Lung Alveolar Epithelial Cell Line Research Articles

Regulation of ACE-2 enzyme by hyperoxia in lung epithelial cells by post-translational modification

Bronchopulmonary Dysplasia (BPD) occurs in premature neonates with respiratory distress who require supplemental oxygen in the first days after birth. BPD involves uniform arrest of alveolar development and variable interstitial cellularity and/or fibroproliferation. Previous studies by our lab showed that the enzyme, angiotensin converting enzyme-2 (ACE-2) and its product Ang1-7 exerting action on the receptor Mas oncogene in what is known as ACE-2/Mas axis is protective to lung cells. We also showed that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas lung injury, an effect caused by ACE-2 enzyme shedding mediated by TNF-alpha-converting enzyme (TACE/ADAM17). However, no reports yet exist about the regulation of ACE-2 in the alveolar epithelia in hyperoxic lung injury. In this study we aim to define the effects of hyperoxic lung injury on the protective ACE-2 enzyme in the human lung alveolar epithelial cell line A549. Cultured A549 cells were exposed to hyperoxia (95% O2) or normoxia (21% O2) for 3 or 7 days in serum-free nutrient media. Cells were lysed and culture media were collected to test for cellular ACE-2 enzymatic activity and for ACE-2, Mas receptor, TACE/ADAM17, and ubiquitin proteins abundance by immunoblotting. Cells were harvested in Trizol for RNA extraction and ACE-2 qRT-PCR. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Total ubiquitinated proteins were increased by hyperoxia treatment, while ACE-2 and Mas receptor proteins abundance and ACE-2 enzymatic activity were decreased significantly in A549 cells exposed to hyperoxia relative to the normoxia controls. The percent decrease in ACE-2 activity corresponded with increased time of hyperoxic gas exposure. However, in contrast to our data from lung fibroblasts, no significant change was noted in ACE-2 protein released into the media or in ACE-2 mRNA levels by the hyperoxic treatment. Ubiquitin immunoreactive bands were detectable in the ACE-2 immunoprecipitate. These data suggest that hyperoxic exposure of the lung epithelial cells decreases the protective enzyme ACE-2 by cell type specific mechanisms independent of shedding by TACE/ADAM17. The data also suggest a regulatory level of ACE-2 downstream of transcription may involve ACE-2 ubiquitination and targeting for degradation.

Idebenone has preventative and therapeutic effects on pulmonary fibrosis via preferential suppression of fibroblast activity

Alveolar epithelial injury induced by reactive oxygen species (ROS) and abnormal collagen production by activated fibroblasts (myofibroblasts) is involved in the onset and exacerbation of idiopathic pulmonary fibrosis (IPF). Compared with alveolar epithelial cells, lung fibroblasts, especially myofibroblasts, exhibit an apoptosis-resistance phenotype (apoptosis paradox) that appears to be involved in IPF pathogenesis. Thus, we screened for chemicals eliciting preferential cytotoxicity of LL29 cells (lung fibroblasts from an IPF patient) compared with A549 cells (human lung alveolar epithelial cell line) from medicines already in clinical use. We identified idebenone, a synthetic analogue of coenzyme Q10 (CoQ10, an antioxidant) that has been used clinically as a brain metabolic stimulant. Idebenone induced cell growth inhibition and cell death in LL29 cells at a lower concentration than in A549 cells, a feature that was not observed for other antioxidant molecules (such as CoQ10) and two IPF drugs (pirfenidone and nintedanib). Administration of idebenone prevented bleomycin-induced pulmonary fibrosis and increased pulmonary ROS levels. Importantly, idebenone also improved pulmonary fibrosis and lung function when administered after the development of fibrosis, whereas administration of CoQ10 similarly prevented bleomycin-induced pulmonary fibrosis, but had no effect after its development. Administration of idebenone, but not CoQ10, suppressed bleomycin-induced increases in lung myofibroblasts. In vitro, treatment of LL29 cells with idebenone, but not CoQ10, suppressed TGF-β–induced collagen production. These results suggest that in addition to antioxidant activity, idebenone exerts inhibitory activity on the function of lung fibroblasts, with the former activity being preventative and the latter therapeutic for bleomycin-induced fibrosis. Thus, we propose that idebenone may be more therapeutically beneficial for IPF patients than current treatments.

Protective potential of different compounds and their combinations with MESNA against sulfur mustard-induced cytotoxicity and genotoxicity

The purpose of this study was to evaluate the efficacy of potential candidate molecules or their combinations against strong alkylation agent sulfur mustard (SM) on the human lung alveolar epithelial cell line A-549. Candidate molecules were chosen on the basis of their previously observed protective effects in vitro. The tested compounds, including antioxidants, sulfhydryl or other sulfur-containing molecules, nitrogen-containing molecules, PARP inhibitors and a NO synthase inhibitor, were applicated 30min before SM treatment. The efficiency of candidate molecules to protect cells against DNA damage and cell death induced by SM was determined using single-cell gel electrophoresis (comet assay) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction by viable cells. The damage of DNA was assessed 1 and 24h after dose 50μM SM. Cell survival was assessed 24 and 72h after the exposure. To achieve maximal cytoprotection, combinations of selected compounds with sodium 2-mercaptoethane sulphonate (MESNA) were tested. We found significant protective effects by several drugs used individually and also in combination with MESNA. High protection was achieved by sodium thiosulphate, which was further potentiated when combined with MESNA. Most of the selected compounds or mixture provided only moderate genoptotection without having any effect towards cell viability.

Dexmedetomidine attenuates oxidative stress induced lung alveolar epithelial cell apoptosis in vitro.

Background. Oxidative stress plays a pivotal role in the lung injuries of critical ill patients. This study investigates the protection conferred by α 2 adrenoceptor agonist dexmedetomidine (Dex) from lung alveolar epithelial cell injury induced by hydrogen peroxide (H2O2) and the underlying mechanisms. Methods. The lung alveolar epithelial cell line, A549, was cultured and then treated with 500 μM H2O2 with or without Dex (1 nM) or Dex in combination with atipamezole (10 nM), an antagonist of α 2 receptors. Their effect on mitochondrial membrane potential (Δψ m), reactive oxygen species (ROS), and the cell cycle was assessed by flow cytometry. Cleaved-caspases 3 and 9, BAX, Bcl-2, phospho-mTOR (p-mTOR), ERK1/2, and E-cadherin expression were also determined with immunocytochemistry. Results. Upregulation of cleaved-caspases 3 and 9 and BAX and downregulation of Bcl-2, p-mTOR, and E-cadherin were found following H2O2 treatment, and all of these were reversed by Dex. Dex also prevented the ROS generation, cytochrome C release, and cell cycle arrest induced by H2O2. The effects of Dex were partially reversed by atipamezole. Conclusion. Our study demonstrated that Dex protected lung alveolar epithelial cells from apoptotic injury, cell cycle arrest, and loss of cell adhesion induced by H2O2 through enhancing the cell survival and proliferation.

The alpha-1-antitrypsin gene promoter in human A549 lung derived cells, and a novel transcription initiation site

Alpha-1-antitrypsin (AAT), also called serine proteinase inhibitor A1 (Serpin A1), is the most abundant serpin in human plasma. A major physiological role of AAT is to protect the lung from the destructive effects of excess uninhibited neutrophil elastase. During inflammation, circulating levels of AAT may increase twofold-to-threefold as part of the acute-phase response. The liver is the main contributor to this increase. However, local synthesis may provide an important mechanism for controlling neutrophil elastase activity at sites of inflammation, and previous studies have shown a marked increase in production after cytokine stimulation. In the current study we report a distinct transcription initiation site for AAT expression in the lung alveolar epithelial cell line A549, which is located nine bases upstream of the previously mapped full-length monocyte transcription start-site, and show using site-directed mutagenesis that two Sp1 sites and a putative TATA box are functional. EMSA experiments provide evidence for Sp1 and Sp3 binding to these two Sp1 sites. We have also mapped the minimal promoter region and a cell-specific element essential for expression in A549 cells, both of which reside in an 865bp fragment upstream of the transcription start-site. Understanding the mechanisms of AAT gene regulation in a lung-derived cell line has important implications for understanding the control of localised lung tissue damage which occurs as a result of excess proteolytic activity.

Chemoprevention and inhibition of P‐glycoprotein in cancer cells by Chinese medicinal herbs

Many of the herbal extracts used in the Chinese clinical medical routine inhibit the growth of tumor cells. In the present work, extracts of 12 selected herbs were prepared with methanol, chloroform, ethyl acetate and water, and the effects of these on the multidrug resistance (MDR) and P-glycoprotein of mouse lymphoma cells transfected with the human mdr1 gene and on a human lung alveolar epithelial cell line were investigated. The extracts were tested for antiproliferative effects, and the reversal of MDR in mouse lymphoma cells. The possible chemopreventive effect of the chloroform extracts was studied on the expression of cytomegalovirus (CMV) immediate-early (IE) antigen in human lung cancer cells (A549). The antimicrobial effects of the extracts were tested on some representative micro-organisms. Certain of the chloroform extracts of the plant materials were the most effective compounds on the reversal of MDR. Two of the chloroform extracts enhanced the antiproliferative effect of doxorubicin on MDR mouse lymphoma cells. The selected extracts did not show any antibacterial effect with the agar diffusion method. Certain chloroform extracts decreased the intermediate IE antigen expression of CMV in A459 cells.

Caveolin‐1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial cells

The P2X7 receptor has recently been described as a marker for lung alveolar epithelial type I cells. Here, we demonstrate both the expression of P2X7 protein and its partition into lipid rafts in the mouse lung alveolar epithelial cell line E10. A significant degree of colocalization was observed between P2X7 and the raft marker protein Caveolin-1; also, P2X7 protein was associated with caveolae. A marked reduction in P2X7 immunoreactivity was observed in lung sections prepared from Caveolin-1-knockout mice, indicating that Caveolin-1 expression was required for full expression of P2X7 protein. Indeed, suppression of Caveolin-1 protein expression in E10 cells using short hairpin RNAs resulted in a large reduction in P2X7 protein expression. Our data demonstrate a potential interaction between P2X7 protein and Caveolin-1 in lipid rafts, and provide a basis for further functional and biochemical studies to probe the physiologic significance of this interaction.

Identification of a novel adhesion molecule involved in the virulence of Legionella pneumophila.

Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.

Molecular mechanisms of lipopolysaccharide induced ICAM-1 expression in A549 cells.

Lung intercellular adhesion molecule-1 (ICAM-1) expression is increased by LPS or hyperoxia on type II cells in vivo. The goals of the present study were to determine the mechanisms of ICAM-1 expression in a lung alveolar epithelial cell line (A549) exposed to lipopolysaccharide (LPS). A549 cells, a transformed human cell line with characteristics of alveolar epithelial cells, were used. Cells were exposed to LPS, TNF-alpha, IL-1beta, or media alone for up to 12 h. Northern blot analyses were done to determine mRNA expression of ICAM-1 after exposures. Protein binding to NF-kappaB sequences were determined by gel mobility shift assays and super-shift analysis. ICAM-1 mRNA expression was induced in A549 cells with exposure to LPS for 1 to 4 h, and was diminished to baseline at 8 h, and the inductions were independent of TNF-alpha and IL-1beta expression. Nuclear protein extracts from LPS-exposed cells bound to a NF-kappaB sequence and the timing of increased binding correlated closely with ICAM-1 mRNA induction. Super-shift studies indicated that p65 was involved in the binding to the NF-kappaB sequence and p50 was not. LPS inducibility of ICAM-1 mRNA in A549 cells is independent of TNF- and IL-1 in A549 cells, and the similar time course of mRNA induction and NF-kappaB activation suggest the induction of ICAM-1 is mediated, in part, by NF-kappaB.

Relationship between surface properties and cellular responses to crystalline silica: studies with heat-treated cristobalite.

A fibrogenic sample of cristobalite dust, CRIS (crystalline silica of mineral origin), was heated to 1300 degrees C (CRIS-1300) to relate induced physicochemical modifications to cytotoxicity. Heating did not affect dust micromorphology and crystallinity, except for limited sintering and decreased surface area of CRIS-1300. Thermal treatments deeply affected surface properties. Electron paramagnetic resonance showed surface radicals progressively annealed by heating, mostly disappearing at >/=800 degrees C. Surface hydrophilicity or hydrophobicity, evaluated with water vapor adsorption, still showed some hydrophilic patches in CRIS-800, but CRIS-1300 was fully hydrophobic. Heating modified the biological activity of cristobalite. Cytotoxicity, tested on proliferating cells of the mouse monocyte macrophage cell line J774, showed that CRIS was cytotoxic and CRIS-800 was still cytotoxic, but CRIS-1300 was substantially inert. Cytotoxicity of CRIS to the rat lung alveolar epithelial cell line, AE6, as measured by colony forming efficiency, was greatly reduced for CRIS-800 and eliminated for CRIS-1300. The rate of lactate dehydrogenase release by rat alveolar macrophages was lowered for CRIS-800, and release was completely inactivated for CRIS-1300. The absence of surface radicals and the onset of hydrophobicity may both account for the loss of cytotoxicity upon heating. Differences observed between CRIS-800 and CRIS-1300, both fully deprived of surface radicals, indicate that hydrophobicity is at least one of the surface properties determining the cytotoxic potential of a dust.

Effect of matrigel on development of A549 lung epithelial cells

Extracellular matrix components are known to influence the growth and differentiation of cultured cells, often causing them to behave more like their in vivo counterparts than cells grown on plastic. To develop an in vitro model in which to study the interaction of pathogenic microorganisms with the lung epithelium, we have undertaken a morphological study of the effects of extracellular matrix components on the human lung alveolar epithelial cell line A549. A549 cells were grown for varying amounts of time on plastic; Costar polycarbonate membrane filter inserts; or on Matrigel coated polycarbonate filter inserts. Cells were fixed for 1 h in 2% glutaraldehyde in 0.1 M cacodylate buffer, post-fixed in 1 % OsO4 for 45 min, dehydrated in ascending ethanol, and embedded in Spurr’s resin.A549 cells grown on a plastic slide formed an even monolayer with cells connected by wellformed junctional complexes or separated by interdigitating microvilli. The cytoplasm showed many polyribosomes, rough endoplasmic reticulum, small golgi complexes, mitochondria, occasional lysosomes and bundles of microfilaments.