Epac agonist improves barrier function in iPSC-derived endothelial colony forming cells for whole organ tissue engineering

Efforts to decipher chronic lung disease and to reconstitute functional lung tissue through regenerative medicine have been hampered by an incomplete understanding of cell-cell interactions governing tissue homeostasis. Because the structure of mammalian lungs is highly conserved at the histologic level, we hypothesized that there are evolutionarily conserved homeostatic mechanisms that keep the fine architecture of the lung in balance. We have leveraged single-cell RNA sequencing techniques to identify conserved patterns of cell-cell cross-talk in adult mammalian lungs, analyzing mouse, rat, pig, and human pulmonary tissues. Specific stereotyped functional roles for each cell type in the distal lung are observed, with alveolar type I cells having a major role in the regulation of tissue homeostasis. This paper provides a systems-level portrait of signaling between alveolar cell populations. These methods may be applicable to other organs, providing a roadmap for identifying key pathways governing pathophysiology and informing regenerative efforts.

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“…Samples were imaged with confocal microscopy (Leica SP5). Detailed staining protocol can be found in the study of Yuan et al (47).…”

Section: Immunostaining and Imagingmentioning

confidence: 99%

Single-cell connectomic analysis of adult mammalian lungs

et al. 2019

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“…An Epac agonist, 007-AM was applied to collagen coatings, significantly increasing JAM-A and Rap1 gene expression, rearranging ZO-1 and the actin cytoskeleton cortically, and decreasing barrier permeability. 007-AM has recently been established as a longlasting treatment for endothelial barriers within bioengineered lungs [7] and treatment of vascular permeability within acute lung injury [15,68]. These data now confirm that Epac activation and laminin-enriched ECM coatings can both benefit the epithelial barriers to produce functional lung biomaterials and potentially serve as therapies for edema-related diseases.…”

Section: Discussionmentioning

confidence: 74%

“…Analysis of ZO-1 immunofluorescent staining at the cell junction showed that there was a significant decrease in the number of breaks in the ZO-1 junction staining per cell and a more linear junction morphology when cells were cultured on laminin. ZO-1 recruitment to the cell junction in an organized morphology is a strong correlation to alveolar permeability because it is required for assembly of AJs, TJs, and the actin cytoskeleton [7,47,[57][58][59].…”

Section: Discussionmentioning

confidence: 99%

“…Most of the research to date has focused on assessments of stem cell differentiation and attachment [1][2][3][4] or mimicking lung development during regeneration [5,6]. More recently, researchers in this field have examined recellularization on a smaller scale by evaluating endothelial barriers [7]; however, there is little research on epithelial barrier formation with recellularized lungs. This research takes a unique approach to re-epithelialization of whole organ scaffolds by examining alveolar junction formation on a cellular level to then inform future recellularization strategies.…”

Section: Introductionmentioning

confidence: 99%

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

Young

Shankar

Tho

et al. 2019

Preprint

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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.

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“…One approach to measure the integrity of the lung vascular niche is the transepithelial/transendothelial electrical resistance (TEER) measurement in a Transwell R assay. This method measures the integrity of TJ dynamics in cell culture models of endothelial and epithelial monolayers (Figure 1) (Neuhaus et al, 2012;Srinivasan et al, 2015;Luan et al, 2018;Yuan et al, 2019). TEER measurements have been used to assess the integrity in such systems as the blood-brain barrier (BBB), gastrointestinal (GI) tract, and pulmonary alveolar septa.…”

Section: Establishment Of An In Vitro Model To Study the Alveolar Walmentioning

confidence: 99%