Revealing the Complexity of the Epicardial Secretome: Characterization and Functional Assessment of Epicardium-Derived Extracellular Vesicles and Matrix

Abstract

Aim: The epicardium, an epithelial layer covering the heart, plays pivotal roles in embryonic heart development and responses to cardiac damage. Epicardial secreted molecular agents are known to be involved in the regulation of these phenomena, but how this regulation occurs is poorly understood. In this study, we have investigated extracellular vesicle (EV) and extracellular matrix (ECM) components of epicardial secretions using a continuous mouse embryonic epicardial-derived cell (EPIC) line. Methods and results: Epicardial-derived EVs were isolated using differential ultracentrifugation from EPIC cultured at 1% (EVs-H1%), 5% (EVs-H5%), and 21% oxygen (EVs-N). EVs protein content was determined by tandem mass tag (TMT) proteomic analysis. The results showed that epicardial-derived EVs cargo is sensitive to the oxygen level of their parenteral cells, increasing their content on glycolytic proteins. Moreover, hypoxic-derived EVs were found to both increase EPIC proliferation and affect the metabolism of Primary Human Umbilical Vein Endothelial Cells (HUVECs). On the other hand, epicardial-derived extracellular matrix (EPIC-ECM) was characterized by subjecting decellularized EPIC to shotgun proteomics and comparing it to decellularized perinatal hearts and Matrigel. We found that EPIC-ECM composition closely resembles that of embryonic cardiac tissue. Although the structural and basement membrane-associated proteins of EPIC-ECM were similar to those found in Matrigel, EPIC-ECM exhibited higher protein diversity and was a more potent inducer of HUVEC proliferation. Conclusion: This work represents the first comprehensive and systematic proteomic analysis of two important components of the epicardial-derived secretome. Our experiments reveal that the epicardium responds to hypoxia by secreting EVs capable of modifying the metabolic responses of surrounding cells. Furthermore, EPIC-ECM promotes endothelial cell proliferation. These findings demonstrate the significant signaling abilities of the epicardial secretome, consistent with reports of endothelial responses following cardiac ischemic damage.