Abstract-Recent work has demonstrated the importance of the epicardium in the development of the heart. During embryogenesis, these epithelial cells provide the progenitors for the epicardium, coronary smooth muscle, endothelium, and cardiac fibroblasts. The epicardium sends important signals to the developing myocardium. Still, analysis of these epithelial cells has lagged behind that of other cardiac cell types largely because of the lack of a defined experimental cell system in which epicardial cell differentiation can be studied. The present report examines the developmental potential of a cell line derived from rat epicardial mesothelial cells. These analyses demonstrate that the cell line retains many characteristics of the intact epithelium, including the ability to form a polarized epithelium and express many epicardial genes. Our data show for the first time that these cells retain the ability to produce mesenchyme in response to specific growth factors and, importantly, to generate smooth muscle cells. Thus, this study provides evidence that these cells can serve as an important model system for the analysis of the cellular and molecular mechanisms that govern epicardial development and function.

Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for treating segmental bone defects. Thus, understanding the earliest events in this process potentially would allow us to design more targeted therapies to either block or enhance this process. Using a murine model of HO induced by delivery of adenovirus-transduced cells expressing bone morphogenetic protein 2 (BMP-2), we show here that one of the earliest stages in this process is the establishment of new vessels prior to the appearance of cartilage. As early as 48 hours after induction of HO, we observed the appearance of brown adipocytes expressing vascular endothelial growth factors (VEGFs) simultaneous with endothelial progenitor replication. This was determined by using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of nuclear-localized yellow fluorescent protein (YFP). Expression of this marker has been shown previously to correlate with the establishment of new vasculature, and the nuclear localization of YFP expression allowed us to quantify changes in endothelial cell numbers. We found a significant increase in Flk1-H2B::YFP cells in BMP-2-treated animals compared with controls. The increase in endothelial progenitors occurred 3 days prior to the appearance of early cartilage. The data collectively suggest that vascular remodeling and growth may be essential to modify the microenvironment and enable engraftment of the necessary progenitors to form endochondral bone. © 2010 American Society for Bone and Mineral Research.

While Blood vessel epicardial substance (Bves) confers adhesive properties, the molecular mechanism of regulating this activity is unknown. No predicted functional motifs in this highly conserved integral membrane protein, other than the transmembrane domain, have been identified. Here, we report for the first time that Bves interacts with itself through an intracellular interaction domain that is essential for its intercellular adhesion activity. Glutathion-S-transferase (GST) pull-down and SPOTs analyses mapped this domain to amino acids 268-274 in the intracellular C-terminus. Site-directed mutagenesis revealed that lysines 272 and 273 are essential for homodimerization and cell adhesion. Human corneal cells transfected with wild-type Bves trafficked the protein to the cell surface, assembled junction complexes and formed epithelial sheets. In contrast, cells expressing Bves mutated at these positions did not form continuous epithelial sheets or maintain junctional proteins such as ZO-1 and E-cadherin at the membrane. A dramatic reduction in transepithelial electrical resistance was also observed indicating a functional loss of tight junctions. Importantly, expression of mutated Bves in epithelial cells promoted the transformation of cells from an epithelial to a mesenchymal phenotype. This study is the first to demonstrate the essential nature of any domain within Bves for maintenance of epithelial phenotype and function.

Abstract-Development of the coronary vascular system is an interesting model in developmental biology with major implications for the clinical setting. Although coronary vessel development is a form of vasculogenesis followed by angiogenesis, this system uses several unique developmental processes not observed in the formation of other blood vessels. This review summarizes the literature that describes the development of the coronary system, highlighting the unique aspects of coronary vessel development. It should be noted that many of the basic mechanisms that govern vasculogenesis in other systems have not been analyzed in coronary vessel development. In addition, we present recent advances in the field that uncover the basic mechanisms regulating the generation of these blood vessels and identify areas in need of additional studies. A Brief Description of Coronary Vessel DevelopmentCoronary vessel development is an example of vasculogenesis followed by angiogenesis with unique variations specific to heart development. Vasculogenesis has been described as the de novo generation of blood vessels, whereas angiogenesis can be thought of as the generation of capillaries, veins, and arteries from preexisting vessels. The process begins with the delivery of vasculogenic cell types to the surface of the heart after beating has begun. These cells must then disperse throughout the heart; differentiate into endothelial cells, smooth muscle cells, pericytes, and fibroblasts; subsequently form arteries, veins, and capillaries; and finally connect to the aorta and coronary sinus. Delivery of a population of cells to an existing organ requires dynamic cellular events, and coordination of cell movement with the precise timing of delivery, commitment, and differentiation is critical for proper vessel formation and organ development. Originally, researchers thought that coronary vascular progenitors were derived from the cardiac mesoderm, like the other cell types in the myocardium and endocardium. 1,2 These data were first challenged by Manasek, 3 who demonstrated that the heart tube is initially composed of only endocardium and myocardium and that the epicardium arises from extracardiac regions. In more recent years, several studies have determined that progenitors of the epicardium and coronary vascular system are derived from extracardiac tissue, the proepicardial organ (PEO; Figure 1). Although the earliest location of these progenitors is still in debate, they appear to arise from splanchnic mesoderm. 4 -6 The PEO is associated with the septum transversum that grows from the dorsal body wall ventrally to divide the embryonic coelom into the pleuropericardial and peritoneal cavities and forms a portion of the diaphragm. 4,[7][8][9] However, the PEO is a transient structure that consists of a single epithelial layer folded into a shape resembling a grapelike cluster. In chicken (HH stage 17) and mouse (E9.5) embryos, the PEO migrates to and then over the surface of the heart to form the primitive epicardium (Figures 1 th...

The Transforming growth factor β (Tgf-β) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-β signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta.