Fearnley Gareth W, Smith Gina A, Odell Adam F, Latham Antony M, Wheatcroft Stephen B, Harrison Michael A, Tomlinson Darren C, Ponnambalam Sreenivasan
Endothelial Cell Biology Unit, School of Molecular & Cellular Biology, University of Leeds, Leeds, United Kingdom.
Division of Diabetes and Cardiovascular Research, Faculty of Medicine & Health, University of Leeds, Leeds, United Kingdom.
Methods Enzymol. 2014;535:265-92. doi: 10.1016/B978-0-12-397925-4.00016-X.
The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function.
血管内皮生长因子A（VEGF-A）是一种多功能细胞因子，可刺激血管生成、血管修复和再生。VEGF-A与VEGF受体酪氨酸激酶（VEGFRs）结合，并刺激细胞内信号传导，从而导致血管生理变化。这一现象的一个重要方面是VEGFR运输和细胞内信号传导的时空协调，以确保VEGFR在不同细胞器中的驻留与下游细胞输出相关联。在这里，我们描述了一系列实验，以评估VEGF-A刺激的来自细胞内区室（如内体-溶酶体系统）的细胞内信号传导的影响。这些实验包括原代人内皮细胞的初始分离和表征、进行反向遗传学分析蛋白质功能；用于研究受体运输、信号传导和蛋白水解的方法；以及用于测量细胞迁移、增殖和管状形成变化的实验。我们实验室进行的研究已举例说明了这些实验中的每一个。总之，我们描述了研究VEGF-A在内皮细胞功能中作用的必要技术。
