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Laminin-driven Epac/Rap1 regulation of epithelial barriers on decellularized matrix

Bethany M. Young, Keerthana Shankar, Cindy K. Tho, Amanda R. Pellegrino, Rebecca L. Heise
doi: https://doi.org/10.1101/612325
Bethany M. Young
1Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219
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Keerthana Shankar
1Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219
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Cindy K. Tho
1Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219
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Amanda R. Pellegrino
2Department of Biomedical Engineering and Nursing, Duquesne University, 600 Forbes Ave, Pittsburg, Pennsylvania 15282
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Rebecca L. Heise
1Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219
3Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, 1101 East Marshall St, Richmond, Virginia 23298
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  • For correspondence: rlheise@vcu.edu
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ABSTRACT

Decellularized tissues offer a unique tool for developing regenerative biomaterials or in vitro platforms for the study of cell-extracellular matrix (ECM) interactions. One main challenge associated with decellularized lung tissue is that ECM components can be stripped away or altered by the detergents used to remove cellular debris. Without characterizing the composition of lung decellularized ECM (dECM) and the cellular response caused by the altered composition, it is difficult to utilize dECM for regeneration and specifically, engineering the complexities of the alveolar-capillary barrier. This study takes steps towards uncovering if dECM must be enhanced with lost ECM proteins to achieve proper epithelial barrier formation. To achieve this, epithelial barrier function was assessed on dECM coatings with and without the systematic addition of several key basement membrane proteins. After comparing barrier function on collagen, fibronectin, laminin, and dECM in varying combinations as an in vitro coating, the alveolar epithelium exhibited superior barrier function when dECM was supplemented with laminin as evidenced by trans-epithelial electrical resistance (TEER) and permeability assays. Increased barrier resistance with laminin addition was associated with upregulation of Claudin-18, E- cadherin, and junction adhesion molecule (JAM)-A, and stabilization of zonula occludens (ZO)-1 at junction complexes. The Epac/Rap1 pathway was observed to play a role in the ECM-mediated barrier function determined by protein expression and Epac inhibition. These findings reveal potential ECM coatings and molecular therapeutic targets for improved regeneration with decellularized scaffolds or edema related pathologies.

  • Abbreviations

    ECM
    extracellular matrix
    dECM
    decellularized extracellular matrix
    TEER
    trans-epithelial electrical resistance
    JAM
    junction adhesion molecule
    ZO
    zonula occludens
    TJ
    tight junction
    AJ
    adherens junction
    PKA
    Protein kinase A
    Epac
    exchange protein directly activated by cAMP
    cAMP
    cyclic AMP
    AF-6
    Afadin
    EMT
    Epithelial to mesenchymal transition
    GAGs
    glycosaminoglycans
    Rap
    repressor activator protein
    SDC
    Sodium Deoxycholate
    BESC
    basal epithelial stem cell
    MLE12s
    mouse alveolar epithelial cells
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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    Posted April 18, 2019.
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    Laminin-driven Epac/Rap1 regulation of epithelial barriers on decellularized matrix
    Bethany M. Young, Keerthana Shankar, Cindy K. Tho, Amanda R. Pellegrino, Rebecca L. Heise
    bioRxiv 612325; doi: https://doi.org/10.1101/612325
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    Laminin-driven Epac/Rap1 regulation of epithelial barriers on decellularized matrix
    Bethany M. Young, Keerthana Shankar, Cindy K. Tho, Amanda R. Pellegrino, Rebecca L. Heise
    bioRxiv 612325; doi: https://doi.org/10.1101/612325

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