Abstract-Cardiac malformations constitute the most common birth defects, of which heart septal and valve defects are the most frequent forms diagnosed in infancy. These cardiac structures arise from the endocardial cushions through dynamic interactions between cells and the extracellular matrix (cardiac jelly). Targeted deletion of the hyaluronan synthase-2 (Has2) gene in mice results in an absence of hyaluronan (HA), cardiac jelly, and endocardial cushions, a loss of vascular integrity, and death at embryonic day 9.5. Despite the requirements for Has2 and its product, HA, in the developing heart, little is known about the normal processing and removal of HA during development. Cell culture studies show that HA obtains new bioactivity after depolymerization into small oligosaccharides. We previously showed reduction in Has2 expression and diminished presence of HA at later stages of heart development as tissue remodeling formed the leaflets of the cardiac valves. Here we show that small oligosaccharide forms of HA (o-HA) act antagonistically to developmental epithelial-to-mesenchymal transformation (EMT), which is required to generate the progenitor cells that populate the endocardial cushions. We further show that o-HA induces vascular endothelial growth factor (VEGF), which acts as a negative regulator of EMT. This is the first report illustrating a functional link between oligosaccharide HA and VEGF. Collectively, our data indicate that following endocardial cell EMT, native HA is likely processed to o-HA, which stimulates VEGF activity to attenuate cardiac developmental EMT. (Circ Res. 2006;99:583-589.)

In the mammalian system the proepicardium (PE) arises from mesothelium of the septum transversum before translocation to the heart where it forms the epicardium and progenitor cells of the coronary vessels. Despite its importance, the process in which PE cells translocate to the myocardium in mammals is not well defined. The current paradigm states that cellular cysts of PE float across the pericardial space and contact the outer surface of the myocardium. This mechanism does not provide a satisfactory explanation for the directionality or localization of PE migration. To better define PE migration, we performed a detailed study of mouse PE development. We provide thorough documentation that redefines the size of the PE migratory field and the mechanism of migration. Our new model incorporates differential growth and direct contact between multicellular PE villi and the myocardium as mechanisms in formation of the epicardium. Developmental Dynamics 237:145-152, 2008.

The epicardium plays critical roles as protector of the myocardium as well as provider of fibroblasts and coronary vessel precursors. Unlike the myocardium and endocardium, the epicardium does not arise from the cardiac mesoderm. Instead, it originates from the proepicardium (PE). The PE develops from mesothelium of the septum transversum and then translocates to the heart. Despite the vital roles for the epicardium during cardiovascular development, the process in which the PE colonizes the myocardium in mammals is not well defined. The current paradigm states that cellular PE cysts float across the pericardial space and contact the outer surface of the myocardium at the atrial‐ventricular sulcus to initiate formation of the epicardium. However, this definition does not provide a satisfactory explanation for the directionality or localization of PE migration. The goal of our study is to establish a better understanding of the mechanism for PE relocation from the septum transversum to the myocardium during murine embryogenesis. We have performed detailed histology and immunohistochemistry experiments combined with confocal and electron microscopy to define the timing, size, and mechanics of PE migration. We provide evidence that redefines the developmental window of time for PE migration, the size of the PE forming field, and the complex physical mechanism for epicardial development.