CCBE1 is essential for coronary vessel formation, independent of their embryonic origin, and is also necessary for peritruncal vessel growth and proper CA stem patterning. Developmental Dynamics 247:1135-1145, 2018. © 2018 Wiley Periodicals, Inc.

Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but its role in early cardiac commitment remains unknown. Here we demonstrate that during mouse embryonic stem cell (ESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from ESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 activity by shRNA knockdown or blockade with a neutralizing antibody results in impaired differentiation of embryonic stem cells along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. In addition, knockdown of Ccbe1 leads to smaller embryoid bodies. Collectively, our results show that CCBE1 is essential for the commitment of cardiac mesoderm and consequently, for the formation of cardiac myocytes in differentiating mouse ESCs.

The collagen- and calcium-binding EGF-like domains 1 (CCBE1) is a secreted protein extensively described as indispensable for lymphangiogenesis during development enhancing VEGF-C signaling. In human patients, mutations in CCBE1 have been found to cause Hennekam syndrome, an inherited disease characterized by malformation of the lymphatic system that presents a wide variety of symptoms such as primary lymphedema, lymphangiectasia, and heart defects. Importantly, over the last decade, an essential role for CCBE1 during heart development is being uncovered. In mice, Ccbe1 expression was initially detected in distinct cardiac progenitors such as first and second heart field, and the proepicardium. More recently, Ccbe1 expression was identified in the epicardium and sinus venosus (SV) myocardium at E11.5–E13.5, the stage when SV endocardium–derived (VEGF-C dependent) coronary vessels start to form. Concordantly, CCBE1 is required for the correct formation of the coronary vessels and the coronary artery stem in the mouse. Additionally, Ccbe1 was found to be enriched in mouse embryonic stem cells (ESC) and revealed as a new essential gene for the differentiation of ESC-derived early cardiac precursor cell lineages. Here, we bring an up-to-date review on the role of CCBE1 in cardiac development, function, and human disease implications. Finally, we envisage the potential of this molecule’s functions from a regenerative medicine perspective, particularly novel therapeutic strategies for heart disease.