Isolation of Murine Valve Endothelial Cells

Abstract

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms. Video LinkThe video component of this article can be found at

Growth and maturation of heart valves leads to changes in endothelial cell distribution, impaired function, decreased metabolism and reduced cell proliferation

“…(27) Analysis revealed several mRNAs differentially expressed between each time point, with the largest change observed between embryonic and aging adults, and the smallest between young and aging adults (Figures 7A, B). This approach also revealed mRNAs that were unique (Supplementary Table 1) and common (Supplementary Table 2) to VEC populations at one, or more time points (Figure 7B), as well as distinct gene clustering patterns (Figure 7C) at each stage (see Supplementary Figure 1 for validation).…”

“…(27) Briefly, valvular tissue from both semilunar and atrioventricular valves was dissected and dissociated using collagenase IV for 7 mins at 37°C. The supernatant, containing the dissociated cells was collected and kept on ice.…”

“…Isolated VECs from PN and young adult mice, as described previously in 2.1 and by our lab(27). Cells were then pelleted, resuspended in 4% paraformaldehyde for 10 minutes at 37°C, and chilled on ice for one minute.…”

“…VECs from embryonic, PN, young adult, and aging adult (12–15 months) mice were isolated as previously described by our lab(27) and described in 2.1.1. Isolated cells were stored in Trizol and submitted for RNA extraction and RNA sequencing at Ocean Ridge Biosciences (Palm Beach Gardens, Florida, USA).…”

Cross Talk between NOTCH Signaling and Biomechanics in Human Aortic Valve Disease Pathogenesis

“…Another limitation of this study is the use DAPT as a non-specific inhibitor of NOTCH signaling, as this may lead to significant off-target effects such as the inhibition of the Erb-B4 signaling pathways [ 82 ]. Additionally, valvular endothelial cell isolation is technically difficult and not logistically feasible for large-scale studies; however, recent literature has begun to develop new and promising protocols aimed at achieving pure isolations [ 83 ]. Moreover, HUVECs have been shown to demonstrate a biological interplay with valvular interstitial cells in vitro , serving as a widely accepted surrogate [ 73 ].…”