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

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, Ecadherin, 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.

A Two-Step Bioreactor for Decellularized Lung Epithelialization

“…This study also shows short-term cell distribution over 3 days and, therefore, does not provide context for cell migration, proliferation, or differentiation. Extending the culture duration with the addition of recent bioreactor upgrades [Gilpin et al, 2014a;Nichols et al, 2014], scaffold pre-conditioning [Gilpin et al, 2017;Nichols et al, 2018;Young et al, 2019], multiple cell lineages and differentiation techniques [Ghaedi et al, 2014;Gilpin et al, 2014b;Stabler et al, 2016], and other practices for long-term tissue culture, this system can propel the study of more complex tissue transplantation challenges such as cellular differentiation, barrier integrity, longer-term function by starting with an efficient seeding mechanism.…”

“…MLE12 Cell Attachment Rate Assay 250,000 cells/cm 2 were seeded onto TCP coated with 0.1 mg/ mL porcine lung dECM prepared by protocols previously published by our laboratory [Link et al, 2017;Pouliot et al, 2016;Young et al, 2019Young et al, , 2020. Every 20 min for 4 h, unadhered cells and media were aspirated from a well and rinsed gently before fixation with 4% PFA.…”

Salvia miltiorrhiza Injection Alleviates LPS‐Induced Acute Lung Injury by Adjusting the Balance of MMPs/TIMPs Ratio

“…e ECM is the main structural component of the alveolar-capillary membrane, which not only plays a mechanical-supporting and mechanical-connecting role between cells but is also important in maintaining normal Evidence-Based Complementary and Alternative Medicine tissue structure and function [9,32,33]. Previous work has shown that ECM metabolic disorders are involved in the pathophysiological process of ALI and chronic lung injury [34,35].…”

Cell Communications among Microorganisms, Plants, and Animals: Origin, Evolution and Interplays

“…In animals, cells are in contact with each other not only directly through these adhesion proteins but also indirectly via the extracellular matrix, ECM, kind of an extracellular "glue," which includes proteins such as collagens, fibronectins, or laminins [89][90][91]. Various adhesion proteins such as cadherins, integrins, CAM (Cell Adhesion Molecules), selectins, etc., participate in these direct or indirect contacts between cell surfaces [92][93][94].…”