epithelial to mesenchymal transition to give rise, via vasculogenesis and angiogenesis, to the initial vascular network However, lineage tracing studies showed that the postnatal vasculature may arise, in part, de novo from lineage conversion rather than mere expansion of the preexisting network.47 The most important characteristic of the vascular smooth muscle cell (VSMC), which has been referred to as the engine of the vasculature,48 is its viscoelastic nature, allowing the cell to respond to deformation, load, stress development, and maintenance As such, vessel tone can be modulated in response to internal and external environments As is the case for the cardiomyocyte, where the myosin motor and its interaction with actin are essential for cellular function, actin and myosin interplay underlies VSMC function Both of these proteins are represented by cell type specific isoforms, αsmooth muscle actin49 and smooth muscle myosin.50 Thus both cardiomyocytes and VSMCs depend on their respective molecular motors for normal and sustained contractile function VSMC proliferation/migration/accumulation is a determining factor in smooth muscle cell accumulation in the intima and thus is key to normal development as well as to pathogenic processes such as atherosclerosis But, as our tools for lineage and cell tracing continue to improve (Fig 5.5), even this accepted dogma51 is coming under renewed scrutiny Dedifferentiation and proliferation of mature smooth muscle cells, migration, and proliferation of stem and/or progenitor cells, or even resident stem cell populations in the vessel wall, are all under active investigation, highlighting the uncertainty of the origin and resident niches of VSMC progenitors FIG 5.5 Lineage tracing (or cell tracing) refers to the process in which early progenitors or distinct populations of cells and their descendants can be visualized in a heritable, permanent fashion At least 15 variations on the theme now exist and allow the descendants of a cell population or even individual cells to be followed Aspects of a widely used approach are shown, in which a specific-cell-type promoter (in this case, one that is active in progenitor cells) elicits the action of a recombinase such that expression of an easily identifiable marker (e.g., green fluorescent protein or β-galactosidase) occurs, thereby marking the cell As the recombination event is permanent and is transmitted to the cells’ progeny, the cell lineage of the resultant cell population or organ region is marked, allowing one to determine the cellular origin or origins of the structure of interest By coupling the promoter driving cre expression to a pharmacologically sensitive transcriptional element such as tet or tamoxifen-sensitive regulatory sequences, one can control the time of induction as well using exogenously applied drugs One of the more generally useful systems is shown When this promoter is used to drive Cre recombinase activity, a stop signal can be recombined out of a sequence, resulting in synthesis of the reporter, but only when tamoxifen is added The recombinase, Cre, when present, acts at loxP sites to excise the DNA The ROSA26 lacZ-loxP reporter strain contains a Cre-dependent, loxP-inactivated lacZ cDNA cassette targeted within the ubiquitously expressed ROSA26 locus Cremediated recombination of this allele deletes neomycin and a series of polyadenylation sequences, resulting in the juxtaposition of a splice acceptor site and the lacZ cDNA, leading to expression of lacZ, which is easily detectable as blue-staining material using light microscopy neo; Neomycin resistance gene (From Buckingham ME, Meilhac SM Tracing cells for tracking cell lineage and clonal behavior Dev Cell 2011;21:394– 409.) Together with VSMCs, pericytes make up the so-called mural cells of the heart This cell type, which is characteristically found in the terminal capillaries, is relatively underexplored in terms of its cardiovascular function, although its critical roles in maintaining and changing capillary circumference and impacting on coagulation in the brain are well documented.52–54 Pericytes are enveloped in the extracellular matrix and envelop the terminal endothelial-derived microvasculature in the heart They have a star-shaped central body with long processes that enwrap the associated endothelial cells Like VSMCs, they express α smooth muscle actin but at approximately one-tenth the level The morphology and potential function of these cells in cardiac physiology have been recently reviewed.55,56 While some estimates for the population are as high as 10%,19 sophisticated lineage analyses indicate that approximately 6% of the cells present in the LV are mural cells.20 Endothelial Cells On the basis of endothelial cell-specific CD31 staining, analyses of healthy human hearts showed that endothelial cells make up 47% to 60% of total cardiac cells,20 and serve diverse roles These cobblestone-shaped cells serve as protective plates in forming vessel walls, safeguarding blood transport, helping to control vascular permeability and, through signaling to VSMCs, regulating vascular tone They are thus the defining cell type for the vascular system in both the heart and the general body plan The endothelial cells that line the inner vessel walls are not inert structural entities, but rather actively signal to two major systems in the heart (and general body): the vascular and immune systems Structurally, the endothelial cells comprise the lining of the vasculature and, for the larger vessels, are in intimate contact with the mural cells Accordingly, they are a major cell mediator in maintaining hemostasis and their barrier function is critical as cell-cell junctions must be tightly controlled and quickly repaired in the event of trauma In cooperation with platelets, the endothelium initiates the coagulation cascade responsible for blood clot formation that plugs the breach Of course, when inappropriate coagulation occurs either temporally or topologically, the result can be fatal with the clot occluding a critical vessel conduit At baseline then, endothelial cells are normally in an anticoagulant state but are highly responsive to both circulating factors and various procoagulants, such as von Willebrand factor and tissue factor, which are expressed in subendothelial areas on the extracellular matrix and become unmasked upon vessel injury Morphogenesis of the vasculature begins with the early appearance of the preendothelial cell, the angioblast in the mesoderm at CS 8 to CS 10.57,58 The