Proteomics Analysis of Extracellular Vesicles from In Vitro Models of the Human Airway.

Extracellular vesicle (EV)-mediated intercellular communication significantly influences pulmonary cell health and disease outcomes, yet in vitro methods to investigate these pathophysiological mechanisms are limited. We hypothesize that organotypic models of the airway can be leveraged to investigate EV-mediated intercellular signaling, focusing on EV proteomic content as a case study. in this study, two in vitro airway culture models were evaluated using mass spectrometry-based proteomics: a tri-culture model consisting of alveolar epithelial, fibroblast, and lung microvascular endothelial cells and a co-culture model consisting of alveolar epithelial and fibroblast cells. EVs isolated from the tri-culture model showed enrichment for EV proteins regulating RNA-to-protein translation. EVs isolated from the co-culture model were enriched with EV biogenesis and extracellular matrix signaling proteins. These model-specific differences suggest that different pulmonary cell types uniquely affect EV composition and the biological pathways influenced by the EV proteome in recipient cells. These findings inform future study designs surrounding EV-related pulmonary disease pathogenesis and therapeutics.