Anita F. Austin

Transforming growth factor‐β stimulates epithelial–mesenchymal transformation in the proepicardium

The proepicardium (PE) migrates over the heart and forms the epicardium. A subset of these PE‐derived cells undergoes epithelial–mesenchymal transformation (EMT) and gives rise to cardiac fibroblasts and components of the coronary vasculature. We report that transforming growth factor‐β (TGFβ) 1 and TGFβ2 increase EMT in PE explants as measured by invasion into a collagen gel, loss of cytokeratin expression, and redistribution of ZO1. The type I TGFβ receptors ALK2 and ALK5 are both expressed in the PE. However, only constitutively active (ca) ALK2 stimulates PE‐derived epithelial cell activation, the first step in transformation, whereas caALK5 stimulates neither activation nor transformation in PE explants. Overexpression of Smad6, an inhibitor of ALK2 signaling, inhibits epithelial cell activation, whereas BMP7, a known ligand for ALK2, has no effect. These data demonstrate that TGFβ stimulates transformation in the PE and suggest that ALK2 partially mediates this effect. Developmental Dynamics 235:50–59, 2006.

Primary and immortalized mouse epicardial cells undergo differentiation in response to TGFβ

Cells derived from the epicardium are required for coronary vessel development. Transforming growth factor β (TGFβ) induces loss of epithelial character and smooth muscle differentiation in chick epicardial cells. Here, we show that epicardial explants from embryonic day (E) 11.5 mouse embryos incubated with TGFβ1 or TGFβ2 lose epithelial character and undergo smooth muscle differentiation. To further study TGFβ Signaling, we generated immortalized mouse epicardial cells. Cells from E10.5, 11.5, and 13.5 formed tightly packed epithelium and expressed the epicardial marker Wilms tumor 1 (WT1). TGFβ induced the loss of zonula occludens‐1 (ZO‐1) and the appearance of SM22α and calponin consistent with smooth muscle differentiation. Inhibition of activin receptor‐like kinase (ALK) 5 or p160 rho kinase activity prevented the effects of TGFβ while inhibition of p38 mitogen activated protein (MAP) kinase did not. These data demonstrate that TGFβ induces epicardial cell differentiation and that immortalized epicardial cells provide a suitable model for differentiation. Developmental Dynamics 237:366–376, 2008.

TGFβ2-Mediated Production of Hyaluronan is Important for the Induction of Epicardial Cell Differentiation and Invasion

In the developing heart, the epicardium is a major source of progenitor cells that contribute to the formation of the coronary vessel system. These epicardial progenitors give rise to the different cellular components of the coronary vasculature by undergoing a number of morphological and physiological changes collectively known as epithelial to mesenchymal transformation (EMT). However, the specific signaling mechanisms that regulate epicardial EMT are yet to be delineated. In this study we investigated the role of TGFβ2 and hyaluronan (HA) during epicardial EMT and how signals from these two molecules are integrated during this important process. Here we show that TGFβ2 induces MEKK3 activation, which in turn promotes ERK1/2 and ERK5 phosphorylation. TGFβ2 also increases Has2 expression and subsequent HA production. Nevertheless, inhibition of MEKK3 kinase activity, silencing of ERK5 or pharmacological disruption of ERK1/2 activation significantly abrogates this response. Thus, TGFβ2 promotes Has2 expression and HA production through a MEKK3/ERK1/2/5-dependent cascade. Furthermore, TGFβ2 is able to induce epicardial cell invasion and differentiation but not proliferation. However, inhibition of MEKK3-dependent pathways, degradation of HA by hyaluronidases or blockade of CD44, significantly impairs the biological response to TGFβ2. Taken together, these findings demonstrate that TGFβ2 activation of MEKK3/ERK1/2/5 signaling modulates Has2 expression and HA production leading to the induction of EMT events. This is an important and novel mechanism showing how TGFβ2 and HA signals are integrated to regulate changes in epicardial cell behavior.

Involvement of the MEKK1 signaling pathway in the regulation of epicardial cell behavior by hyaluronan.

During embryonic development, cells comprising the outermost layer of the heart or epicardium play a critical role in the formation of the coronary vasculature. Thus, uncovering the molecular mechanisms that govern epicardial cell behavior is imperative to better understand the etiology of cardiovascular diseases. In this study, we investigated the function of hyaluronan (HA), a major component of the extracellular matrix, in the modulation of epicardial signaling. We show that stimulation of epicardial cells with high molecular weight HA (HMW-HA) promotes the association of MEKK1 with the HA receptor CD44 and induces MEKK1 phosphorylation. This leads to the activation of two distinct pathways, one ERK-dependent and another NFkappaB-dependent. Furthermore, HMW-HA stimulates epicardial cells to differentiate and invade, as suggested by increased vimentin expression and enhanced invasion through a collagen matrix. Blockade of CD44, transfection with a kinase-inactive MEKK1 construct or the use of ERK1/2 and NFkappaB inhibitors significantly abrogates the invasive response to HMW-HA. Together, these findings suggest an important role for HA in the regulation of epicardial cell fate via activation of MEKK1 signaling cascades.

Expression of Lymphatic Markers During Avian and Mouse Cardiogenesis

The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear‐localized transcription factor Prox‐1 identified a sheet of Prox‐1‐positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just before septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE‐1, VEGFR‐3, and podoplanin. Before the Prox‐1‐positive cells were detected in the mouse epicardium, LYVE‐1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE‐1/CD44‐positive mesenchymal cells to Prox‐1‐positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE‐1/PECAM/VEGFR‐3‐positive vessels within the embryonic and adult myocardium, which remained Prox‐1/podoplanin‐negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox‐1 also exhibiting nuclear‐localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox‐1‐positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE‐1‐positive cells in the epicardium that may be incorporated into the Prox‐1‐positive lymphatic vasculature, and (3) LYVE‐1‐positive cells/vessels in the myocardium that do not become Prox‐1‐positive even in the adult heart. Anat Rec, 2010.