During embryogenesis, the formation of blood vessels proceeds by both vasculogenesis and angiogenesis. Both processes appear to be finely regulated. To date, factors and genes involved in the negative regulation of embryonic vasculogenesis remain largely unknown. Angiostatin is a proteolytic fragment of plasminogen that acts as an inhibitor of angiogenesis. In this study, we analyzed the potential role of angiostatin during early stages of embryonic stem (ES) cell endothelial in vitro differentiation, as a model of vasculogenesis. We found an early expression of the known angiostatin binding sites (angiomotin, alphav integrin and c-met oncogene) during ES cell differentiation. Nevertheless, we did not detect any significant effect of angiostatin on mesoderm induction and on differentiation commitment into cells of the endothelial lineage. In both control and angiostatin-treated conditions, the temporal and extent of formation of the Flk1 positive and Flk-1/CD31 (PECAM-1) positive cell populations were not significantly different. Quantitative RT-PCR experiments of endothelial gene expression (Flk-1, PECAM-1 and tie-2) confirm a lack of interference with early steps of endothelial differentiation in embryoid bodies. No evidence for an angiostatin effect on endothelial cord-like formation could be detected at later differentiation stages. On the other hand, angiostatin inhibits vascular endothelial growth factor-induced endothelial sprouting from embryoid bodies cultured in three dimensional type I collagen gels. Taken together, these findings support a selective inhibitory effect on the sprouting angiogenesis response for angiostatin during embryonic vascular development.
(c) 2007 Wiley-Liss, Inc.