Abstract
SummaryBlood vessels are formed through vasculogenesis, followed by remodeling of the endothelial network through angiogenesis. Many events that occur during embryonic vascular development are recapitulated during adult neoangiogenesis, which is critical to tumor growth and metastasis. Current antiangiogenic tumor therapies, based largely on targeting the vascular endothelial growth factor pathway, show limited clinical benefits, thus necessitating the discovery of alternative targets. Here we report the development of a robust embryonic stem cell-based vascular differentiation assay amenable to small-molecule screens to identify novel modulators of angiogenesis. In this context, RSK and TTK were identified as angiogenic modulators. Inhibition of these pathways inhibited angiogenesis in embryoid bodies and human umbilical vein endothelial cells. Furthermore, inhibition of RSK and TTK reduced tumor growth, vascular density, and improved survival in an in vivo Lewis lung carcinoma mouse model. Our study suggests that RSK and TTK are potential targets for antiangiogenic therapy, and provides an assay system for further pathway screens.
Highlights
Pluripotent embryonic stem cells (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)
Development of a Robust, and Reproducible Vascular Differentiation Assay Using ESCs We have previously reported the generation of ESCs whereby EGFP was inserted into the Flk-1 locus, and showed that this reporter faithfully recapitulates all areas of FLK-1 expression (Ema et al, 2006)
There was no significant difference in the number of FLK-1 positive (FLK-1+) sprouts between embryoid body (EB) treated with Vascular endothelial growth factor (VEGF) only and EBs treated with VEGF in the presence of one or more of the previously established angiogenic growth factors (Feraud et al, 2001) (Figure S1A), suggesting that VEGF alone accounts for the majority of the angiogenic response and is the only factor required in our assay
Summary
Pluripotent embryonic stem cells (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). Impaired vascular development was reported for mutations in ANG/TIE, platelet-derived growth factor (PDGF), transforming growth factor b (TGF-b), EFN, HH, and PLXN/SEMA signaling pathways (reviewed by Rossant and Howard, 2002)
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