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    • br Discussion br Experimental Procedures br Author

    2018-10-24


    Discussion
    Experimental Procedures
    Author Contributions
    Acknowledgments We thank members of the Azrieli Center for Stem Cells and Genetic Research at the Hebrew University for their contribution, and especially Mordecai Peretz for assistance with the figures, and Ido Sagi for critical reading of the manuscript and assistance with the graphical design. N.B. is the Herbert Cohn Chair in Cancer Research. This work was partially supported by the Israel Science Foundation (grant number 269/12), by the Israel Science Foundation – Morasha Foundation (grant number 1252/12), by the Rosetrees Trust, and by the Azrieli Foundation.
    Introduction Pluripotent embryonic stem bace inhibitor (ESCs) provide essential tools for understanding mammalian developmental processes, as they can differentiate in vitro into many tissues in a normal developmental manner (Keller, 2005; Solter, 2006). These cells are amenable to high-throughput screens using RNAi or small-molecule libraries to dissect molecular pathways (Ding and Buchholz, 2006; Xu et al., 2008). Early vascular and hematopoietic differentiation of ESCs has been extensively studied (Keller, 2005), making these pathways particularly attractive for large-scale screens. Blood vessels are first formed through vasculogenesis, whereby angioblasts (endothelial precursors) aggregate in the developing embryo to form a primitive network of endothelial tubes. This network is later remodeled through a complex process termed angiogenesis, which includes sprouting of new blood vessels, to form the mature circulatory network (Rossant and Howard, 2002). Major breakthroughs in our understanding of vascular development and remodeling have arisen from characterization of vascular mutant phenotypes in mice. Vascular endothelial growth factor (VEGF), acting through the FLK-1/VEGF receptor 2 (VEGFR2), is crucial for blood vessel formation and development (Carmeliet et al., 1996; Shalaby et al., 1995). NOTCH/DLL4 signaling plays a critical role in branching/sprouting morphogenesis, whereby loss of NOTCH signaling leads to excess tip cell formation and non-productive vessel development (Hellstrom et al., 2007). Impaired vascular development was also reported for mutations in ANG/TIE, platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), EFN, HH, and PLXN/SEMA signaling pathways (reviewed by Rossant and Howard, 2002). Many signaling pathways required during embryonic vascular development are also essential during adult neoangiogenesis (Carmeliet, 2003). Adult neovascularization occurs in many physiological and pathological settings, such as wound healing (Ruiter et al., 1993), recovery from myocardial infarction (Chung et al., 2002), tumor growth, and metastasis (Ruiter et al., 1993). There is increasing interest in using modulators of angiogenesis to treat cancer (Ferrara, 2004). Currently antiangiogenic therapy has two opposing target pathways, the VEGF/FLK-1 and DLL4/NOTCH pathways (Kuhnert et al., 2011). The new generation of antiangiogenic drugs that have arisen from an understanding of vascular developmental biology, such as bevacizumab (anti-VEGF) (Ferrara et al., 2005), have demonstrated some efficacy in cancer patients, but cause serious side effects and frequent relapses (Kerbel, 2008). Similar results have been obtained from inhibition of the NOTCH/DLL4 pathway (Andersson and Lendahl, 2014), thus necessitating the discovery of alternative therapeutic targets.
    Results
    Discussion We have developed an unbiased, robust, and reproducible three-dimensional EB-based vascular differentiation assay that is amenable to screening for modulators of angiogenesis. The EB-based vascular differentiation assay in collagen matrix offers advantages over the widely used HUVEC/Matrigel assay as well other in vitro angiogenic models in that it uniquely allows the study of both vasculogenesis and angiogenesis (Feraud et al., 2001). The EB assay, unlike the HUVEC assay, models the complex in vivo interactions between endothelial cells and their support cells, which is essential for recapitulating normal vessel formation (Feraud et al., 2001). The assay is sensitive to both increases and decreases in vessel sprouting as well as reading out morphological changes in vessel shape, as exemplified by an additional screen that showed treatment with all-trans retinoic acid resulted in the ballooning of vascular sprouts (Figures S1F and S1G).