Toren Finkel is supported by the Leducq Foundation (Transatlantic Network of Excellence Award) and the Progeria Research Foundation. by chemical or physical stimuli, with the most widely used being the application of transforming growth factor-beta (TGF-) for 5C8 days. Again, while a lack of standardization is problematic, an increasing tendency has been to use TGF- with an additional stimulus, such as interleukin (IL)-1 (7) or hydrogen peroxide (H2O2) (8). These Gamitrinib TPP hexafluorophosphate models have the advantage of providing a convenient and controllable environment to test novel factors Lepr and study molecular aspects of EndMT. They also provide a supply of cells that have undergone EndMT, which can be studied in downstream molecular and functional assays. However, a major limitation is usually that cell culture conditions (e.g. media, supplements) impact the extent and phenotype of EndMT. EndMT models At present, 3 principal methods are used for studying EndMT (9). Such mice are able to activate Cre-recombinase, which can be placed under the control of an endothelial-specific gene (e.g. (and also (12). In addition, due to its potential to resolve EC signatures while concurrently showing mesenchymal gene upregulation at the single cell level, it is anticipated that single cell RNA-sequencing will be another useful tool for studying EndMT in human samples. Furthermore, RNA-sequencing holds promise for providing insights on EC plasticity, which is the ability of an EC to switch its identity, including to additional phenotypes other than mesenchymal cells and also, having changed identity, to revert back to an EC state (see review (13)). Cellular and molecular analysis of EndMT A diverse selection of readouts has been used to demonstrate EndMT, but obligatory characteristics are either: i) reduced expression of endothelial genes/proteins; ii) increased expression of mesenchymal genes/proteins, or iii) ideally, both of these. Typically, most investigators present 2C3 each of endothelial and mesenchymal genes/proteins. Common examples include: Endothelial; CD31, VE-Cadherin, endothelial nitric oxide synthase (NOS3); Mesenchymal; -easy muscle actin (-SMA), calponin, SM22, versican. However, there is no agreement on which genes/proteins should be studied, or how many, and the level of change required. Additional features that are sometimes also studied include increased expression of EndMT-associated transcription factors such as TWIST, SMAD3, ZEB2, SNAI1 and SNAI2. Looking ahead, we propose that future studies should seek to provide more comprehensive transcriptomic and proteomic profiles of any proposed EndMT phenomenon. Furthermore, in any individual cell or cell population undergoing EndMT, gradations of EndMT exist (i.e. partial versus more complete EndMT, reversible, transient etc.), and there may be relative differences in the extent of endothelial gene/protein downregulation versus mesenchymal upregulation (12). This heightens the importance of a complete portrayal of EndMT using high-throughput techniques, whereby the balance of endothelial gene/protein downregulation versus mesenchymal upregulation is usually fully appreciated. Functional and phenotypic cell changes during EndMT are also fundamental to this process and, thereby, to a definition of EndMT. Accordingly, studies of EndMT are increasingly demonstrating relevant changes in phenotypic traits (Table 1). However, yet again, no standardization exists. Indeed, reaching a consensus on these functional cellular aspects Gamitrinib TPP hexafluorophosphate may be challenging, because certain EndMT-related phenotypic features may be important in Gamitrinib TPP hexafluorophosphate specific contexts, but irrelevant or even opposing in others. For example, reduced tubule formation (i.e. angiogenesis) has been associated with EndMT (14,15), but as an apparent paradox, at least partial EndMT is necessary for angiogenesis (16). Highlighting this paradox at a molecular level, the transcription factor SNAI2 is expressed in angiogenic ECs and mediates angiogenesis (16), but Gamitrinib TPP hexafluorophosphate SNAI2 is also a key mediator of EndMT (11). A full reconciliation of these functional aspects will likely remain challenging until more is usually comprehended about EndMT as a whole. Table 1. Functional studies to support an altered cellular phenotype with EndMT. from endothelial progenitors. The endocardium likely has heterogeneous origins, arising from endocardial-myocardial heart field progenitors (17,18,20C22), and also cells that migrate in from the yolk sac mesoderm (an extra-embryonic Gamitrinib TPP hexafluorophosphate tissue) (19). Some endocardial cells express markers of hemogenic endothelium, perhaps reflecting their origins from yolk sac hemangioblasts (a common progenitor of blood and vessels) (19) and the activation of the hematopoietic program within embryonic endothelium (23). As the heart develops, the endocardium retains remarkable cellular plasticity. For example, the endocardium associated with the forming ventricles undergoes a process that resembles angiogenic sprouting in developing vascular beds (24), leading to the formation of endocardial domes which, together with myocardium and extracellular matrix (ECM), define the morphological.