Endothelial Cell Lineage Analysis Does Not Provide Evidence for EMT in Adult Valve Homeostasis and Disease

Epithelial-to-mesenchymal transition (EMT) enables stationary epithelial cells to exhibit migratory behavior and is the key step that initiates heart valve development. Recent studies suggest that EMT is reactivated in the pathogenesis of myxomatous valve disease (MVD), a condition that involves the progressive degeneration and thickening of valve leaflets. These studies have been limited to in vitro experimentation and reliance on histologic costaining of epithelial and mesenchymal markers as evidence of EMT in mouse and sheep models of valve disease. However, longitudinal analysis of cell lineage origins and potential pathogenic or reparative contributions of newly generated mesenchymal cells have not been reported previously. In this study, a genetic lineage tracing strategy was pursued by irreversibly labeling valve endothelial cells in the Osteogenesis imperfecta and Marfan syndrome mouse models to determine whether they undergo EMT during valve disease. Tie2-CreER(T2) and Cdh5(PAC)-CreER(T2) mouse lines were used in combination with colorimetric and fluorescent reporters for longitudinal assessment of endothelial cells. These lineage tracing experiments showed no evidence of EMT during adult valve homeostasis or valve pathogenesis. Additionally, CD31 and smooth muscle -actin (SMA) double-positive cells, used as an indicator of EMT, were not detected, and levels of EMT transcription factors were not altered. Interestingly, contrary to the endothelial cell-specific Cdh5(PAC)-CreER(T2) driver line, Tie2-CreER(T2) lineage-derived cells in diseased heart valves also included CD45+ leukocytes. Altogether, our data indicate that EMT is not a feature of valve homeostasis and disease but that increased immune cells may contribute to MVD. Anat Rec, 302:125-135, 2019. (c) 2018 Wiley Periodicals, Inc.