Cyclic stretch attenuates effects of hyperoxia on cell proliferation and viability in human alveolar epithelial cells

The treatment of severe lung disease often requires the use of high concentrations of oxygen coupled with the need for assisted ventilation, potentially exposing the pulmonary epithelium to both reactive oxygen species and nonphysiological cyclic stretch. Whereas prolonged hyperoxia is known to cause increased cell injury, cyclic stretch may result in either cell proliferation or injury depending on the pattern and degree of exposure to mechanical deformation. How hyperoxia and cyclic stretch interact to affect the pulmonary epithelium in vitro has not been previously investigated. This study was performed using human alveolar epithelial A549 cells to explore the combined effects of cyclic stretch and hyperoxia on cell proliferation and viability. Under room air conditions, cyclic stretch did not alter cell viability at any time point and increased cell number after 48 h compared with unstretched controls. After exposure to prolonged hyperoxia, cell number and [3H]thymidine incorporation markedly decreased, whereas evidence of oxidative stress and nonapoptotic cell death increased. The combination of cyclic stretch with hyperoxia significantly mitigated the negative effects of prolonged hyperoxia alone on measures of cell proliferation and viability. In addition, cyclic stretch resulted in decreased levels of oxidative stress over time in hyperoxia-exposed cells. Our results suggest that cyclic stretch, as applied in this study, can minimize the detrimental effects of hyperoxia on alveolar epithelial A549 cells.

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Silica directly increases permeability of alveolar epithelial cells

Journal of Applied Physiology ◽

10.1152/jappl.1990.68.4.1354 ◽

1990 ◽

Vol 68 (4) ◽

pp. 1354-1359 ◽

Cited By ~ 11

Author(s):

R. K. Merchant ◽

M. W. Peterson ◽

G. W. Hunninghake

Keyword(s):

Epithelial Cells ◽

Cell Injury ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelium ◽

Alveolar Epithelial Cells ◽

Dependent Manner ◽

Alveolar Epithelial ◽

Lactate Dehydrogenase Release ◽

Capillary Membrane

Alveolar epithelial cell injury and increased alveolar-capillary membrane permeability are important features of acute silicosis. To determine whether silica particles contribute directly to this increased permeability, we measured paracellular permeability of rat alveolar epithelium after exposure to silica, in vitro, using markers of the extracellular space. Silica (Minusil) markedly increased permeability in a dose- and time-dependent manner. This was not the result of cytolytic injury, because lactate dehydrogenase release from monolayers exposed to silica was not increased. Pretreatment of the silica with serum, charged dextrans, or aluminum sulfate blocked the increase in permeability. Scanning electron microscopy demonstrated adherence of the silica to the surface of the alveolar epithelial cells. Thus silica can directly increase permeability of alveolar epithelium.

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Pneumocystis Pneumonia Increases the Susceptibility of Mice to Sublethal Hyperoxia

Infection and Immunity ◽

10.1128/iai.71.10.5970-5978.2003 ◽

2003 ◽

Vol 71 (10) ◽

pp. 5970-5978 ◽

Cited By ~ 7

Author(s):

James M. Beck ◽

Angela M. Preston ◽

Steven E. Wilcoxen ◽

Susan B. Morris ◽

Eric S. White ◽

...

Keyword(s):

T Cells ◽

Epithelial Cell ◽

Cell Injury ◽

Alveolar Epithelial Cell ◽

Pulmonary Inflammation ◽

Alveolar Epithelial Cells ◽

Alveolar Epithelial ◽

Epithelial Cell Injury

ABSTRACT Patients with Pneumocystis pneumonia often develop respiratory failure after entry into medical care, and one mechanism for this deterioration may be increased alveolar epithelial cell injury. In vitro, we previously demonstrated that Pneumocystis is not cytotoxic for alveolar epithelial cells. In vivo, however, infection with Pneumocystis could increase susceptibility to injury by stressors that, alone, would be sublethal. We examined transient exposure to hyperoxia as a prototypical stress that does cause mortality in normal mice. Mice were depleted of CD4+ T cells and inoculated intratracheally with Pneumocystis. Control mice were depleted of CD4+ T cells but did not receive Pneumocystis. After 4 weeks, mice were maintained in normoxia, were exposed to hyperoxia for 4 days, or were exposed to hyperoxia for 4 days followed by return to normoxia. CD4-depleted mice with Pneumocystis pneumonia demonstrated significant mortality after transient exposure to hyperoxia, while all uninfected control mice survived this stress. We determined that organism burdens were not different. However, infected mice exposed to hyperoxia and then returned to normoxia demonstrated significant increases in inflammatory cell accumulation and lung cell apoptosis. We conclude that Pneumocystis pneumonia leads to increased mortality following a normally sublethal hyperoxic insult, accompanied by alveolar epithelial cell injury and increased pulmonary inflammation.

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Nanomaterials induce different levels of oxidative stress, depending on the used model system: Comparison of in vitro and in vivo effects

10.21203/rs.3.rs-57664/v1 ◽

2020 ◽

Author(s):

Isabel Karkossa ◽

Anne Bannuscher ◽

Bryan Hellack ◽

Wendel Wohlleben ◽

Julie Laloy ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Alveolar Macrophages ◽

Model System ◽

Alveolar Epithelial Cells ◽

Model Systems ◽

Alveolar Epithelial ◽

Different Levels

Abstract Background The immense variety and constant development of nanomaterials (NMs) raise the demand for a facilitated risk assessment, for which knowledge on NMs mode of actions (MoAs) is required. For this purpose, a comprehensive data basis is of paramountcy that can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is indispensable to follow the 3R concept and to master the high number of NMs. In the present study, we aimed at comparing NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data evaluation strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For in vitro investigations, alveolar epithelial cells and alveolar macrophages were treated with different doses of NMs, and the results were compared to effects on rat lungs after short-term inhalations and instillations at varying doses with and without a recovery period.Results Since the production of reactive oxygen species (ROS) is described to be a critical biological effect of NMs, and enrichment analyses confirmed oxidative stress as a significant effect upon NM treatment in vitro in the present study, we focused on different levels of oxidative stress. Thus, we found opposite changes for proteins and metabolites that are related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, illustrating that NMs MoAs depend on the used model system. Interestingly, in vivo, pathways related to inflammation were affected to a greater extent than oxidative stress responses. Hence, the assignment of the observed effects to the levels of oxidative stress was different in vitro and in vivo as well. However, the overall classification of “active” and “passive” NMs was consistent in vitro and in vivo.Conclusions The consistent classification indicates both tested cell lines to be suitable for NM toxicity assessment even though the induced levels of oxidative stress strongly depend on the used model systems. Thus, the here presented results highlight that model systems need to be carefully revised to decipher the extent to which they can replace in vivo testing.

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Alveolar epithelial cell injury with Epstein-Barr virus upregulates TGFβ1 expression

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00376.2007 ◽

2008 ◽

Vol 295 (3) ◽

pp. L451-L460 ◽

Cited By ~ 28

Author(s):

Andrea P. Malizia ◽

Dominic T. Keating ◽

Sinead M. Smith ◽

Dermot Walls ◽

Peter P. Doran ◽

...

Keyword(s):

Cell Proliferation ◽

Epithelial Cell ◽

Epithelial Cells ◽

Epstein Barr Virus ◽

Cell Injury ◽

Alveolar Epithelial Cells ◽

Alveolar Epithelial ◽

Barr Virus ◽

Epstein Barr ◽

Epithelial Cell Injury

Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation, and ECM protein deposition. Epstein-Barr virus (EBV) has previously been localized to alveolar epithelial cells of IPF patients and is associated with a poor prognosis. In this study, we utilized a microarray-based differential gene expression analysis strategy to identify molecular drivers of EBV-associated lung fibrosis. Two cell lines, primary human alveolar epithelial cells type 2 and A549 cells, were infected with EBV. EBV lytic phase induction increased active and total transforming growth factor-β1 (TGFβ1) transcript expression in association with reduced cell proliferation and increased caspase 3/7 activity. Exposing EBV-infected cells to ganciclovir resulted in TGFβ1 deregulation and reduced expression of EBV early response genes, BRLF1 and BZLF1. We targeted the BRLF1 and BZLF1 gene products, Rta and Zta, by silencing RNA, and this resulted in the normalization of TGFβ1 transcript and cell proliferation levels. Our study using a viral cell line model complements existing human and animal model data and further provides evidence to suggest that viral epithelial cell injury may play a role in IPF.

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Effect of cigarette smoke and its condensates on alveolar epithelial cell injury in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.266.1.l92 ◽

1994 ◽

Vol 266 (1) ◽

pp. L92-L100 ◽

Cited By ~ 18

Author(s):

S. Lannan ◽

K. Donaldson ◽

D. Brown ◽

W. MacNee

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Cigarette Smoke ◽

Cell Injury ◽

Cell Attachment ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelial Cells ◽

Type Ii ◽

Alveolar Epithelial

The oxidant-antioxidant balance in the airspaces of the lungs may be critical in protecting the lungs from the effects of cigarette smoke. We studied the effect of cigarette smoke and its condensates on the detachment, attachment, and proliferation of the A549 human alveolar epithelial cell line, in an in vitro model of cell injury and regeneration and the protective effects of antioxidants. Whole and vapor phase cigarette smoke decreased 51Cr-labeled A549 cell attachment, increased cell detachment, and decreased cell proliferation, as assessed by [3H]thymidine uptake. Freshly isolated rat type II alveolar epithelial cells showed an enhanced susceptibility to smoke-induced cell lysis when compared with the A549 cell line. Reduced glutathione (GSH) (400 microM) protected against the effects of cigarette smoke exposure on cell attachment, proliferation, and detachment. Depletion of intracellular GSH with buthionine sulfoxamine enhanced the epithelial cell detachment injury produced by smoke condensates. We conclude that cigarette smoke and its condensates cause an oxidant-induced injury to A549 human type II alveolar epithelial cells. Both intra- and extracellular GSH have important roles in protecting epithelial cells from the injurious effects of cigarette smoke.

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Nanomaterials induce different levels of oxidative stress, depending on the used model system: Comparison of in vitro and in vivo effects

10.21203/rs.3.rs-57664/v2 ◽

2021 ◽

Author(s):

Isabel Karkossa ◽

Anne Bannuscher ◽

Bryan Hellack ◽

Wendel Wohlleben ◽

Julie Laloy ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Alveolar Macrophages ◽

Model System ◽

Alveolar Epithelial Cells ◽

Model Systems ◽

Alveolar Epithelial ◽

Different Levels

Abstract Background: The immense variety and constant development of nanomaterials (NMs) raise the demand for a facilitated risk assessment, for which knowledge on NMs mode of actions (MoAs) is required. For this purpose, a comprehensive data basis is of paramountcy that can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is indispensable to follow the 3R concept and to master the high number of NMs. In the present study, we aimed at comparing NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data evaluation strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For in vitro investigations, rat alveolar epithelial cells (RLE-6TN) and rat alveolar macrophages (NR8383) were treated with different doses of NMs, and the results were compared to effects on rat lungs after short-term inhalations and instillations at varying doses with and without a recovery period.Results: Since the production of reactive oxygen species (ROS) is described to be a critical biological effect of NMs, and enrichment analyses confirmed oxidative stress as a significant effect upon NM treatment in vitro in the present study, we focused on different levels of oxidative stress. Thus, we found opposite changes for proteins and metabolites that are related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, illustrating that NMs MoAs depend on the used model system. Interestingly, in vivo, pathways related to inflammation were affected to a greater extent than oxidative stress responses. Hence, the assignment of the observed effects to the levels of oxidative stress was different in vitro and in vivo as well. However, the overall classification of “active” and “passive” NMs was consistent in vitro and in vivo.Conclusions: The consistent classification indicates both tested cell lines to be suitable for NM toxicity assessment even though the induced levels of oxidative stress strongly depend on the used model systems. Thus, the here presented results highlight that model systems need to be carefully revised to decipher the extent to which they can replace in vivo testing.

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TGF-β-Induced α-SMA Expression is Mediated by C/EBPβ Acetylation in Human Alveolar Epithelial Cells In Vitro

10.21203/rs.3.rs-76632/v1 ◽

2020 ◽

Author(s):

Hui Ding ◽

Jinjun Chen ◽

Jingping Qin ◽

Ruhua Chen ◽

Zili Yi

Keyword(s):

A549 Cells ◽

Alveolar Epithelial Cells ◽

Collagen I ◽

Alveolar Epithelial ◽

Matrix Deposition ◽

Extracellular Matrix Deposition ◽

Vitro Model ◽

Relationship Of ◽

Tgf Β1

Abstract This study sought to determine whether binding of acetylated C/EBPβ to α-SMA promoter could affect its activity and was essential for EMT and extracellular matrix deposition in IPF using in vitro model. The expression of EMT and C/EBPβ in A549 cells with TGF-β as pulmonary fibrotic model were detected by western blotting and qPCR. Collagen-I expression using ELISA was performed. The luciferase activity was used to examine the activity of C/EBPβ. Knockdown of C/EBPβ was performed by siRNA. We also investigated the effect of deacetylation of C/EBPβ on EMT using SIRT1. The binding ability of C/EBPβ with α-SMA promoter was affirmed via ChIP and EMSA. The relationship of α-SMA and acetylated C/EBPβ was investigated by Co-IP. SiRNA-mediated knockdown of C/EBPβ in A549 cells attenuated TGF-β1-induced myofibroblast differentiation and ECM deposition. The extent of association between acetylated C/EBPβ and α-SMA promoter was dynamically monitored. It was confirmed that deacetylation of C/EBPβ in A549 cells successfully ameliorated TGF-β1-induced EMT, as shown by reduction in α-SMA expression and excessive collagen-I accumulation. The EMT and fibrotic effect of TGF-β1 dependent on acetylated C/EBPβ-mediated regulation of α-SMA gene activity. C/EBPβ acetylation may play a central role in pulmonary fibrosis.

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TGF-β-induced α-SMA expression is mediated by C/EBPβ acetylation in human alveolar epithelial cells in vitro

10.21203/rs.3.rs-17120/v1 ◽

2020 ◽

Author(s):

Hui Ding ◽

Jinjun Chen ◽

Jingping Qin ◽

Ruhua Chen ◽

Zili Yi

Keyword(s):

A549 Cells ◽

Alveolar Epithelial Cells ◽

Collagen I ◽

Alveolar Epithelial ◽

Matrix Deposition ◽

Luciferase Activity Assay ◽

Extracellular Matrix Deposition ◽

Vitro Model ◽

Tgf Β1

Abstract Background This study sought to determine whether binding of acetylated C/EBPβ to α-SMA promoter could affect its activity and was essential for EMT and extracellular matrix deposition in IPF using in vitro model. Methods Through western blotting, the expression of EMT and C/EBPβ were detected in A549 cells with TGF-β as pulmonary fibrotic model in vitro. Moreover, the expression of C/EBPβ mRNA via Real Time-PCR and Collagen-I expression using ELISA were performed. The luciferase activity assay was used to examine the activity of C/EBPβ. The knockdown expression of C/EBPβ gene was prepared in A549 cells with C/EBPβ siRNA. We also investigated the effect of deacetylation of C/EBPβ on EMT using SIRT1. The binding ability of C/EBPβ with the α-SMA promoter was affirmed via ChIP and EMSA. Furthermore, the relationship between α-SMA and acetylated C/EBPβ was investigated using the co-immunoprecipitation. Results SiRNA-mediated knockdown of C/EBPβ in A549 cells attenuated TGF-β1-induced myofibroblast differentiation and ECM deposition. The extent of association between acetylated C/EBPβ and the α-SMA promoter was dynamically monitored. Furthermore, it was confirmed that deacetylation of C/EBPβ in A549 cells successfully ameliorated TGF-β1-induced EMT, as shown by reduction in α-SMA expression and excessive collagen-I accumulation. Conclusions Collectively, our data suggested that the EMT and fibrotic effect of TGF-β1 could be dependent on acetylated C/EBPβ-mediated regulation of α-SMA gene activity. This thus suggests that C/EBPβ acetylation may play a central role in pulmonary fibrosis.

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Nuclear Factor-kappaB Regulates the Transcription of NADPH Oxidase 1 in Human Alveolar Epithelial Cells

10.21203/rs.3.rs-132252/v1 ◽

2020 ◽

Author(s):

Weijing Wu ◽

Li Li ◽

Xiaoshan Su ◽

ZHIXING ZHU ◽

Xiaoping Lin ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

A549 Cells ◽

Alveolar Epithelial Cells ◽

Ros Generation ◽

Alveolar Epithelial ◽

Tnf Α ◽

Potential Regulatory Role ◽

Human Alveolar Epithelial Cells

Abstract Objective Acute lung injury (ALI) is characterized by inflammation and oxidative stress. Nuclear factor-kappaB (NF-κB) mediates the expression of various inflammation-related genes, including the NADPH oxidase family. This study aimed to identify the potential regulatory role of NF-𝜅B on NADPH oxidases in TNF-α-induced oxidative stress in human alveolar epithelial cells. Methods Type II alveolar epithelial cell-derived A549 cells were treated with TNF-α for 24 hours to establish ALI cell models. RT-PCR, western blot, DCFH-DA ROS assay, Alibaba 2.1 online analysis, electrophoretic mobility shift assays and luciferase reporter analysis were employed to identify the potential regulatory role of NF-𝜅B on NADPH oxidases in TNF-α-induced oxidative stress in human alveolar epithelial cells. Results The expression of NF-κB/p65 was notably upregulated in TNF-α-stimulated A549 cells. NF-κB knockdown by siRNA significantly inhibited the TNF-α-induced ROS generation. Moreover, NF-𝜅B/p65 siRNA could inhibite the activation of NOX1, NOX2 and NOX4 mRNA and protein expression in TNF-α-stimulated A549 cells. The next study demonstrated that NF-𝜅B activated the transcription of NOX1 by binding to the -261 to -252 bp (NOX1/κB2, TAAAAATCCC) region of NOX1 promoter in TNF-α-stimulated A549 cells. Conclusion Our data demonstrated that NF-κB can aggravate TNF-α-induced ALI by regulating the activation of ROS generation and the expression of NOX1, NOX2 and NOX4. Moreover, NF-𝜅B could promote the NOX1 transcriptional activity via binding its promoter in TNF-α-stimulated A549 cells.

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Stretch induces cytokine release by alveolar epithelial cells in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.1.l167 ◽

1999 ◽

Vol 277 (1) ◽

pp. L167-L173 ◽

Cited By ~ 98

Author(s):

Nicholas E. Vlahakis ◽

Mark A. Schroeder ◽

Andrew H. Limper ◽

Rolf D. Hubmayr

Keyword(s):

Epithelial Cells ◽

Cell Damage ◽

A549 Cells ◽

Alveolar Epithelium ◽

Alveolar Epithelial Cells ◽

Structural Cell ◽

Alveolar Epithelial ◽

Macrophage Colony ◽

Factor Α

Mechanical ventilation can injure the lung, causing edema and alveolar inflammation. Interleukin-8 (IL-8) plays an important role in this inflammatory response. We postulated that cyclic cell stretch upregulates the production and release of IL-8 by human alveolar epithelium in the absence of structural cell damage or paracrine stimulation. To test this hypothesis, alveolar epithelial cells (A549 cells) were cultured on a deformable silicoelastic membrane. When stretched by 30% for up to 48 h, the cells released 49 ± 34% more IL-8 ( P < 0.001) than static controls. Smaller deformations (20% stretch) produced no consistent increase in IL-8. Stretch of 4 h duration increased IL-8 gene transcription fourfold above baseline. Stretch had no effect on cell proliferation, cell viability as assessed by51Cr release assay, or the release of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α. We conclude that deformation per se can trigger inflammatory signaling and that alveolar epithelial cells may be active participants in the alveolitis associated with ventilator-induced lung injury.

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