Fifty-four percent of all cardiovascular disease in the United States effects the coronary arteries (American Heart Association, 2004). A detailed understanding of the cell populations and molecules that regulate coronary vessel development will be required to reveal novel drug targets and therapeutic strategies to direct the repair or remodeling of coronary vessels in adults. The importance of cells derived from the proepicardium (PE) and epicardium (EP) in the formation of the coronary vessels is well established (reviewed in Olivey et al.,2004; Tomanek,2005). During development these cells give rise to both the endothelial and smooth muscle components of the coronary vessels. In chick embryos, the PE arises from mesothelial cells along the caudal border of the pericardial cavity (Ho and Shimada,1978). The PE contacts the heart at the atrioventricular groove and migrates to the heart by means of an extracellular matrix bridge (Nahirney et al.,2003) between the myocardium and the PE. These cells maintain polarity while migrating as an intact epithelium with the luminal surface in contact with the myocardium. In mammals clusters of PE cells detach as vesicles that are transferred to the heart by means of the pericardial fluid (Komiyama et al.,1987; Kuhn and Liebherr,1988). In both avians and mammals, after contacting the myocardium, a fraction of cells undergo epithelial–mesenchymal transformation (EMT) and migrate into the subepicardial space. A subset of these cells continues into the compact zone of the myocardium (Mikawa and Fischman,1992; Poelmann et al.,1993). Coronary vessel formation begins as angioblasts coalesce to form a primitive vascular plexus in the subepicardial space and myocardium. These nascent endothelial tubes form larger vessels to become the coronary arteries and veins that attach to the ascending aorta and the right atrium. Once attached, these vessels recruit PE-derived cells to form the smooth muscle and fibroblast components of the vascular network. Therefore, precursor cells are delivered to the heart by the PE and form coronary vessels by a process of vasculogenesis (Munoz-Chapuli et al.,2002). In zebrafish, epicardial cells are able to reinitiate this developmental program and contribute to the genesis of new coronary vessels in injured myocardium (Lepilina et al.,2006; Poss,2007).
Studies of EMT in explanted PE (Mikawa and Gourdie,1996) and EP (Dettman et al.,1998) have identified factors that regulate EMT. For example, both vascular-derived endothelial growth factor (VEGF) and fibroblast growth factor (FGF) stimulate EMT of epicardial cells in vitro (Morabito et al.,2001). Recently, we showed that transforming growth factor β (TGFβ) induces EMT and smooth muscle differentiation in chick epicardial explants (Compton et al.,2006). TGFβ ligands are abundantly expressed in the developing heart (Akhurst et al.,1990; Pelton et al.,1991; Dickson et al.,1993; Jakowlew et al.,1994; Molin et al.,2003) and are known to play a prominent role in stimulating EMT (Kalluri and Neilson,2003) and smooth muscle differentiation (Owens et al.,2004). Three ligands, TGFβ1, TGFβ2, and TGFβ3 (Roberts and Sporn,1990; Sanford et al.,1997; Hu et al.,1998), bind four cell surface proteins. These include two transmembrane serine/threonine kinase receptors, the type I TGFβ receptor (TGFβR1) and the type II TGFβ receptor (TGFβR2; Lin et al.,1992; Ebner et al.,1993; Bassing et al.,1994). In the canonical signaling pathway (Shi and Massague,2003) ligand binding to TGFβR2 results in recruitment of the TGFβR1, activin receptor-like kinase (ALK) 5, to the complex. The constitutively active kinase of TGFβR2 phosphorylates and activates the kinase domain of ALK5, which subsequently phosphorylates and activates downstream receptor associated (R-) Smads 2 and 3 (Kretzschmar and Massague,1998). These activated R-Smads complex with Smad 4 and translocate into the nucleus to alter gene transcription. TGFβ can activate additional downstream effectors including RhoA (Bhowmick et al.,2001a,2003; Edlund et al.,2002; Masszi et al.,2003; Deaton et al.,2005), Ras (Ward et al.,2002), mitogen activated protein (MAP) kinases (Bhowmick et al.,2001b; Bakin et al.,2002; Xie et al.,2004; Deaton et al.,2005), and PI3K/AKt (Bakin et al.,2000), although the mechanisms by which TGFβ regulates these effectors is less well described. A second class of TGFβ binding proteins contains two transmembrane proteins termed the type III TGFβ receptor (TGFβR3), or betaglycan, and endoglin. Both TGFβR3 and endoglin contain a short, highly conserved intracellular domains with no apparent signaling function (Lopez-Casillas et al.,1991; Wang et al.,1991; Cheifetz et al.,1992). Mutations in the endoglin gene are linked to human hereditary hemorrhagic telangiectasia (McAllister et al.,1994), whereas targeted inactivation of the gene encoding TGFβR3 has been shown to result in embryonic death at E14.5 associated with a failure of coronary vessel development (Compton et al.,2007).
TGFβ induced EMT in both PE and EP explants from chick embryos (Compton et al.,2006; Olivey et al.,2006). Experiments using EP explants cultured with the addition of growth factor, specific small molecule inhibitors, and adenoviral gene transfer demonstrated that TGFβ-stimulated loss of epithelial character was accompanied by smooth muscle differentiation (Compton et al.,2006). These effects of TGFβ are dependent on ALK5 kinase activity, and ALK5 kinase activity is sufficient to induce EMT and invasion of a three-dimensional matrix by epicardial cells. Overexpression of Smad 3 is not sufficient to induce cell invasion. The loss of epithelial character in response to TGFβ requires the activity of the downstream effectors, p160 rho kinase and p38 MAP kinase. Induction of smooth muscle differentiation requires p160 rho kinase but not p38 MAP kinase.
To determine whether TGFβ signaling pathways play a similar role in regulating epicardial cells in mammals, we characterized mouse epicardial cell explants. We have defined culture conditions and demonstrated the ability to use adenoviral gene transfer to introduce genes of interest into mouse explants. We determined that, similar to the chick, the addition of TGFβ1 or TGFβ2 causes the loss of epithelial character and smooth muscle differentiation in mouse epicardial cells. The type I TGFβ receptor ALK5 is both required and sufficient to mediate EMT and smooth muscle differentiation in mouse epicardial cells. Immortalized epicardial cells were generated using a transgenic mouse where the large T antigen is temperature regulated (Jat et al.,1991). Immortalized epicardial cells retain the expression of the epicardial cell marker WT1 and respond to TGFβ in a manner indistinguishable from primary epicardial cells. These data demonstrate that TGFβ regulates epicardial cell differentiation in the mouse and that immortalized epicardial cells may be used as a model system to study smooth muscle differentiation.