Cell biological analyses of vascular remodeling by using an in vitro differentiation system of ES cells

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The mechanism by which primary plexus and subsequent vascular tree which consists of large and small vessels form in the process of vertebrate embryogenesis is largely unknown. In vitro differentiation system of murine ES cells serves as a means to break down the complicated processes of vascular development into cellular events which can be monitored in real time. We have developed a culture system in which endothelial cells differentiate from ES cells via lateral mesodermal cells as intermediate precursors. ES cell-derived endothelial cell colonies generated on OP9 stromal cell layer have been used as an in vitro model to examine the behavior of individual endothelial cells in response to various angiogenic stimuli such as VEGF-A/VEGFR-2 and VEGF-C/VEGFR-3 signals. The VEGFR-2 signal was shown to induce elongation and dispersion of endothelial cells while the VEGFR-3 signal maintains integrity of cell-cell adhesion by modulating the VEGFR-2 signal. We also identified a transcription factor FOXO1 as a regulatory molecule of the elongation reaction of endothelial cells in response to the VEGFR-2 signal. Time-lapse analyses of ES cell-derived endothelial cell colonies showed that endothelial cells are moving around inside the colonies. Integrity of the VE-cadherin-based adherens junction was maintained despite the high motility of endothelial cells. Stimulation of pre-formed endothelial cell colonies with high dose of VEGF-A resulted in elongation of endothelial cells which is accompanied with restraint of cell movement. Suppression of endothelial cell movement was also observed on the colonies stimulated with angiopoietin-1, although individual cells lost their polarity and became rather round compared to the elongation induced by VEGF-A. Modulation of endothelial cell motility might be an important function of the angiogenic growth factors and it will provide a clue to how those factors regulate vascular development at the cellular level.