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

脊椎动物胚胎发生过程中，初级血管丛和随后由大小血管组成的血管树的形成机制在很大程度上是未知的。小鼠ES细胞的体外分化系统是一种将复杂的血管发育过程分解为可以真实的实时监测的细胞事件的手段。我们已经开发了一种培养系统，其中内皮细胞分化的ES细胞通过侧中胚层细胞作为中间前体。在OP 9基质细胞层上产生的ES细胞衍生的内皮细胞集落已被用作体外模型，以检查个体内皮细胞响应于各种血管生成刺激物如VEGF-A/VEGFR-2和VEGF-C/VEGFR-3信号的行为。显示VEGFR-2信号诱导内皮细胞的伸长和分散，而VEGFR-3信号通过调节VEGFR-2信号维持细胞-细胞粘附的完整性。我们还确定了一个转录因子FOXO 1作为一个调节分子的延伸反应的内皮细胞的VEGFR-2信号。ES细胞衍生的内皮细胞集落的延时分析显示，内皮细胞在集落内四处移动。尽管内皮细胞具有高运动性，但基于VE-钙粘蛋白的粘附连接的完整性得以维持。用高剂量的VEGF-A刺激预先形成的内皮细胞集落导致内皮细胞伸长，这伴随着细胞运动的抑制。在用血管生成素-1刺激的集落上也观察到内皮细胞运动的抑制，尽管与VEGF-A诱导的伸长相比，单个细胞失去了它们的极性并且变得相当圆。调节内皮细胞运动可能是血管生成因子的一个重要功能，这将为这些因子如何在细胞水平上调节血管发育提供线索。

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.