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Local Influence of Cell Viability on Stretch-Induced Permeability of Alveolar Epithelial Cell Monolayers

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Abstract

Ventilator induced lung injury, often attributed to over-distension of the alveolar epithelial cell layer, can trigger loss of barrier function. Alveolar epithelial cell monolayers can be used as an idealized in vitro model of the pulmonary epithelium, with cell death and tight junction disruption and permeability employed to estimate stretch-induced changes in barrier function. We adapted a method published for vascular endothelial permeability, compare its sensitivity with our previously published method, and determine the relationship between breeches in barrier properties after stretch and regions of cell death. After 4–5 days in culture, primary rat alveolar epithelial cells seeded on plasma-treated polydimethylsiloxane membrane coated with biotin-labeled fibronectin or fibronectin alone were stretched in the presence of FITC-tagged streptavidin (biotin-labeled membrane) or BODIPY-ouabain. We found that the FITC-labeling method was a more sensitive indicator of permeability disruption, with significantly larger positively stained areas visible in the presence of stretch and with ATP production inhibitor Antimycin-A. Triple-stained images with Hoescht (nuclei), Ethidium Homodimer (EthD, damaged cell nuclei) and FITC (permeable regions) were used to determine that intact cells were positioned closer to damaged cells in permeable regions than in non-permeable regions. We concluded that local cell death may be an important contributor to barrier integrity.

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Acknowledgements

This research was supported by NIH R01-HL57204 and the Stephenson Fund of the University of Pennsylvania.

Conflict of interest

The authors (Song, Davis, Lawrence, and Margulies) have no conflicts of interest to disclose.

Ethical Standards

No human studies were conducted for this research. The animal was protocol approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).

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Authors and Affiliations

  1. Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 South 33rd Street, Philadelphia, PA, 19104-6321, USA

    M. J. Song, C. I. Davis, G. G. Lawrence & S. S. Margulies

Authors
  1. M. J. Song
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  2. C. I. Davis
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  3. G. G. Lawrence
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  4. S. S. Margulies
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Corresponding author

Correspondence to S. S. Margulies.

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Associate Editor Michael R. King oversaw the review of this article.

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Figure S1

Distribution of FITC-streptavidin–biotin on cell-free PDMS membranes with and without O2-plasma treatment. Representative images 5 min after 25ug/ml of FITC-streptavidin was added to biotin (40 nM) labeled fibronectin coated PDMS membranes. Bars = 100 µm. Supplementary material 1 (TIFF 3594 kb)

12195_2015_405_MOESM2_ESM.tif

Percent of image field above unstretched monolayers. All values provided as mean ± SEM. Supplementary material 2 (TIFF 133 kb)

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Song, M.J., Davis, C.I., Lawrence, G.G. et al. Local Influence of Cell Viability on Stretch-Induced Permeability of Alveolar Epithelial Cell Monolayers. Cel. Mol. Bioeng. 9, 65–72 (2016). https://doi.org/10.1007/s12195-015-0405-8

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