Differential Regulation of Bone Marrow-Derived Endothelial Progenitor Cells and Endothelial Outgrowth Cells by the Notch Signaling Pathway

et al. (2012) Differential Regulation of Bone Marrow-Derived Endothelial Progenitor Cells and Endothelial Outgrowth Cells by the Notch Signaling Pathway. PLoS ONE 7(10): e43643. doi:10.1371/journal.pone.0043643 Differential Regulation of Bone Marrow-Derived Endothelial Progenitor Cells and Endothelial Outgrowth Cells by the Notch Signaling Pathway Jing-Yuan Chen 0 Lei Feng 0 Hai-Long Zhang 0 Jun-Chang Li 0 Xin-Wei Yang 0 Xiu-Li Cao 0 Li Liu 0 Hong-Yan Qin 0 Ying-Min Liang 0 Hua Han 0 Pieter H. Reitsma, Leiden University Medical Center, The Netherlands 0 1 Department of Hematology, Tangdu Hospital, Fourth Military Medical University , Xi'an , People's Republic of China, 2 State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, Fourth Military Medical University , Xi'an , People's Republic of China Endothelial progenitor cells (EPCs) are heterogeneous populations of cells that participate in vasculogenesis and promote tissue regeneration. However the different roles of EPC populations in vasculogenesis and tissue regeneration, as well as their regulation and mechanisms remain elusive. In the present study, we cultured bone marrow (BM)-derived early EPCs (EEPCs) and endothelial outgrowth cells (EOCs), and investigated their roles in liver regeneration and their regulation by the Notch signaling pathway. We found that Notch signaling exhibited different effects on the proliferation and migration of EEPCs and EOCs. Our results also showed that while EEPCs failed to form vessel-like structures in a three dimensional sprouting model in vitro, EOCs could sprout and form endothelial cords, and this was regulated by the Notch signaling. We further showed that, by using a conditional knockout model of RBP-J (the critical transcription factor mediating Notch signaling), Notch signaling differentially regulates EEPCs and EOCs. In a partial hepatectomy (PHx) model, EEPCs Notchdependently benefitted liver regeneration with respect to liver function and hepatocyte proliferation and apoptosis. In contrast, EOCs appeared not directly involved in the recovery of liver function and the increase of hepatocytes. These data suggested that the RBP-J-mediated Notch signaling differentially regulated the two types of EPCs, which showed different roles in liver regeneration. - Funding: This work was supported by grants from the National Natural Science Foundation of China (30830067, 30871090, 81072972, 30873229) and the Ministry of Science and Technology of China (2009CB521706, 2011ZXJ09101-02C). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Endothelial progenitor cells (EPCs) are progenitor cells derived from mesodermal progenitor cells in early embryogenesis, and are responsible for initial vascularization in both embryo body and extra-embryonic tissues through a process defined as vasculogenesis [1,2]. In the past decade it has been recognized that EPCs also exist in adult tissues, mostly in bone marrow (BM), and take part in neovascularization at the sites of ischemia in disease models. EPCs can be mobilized from BM and can home to wounded tissues [3,4], where they can differentiate into endothelial cells (EC) to directly participate in vasculogenesis, and/or to produce angiogenic factors to contribute to vascular remodeling. Moreover, a large body of evidence has suggested that EPCs have therapeutic benefits in the treatment of ischemic diseases [5]. For example, several groups have shown the roles of EPC in liver regeneration and in the therapy of liver cirrhosis [6,7]. However, the effects of EPCs on the repair of tissue damages appear varied as reported by researchers in different sets of preclinical and clinical studies [8]. This inconsistency is at least partially attributable to the heterogeneous nature of EPCs [9]. EPCs in BM or just entering the peripheral blood express stem cell markers such as CD34 and CD133, together with VEGFR2 (KDR). Along with in vitro culturing and maturation, the cells gradually lost stem cell markers, and begin to express EC-specific antigens such as platelet endothelial cell adhesion molecule 1 (PECAM-1 or CD31) and VE-cadherin, among others [10]. Other researchers have suggested that EPCs is composed of endothelial lineage cells at different differentiation stages [11]. Two types of EPCs have been identified from in vitro cultured EPCs, which are supposed to have different cellular origins [12,13]. Early EPCs (EEPCs) are spindle-like in shape, and have limited proliferative potential and can be cultivated no more than 4 weeks in vitro. Endothelial outgrowth cells (EOCs) or late EPCs, in contrast, have a cobblestone-like appearance and maintain a high proliferative potential. EEPCs are myeloid endothelial progenitor cells, originating from CD14+ monocytic cells, while OECs are derived from CD142 cells. But further defining different subpopulations of EPCs and understanding their roles and mechanisms in vascularization is still required. EOCs and EEPCs can be involved in the formation of new blood vessels through different mechanisms such as differentiating into ECs or producing angiogenic cytokines [1417]. Signals regulating their mobilization and functions have been elusive. Among the molecules identified so far, such as angiogenic factors [18], integrins [19] and adhesion molecules [20], the stromaderived factor (SDF)-1a-CXCR4-mediated signaling plays an important role in the trafficking and the homing of EPCs [2125]. SDF-1a induced by hypoxia inducible factor (Hif)-1a enhances the adhesion, migration, and homing of circulating CXCR4-positive EPCs to ischemic tissues [22,26]. Another important signaling pathway in EPCs is the Notch receptor-mediated signaling. The Notch pathway is highly conserved in evolution, and plays an essential role in cell fate determination in multiple lineages of stem and progenitor cells [27]. There are five Notch ligands (Jagged1, 2, and Delta-like [Dll]1, 3, 4) and four Notch receptors (Notch14) in mammals. Ligand binding triggers proteolytic cleavages of Notch receptors, releasing the Notch intracellular domain (NICD) to translocate into the nucleus, where NICD associates with the transcription factor RBP-J and recruits other co-activators to activate target gene expression [28]. Kwon et al [29] have shown that the Jagged1-mediated Notch signaling promote adult neovascularization by regulating the function of EPCs. We have also found that Notch-RBP-J signaling regulates the mobilization, migration and function of EPCs through the expression of CXCR4 [30]. However, the roles of the Notch signaling pathway in different subpopulations of EPCs, namely EEPCs and EOCs, have not yet been revealed. In this study, we accessed this question by using in vitro cultured EPC (...truncated)