Choroid Sprouting Assay: An Ex Vivo Model of Microvascular Angiogenesis

Angiogenesis of the microvasculature is central to the etiology of many diseases including proliferative retinopathy, age-related macular degeneration and cancer. A mouse model of microvascular angiogenesis would be very valuable and enable access to a wide range of genetically manipulated tissues that closely approximate small blood vessel growth in vivo. Vascular endothelial cells cultured in vitro are widely used, however, isolating pure vascular murine endothelial cells is technically challenging. A microvascular mouse explant model that is robust, quantitative and can be reproduced without difficulty would overcome these limitations. Here we characterized and optimized for reproducibility an organotypic microvascular angiogenesis mouse and rat model from the choroid, a microvascular bed in the posterior of eye. The choroidal tissues from C57BL/6J and 129S6/SvEvTac mice and Sprague Dawley rats were isolated and incubated in Matrigel. Vascular sprouting was comparable between choroid samples obtained from different animals of the same genetic background. The sprouting area, normalized to controls, was highly reproducible between independent experiments. We developed a semi-automated macro in ImageJ software to allow for more efficient quantification of sprouting area. Isolated choroid explants responded to manipulation of the external environment while maintaining the local interactions of endothelial cells with neighboring cells, including pericytes and macrophages as evidenced by immunohistochemistry and fluorescence-activated cell sorting (FACS) analysis. This reproducible ex vivo angiogenesis assay can be used to evaluate angiogenic potential of pharmacologic compounds on microvessels and can take advantage of genetically manipulated mouse tissue for microvascular disease research.

微血管血管生成是包括增生性视网膜病变、年龄相关性黄斑变性及癌症在内的多种疾病的核心病因学机制。构建微血管血管生成的小鼠模型极具研究价值，可获取一系列与体内小血管生长过程高度相似的基因工程改造组织。体外培养的血管内皮细胞虽应用广泛，但分离纯化小鼠血管内皮细胞在技术上仍颇具挑战。一种稳定可靠、可定量且易于重现的小鼠微血管外植体模型，便可有效克服上述技术局限。本研究针对源自眼球后部微血管床——脉络膜的器官型微血管血管生成小鼠及大鼠模型，开展了表征工作并优化其重现性。我们分别分离了C57BL/6J、129S6/SvEvTac小鼠及斯波雷格-道利（Sprague Dawley）大鼠的脉络膜组织，并将其置于基质胶（Matrigel）中培养。同一遗传背景下不同个体来源的脉络膜样本，其血管出芽情况无显著差异。以对照组为基准归一化的出芽面积，在独立重复实验间具有高度稳定性与重现性。我们基于ImageJ软件开发了半自动化宏程序，以更高效地完成出芽面积的定量分析。分离得到的脉络膜外植体可响应外界环境干预，同时维持内皮细胞与周细胞、巨噬细胞等邻近细胞的局部相互作用，该结果通过免疫组织化学及荧光激活细胞分选术（fluorescence-activated cell sorting，FACS）分析得以验证。该重现性优异的离体血管生成实验模型，可用于评估药物化合物对微血管的血管生成调控潜能，同时可借助基因工程改造的小鼠组织开展微血管疾病相关研究。