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In vitro lung research requires appropriate cell culture models that adequately mimic in vivo structure and function. Previously, researchers extensively used commercially available and easily expandable A549 and NCI-H441 cells, which replicate some but not all features of alveolar epithelial cells. Specifically, these cells are often restricted by terminally altered expression while lacking important alveolar epithelial characteristics. Of late, human primary alveolar epithelial cells (hPAEpCs) have become commercially available but are so far poorly specified. Here, we applied a comprehensive set of technologies to characterize their morphology, surface marker expression, transcriptomic profile, and functional properties. At optimized seeding numbers of 7,500 cells per square centimeter and growth at a gas-liquid interface, hPAEpCs formed regular monolayers with tight junctions and amiloride-sensitive transepithelial ion transport. Electron microscopy revealed lamellar body and microvilli formation characteristic for alveolar type II cells. Protein and single-cell transcriptomic analyses revealed expression of alveolar type I and type II cell markers; yet, transcriptomic data failed to detect NKX2-1, an important transcriptional regulator of alveolar cell differentiation. With increasing passage number, hPAEpCs transdifferentiated toward alveolar-basal intermediates characterized as SFTPC - , KRT8 high , and KRT5 - cells. In spite of marked changes in the transcriptome as a function of passaging, Uniform Manifold Approximation and Projection plots did not reveal major shifts in cell clusters, and epithelial permeability was unaffected. The present work delineates optimized culture conditions, cellular characteristics, and functional properties of commercially available hPAEpCs. hPAEpCs may provide a useful model system for studies on drug delivery, barrier function, and transepithelial ion transport in vitro .

Our previous study showed that in adult mice, conditional Nedd4-2 -deficiency in club and alveolar epithelial type II (AE2) cells results in impaired mucociliary clearance, accumulation of Muc5b and progressive, terminal pulmonary fibrosis within 16 weeks. In the present study, we investigated ultrastructural alterations of the alveolar epithelium in relation to interstitial remodeling in alveolar septa as a function of disease progression. Two, eight and twelve weeks after induction of Nedd4-2 knockout, lungs were fixed and subjected to design-based stereological investigation at the light and electron microscopic level. Quantitative data did not show any abnormalities until 8 weeks compared to controls. At 12 weeks, however, volume of septal wall tissue increased while volume of acinar airspace and alveolar surface area significantly decreased. Volume and surface area of alveolar epithelial type I cells were reduced, which could not be compensated by a corresponding increase of AE2 cells. The volume of collagen fibrils in septal walls increased and was linked with an increase in blood-gas barrier thickness. A high correlation between parameters reflecting interstitial remodeling and abnormal AE2 cell ultrastructure could be established. Taken together, abnormal regeneration of the alveolar epithelium is correlated with interstitial septal wall remodeling.

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.

Background: Hyperoxia downregulates the tight junction (TJ) proteins of the alveolar epithelium and leads to barrier dysfunction. Previous study has showed that STE20/SPS1-related proline/alanine-rich kinase (SPAK) interferes with the intestinal barrier function in mice. The aim of the present study is to explore the association between SPAK and barrier function in the alveolar epithelium after hyperoxic exposure., Methods: Hyperoxic acute lung injury (HALI) was induced by exposing mice to > 99% oxygen for 64 h. The mice were randomly allotted into four groups comprising two control groups and two hyperoxic groups with and without SPAK knockout. Mouse alveolar MLE-12 cells were cultured in control and hyperoxic conditions with or without SPAK knockdown. Transepithelial electric resistance and transwell monolayer permeability were measured for each group. In-cell western assay was used to screen the possible mechanism of p-SPAK being induced by hyperoxia., Results: Compared with the control group, SPAK knockout mice had a lower protein level in the bronchoalveolar lavage fluid in HALI, which was correlated with a lower extent of TJ disruption according to transmission electron microscopy. Hyperoxia down-regulated claudin-18 in the alveolar epithelium, which was alleviated in SPAK knockout mice. In MLE-12 cells, hyperoxia up-regulated phosphorylated-SPAK by reactive oxygen species (ROS), which was inhibited by indomethacin. Compared with the control group, SPAK knockdown MLE-12 cells had higher transepithelial electrical resistance and lower transwell monolayer permeability after hyperoxic exposure. The expression of claudin-18 was suppressed by hyperoxia, and down-regulation of SPAK restored the expression of claudin-18. The process of SPAK suppressing the expression of claudin-18 and impairing the barrier function was mediated by p38 mitogen-activated protein kinase (MAPK)., Conclusions: Hyperoxia up-regulates the SPAK-p38 MAPK signal pathway by ROS, which disrupts the TJ of the alveolar epithelium by suppressing the expression of claudin-18. The down-regulation of SPAK attenuates this process and protects the alveolar epithelium against the barrier dysfunction induced by hyperoxia.

Alveolar type II (ATII) epithelial cells contain lamellar bodies (LBs) which synthesize and store lung surfactants. In animals, the inhibition or knockout of leucine-rich repeat kinase 2 (LRRK2) causes abnormal enlargement of LBs in ATII cells. This effect of LRRK2 inhibition in lung is largely accepted as being mediated directly through blocking of the kinase function; however, downstream consequences in the lung remain unknown. In this work we established an in vitro alveolar epithelial cell (AEC) model that recapitulates the in vivo phenotype of ATII cells and developed an assay to quantify changes in LB size in response to LRRK2 inhibitors. Culture of primary human AECs at the air-liquid interface on matrigel and collagen-coated transwell inserts in the presence of growth factors promoted the LB formation and apical microvilli and induced expression of LRRK2 and ATII cell markers. Treatment with a selective LRRK2 inhibitor resulted in pharmacological reduction of phospho-LRRK2 and a significant increase in LB size; effects previously reported in lungs of non-human primates treated with LRRK2 inhibitor. In summary, our human in vitro AEC model recapitulates the abnormal lung findings observed in LRRK2-perturbed animals and holds the potential for expanding current understanding of LRRK2 function in the lung., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bronchopulmonary dysplasia (BPD), also known as chronic lung disease, is one of the most common respiratory diseases in premature new‑born humans. Mitochondria are not only the main source of reactive oxygen species but are also critical for the maintenance of homeostasis and a wide range of biological activities, such as producing energy, buffering cytosolic calcium and regulating signal transduction. However, as a critical quality control method for mitochondria, little is known about the role of mitophagy in BPD. The present study assessed mitochondrial function in hyperoxia‑exposed alveolar type II (AT‑II) cells of rats during lung development. New‑born Sprague‑Dawley rats were divided into hyperoxia (85% oxygen) and control (21% oxygen) groups. Histopathological and morphological properties of the lung tissues were assessed at postnatal days 1, 3, 7 and 14. Ultrastructural mitochondrial alteration was observed using transmission electron microscopy and the expression of the mitophagy proteins putative kinase (PINK)1, Parkin and Nip3‑like protein X (NIX) in the lung tissues was evaluated using western blotting. Immunofluorescence staining was used to determine the co‑localisation of PINK1 and Parkin. Real‑time analyses of extracellular acidification rate and oxygen consumption rate were performed using primary AT‑II cells to evaluate metabolic changes. Mitochondria in hyperoxia‑exposed rat AT‑II cells began to show abnormal morphological and physiological features. These changes were accompanied by decreased mitochondrial membrane potential and increased expression levels of PINK1‑Parkin and NIX. Increased binding between a mitochondria marker (cytochrome C oxidase subunit IV isoform I) and an autophagy marker (microtubule‑associated protein‑1 light chain‑3B) was observed in primary AT‑II cells and was accompanied by decreased mitochondrial metabolic capacity in model rats. Thus, mitophagy mediated by PINK1, Parkin and NIX in AT‑II cells occurred in hyperoxia‑exposed new‑born rats. These findings suggested that the accumulation of dysfunctional mitochondria may be a key factor in the pathogenesis of BPD and result in attenuated alveolar development.

To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology.

The term baby presented with respiratory distress with X-ray pictures consistent as hyaline membrane disease (HMD). Baby was ventilated and treated with surfactant. Because of the persistence of high ventilation needs with X-ray pictures consistent with HMD with a transient response to surfactant every time, the possibility of an inherited disorder of surfactant metabolism was kept. Whole-exome sequencing revealed a novel homozygous missense mutation in the gene for ATP binding cassette transporter protein A3. The baby died after 100 days of ventilation., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic, with increasing deaths worldwide. To date, documentation of the histopathological features in fatal cases of the disease caused by SARS-CoV-2 (COVID-19) has been scarce due to sparse autopsy performance and incomplete organ sampling. We aimed to provide a clinicopathological report of severe COVID-19 cases by documenting histopathological changes and evidence of SARS-CoV-2 tissue tropism., Methods: In this case series, patients with a positive antemortem or post-mortem SARS-CoV-2 result were considered eligible for enrolment. Post-mortem examinations were done on 14 people who died with COVID-19 at the King County Medical Examiner's Office (Seattle, WA, USA) and Snohomish County Medical Examiner's Office (Everett, WA, USA) in negative-pressure isolation suites during February and March, 2020. Clinical and laboratory data were reviewed. Tissue examination was done by light microscopy, immunohistochemistry, electron microscopy, and quantitative RT-PCR., Findings: The median age of our cohort was 73·5 years (range 42-84; IQR 67·5-77·25). All patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease including diabetes and obesity. The major pulmonary finding was diffuse alveolar damage in the acute or organising phases, with five patients showing focal pulmonary microthrombi. Coronavirus-like particles were detected in the respiratory system, kidney, and gastrointestinal tract. Lymphocytic myocarditis was observed in one patient with viral RNA detected in the tissue., Interpretation: The primary pathology observed in our cohort was diffuse alveolar damage, with virus located in the pneumocytes and tracheal epithelium. Microthrombi, where observed, were scarce and endotheliitis was not identified. Although other non-pulmonary organs showed susceptibility to infection, their contribution to the pathogenesis of SARS-CoV-2 infection requires further examination., Funding: None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The new coronavirus SARS-CoV-2, first identified in Wuhan, China in December, 2019, can cause Severe Acute Respiratory Syndrome (SARS) with massive alveolar damage and progressive respiratory failure. We present the relevant autopsy findings of the first patient known to have died from COVID19 pneumonia in Spain, carried out on the 14 th of February, 2020, in our hospital (Hospital Arnau de Vilanova-Lliria, Valencia). Histological examination revealed typical changes of diffuse alveolar damage (DAD) in both the exudative and proliferative phase of acute lung injury. Intra-alveolar multinucleated giant cells, smudge cells and vascular thrombosis were present. The diagnosis was confirmed by reverse real-time PCR assay on a throat swab sample taken during the patient's admission. The positive result was reported fifteen days subsequent to autopsy., (Copyright © 2020 Sociedad Española de Anatomía Patológica. Publicado por Elsevier España, S.L.U. All rights reserved.)

We describe the implementation of a COVID-19 Autopsy Programme in our Hospital, report the main findings from the first autopsy of the programme and briefly review the reports of lung pathology of these patients., (Copyright © 2020. Publicado por Elsevier España, S.L.U.)

Mycoplasma gallisepticum (MG) can cause chronic respiratory disease (CRD) in chickens. While several studies have reported the inflammatory functions of microRNAs during MG infection, the mechanism by which exosomal miRNAs regulate MG-induced inflammation remains to be elucidated. The expression of exosome-microRNA derived from MG-infected chicken type II pneumocytes (CP-II) was screened, and the target genes and function of differentially expressed miRNAs (DEGs) were predicted. To verify the role of exosomal gga-miR-451, Western blot, ELISA and RT-qPCR were used in this study. The results showed that a total of 722 miRNAs were identified from the two exosomal small RNA (sRNA) libraries, and 30 miRNAs (9 up-regulated and 21 down-regulated) were significantly differentially expressed. The target miRNAs were significantly enriched in the treatment group, such as cell cycle, Toll-like receptor signalling pathway and MAPK signalling pathway. The results have also confirmed that gga-miR-451-absent exosomes derived from MG-infected CP-II cells increased inflammatory cytokine production in chicken fibroblast cells (DF-1), and wild-type CP-II cell-derived exosomes displayed protective effects. Collectively, our work suggests that exosomes from MG-infected CP-II cells alter the dynamics of the DF-1 cells, and may contribute to pathology of the MG infection via exosomal gga-miR-451 targeting YWHAZ involving in inflammation., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Gas exchange in the lung takes place via the air-blood barrier in the septal walls of alveoli. The tissue elements that oxygen molecules have to cross are the alveolar epithelium, the interstitium and the capillary endothelium. The epithelium that lines the alveolar surface is covered by a thin and continuous liquid lining layer. Pulmonary surfactant acts at this air-liquid interface. By virtue of its biophysical and immunomodulatory functions, surfactant keeps alveoli open, dry and clean. What needs to be added to this picture is the glycocalyx of the alveolar epithelium. Here, we briefly review what is known about this glycocalyx and how it can be visualized using electron microscopy. The application of colloidal thorium dioxide as a staining agent reveals differences in the staining pattern between type I and type II alveolar epithelial cells and shows close associations of the glycocalyx with intraalveolar surfactant subtypes such as tubular myelin. These morphological findings indicate that specific spatial interactions between components of the surfactant system and those of the alveolar epithelial glycocalyx exist which may contribute to the maintenance of alveolar homeostasis, in particular to alveolar micromechanics, to the functional integrity of the air-blood barrier, to the regulation of the thickness and viscosity of the alveolar lining layer, and to the defence against inhaled pathogens. Exploring the alveolar epithelial glycocalyx in conjunction with the surfactant system opens novel physiological perspectives of potential clinical relevance for future research., Competing Interests: The authors declare no conflict of interest.

Acute lung injury (ALI) is characterized by enhanced permeability of the air-blood barrier, pulmonary edema, and hypoxemia. MicroRNA-21 (miR-21) was shown to be involved in pulmonary remodeling and the pathology of ALI, and we hypothesized that miR-21 knock-out (KO) reduces injury and remodeling in ALI. ALI was induced in miR-21 KO and C57BL/6N (wildtype, WT) mice by an intranasal administration of 75 µg lipopolysaccharide (LPS) in saline ( n = 10 per group). The control mice received saline alone ( n = 7 per group). After 24 h, lung function was measured. The lungs were then excised for proteomics, cytokine, and stereological analysis to address inflammatory signaling and structural damage. LPS exposure induced ALI in both strains, however, only WT mice showed increased tissue resistance and septal thickening upon LPS treatment. Septal alterations due to LPS exposure in WT mice consisted of an increase in extracellular matrix (ECM), including collagen fibrils, elastic fibers, and amorphous ECM. Proteomics analysis revealed that the inflammatory response was dampened in miR-21 KO mice with reduced platelet and neutrophil activation compared with WT mice. The WT mice showed more functional and structural changes and inflammatory signaling in ALI than miR-21 KO mice, confirming the hypothesis that miR-21 KO reduces the development of pathological changes in ALI.

The lung consists of branched structures that are anatomically, developmentally and functionally divided into airway and alveolar regions. Each region contributes to lung-specific functions involving a defense system and gas exchange, and their dysfunction can cause fatal lung diseases. In the alveolar region, the cuboidal alveolar type 2 (AT2) cells account for 90% of the alveolar epithelial cells and serve as the tissue stem cells secreting pulmonary surfactant, and flattened alveolar type I (AT1) cells cover most of the alveolar surface directly contributing to gas exchange adjacent to capillary vessels. It has been difficult to culture alveolar epithelial cells in vitro, as the lineage-specific features of those cells are rapidly lost in a conventional two-dimensional culture setting. The culture of alveolar organoids (AOs) is an emerging technique that can help maintain the features of alveolar epithelial cells in vitro, and their application to human disease modeling is eagerly awaited. We herein describe our method of generating and culturing alveolar epithelial cells and AOs derived from human induced pluripotent stem cells (iPSCs). iPSCs derived from lung disease patients, including those with rare genetic diseases, will make help elucidate the disease mechanism and hopefully identify therapeutic targets., (© 2020 Elsevier Inc. All rights reserved.)

High surface tension at the alveolar air-liquid interface is a typical feature of acute and chronic lung injury. However, the manner in which high surface tension contributes to lung injury is not well understood. This study investigated the relationship between abnormal alveolar micromechanics, alveolar epithelial injury, intra-alveolar fluid properties and remodeling in the conditional surfactant protein B (SP-B) knockout mouse model. Measurements of pulmonary mechanics, broncho-alveolar lavage fluid (BAL), and design-based stereology were performed as a function of time of SP-B deficiency. After one day of SP-B deficiency the volume of alveolar fluid V(alvfluid,par) as well as BAL protein and albumin levels were normal while the surface area of injured alveolar epithelium S(AEinjure,sep) was significantly increased. Alveoli and alveolar surface area could be recruited by increasing the air inflation pressure. Quasi-static pressure-volume loops were characterized by an increased hysteresis while the inspiratory capacity was reduced. After 3 days, an increase in V(alvfluid,par) as well as BAL protein and albumin levels were linked with a failure of both alveolar recruitment and airway pressure-dependent redistribution of alveolar fluid. Over time, V(alvfluid,par) increased exponentially with S(AEinjure,sep). In conclusion, high surface tension induces alveolar epithelial injury prior to edema formation. After passing a threshold, epithelial injury results in vascular leakage and exponential accumulation of alveolar fluid critically hampering alveolar recruitability.

Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis.

In case of a life-threatening, stressful event, the body prepares for an emergency. Indeed, the lung is unique in which alveolar cells are constantly exposed to physical and chemical stresses. This study aimed to study the impact of immobilization stress on the blood-air barrier and how it initiate and maintain an inflammatory response, plus determining the resolution of lung inflammation and repair. There was a significant increase in the plasma levels of stress markers "corticosterone and catecholamines" with a decrease in surfactant protein A (a lung-injury marker). Chronic stress produced a significant increase in the pulmonary oxidative and inflammatory markers malondialdehyde, tumor necrosis factor α, and induced nitric oxide synthase when compared with that of acute stress. Both stresses provoked marked pulmonary morphological and ultrastructural changes with a significant increase in caspase-3 immunoexpression. There was increasing evidence of lung's capacity for repair. This process involved edema resolution, cell proliferation, and tissue remodeling in improving the lung-injury, oxidative, and inflammatory markers., (© 2018 Wiley Periodicals, Inc.)

In human idiopathic pulmonary fibrosis (IPF), collapse of distal airspaces occurs in areas of the lung not (yet) remodeled. Mice lungs overexpressing transforming growth factor-β1 (TGF-β1) recapitulate this abnormality: surfactant dysfunction results in alveolar collapse preceding fibrosis and loss of alveolar epithelial type II (AE2) cells' apical membrane surface area. Here we examined whether surfactant dysfunction-related alveolar collapse due to TGF-β1 overexpression is linked to septal wall remodeling and AE2 cell abnormalities. Three and 6 days after gene transfer of TGF-β1, mice received either intratracheal surfactant (Surf-groups: Curosurf®, 100 mg/kg bodyweight) or 0.9% NaCl (Saline-groups). On days 7 (D7) and 14 (D14), lung mechanics were assessed followed by design-based stereology at light and electron microscopic level to quantify structures. Compared with Saline, Surf showed significantly improved tissue elastance, increased numbers of open alveoli, as well as reduced alveolar size heterogeneity on D7. Deterioration in lung mechanics was highly correlated to the loss of open alveoli. On D14, lung mechanics, number of open alveoli, and alveolar size heterogeneity remained significantly improved in the Surf-group. Volumes of extracellular matrix and collagen fibrils in septal walls were significantly reduced, whereas the apical membrane surface area of AE2 cells was increased in Surf compared with Saline. In remodeled tissue with collapsed alveoli, three-dimensional reconstruction of AE2 cells based on scanning electron microscopy array tomography revealed that AE2 cells were trapped without contact to airspaces in the TGF-β1 mouse model. Similar observations were made in human IPF. Based on correlation analyses, the number of open alveoli and of alveolar size heterogeneity were highly linked with the loss of apical membrane surface area of AE2 cells and deposition of collagen fibrils in septal walls on D14. In conclusion, surfactant replacement therapy stabilizes alveoli and prevents extracellular matrix deposition in septal walls in the TGF-β1 model.

Although mammalian target of rapamycin inhibitors (mTORi) are used to treat various malignancies, they frequently induce active alveolitis and dyslipidemia. Abnormal lipid metabolism affects alveolar surfactant function and results in pulmonary disorders; however, the pathophysiology of lung injury and its relationship with lipid metabolism remain unknown. We investigated the relationship between lipid metabolism and alveolar epithelial injury, focusing on peroxisome proliferator-activated receptor-γ (PPAR-γ) as a lipid stress-related factor in mTORi-induced lung injury. We clinicopathologically examined three patients with mTORi-induced lung injury. We constructed an mTORi injury mouse model using temsirolimus in mice (30 mg/kg/day), with the vehicle control and bleomycin injury groups. We also constructed a cultured alveolar epithelial cell injury model using temsirolimus (0-40 μM) in the mouse lung epithelial cell line MLE-12 and performed analysis with or without pioglitazone (PPAR-γ agonist) treatment. All three patients had dyslipidemia and lung lesions of hyperplastic pneumocytes with foamy and enlarged changes. In the mouse model, temsirolimus induced significantly higher levels of total cholesterol and free fatty acids in serum and higher levels of surfactant protein D in serum and BAL fluid with an increase in inflammatory cytokines in the lung compared to control. Temsirolimus also induced hyperplastic foamy pneumocytes with increased lipid-associated spots and larger round electron-lucent bodies compared to the control or bleomycin groups in microscopic analyses. Multiple lipid-associated spots within the cytoplasm were also induced by temsirolimus administration in MLE-12 cells. Temsirolimus downregulated PPAR-γ expression in mouse lung and MLE-12 cells but upregulated cleaved caspase-3 in MLE-12 cells. Pioglitazone blocked the upregulated cleaved caspase-3 expression in MLE-12 cells. The pathogenesis of mTORi-induced lung disease may be involved in alveolar epithelial injury, via lipid metabolic stress associated with downregulated PPAR-γ expression. Focusing on the relationship between lipid metabolic stress and alveolar epithelial injury represents a potentially novel approach to the study of pulmonary damage.

Bronchopulmonary dysplasia (BPD) seriously affects the health and prognosis of children, but the efficacy of treatments is poor. The present study aimed to examine the effects of C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4) and microtubule‑associated protein light chain 3β (LC3B), and the interaction between CHOP and LC3B, in newborn rats with BPD. At 1, 7, 14 and 21 days, the rats in the model [fraction of inspired oxygen (FiO2)=80‑85%] and control groups (FiO2=21%) were randomly sacrificed, and lung samples were collected. Alveolar development was evaluated according to the radial alveolar count (RAC) and alveolar septum thickness. Ultrastructural changes were observed by transmission electron microscopy (TEM), the expression levels of CHOP, ATF4 and LC3B were determined by immunohistochemistry, and western blot and reverse transcription‑quantitative polymerase chain reaction analyses. The co‑localization of CHOP and LC3B in lung tissues was determined by immunofluorescence. The results showed that, compared with the control group, alveolarization arrest was present in the model group. The TEM observations revealed that, at 14 days, type II alveolar epithelial cell (AECII) lamellar bodies were damaged, with an apparent dilation of the endoplasmic reticulum (ER) and autophagy in cells within the model group. Between days 7 and 14, the protein levels of ATF4, CHOP and LC3B were significantly increased in the model group. The mRNA levels of CHOP and LC3B were lower at days 7‑21. CHOP and LC3B were co‑localized in the cells of the lung tissues at day 14 in the model group. Pearson's correlation analysis showed that the protein levels of CHOP and LC3B‑II were positively correlated in the model groups. As in previous studies, the present study demonstrated that BPD damaged the AECII cells, which exhibited detached and sparse microvilli and the vacuolization of lamellar bodies. In addition, it was found that the ER was dilated, with autophagosomes containing ER and other organelles in AECII cells; the expression levels of CHOP and LC3B‑II were upregulated. CHOP and LC3B‑II may have joint involvement in the occurrence and development of BPD.

The nuclear membrane acts as a mechanosensor that drives cellular responses following changes in the extracellular environment. Mechanically ventilated lungs are exposed to an abnormally high mechanical load that may result in clinically relevant alveolar damage. We report that mechanical ventilation in mice increased the expression of Lamin-A, a major determinant of nuclear membrane stiffness, in alveolar epithelial cells. Lamin-A expression increased and nuclear membrane compliance decreased in human bronchial epithelial cells after a mechanical stretch stimulus and in a murine model of lung injury after positive-pressure ventilation. Reducing Lamin-A maturation by depletion of the protease-encoding gene Zmpste24 preserved alveolar nuclear membrane compliance after mechanical ventilation in mice. Ventilator-induced proapoptotic gene expression changes and lung injury were reduced in mice lacking Zmpste24 compared to wild-type control animals. Similarly, treatment with the human immunodeficiency virus protease inhibitors lopinavir and ritonavir reduced the accumulation of Lamin-A at nuclear membranes and preserved nuclear membrane compliance after mechanical ventilation, mimicking the protective phenotype of Zmpste24 -/- animals. These results show that the pathophysiological response to lung mechanical stretch is sensed by the nuclear membranes of lung alveolar cells, and suggest that protease inhibitors might be effective in preventing ventilator-induced lung injury., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In this study, we describe the isolation and immortalisation of primary murine alveolar epithelial cells (mAEpC), as well as their epithelial differentiation and barrier properties when grown on Transwell® inserts. Like human alveolar epithelial cells (hAEpC), mAEpC transdifferentiate in vitro from an alveolar type II (ATII) phenotype to an ATI-like phenotype and exhibit features of the air-blood barrier, such as the establishment of a thin monolayer with functional tight junctions (TJs). This is demonstrated by the expression of TJ proteins (ZO-1 and occludin) and the development of high transepithelial electrical resistance (TEER), peaking at 1800Ω ·cm². Transport across the air-blood barrier, for general toxicity assessments or preclinical drug development, is typically studied in mice. The aim of this work was the generation of novel immortalised murine lung cell lines, to help meet Three Rs requirements in experimental testing and research. To achieve this goal, we lentivirally transduced mAEpC of two different mouse strains with a library of 33 proliferation-promoting genes. With this immortalisation approach, we obtained two murine alveolar epithelial lentivirus-immortalised (mAELVi) cell lines. Both showed similar TJ protein localisation, but exhibited less prominent barrier properties (TEERmax ~250Ω·cm²) when compared to their primary counterparts. While mAEpC demonstrated their suitability for use in the assessment of paracellular transport rates, mAELVi cells could potentially replace mice for the prediction of acute inhalation toxicity during early ADMET studies., (2018 FRAME.)

Hyperoxia‑induced acute lung injury (HALI) as one of the most common complications in patents on mechanical ventilation, and there are no efficient methods to overcome this at present. It was hypothesized that microRNA 21‑5p(miR‑21‑5p) can promote the survival of type II alveolar epithelial cells (AECII), alleviating HALI. The present study aimed to combine gene chip analysis with the overexpression miR‑21‑5p to develop a novel therapeutic option for HALI. It was found that AECII apoptosis was an important pathogenic event in the development of HALI, and the overexpression of miR‑21‑5p prevented HALI, associated with reducing AECII apoptosis. These results were obtained using adenoviral/lentiviral vectors, which overexpressed miR‑21‑5p, to transfect AECII cells in vitro and in vivo. It was found that the overexpression of miR‑21‑5p reduced the apoptotic rate of the AECII cells. In addition, miR‑21‑5p decreased the ratio of B‑cell lymphoma 2 (Bcl‑2)‑associated X protein/Bcl‑2 and the expression of caspase‑3. It was also revealed that the overexpression of miR‑21‑5p alleviated acute lung injury in adult rats exposed to a hyperoxic environment. These results suggest that miR‑21‑5p may become a novel therapeutic option for patients with HALI, by protecting AECII cells from apoptosis.

Zinc oxide nanoparticles (ZnONPs) widely used in various products, have been concerned with its impact on human health, in particular, on the risk of pulmonary toxicity. Our previous study indicated that ZnONPs could harness autophagy and impair the autophagic flux, which was positively linked to ZnONPs-induced toxicity. The objective of this study was to investigate whether ZnONPs-induced impairment of autophagic flux and cell death in lung epithelial cells is related to the size of ZnONPs. We demonstrate that ZnONPs with the average size of 50 nm could induce toxic effects in A549 lung epithelial cells, including accumulation of autophagosomes (the elevation of LC3B-II/LC3B-I ratio), impaired autophagic flux (the increase of p62 expression), the release of intracellular zinc ions (the increase of FluoZin-3 signal and ZnT1 mRNA expression), mitochondrial damage (the decrease of TMRE signal), lysosomal dysfunction (the aberrant expression of LAMP-2), oxidative stress (the increase of DCFH-DA signal and HO-1 expression) and cell death. Interestingly, ZnONPs with the average size of 200 nm failed to induce autophagy-mediated toxicity. Taken together, our results indicate that the size of ZnONPs is closely correlated with its toxicity, which is probably mediated by induction of impaired autophagic flux. This finding provides an insight into better understating of ZnONPs-associated toxicity, and mitigating the risk to humans and allowing the safer application., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Alterations to the pulmonary surfactant system have been observed consistently in ventilation-induced lung injury (VILI) including composition changes and impairments in the surface tension reducing ability of the isolated extracellular surfactant. However, there is limited information about the effects of VILI on the intracellular form of surfactant, the lamellar body. It is hypothesized that VILI leads to alterations of lamellar body numbers and function. To test this hypothesis, rats were randomized to one of three groups, nonventilated controls, control ventilation, and high tidal volume ventilation (VILI). Following physiological assessment to confirm lung injury, isolated lamellar bodies were tested for surfactant function on a constrained sessile drop surfactometer. A separate cohort of animals was used to fix the lungs followed by examination of lamellar body numbers and morphology using transmission electron microscopy. The results showed an impaired ability of reducing surface tension for the lamellar bodies isolated from the VILI group as compared with the two other groups. The morphological assessment revealed that the number, and the relative area covered by, lamellar bodies were significantly decreased in animals with VILI animals as compared with the other groups. It is concluded that VILI causes significant alterations to lamellar bodies. It is speculated that increased secretion causes a depletion of lamellar bodies that cannot be compensated by de novo synthesis of surfactant in these injured lungs., (Copyright © 2017 the American Physiological Society.)

Objective To observe the effect of Xinfeng Capsule (XFC) on Notch/Jagged-HES of type II alveolar epithelial cells (AECII). Methods Rats were divided for four groups: normal control (NC) group, model control (MC) group, leflunomide (LEF) group, XFC group, with 10 rats in each group. Complete Freund's adjuvant (CFA) was injected in the right foot plantar skin of each rat except for the NC group. After adjuvant arthritis was successfully induced, LEF group was given LEF (0.5 mg/100 g), and XFC group was treated with XFC (0.034 g/100 g), once a day from the 13th day to the 42th day. The NC and MC groups were given normal saline instead. Swelling degree (SD), arthritis index (AI) and pulmonary function were observed. AECII was observed by transmission electron microscopy (TEM). The expressions of transforming growth factor β1 (TGF-β1), Notch1, Notch3, Jagged1 and HES1 proteins in AECII were detected by Western blotting. Results The pulmonary function parameters such as forced expiratory volume in 1 second (FEV1), maximum expiratory flow rate at 50% FVC (FEF 50 ), instantaneous flow at 75% of expired volume (FEF 75 ), peak expiratory flow (PEF) in the MC group were significantly lower than those in the NC group, and the expressions of TGF-β1, Notch1, Notch3, Jagged1 and HES1 in AECII increased. The ultrastructure of AECII was damaged. Compared with the MC group, FEV1, FEF 50 , FEF 75 and PEF increased, and TGF-β1, Notch1, Notch3, Jagged1 and HES1 decreased in the XFC group. Compared with LEF group, the lung function was better in XFC group. Conclusion XFC can inhibit pulmonary fibrosis and improve pulmonary function by down-regulating TGF-β1, Notch1, Notch3, Jagged1 and HES1 in rats with adjuvant arthritis.

The development of multiple organ failure in patients with hemorrhagic shock is significantly influenced by patient age. Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which coordinates metabolic repair during cellular stress. We investigated whether AMPK-regulated signaling pathways are age-dependent in hemorrhage-induced lung injury and whether AMPK activation by 5-amino-4-imidazole carboxamide riboside (AICAR) affords lung protective effects. Male C57/BL6 young mice (3-5 mo), mature adult mice (9-12 mo), and young AMPKα1 knockout mice (3-5 mo) were subjected to hemorrhagic shock by blood withdrawing, followed by resuscitation with shed blood and lactated Ringer's solution. Plasma proinflammatory cytokines were similarly elevated in C57/BL6 young and mature adult mice after hemorrhagic shock. However, mature adult mice exhibited more severe lung edema and neutrophil infiltration, and higher mitochondrial damage in alveolar epithelial type II cells, than did young mice. No change in autophagy was observed. At molecular analysis, the phosphorylation of the catalytic subunit AMPKα1 was associated with nuclear translocation of peroxisome proliferator-activated receptor γ co-activator-α in young, but not mature, adult mice. Treatment with AICAR ameliorated the disruption of lung architecture in mice of both ages; however, effects in mature adult mice were different than young mice and also involved inhibition of nuclear factor-κB. In young AMPKα1 knockout mice, AICAR failed to improve hypotension and lung neutrophil infiltration. Our data demonstrate that during hemorrhagic shock, AMPK-dependent metabolic repair mechanisms are important for mitigating lung injury. However, these mechanisms are less competent with age.

The pulmonary delivery of nanoparticles (NPs) is a promising approach in nanomedicine. For the efficient and safe use of inhalable NPs, understanding of NP interference with lung surfactant metabolism is needed. Lung surfactant is predominantly a phospholipid substance, synthesized in alveolar type II cells (ATII), where it is packed in special organelles, lamellar bodies (LBs). In vitro and in vivo studies have reported NPs impact on surfactant homeostasis, but this phenomenon has not yet been sufficiently examined. We showed that in ATII-like A549 human lung cancer cells, silica-coated superparamagnetic iron oxide NPs (SiO 2 -SPIONs), which have a high potential in medicine, caused an increased cellular amount of acid organelles and phospholipids. In SiO 2 -SPION treated cells, we observed elevated cellular quantity of multivesicular bodies (MVBs), organelles involved in LB biogenesis. In spite of the results indicating increased surfactant production, the cellular quantity of LBs was surprisingly diminished and the majority of the remaining LBs were filled with SiO 2 -SPIONs. Additionally, LBs were detected inside abundant autophagic vacuoles (AVs) and obviously destined for degradation. We also observed time- and dose-dependent changes in mRNA expression for proteins involved in lipid metabolism. Our results demonstrate that non-cytotoxic concentrations of SiO 2 -SPIONs interfere with surfactant metabolism and LB biogenesis, leading to disturbed ability to reduce hypophase surface tension. To ensure the safe use of NPs for pulmonary delivery, we propose that potential NP interference with LB biogenesis is obligatorily taken into account.

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 (Gab1 Δ/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 Gab1 Δ/Δ mice shows no apparent pathological alterations or inflammation. However, Gab1 Δ/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 Gab1 Δ/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 (Gab1 ΔShp2 , Gab1 ΔPI3K ), 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., (Copyright © 2016 the American Physiological Society.)

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., Competing Interests: Public Health England, the primary funder of this work and employer of five of the authors is a "not for profit" supplier of the subject of the manuscript: the A549 Cell Line. The authors have declared that no other competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Sulfur mustard (SM), a bifunctional alkylating agent that causes severe lung damage, is a significant threat to both military and civilian populations. The mechanisms mediating the cytotoxic effects of SM are unknown and were investigated in this study. The purpose of this study was to establish a rat model of SM-induced lung injury to observe the resulting changes in the lungs. Male rats (Sprague Dawley) were anesthetized, intratracheally intubated, and exposed to 2 mg/kg of SM by intratracheal instillation. Animals were euthanized 6, 24, 48, and 72 h post-exposure, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. Exposure of rats to SM resulted in rapid pulmonary toxicity, including partial bronchiolar epithelium cell shedding, focal ulceration, and an increased amount of inflammatory exudate and number of cells in the alveoli. There was also evidence that the protein content and cell count of BALF peaked at 48 h, and the alveolar septum was widened and filled with lymphocytes. SM exposure also resulted in partial loss of type I alveolar epithelial cell membranes, fuzzy mitochondrial cristae, detachment and dissociation of ribosomes attached to the surface of rough endoplasmic reticulum, cracked, missing, and disorganized microvilli of type II alveolar epithelial cells, and increased apoptotic cells in the alveolar septum. The propylene glycol control group, however, was the same as the normal group. These data demonstrate that the mechanism of a high concentration of SM (2 mg/kg) induced acute lung injury include histologic changes, inflammatory reactions, apoptosis, oxidative stress, and nuclear DNA damage; the degree of injury is time dependent., (© The Author(s) 2014.)

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air-liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air-liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air-liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air-liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air-liquid interface., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is required in the biosynthesis of pulmonary surfactant. This short communication describes our assessment of LPCAT1 mRNA levels in human amniotic fluid. We found a direct correlation between LPCAT1 mRNA copies and the amniotic fluid lamellar body count (LBC). This finding corroborates an association between LPCAT1 and surfactant phospholipid biosynthesis in humans. It may provide a model for future research in perinatal medicine.

Background: Bronchopulmonary dysplasia (BPD) is a common complication in preterm infants that involves the downregulation of tight junction (TJ) proteins. However, the mechanism underlying downregulation of the expression of TJ proteins during at the early stages of hyperoxia-induced BPD remains to be understood. Here, we aimed to identify the role of caveolin-1 (Cav-1) in hyperoxia-induced pulmonary epithelial barrier breakdown., Methods: First, we established an in vitro pulmonary epithelial barrier models using primary type II alveolar epithelial cells (AEC-II) from newborn rats. AEC-II was assigned to the hyperoxic (85 % O2/5 % CO2) or normoxic (21 % O2/5 % CO2) groups. Second, AEC-II was transfected with Cav-1-siRNA to downregulate Cav-1 under normoxic exposure. Third, AEC-II was transfected with a cDNA encoding Cav-1 to upregulate Cav-1 expression under hyperoxic exposure. Then, expression levels of Cav-1 and TJ proteins were examined by immunofluorescence staining, reverse transcription-polymerase chain reaction, and Western blotting. The TJ structures visualized using a transmission electron microscope, and transepithelial resistance and apparent permeability coefficient of fluorescein isothiocyanate-dextran, which are indicators of barrier function, were measured., Results: Our data showed that exposure to hyperoxia disrupted the structure and function of the pulmonary epithelial barrier and decreased the ZO-1, occludin, claudin-4, and Cav-1 expression levels. Moreover, Cav-1 knockdown attenuated the expression of the other three genes and disrupted pulmonary epithelial barrier structure and function under normoxic exposure. However, Cav-1 upregulation markedly antagonized the hyperoxia-induced pulmonary epithelial barrier destruction and TJ protein loss., Conclusions: This is the first study to present evidence illustrating the novel role of Cav-1 downregulation-mediated TJ protein loss in pulmonary epithelial barrier destruction during BPD.

Background: Heterozygous mutations of SFTPC, the gene-encoding surfactant protein C (SP-C), result in interstitial lung disease (ILD). However, characterization of mutations located in the mature domain of precursor SP-C (proSP-C) is limited. This study examined the molecular pathogenesis of such a mutation of ILD., Methods: We employed sequencing of SFTPC and established A549 cells stably expressing several proSP-C mutants. Histopathology and transmission electron microscopy (TEM) of lung tissue from a pediatric patient with ILD were assessed. Effects of mutant proSP-C were evaluated by western blotting, immunofluorescence, and TEM., Results: Sequencing of SFTPC revealed a novel heterozygous mutation, c.163C>T (L55F). In lung tissue, abnormal localization of proSP-C was observed by immunohistochemistry, and small and dense lamellar bodies (LBs) in type II alveolar epithelial cells (AECs) were detected by TEM. TEM of A549 cells stably expressing proSP-C(L55F) displayed abnormal cytoplasmic organelles. ProSP-C(L55F) exhibited a band pattern similar to that of proSP-C(WT) for processed intermediates. Immunofluorescence studies demonstrated that proSP-C(L55F) partially colocalized in CD63-positive cytoplasmic vesicles of A549 cells, which was in contrast to proSP-C(WT)., Conclusion: We detected a novel c.163C>T mutation located in the mature domain of SFTPC associated with ILD that altered the subcellular localization of proSP-C in A549 cells.

Objective. To determine whether bile acids (BAs) affect respiratory functions through the farnesoid X receptor (FXR) expressed in the lungs and to explore the possible mechanisms of BAs-induced respiratory disorder. Methods. Primary cultured alveolar epithelial type II cells (AECIIs) of rat were treated with different concentrations of chenodeoxycholic acid (CDCA) in the presence or absence of FXR inhibitor Z-guggulsterone (GS). Then, expression of FXR in nuclei of AECIIs was assessed by immunofluorescence microscopy. And ultrastructural changes of the cells were observed under transmission electron microscope and analyzed by Image-Pro Plus software. Results. Morphologic damage of AECIIs was exhibited in high BAs group in vitro, with high-level expression of FXR, while FXR inhibitor GS could attenuate the cytotoxicity of BAs to AECIIs. Conclusions. FXR expression was related to the morphologic damage of AECIIs induced by BAs, thus influencing respiratory functions.

Background: Engineered nanoparticles (NP) are being developed for inhaled drug delivery. This route is non-invasive and the major target; alveolar epithelium provides a large surface area for drug administration and absorption, without first pass metabolism. Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery. We explored the differential effect of neutral, cationic and anionic polystyrene latex NPs on the target cells of the human alveolus, using primary human alveolar macrophages (MAC) and primary human alveolar type 2 (AT2) epithelial cells and a unique human alveolar epithelial type I-like cell (TT1). We hypothesized that the bioreactivity of the NPs would relate to their surface chemistry, charge and size as well as the functional role of their interacting cells in vivo., Methods: Amine- (ANP) and carboxyl- surface modified (CNP) and unmodified (UNP) polystyrene NPs, 50 and 100 nm in diameter, were studied. Cells were exposed to 1-100 μg/ml (1.25-125 μg/cm(2); 0 μg/ml control) NP for 4 and 24 h at 37 °C with or without the antioxidant, N-acetyl cysteine (NAC). Cells were assessed for cell viability, reactive oxygen species (ROS), oxidised glutathione (GSSG/GSH ratio), mitochondrial integrity, cell morphology and particle uptake (using electron microscopy and laser scanning confocal microscopy)., Results: ANP-induced cell death occurred in all cell types, inducing increased oxidative stress, mitochondrial disruption and release of cytochrome C, indicating apoptotic cell death. UNP and CNP exhibited little cytotoxicity or mitochondrial damage, although they induced ROS in AT2 and MACs. Addition of NAC reduced epithelial cell ROS, but not MAC ROS, for up to 4 h. TT1 and MAC cells internalised all NP formats, whereas only a small fraction of AT2 cells internalized ANP (not UNP or CNP). TT1 cells were the most resistant to the effects of UNP and CNP., Conclusion: ANP induced marked oxidative damage and cell death via apoptosis in all cell types, while UNP and CNP exhibited low cytotoxicity via oxidative stress. MAC and TT1 cell models show strong particle-internalization compared to the AT2 cell model, reflecting their cell function in vivo. The 50 nm NPs induced a higher bioreactivity in epithelial cells, whereas the 100 nm NPs show a stronger effect on phagocytic cells.

This work presents the synthesis and characterization of Au nanostars (AuNSs) and demonstrates their application as surface enhanced Raman scattering (SERS)-activity tags for cellular imaging and sensing. Nile blue A (NBA) and bovine serum albumin (BSA) were used as Raman reporter molecules and capping materials, respectively. The SERS-activity tags were tested on human lung adenocarcinoma cell (A549) and alveolar type II cell (AT II) and found to present a low level of cytotoxicity and high chemical stability. These SERS-activity tags not only can be applied in multiplexed cellular imaging, including dark field imaging, transmission electron microscopy (TEM) and SERS imaging, but also can be used for cellular sensing. The SERS spectra clearly identified cellular important components such as proteins, nucleic acids, lipids, and carbohydrates. This study also shows that endocytosis is the main channel of tags internalized in cells. The AuNSs exhibiting strong surface enhanced Raman effects are utilized in the design of an efficient, stable SERS-activity tag for intracellular applications.

Objective: To detect the ultrastructural changes in rabbits with type II decompression sickness (DCS), and study the therapeutic effects of hyperbaric oxygen (HBO)., Methods: Twenty-seven male New Zealand rabbits were randomly divided equally into the DCS group, HBO treatment group and control group. Experimental models of each group were prepared. Lung apex tissues were harvested to prepare paraffin- and EPON812-embedded tissues., Results: In the DCS group, macroscopic and histological examination revealed severe and rapid damage to lung tissue. Ultrastructural examination revealed exudation of red blood cells in the alveolar space. Type I alveolar epithelial cells exhibited retracted cell processes and swollen mitochondria, and type II cells showed highly swollen mitochondria and decrease in cytoplasmic lamellar bodies. Dilatation and congestion of capillary vessels were accompanied by swelling of endothelial cells and incomplete basement membrane. In the HBO treatment group, the findings were somewhat similar to those in the DCS group, but the extent of damage was lesser. Only a small amount of tiny bubbles could be seen in the blood vessels. Type I alveolar epithelia cells and endothelial cells of the capillaries illustrated slight shortening of cells, swollen cytoplasm and decreased cell processes. Type II alveolar epithelial cells showed slight swelling of the mitochondria, decreased vacuolar degeneration of lamellar bodies, and increase in the number of free ribosomes., Conclusions: Our microscopic and ultrastructural findings confirm that the lung is an important organ affected by DCS. We also confirmed that HBO can alleviate DCS-induced pulmonary damage.

Lung alveolar type II (ATII) cells are specialized in the synthesis and secretion of pulmonary surfactant, a lipid-protein complex that reduces surface tension to minimize the work of breathing. Surfactant synthesis, assembly and secretion are closely regulated and its impairment is associated with severe respiratory disorders. At present, well-established ATII cell culture models are not available. In this work, Decidua-derived Mesenchymal Stem Cells (DMSCs) have been differentiated into Alveolar Type II- Like Cells (ATII-LCs), which display membranous cytoplasmic organelles resembling lamellar bodies, the organelles involved in surfactant storage and secretion by native ATII cells, and accumulate disaturated phospholipid species, a surfactant hallmark. Expression of characteristic ATII cells markers was demonstrated in ATII-LCs at gene and protein level. Mimicking the response of ATII cells to secretagogues, ATII-LCs were able to exocytose lipid-rich assemblies, which displayed highly surface active capabilities, including faster interfacial adsorption kinetics than standard native surfactant, even in the presence of inhibitory agents. ATII-LCs could constitute a highly useful ex vivo model for the study of surfactant biogenesis and the mechanisms involved in protein processing and lipid trafficking, as well as the packing and storage of surfactant complexes.

Background: Lung ischemia-reperfusion injury (LIRI) may occur in the region of the affected lung after reperfusion therapy. Inhaled NO may be useful in treating acute and chronic pulmonary thromboembolism (PTE) due to the biological effect property of NO., Methods: A PTE canine model was established through selectively embolizing blood clots to an intended right lower lobar pulmonary artery. PaO2/FiO2, the mPAP and PVR were investigated at the time points of 2, 4, 6 hours after inhaled NO. Masson's trichrome stain, apoptotic pneumocytes and lung sample ultrastructure were also investigated among different groups., Results: The PaO2/FiO2 in the Inhaled NO group increased significantly when compared with the Reperfusion group at time points of 4 and 6 hours after reperfusion, mPAP decreased significantly at point of 2 hours and the PVR decreased significantly at point of 6 hours after reperfusion. The amounts of apoptotic type II pneumocytes in the lower lobar lung have negative correlation trend with the arterial blood PaO2/FiO2 in Reperfusion group and Inhaled NO group. Inhaled nitric oxide given at 20 ppm for 6 hours can significantly alleviate the LIRI in the model., Conclusions: Dramatic physiological improvements are seen during the therapeutic use of inhaled NO in pulmonary thromboembolism canine model. Inhaled NO may be useful in treating LIRI in acute or chronic PTE by alleviating apoptotic type II pneumocytes. This potential application warrants further investigation.

Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.

Ischemic acute kidney injury (AKI) is a common complication during inpatient hospitalization, and often induces acute lung injury (ALI). A lot of studies have concentrated on the relevance between AKI and ALI, but the underlying mechanisms of AKI- associated ALI have remained unclear until now. One reason is that evidence of the ultrastructural pathology of AKI-associated ALI has been scarce and needed to be accumulated. The aims of present study are to observe ultrastructural changes, and to reveal leukocyte trafficking of ALI induced by ischemic AKI in rats. For this purpose light microscopy (LM) and electron microscopy (EM), as well as morphometric analysis, were employed in present study. LM observations revealed distinct regions of collapsed alveoli, hemorrhage in alveoli, and interstitial edema in AKI-induced ALI. EM examinations provided facts that alveolar epithelial cells, including type I and type II cells, were necrotic, and endothelia cells undergoing apoptosis as well as interstitial cells undergoing necroptosis were noted in AKI lungs. In addition, shrinkage and decreased or disappeared lamellar bodies were evident in alveolar type II cell of AKI rat lungs. Leukocyte numerical density on area (NA) in AKI lungs was significantly more than that in sham lungs. Based on the morphological criteria from EM examinations and morphometric analysis, a conclusion was that necrosis, including necroptosis, and apoptosis were involved in damaged lung induced by AKI. And inflammation also contributed to acute lung injury of rats with AKI.

Metal oxide NPs are abundantly produced in nanotech industries and are emitted in several combustion processes, suggesting the need to characterize their toxic impact on the human respiratory system. The acute toxicity and the morphological changes induced by copper oxide and titanium dioxide NPs (nCuO and nTiO₂) on the human alveolar cell line A549 are here investigated. Cell viability and oxidative stress have been studied in parallel with NP internalization and cell ultrastructural modifications. TiO₂ NPs were abundantly internalized by cells through the endocytic pathway, even they did not induce cell death and ultrastructural lesions. Only after 24h cells were affected by an abundant NP internalization presenting a consequent altered morphology. High cytotoxicity, oxidative stress and severe ultrastructural damages were produced by nCuO, since cell membrane and mitochondria resulted to be heavily affected, even at early exposure time. nCuO-induced toxicity has been interpreted as a consequence of both NPs reactivity and copper ions dissolution in lysosomal compartments, even the free NPs, scattered throughout all the cell compartments, might contribute to the toxicity. The antioxidant N-acetylcysteine was effective in recovering nCuO exposed cells viability and Bafilomycin A1 inhibited copper ions release in phagolysosomes and significantly rescued cells, suggesting a relevant cytotoxic mechanism relative to oxidative damages and authophagic cell death, together with NP internalization and dissolution. Our results support the previous data reporting CuO NPs are highly cytotoxic and genotoxic, and associate their toxic effects with their cell penetration and interaction with various compartments. In conclusion, the so-called "Trojan horse" mechanism and autophagy, are involved in nCuO-induced cell death, even a further research is needed to explain the events occurring at early exposure time., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Paraquat poisoning is well known for causing multiple organ function failure (MODS) and high mortality. Acute lung injury and advanced pulmonary fibrosis are the most serious complications. Bosentan is a dual endothelin receptor antagonist. It plays an important role in treating PF. There is no related literature on the use of bosentan therapy for paraquat poisoning., Objective: To study the use of bosentan to treat acute lung injury and pulmonary fibrosis as induced by paraquat., Method: A total of 120 adult Wister male rats were randomly assigned to three groups: the paraquat poisoning group (rats were intragastrically administered with paraquat at 50 mg/kg body weight once at the beginning); the bosentan therapy group (rats were administered bosentan at 100 mg/kg body weight by intragastric administration half an hour after paraquat was administered, then the same dose was administered once a day); and a control group (rats were administered intragastric physiological saline). On the 3rd, 7th, 14th, and 21st days following paraquat exposure, rats were sacrificed, and samples of lung tissue and venous blood were collected. The levels of transforming growth factor-β1 (TGF-β1), endothelin-1 (ET-1), and hydroxyproline (HYP) in the plasma and lung homogenate were determined. Optical and electronic microscopes were used to examine pathological changes., Result: The TGF-β1, ET-1, and HYP of the paraquat poisoning group were significantly higher than in the control group, and they were significantly lower in the 21st day therapy group than in the paraquat poisoning group on the same day. Under the optical and electronic microscopes, lung tissue damage was observed to be more severe but was then reduced after bosentan was administered., Conclusion: Bosentan can reduce inflammation factor release. It has a therapeutic effect on acute lung injury as induced by paraquat.

Background: Bronchopulmonary dysplasia (BPD) presents a major threat of very preterm birth and treatment options are still limited. Stem cells from different sources have been used successfully in experimental BPD, induced by postnatal hyperoxia., Objectives: We investigated the effect of umbilical cord blood mononuclear cells (MNCs) in a new double-hit mouse model of BPD., Methods: For the double-hit, date mated mice were subjected to hypoxia and thereafter the offspring was exposed to hyperoxia. Human umbilical cord blood MNCs were given intraperitoneally by day P7. As outcome variables were defined: physical development (auxology), lung structure (histomorphometry), expression of markers for lung maturation and inflammation on mRNA and protein level. Pre- and postnatal normoxic pups and sham treated double-hit pups served as control groups., Results: Compared to normoxic controls, sham treated double-hit animals showed impaired physical and lung development with reduced alveolarization and increased thickness of septa. Electron microscopy revealed reduced volume density of lamellar bodies. Pulmonary expression of mRNA for surfactant proteins B and C, Mtor and Crabp1 was reduced. Expression of Igf1 was increased. Treatment with umbilical cord blood MNCs normalized thickness of septa and mRNA expression of Mtor to levels of normoxic controls. Tgfb3 mRNA expression and pro-inflammatory IL-1β protein concentration were decreased., Conclusion: The results of our study demonstrate the therapeutic potential of umbilical cord blood MNCs in a new double-hit model of BPD in newborn mice. We found improved lung structure and effects on molecular level. Further studies are needed to address the role of systemic administration of MNCs in experimental BPD.