The elevation of JAG surface expression in
The elevation of JAG1 surface expression in transitional ECs suggests that it plays a role in phenotypic transition of these cells. Overexpression of activated JAG1 cDNA in cultured human microvascular ECs activated a SMA-promoter driven luciferase and induced trans-differentiation to vascular smooth muscle orexin (Noseda et al., 2004). Elevation of JAG1 during wound healing can function to attenuate activation of Notch signaling (Pedrosa et al., 2015) and expression profiling of CD31 ECs and CD31PDGFRβ derivatives of EndMT revealed markedly reduced expression of genes related to Notch activation (Fig. 3e). Yet JAG1 is likely not the sole actor in reducing Notch pathway activation, as JAG1 knockdown leads to increased, instead of decreased EndMT. Here, we showed that increased Notch activation, as reflected by expression of Notch target DLL4, correlates with the degree of EndMT in hESC-EC monolayers (Fig. 5d and e). Previous work in Drosophila has identified the potential for Delta (DLL4 orthologue) to inhibit Notch on its own cell (de Celis and Bray, 1997; Klein et al., 1997; Micchelli et al., 1997; Miller et al., 2009), and Sprinzak et al. have modeled cis- and trans- activity of human DLL1-Notch1 in live cells using real time fluorescence microscopy (Sprinzak et al., 2010). This system revealed that while the response to trans-DLL1 is graded, inhibitory action of cis-DLL1 is sharp and occurs at a fixed threshold, irrespective of trans-DLL1 activity. Thus, JAG1 may contribute to attenuation of Notch activation in hESC-ECs, but cis-inhibitory activity of DLL4 at a critical threshold of Notch hyper-activation likely accounts for the abrupt shift in Notch signaling status during EndMT.
Given the technical and bioethical hurdles associated with the study of human embryogenesis, a human cell-based model that approximates in vivo cardiogenesis provides unique insight into the cellular/molecular bases of cardiac anomalies. Moreover, mounting evidence supports a role for EndMT in wound healing and a broad array of pathological disease processes, including pulmonary (Arciniegas et al., 2005), intestinal (Rieder et al., 2011), cardiac (Zeisberg et al., 2007) and kidney fibrosis (Zeisberg et al., 2008). EndMT has also been identified as a critical mediator of neo-intima formation following coronary artery bypass grafting (Cooley et al., 2014) and has even been implicated in the advance of atherosclerosis in a mouse model of cardiovascular disease (Chen et al., 2015). Our results establish an in vitro model of embryonic EndMT using hESC-derived ECs, and utilize this platform to integrate EC membrane-bound Notch signaling inputs (DLL4) with pathway activation status and the initiation of EndMT. Understanding of the signaling mechanisms that regulate EC identity and its organization into multicellular vascular channels will be vital to implementing hESC-based vascular applications, and insight into molecular influences that mediate embryonic EndMT may provide fertile ground for therapeutic targeting of pathways that contribute to degenerative disease and organ/tissue fibrosis.
Materials and methods
Introduction Human adipose tissue-derived stromal cells (hASCs) are considered a potential source of adult stem cells (Gimble et al., 2007). hASCs can be easily isolated in large quantities with minimal invasion procedures (Zuk et al., 2002) and maintain the potential of differentiation into adipocytes. These features make these cells ideal candidates for use in cell therapy (Spangenberg et al., 2013). A better understanding of the specific mechanisms that regulate proliferation and differentiation of hASCs may yield information about how stem cells behave into an organism and suggest new strategies for therapy (Zhang et al., 2013). Metabolism, mainly energy metabolism, plays an important role in dictating whether a cell proliferates, differentiates or remains quiescent and some metabolic pathways are known to determinate cell fate (Shyh-Chang et al., 2013). Metabolic modulation of adult stem cells can maintain stem cell potency (Zhang et al., 2013) or direct adult stem cell differentiation into specific cell types (Shyh-Chang et al., 2013).