An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke

COPD is characterized by irreversible lung tissue damage. We hypothesized that lung-derived mesenchymal stromal cells (LMSCs) reduce alveolar epithelial damage via paracrine processes, and may thus be suitable for cell-based strategies in COPD. We aimed to assess whether COPD-derived LMSCs display abnormalities. LMSCs were isolated from lung tissue of severe COPD patients and non-COPD controls. Effects of LMSC conditioned-medium (CM) on H2O2-induced, electric field- and scratch-injury were studied in A549 and NCI-H441 epithelial cells. In organoid models, LMSCs were co-cultured with NCI-H441 or primary lung cells. Organoid number, size and expression of alveolar type II markers were assessed. Pre-treatment with LMSC-CM significantly attenuated oxidative stress-induced necrosis and accelerated wound repair in A549. Co-culture with LMSCs supported organoid formation in NCI-H441 and primary epithelial cells, resulting in significantly larger organoids with lower type II-marker positivity in the presence of COPD-derived versus control LMSCs. Similar abnormalities developed in organoids from COPD compared to control-derived lung cells, with significantly larger organoids. Collectively, this indicates that LMSCs’ secretome attenuates alveolar epithelial injury and supports epithelial repair. Additionally, LMSCs promote generation of alveolar organoids, with abnormalities in the supportive effects of COPD-derived LMCS, reflective of impaired regenerative responses of COPD distal lung cells.

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Loss of Gadd45a does not modify the pulmonary response to oxidative stress

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00355.2004 ◽

2005 ◽

Vol 288 (4) ◽

pp. L663-L671 ◽

Cited By ~ 6

Author(s):

Jason M. Roper ◽

Sean C. Gehen ◽

Rhonda J. Staversky ◽

M. Christine Hollander ◽

Albert J. Fornace ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Epithelial Cells ◽

Oxidative Dna Damage ◽

Genotoxic Stress ◽

Clara Cell ◽

Heme Oxygenase 1 ◽

Alveolar Type ◽

Type I ◽

Type Ii

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage ( Gadd) 45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (−/−) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21Cip1/WAF1 and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (−/−) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.

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Cytotoxic effects of cigarette smoke extract on an alveolar type II cell-derived cell line

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.2.l509 ◽

2001 ◽

Vol 281 (2) ◽

pp. L509-L516 ◽

Cited By ~ 128

Author(s):

Yuma Hoshino ◽

Tadashi Mio ◽

Sonoko Nagai ◽

Hiroyuki Miki ◽

Isao Ito ◽

...

Keyword(s):

Cell Death ◽

Cell Line ◽

Cigarette Smoke ◽

A549 Cells ◽

Cigarette Smoke Extract ◽

Alveolar Type ◽

Type Ii ◽

Cytotoxic Effects ◽

Alveolar Type Ii Cell ◽

Type Ii Cell

Injury of the alveolar epithelium by cigarette smoke is presumed to be an important process in the pathogenesis of smoking-related pulmonary diseases. We investigated the cytotoxic effects of cigarette smoke extract (CSE) on an alveolar type II cell-derived cell line (A549). CSE caused apoptosis at concentrations of 5% or less and necrosis at 10% or more. When CSE was exposed to air before application to A549 cells, the cytotoxic effects were attenuated. CSE caused cell death without direct contact with the cells. Acrolein and hydrogen peroxide, two major volatile factors in cigarette smoke, caused cell death in a similar manner. Aldehyde dehydrogenase, a scavenger of aldehydes, and N-acetylcysteine, a scavenger of oxidants and aldehydes, completely inhibited CSE-induced apoptosis. CSE and acrolein increased intracellular oxidant activity. In conclusion, apoptosis of alveolar epithelial cells may be one of the mechanisms of lung injury induced by cigarette smoking. This cytotoxic effect might be due to an interaction between aldehydes and oxidants present in CSE or formed in CSE-exposed cells.

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Ascorbate uptake by isolated rat alveolar macrophages and type II cells

Journal of Applied Physiology ◽

10.1152/jappl.1983.54.1.208 ◽

1983 ◽

Vol 54 (1) ◽

pp. 208-214 ◽

Cited By ~ 20

Author(s):

V. Castranova ◽

J. R. Wright ◽

H. D. Colby ◽

P. R. Miles

Keyword(s):

Alveolar Macrophages ◽

Membrane Surface ◽

Lung Cells ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Sodium Influx ◽

Saturation Kinetics ◽

Ascorbate Uptake ◽

Isolated Rat

Studies were conducted to measure intracellular ascorbate content and to characterize ascorbate uptake in three fractions of isolated rat pneumocytes (i.e., alveolar macrophages, alveolar type II epithelial cells, and another fraction of small pneumocytes that contains neither macrophages nor type II cells). When cells are incubated in medium containing 0.1 mM ascorbate (i.e., the concentration normally found in plasma), intracellular ascorbate concentrations are 3.2 mM in alveolar macrophages and type II cells and 0.9 mM in other lung cells; ascorbate influx is 1.5 nmol . 10(7) cells-1 . h-1 for alveolar macrophages, 0.24 nmol . 10(7) cells-1 . h-1 for type II cells, and very slow in other pneumocytes. Ascorbate influx displays saturation kinetics in both alveolar macrophages (K1/2 = 2 mM; Vmax = 32.2 nmol . 10(7) cells-1 . h-1) and type II cells (K1/2 = 5 mM; Vmax = 14.2 nmol . 10(7) cells-1 . h-1). After correction for differences in the membrane surface areas of these two types of lung cells, the rates for maximum ascorbate influx (Vmax) are similar in alveolar macrophages and type II cells. In addition, ascorbate uptake by alveolar macrophages and type II cells is dependent on metabolic activity and extracellular sodium. In contrast, ascorbate uptake in other lung cells does not exhibit saturation kinetics and is not dependent on metabolism or sodium. Thus alveolar macrophages and type II cells possess an energy-dependent cotransport system for ascorbate and sodium influx. The high ascorbate content and the existence of a specialized transport mechanism for ascorbate uptake may explain the relative resistance of alveolar macrophages and type II cells to oxidant injury.

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