Notch Signaling Regulates Smooth Muscle Differentiation of Epicardium-Derived Cells

ArticleinCirculation Research 108(7):813-23 · March 2011with23 Reads
DOI: 10.1161/CIRCRESAHA.110.228809 · Source: PubMed
The embryonic epicardium plays a crucial role in the formation of the coronary vasculature and in myocardial development, yet the exact contribution of epicardium-derived cells (EPDCs) to the vascular and connective tissue of the heart, and the factors that regulate epicardial differentiation, are insufficiently understood. To define the role of Notch signaling in murine epicardial development. Using in situ hybridization and RT-PCR analyses, we detected expression of a number of Notch receptor and ligand genes in early epicardial development, as well as during formation of coronary arteries. Mice with epicardial deletion of Rbpj, the unique intracellular mediator of Notch signaling, survived to adulthood and exhibited enlarged coronary venous and arterial beds. Using a Tbx18-based genetic lineage tracing system, we show that EPDCs give rise to fibroblasts and coronary smooth muscle cells (SMCs) but not to endothelial cells in the wild type, whereas in Rbpj-deficient embryos EPDCs form and surround the developing arteries but fail to differentiate into SMCs. Conditional activation of Notch signaling results in premature SMC differentiation of epicardial cells and prevents coronary angiogenesis. We further show that Notch signaling regulates, and cooperates with transforming growth factor β signaling in SM differentiation of EPDCs. Notch signaling is a crucial regulator of SM differentiation of EPDCs, and thus, of formation of a functional coronary system.
    • "The resulting mesenchymal cells invade into the subepicardial space with some cells proceeding to invade into the myocardium as well (reviewed [6]). These epicardial-derived cells differentiate into distinct lineages [7][8][9][10][11], that include cardiac fibroblasts, pericytes, and vascular smooth muscle cells, and support the formation of coronary vessels. Several reports support epicardial contribution to the coronary endothelial cell lineage [12][13][14]. "
    [Show abstract] [Hide abstract] ABSTRACT: The epicardium plays an important role in coronary vessel formation and Tgfbr3-/- mice exhibit failed coronary vessel development associated with decreased epicardial cell invasion. Immortalized Tgfbr3-/- epicardial cells display the same defects. Tgfbr3+/+ and Tgfbr3-/- cells incubated for 72 hours with VEH or ligands known to promote invasion via TGFβR3 (TGFβ1, TGFβ2, BMP2), for 72 hours were harvested for RNA-seq analysis. We selected for genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells when incubated with VEH (604), TGFβ1 (515), TGFβ2 (553), or BMP2 (632). Gene Ontology (GO) analysis of these genes identified dysregulated biological processes consistent with the defects observed in Tgfbr3-/- cells, including those associated with extracellular matrix interaction. GO and Gene Regulatory Network (GRN) analysis identified distinct expression profiles between TGFβ1-TGFβ2 and VEH-BMP2 incubated cells, consistent with the differential response of epicardial cells to these ligands in vitro. Despite the differences observed between Tgfbr3+/+ and Tgfbr3-/- cells after TGFβ and BMP ligand addition, GRNs constructed from these gene lists identified NF-ĸB as a key nodal point for all ligands examined. Tgfbr3-/- cells exhibited decreased expression of genes known to be activated by NF-ĸB signaling. NF-ĸB activity was stimulated in Tgfbr3+/+ epicardial cells after TGFβ2 or BMP2 incubation, while Tgfbr3-/- cells failed to activate NF-ĸB in response to these ligands. Tgfbr3+/+ epicardial cells incubated with an inhibitor of NF-ĸB signaling no longer invaded into a collagen gel in response to TGFβ2 or BMP2. These data suggest that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB signaling is required for epicardial cell invasion in vitro. Our approach successfully identified a signaling pathway important in epicardial cell behavior downstream of TGFβR3. Overall, the genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFβR3 signaling.
    Full-text · Article · Aug 2016
    • "Conditional knockout of β-catenin in the Proepicardial Organ does not affect epicardial cell proliferation or epicardial investment of the myocardium, however there is impairment of myocardial EPDC invasion and differentiation of EPDCs into vascular smooth muscle cells of the coronary arteries [26]. Additionally, Notch mediates EPDC differentiation into vascular smooth muscle cells via the transcription factor RBPJ, and inhibition of epicardial Notch signaling results in impaired maturation of the coronary arteries [27,28]. The above discussion highlights the importance of myocardial derived growth factors and epicardial Wnt/ β-catenin and Notch signaling in the induction of epicardial EMT, and the differentiation of EPDCs into cells of mesenchymal lineage. "
    [Show abstract] [Hide abstract] ABSTRACT: The epicardium is an epithelial monolayer that plays a central role in heart development and the myocardial response to injury. Recent developments in our understanding of epicardial cell biology have revealed this layer to be a dynamic participant in fundamental processes underlying the development of the embryonic ventricles, the coronary vasculature, and the cardiac valves. Likewise, recent data have identified the epicardium as an important contributor to reparative and regenerative processes in the injured myocardium. These essential functions of the epicardium rely on both non-cell autonomous and cell-autonomous mechanisms, with the latter featuring the process of epicardial Epithelial-to-Mesenchymal Transition (EMT). This review will focus on the induction and regulation of epicardial EMT, as it pertains to both cardiogenesis and the response of the myocardium to injury.
    Full-text · Article · Feb 2016
    • "This is consistent with prior studies showing that the EMT process in epicardial cells is independent of Snail1 [37]. E-cadherin (a marker for epithelial identity) or Notch signaling genes (Jagged1, Delta3, Delta4 and Hey1; involved in EMT and coronary devel- opment [38,39]) showed no significant change in the mutant samples (data not shown). Expression level of Hif1a was not increased, suggesting that such abnormalities of coronary vessels are not secondary to hypoxia (data not shown). "
    [Show abstract] [Hide abstract] ABSTRACT: During embryogenesis, hematopoietic cells appear in the myocardium prior to the initiation of coronary formation. However, their role is unknown. Here we investigate whether pre-existing hematopoietic cells are required for the formation of coronary vasculature. As a model of for hematopoietic cell deficient animals, we used Runx1 knockout embryos and Vav1-cre; R26-DTA embryos, latter of which genetically ablates 2/3 of CD45(+) hematopoietic cells. Both Runx1 knockout embryos and Vav1-cre; R26-DTA embryos revealed disorganized, hypoplastic microvasculature of coronary vessels on section and whole-mount stainings. Furthermore, coronary explant experiments showed that the mouse heart explants from Runx1 and Vav1-cre; R26-DTA embryos exhibited impaired coronary formation ex vivo. Interestingly, in both models it appears that epicardial to mesenchymal transition is adversely affected in the absence of hematopoietic progenitors. Hematopoietic cells are not merely passively transported via coronary vessel, but substantially involved in the induction of the coronary growth. Our findings suggest a novel mechanism of coronary growth. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Aug 2015
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