The primitive epicardial epithelium forms after epicardial progenitor cells are transferred from the proepicardium to the myocardial surface [27, 28]. Abnormal primitive epicardium formation is known to affect ventricular myocardium growth and coronary vascular development [29]. Integrins are currently the only type of adhesion molecule that have been shown to be involved in this process [30]. The α4-integrin null mouse displays severe ventricular myocardial thinning and coronary phenotype [31]. Soon after the primitive epicardium forms, epicardial epithelial-to-mesenchymal transition (EMT) is initiated. Epicardial EMT is a finely regulated process which supplies the heart with highly invasive mesenchymal epicardially derived cells (EPDCs) [26]. Multiple molecules participate in triggering EMT. To date, TGFβ (transforming growth factor beta) 1–2 appears to be the strongest epicardial EMT inducer [32–34], as shown by the epicardial deletion of the TGFβ type I receptor ALK5 (TGFBR1), which disrupts epicardial EMT and coronary blood vessel development [35].

Epicardial transcription factors Wt1 and Tbx18, together with canonical WNT effectors such as β-CATENIN and retinoic acid, also participate as effectors of the EMT process by modulating cell adhesion and motility [36, 37]. Potentially, genetic defects in any of the genes encoding for all these molecules (or participating in their biosynthesis) could give rise to CCAs, but due to the importance of primitive epicardial formation and EMT, individuals carrying such a defect are likely to die during gestation. Still, experimental studies using the chick embryo as experimental animal model reveal that a simple delay in epicardial formation and EMT activation is sufficient to generate abnormal coronary patterning and arterioventricular connections or fistulae (CAA group 3) [38, 39], anomalies which are associated with survival to adulthood.

The epicardium does not only contribute cells to the developing coronary vascular system, but it also acts as a signaling center by secreting various morphogens. These epicardial-secreted molecules include retinoic acid, FGF (fibroblast growth factor) 9,16,20, and IGF2 (insulin-like growth factor 2) [26, 40]. Retinoic acid seems to also act in an autocrine fashion supporting the secretory activity of the epicardium [41] which in turn affects coronary development. Although the characterization of this secretome has been carried out with the main goal of identifying epicardial-secreted molecules regulating myocardial compact layer growth, many of these molecules are likely to affect coronary blood vessel development. As a matter of fact, the thin myocardium that characterizes mice mutant epicardial molecules frequently carries aberrant coronary blood vessels [31, 36, 42], reflecting abnormal ventricular patterning and ramification of coronary vasculature (group 2 CCA).

The epicardium also is sensitive to widely distributed molecules like erythropoietin (EPO) [43] and, accordingly, erythropoietin receptor knockouts have severe coronary dysmorphogenesis [44]. Interfering with embryonic epicardial transcriptional regulation, as shown by the molecular characterization of Wt1 (Wilms tumor 1) and Tbx18 mouse mutants [36, 45], reveals anomalies in the expression of secreted molecules such as VEGF-A and ANGIOPOIETIN-1, growth factor receptors (e.g., PDGFRα,β), or mediators like smoothened and patched (components of the hedgehog signal transduction machinery), all of which are known to be involved in coronary blood vessel development [5, 46–48].

The developing myocardium also contributes to coronary development by providing a material substrate for blood vessel formation, as evidenced by the altered coronary patterning found in mice defective for myocardially expressed Vcam–1 (vascular cell adhesion molecule 1) and Vangl–2 (VANGL planar cell polarity protein 2). Of these two genes, Vcam-1 encodes for a cell adhesion molecule which can act as ligand for α4 INTEGRIN-containing cell receptors [49], while VANGL-2 is a molecule involved in the planar cell polarity signaling pathway [50]. Genetic deletion of cofactors involved in the transcriptional regulation of the myocardium like FOG-2 (zinc finger protein, FOG family member 2) also gives rise to anomalous coronary development [51].

Misspecification of coronary cell types and/or poor deployment of mesenchymal EPDCs can alter early coronary vascular formation. Knockout embryos for the gap junction connexin 43 gene, which is expressed by epicardial progenitor (pro-epicardial) cells, have severe defects in coronary vascular patterning [52]. Accordingly, Wt1-null mice present decreased coronary endothelial and ectopic smooth muscle differentiation, eventually impairing coronary formation at midgestation [36]. Abrogation of NOTCH1 signaling (a well-known cell fate determinant) in the epicardium severely impacts coronary morphogenesis [42, 53].

Some other genes such as TCF21 (transcription factor 21) have been reported to be involved in the early specification of fibroblasts from the epicardium, as shown by the epicardial EMT and coronary morphogenesis defects displayed by TCF21 mutants [54]. It is of particular interest to consider here that epicardial progenitor (pro-epicardial) cells seem to carefully balance their developmental fate as a response to local BMP2 (bone morphogenic protein 2) and FGF2 levels [55], suggesting that genetic defects in any component of these signaling pathways may result in defects in coronary development.

The muscular fate of EPDCs is modulated by the subepicardial microenvironment and requires activation of SRF-dependent pool of characteristic smooth muscle genes [56] and p160 RHO-KINASE activation [57]. Both VEGF-A and retinoic acid have been shown to act synergistically to favor early endothelial differentiation from EPDCs at the expenses of smooth muscle cell differentiation [58]. This study proposes, for the first time, a mechanism that explains the physiological delay in the muscularization of developing blood vessels, which is supposed to allow for the extensive remodeling of the primitive coronary vasculature before its maturation and stabilization is initiated. Therefore, premature muscularization of the developing coronary blood vessels may alter the ramification and complexity of adult coronary vasculature, whereas a delay in the formation of the vascular medial wall could result in the formation of aneurism-like defects (Group 2 CCA).