Notch Signaling Regulates Smooth Muscle Differentiation of Epicardium-Derived Cells

Institut für Molekularbiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, Hannover, Germany.
Circulation Research (Impact Factor: 11.02). 03/2011; 108(7):813-23. 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.

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    • "This is in line with findings that Notch has a tumor suppressor function in the skin, as mice lacking Notch1 expression in keratinocytes frequently develop tumors [10]. The Notch1 signaling pathway is evolutionarily conserved from invertebrates to vertebrates and is involved in cell-fate decisions during development [11–14]. Notch1 signaling is activated via juxtacrine binding of an adjacent cell's Delta-like or Jagged ligands with the Notch receptor. "
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    ABSTRACT: Introduction Stem cells from hair follicle have great therapeutic applications in regenerative medicine as sources of cells for transplantation. The differentiation pathway selected by hair follicle stem cells (HFSC) is largely determined by local microenvironmental signals. In this study, human hair follicle stem cells were treated with Notch signaling blocker to explore a new approach to modulate human hair follicle stem cell proliferation and differentiation in vitro. Aim To define the functional consequences of blocking the Notch signaling pathway on the proliferation and differentiation of human HSCs. Material and methods The human hair follicle stem cells were treated with various concentrations of Notch signaling blocker DAPT (24-diamino-5-phenylthiazole). The viability of the cells was investigated with clonogenicity assays. The expression of stem cell markers, cell cycle and cell apoptosis were analysed by flow cytometry. Results Notch blocking leads to promotion of human hair follicle stem cell proliferation and inhibition of differentiation in response to DAPT. The maximum effect of DAPT on the viability of human HFSC was observed at a concentration of 20 µM. We found that DAPT treatment results in downregulation of Hes1 and p21 and upregulation of Wnt10b. Conclusions γ-Secretase inhibitor DAPT has a modulatory effect on the human HFSC. The DAPT may modulate human hair follicle stem cell proliferation and differentiation through regulation of p21 and Wnt-10b.
    Postepy Dermatologii I Alergologii 08/2014; 31(4):201-6. DOI:10.5114/pdia.2014.44002 · 0.85 Impact Factor
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    • "Notch signaling was thought to be involved in the regulation of vascular smooth muscle differentiation during heart valve and cardiac cushion development (22). Subsequent studies have confirmed the involvement of Notch signaling in the EndoMT process (23–25). "
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    ABSTRACT: Endothelial-mesenchymal transition (EndoMT) is a process in which endothelial cells lose their cell-type‑specific characteristics and gain a mesenchymal cell phenotype. The Notch signaling pathway is crucial in the regulation of EndoMT; however, its roles have not been fully studied in vivo. In a previous study, we reported the generation of transgenic mice with a floxed β-geo/stop signal between a CMV promoter and the constitutively active intracellular domain of Notch1 (IC-Notch1) linked with a human placental alkaline phosphatase (hPLAP) reporter (ZAP-IC-Notch1). In this study, we examined the results of activating IC-Notch1 in endothelial cells. ZAP-IC‑Notch1 mice were crossed with Tie2-Cre mice to activate IC-Notch1 expression specifically in endothelial cells. The ZAP-IC-Notch1/Tie2-Cre double transgenic embryos died at E9.5-10.5 with disruption of vasculature and enlargement of myocardium. VE-cadherin expression was decreased and EphrinB2 expression was increased in the heart of these embryos. Mesenchymal cell marker α-smooth muscle actin (SMA) was expressed in IC-Notch1‑expressing endothelial cells. In addition, upregulation of Snail, the key effector in mediating EndoMT, was identified in the cardiac cushion of the double transgenic murine embryo heart. The results of the present study demonstrate that constitutively active Notch signaling promotes EndoMT and differentially regulates endothelial/mesenchymal cell markers during cardiac development.
    International Journal of Molecular Medicine 06/2014; 34(3). DOI:10.3892/ijmm.2014.1818 · 2.09 Impact Factor
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    • "After 4 days, VEGF-A treatment promoted endothelial differentiation of KDR+ precursors and decreased KDR expression, along with limited but detectable differentiation into vascular SMCs (PDGFRα- or smooth muscle myosin heavy chain (SM-MHC)-positive) and cardiomyocytes (cTnT-positive) (Figure 4A-4C). Combined treatment with rh-Dll4 and VEGF-A enhanced differentiation of KDR+ precursors into vascular SMC and cardiomyocyte lineages, and attenuated endothelial differentiation (Figure 4A-4C), which is in keeping with previous reports43,44. In contrast, treatment with DAPT and VEGF-A significantly enhanced the induction of ECs, with maintained KDR expression, while attenuating differentiation into SMCs and cardiomyocytes (Figure 4A-4C). "
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    ABSTRACT: Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC(+)CD31(+)CD34(+)CD14(-)KDR(high) endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR(+) mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.Cell Research advance online publication 9 May 2014; doi:10.1038/cr.2014.59.
    Cell Research 05/2014; 24(7). DOI:10.1038/cr.2014.59 · 12.41 Impact Factor
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