Abstract：
Angiogenesis is the formation of new blood vessels from the existing vasculature. It is a fundamental process in developmental biology but also a pathological event that initiates or aggravates many diseases. In this complex multistep process, endothelial cells are activated by angiogenic stimuli; undergo specialization in response to VEGF/Notch signaling; degrade the basement membrane of the parent vessel; sprout, migrate, and proliferate to form capillary tubes that branch; and ultimately anastomose with adjacent vessels. Here we describe an assay that mimics the invasion step in vitro. Human microvascular endothelial cells are confronted by a VEGF-enriched basement membrane material in a three-dimensional environment that promotes endothelial cell sprouting, tube formation, and anastomosis. After a few hours, endothelial cells have become tip cells, and vascular sprouts can be observed by phase contrast, fluorescence, or time-lapse microscopy. Sprouting endothelial cells express tip cell markers, display podosomes and filopodia, and exhibit cell dynamics similar to those of angiogenic endothelial cells in vivo. This model provides a system that can be manipulated genetically to study physiological or pathological angiogenesis and that can be used to screen compounds for pro-/anti-angiogenic properties. In this chapter, we describe the key steps in setting up this assay.
摘要：
血管生成是从现有的脉管系统形成新的血管。它是发育生物学的基本过程，也是引发或加重许多疾病的病理事件。在这个复杂的多步骤过程中，内皮细胞被血管生成刺激激活；响应VEGF/Notch信号传导而经历特化；降解亲本血管的基底膜；发芽，migrate,并增殖形成分支的毛细管；并最终与相邻血管吻合。在这里，我们描述了一种模拟体外入侵步骤的测定法。人微血管内皮细胞在促进内皮细胞发芽的三维环境中面对富含VEGF的基底膜材料，管形成,和吻合。几个小时后,内皮细胞变成了尖端细胞,血管芽可以通过相位对比观察到，荧光，或延时显微镜。发芽内皮细胞表达尖端细胞标志物，显示足体和丝状体，并表现出与体内血管生成内皮细胞相似的细胞动力学。该模型提供了一种系统，该系统可以通过遗传方式进行操作以研究生理或病理性血管生成，并且可以用于筛选化合物的促/抗血管生成特性。在这一章中,我们描述了建立该测定的关键步骤。
