Azam Salma H, Smith Mitchell, Somasundaram Vivek, Pecot Chad V
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill.
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill.
J Vis Exp. 2018 Feb 16(132):57309. doi: 10.3791/57309.
Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial - mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.
血管生成是指从已有的脉管系统生长出新的血管，它是许多生物学过程的重要组成部分，包括胚胎发生与发育、伤口愈合、肿瘤生长与转移以及眼部和心血管疾病。需要有效的体外模型来重现血管生成的生物学过程，以便恰当地研究这一过程，并确定最终可作为新型治疗策略靶点的调控机制。先前已证明珠粒血管生成试验能够在体外重现内皮细胞芽生的多个阶段。然而，该试验的一个局限性是缺乏内皮细胞与壁细胞的相互作用，而这种相互作用在体内对于内皮细胞功能的分子和表型调控至关重要。此处给出的方案介绍了将壁细胞纳入珠粒血管生成试验的方法，并证明了体外芽生过程中内皮细胞与周细胞紧密关联。该方案还详细介绍了在机制研究中使用小干扰RNA（siRNA）有效沉默内皮细胞中靶基因的方法。总之，该方案提供了一种体外试验，能更恰当地模拟参与芽生血管生成的多种细胞类型，并为治疗评估和血管生成调控机制的新发现提供了一个更具生理相关性的平台。
