-cadherin or gains in N-cadherin and FSP-1, suggest that perinatal nicotine exposure does not result in EMT, but it leads to myogenesis, which predominantly accounts for the lung phenotype seen in perinatally nicotine exposed rat offspring. Pediatr Pulmonol. 2016;51:1142-1150. © 2016 Wiley Periodicals, Inc.

Heparan Sulfate is Shed into the Alveolus after Intratracheal Lung Injury and Facilitates Alveolar Epithelial Growth Factor Signaling and Lung Repair

Heparan sulfate proteoglycans (HSPGs) are proteins covalently bound to heparan sulfate (HS), a linear highly‐sulfated polysaccharide, and are present in both the extracellular matrix and on cell surfaces. During tissue injury, HSPGs are thought to be shed from the injured cell surface; for example, the protein component of the cell membrane‐bound HSPG, syndecan‐1, has been shown to be shed from the respiratory epithelial cell surface into the airway during naphthalene lung injury. However, it remains unknown whether heparan sulfate (HS) is also shed into the airspace during intratracheal (IT) lung injury. Additionally, as HS is known to function as a scaffolding molecule for numerous growth factor ligands and receptors, facilitating ligand‐receptor binding and enhancing signaling, it is important to determine if HS is shed into the airspace during lung injury, as the shed HS may affect growth factor signaling and lung repair.To determine if HS is shed into the airspace during lung injury, we intratracheally instilled lipopolysaccharide (LPS) in mice and performed mass spectrometry for HS in bronchoalveolar (BAL) fluid collected from mice days 2, 4, 6, and 8 after IT LPS instillation. We detected increased BAL HS on days 2 (2.3 fold) and 4 (3.2 fold) after IT LPS instillation in comparison to animals instilled with PBS as control. To determine if the airspace HS enhances lung epithelial growth factor signaling and repair, we tested if exogenous HS enhances ERK signaling in alveolar type II (ATII) epithelial cells, progenitor cells that re‐epithelialize the alveolus after injury. We treated MLE‐12 cells (a mouse ATII cell line) with 0.5μg/ml HS and determined that, while HS inhibits ERK signaling in naïve MLE‐12 cells, HS permits ERK signaling in MLE‐12 cells that are enzymatically devoid of cell‐surface HS. This finding suggests that airspace HS may facilitate growth factor signaling in ATII cells that have shed their cell‐surface HS during injury. Finally, to determine if supplementation with exogenous HS enhances lung repair in vivo, we treated animals with intravenous HS on day 4 after IT LPS instillation and measured outcomes of lung injury. Twenty‐four hours after supplementation, HS reduced BAL total protein by 30%, indicating that exogenous HS supplementation may enhance alveolar epithelial barrier function and lung repair.Support or Funding InformationNHLBI RO1 HL125371

Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease withlimited response to currently available therapies. Alveolar type II (ATII) cells act as progenitor cells in the adult lung, contributing to alveolar repair during pulmonary injury. However, in IPF, ATII cells die and are replaced by fibroblasts and myofibroblasts. In previous preclinical studies, we demonstrated that ATII-cell intratracheal transplantation was able to reduce pulmonary fibrosis. The main objective of this study was to investigate the safety and tolerability of ATII-cell intratracheal transplantation in patients with IPF. We enrolled 16 patients with moderate and progressive IPF who underwent ATII-cell intratracheal transplantation through fiberoptic bronchoscopy. We evaluated the safety and tolerability of ATII-cell transplantation by assessing the emergent adverse sideeffects that appeared within 12months. Moreover, pulmonary function, respiratory symptoms, and disease extent during 12months of follow-up were evaluated. No significant adverse events were associated with the ATII-cell intratracheal transplantation. After 12months of follow-up, there was no deterioration in pulmonary function, respiratory symptoms, or disease extent. Our results support the hypothesis that ATII-cell intratracheal transplantation is safe and well tolerated in patients with IPF. This study opens the door to designing a clinical trial to elucidate the potential beneficial effects of ATII-cell therapy in IPF.