A Dynamic Notch Injury Response Activates Epicardium and Contributes to Fibrosis Repair

Abstract

Transgenic Notch reporter mice express enhanced green fluorescent protein in cells with C-promoter binding factor-1 response element transcriptional activity (CBF1-RE(x)₄-EGFP), providing a unique and powerful tool for identifying and isolating "Notch-activated" progenitors. We asked whether, as in other tissues of this mouse, EGFP localized and functionally tagged adult cardiac tissue progenitors, and, if so, whether this cell-based signal could serve as a quantitative and qualitative biosensor of the injury repair response of the heart. In addition to scattered endothelial and interstitial cells, Notch-activated (EGFP(+)) cells unexpectedly richly populated the adult epicardium. We used fluorescence-activated cell sorting to isolate EGFP(+) cells and excluded hematopoietic (CD45(+)) and endothelial (CD31(+)) subsets. We analyzed EGFP(+)/CD45⁻/CD31⁻ cells, a small (<2%) but distinct subpopulation, by gene expression profiling and functional analyses. We called this mixed cell pool, which had dual multipotent stromal cell and epicardial lineage signatures, Notch-activated epicardial-derived cells (NECs). Myocardial infarction and thoracic aortic banding amplified the NEC pool, increasing fibroblast differentiation. Validating the functional vitality of clonal NEC lines, serum growth factors triggered epithelial-mesenchymal transition and the immobilized Notch ligand Delta-like 1-activated downstream target genes. Moreover, cardiomyocyte coculture and engraftment in NOD-SCID (nonobese diabetic-severe combined immunodeficiency) mouse myocardium increased cardiac gene expression in NECs. A dynamic Notch injury response activates adult epicardium, producing a multipotent cell population that contributes to fibrosis repair.