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

Abstract

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 co-staining 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-CreERT2 and Cdh5(PAC)-CreERT2 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 α-actin (αSMA) 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)-CreERT2 driver line, Tie2-CreERT2 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.

Endothelial-to-Mesenchymal Transition

“…In vitro studies with valve endothelial cells provide supporting evidence for postdevelopment EndMT capability 3 , and thus we speculated that EndMT persists at a low level throughout life in order to replenish valve interstitial cells, major producers of the valve extracellular matrix needed to insure durability and function. In contrast, EndMT is not seen in adult murine valves when analyzed by in vivo endothelial lineage tracing 4 , indicating that species, models and experimental tools are important variables. We found EndMT increased, in vivo, when ovine mitral valve leaflets were exposed to mechanical stretch designed to mimic the tethering imparted on the leaflets when the left ventricle enlarges after myocardial infarction; EndMT occurred with little evident TGFβ or leukocyte infiltration, and coincided with an increase in leaflet area 5,6 .…”

“…Our understanding of EndMT has grown by leaps and bounds with the wealth of new tools and enhanced experimental capabilities. Inducible endothelial lineage tracing in murine models is particularly useful for distinguishing mesenchymal cells derived from embryonic versus post-natal EndMT 4,14 . In vitro studies complement and extend in vivo findings, and enable mechanistic experiments that can sharpen hypotheses for further testing in vivo.…”

Smooth Muscle α-Actin Expression in Mitral Valve Interstitial Cells is Important for Mediating Extracellular Matrix Remodeling

“…Studies of canine myxomatous mitral valve disease have suggested that SMA expression is the product of endothelial-to-mesenchymal transformation (EMT) [ 43 ]. However, in mouse models of myxomatous degeneration, cell tracing experiments have not detected active EMT [ 44 ]. Therefore, we anticipate that SMA is induced by alternative mechanisms in murine mitral valve disease.…”

Biology and Biomechanics of the Heart Valve Extracellular Matrix

“…However, this process is less controlled. While endothelial-to-mesenchymal transitions have been shown to give rise to SMA-positive cells in the embryo, fate map studies in valve disease models suggest that this process does not occur in adult valve disease [ 120 ], but it may mediate disease in canine models [ 121 ]. As there is accumulating data to suggest that the transition of qVICs to aVICs may be one of the early triggers that initiates progressive degeneration, the field has turned to understanding the mechanistic triggers.…”