Angiogenesis - Methods and Protocols
Angiogenesis - Methods and Protocols
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血管生成 - 方法和方案
DOI：
10.1007/978-1-0716-2059-5_13
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Klaourakis K
中科院分区：
文献类型：
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作者：
Klaourakis K
淋巴管系统的发育和成熟对于器官功能是必不可少的，淋巴管系统的破坏会导致严重的表型。例如，心脏淋巴管故障会导致心肌水肿、持续性炎症和心输出量减少。因此，从胎儿发育早期到成年，研究心脏淋巴管的形成和生长过程是非常重要的。在小鼠的心脏中，淋巴管在出生后继续发育和扩张，并在出生后至少2周出现广泛的生长和图案。在这里，我们描述了一种用于整体、多视角成像和量化淋巴管参数的方案，包括小鼠出生后心脏中的淋巴管连接数量(即分支密度)、血管长度和血管终点数。该方案基于针对淋巴管内皮细胞(LECs)关键标志物的可靠抗体的使用，特别是糖蛋白淋巴管内皮细胞透明质酸受体1(LYVE1)、血管内皮生长因子受体3(VEGFR3；也称为FMS相关受体酪氨酸激酶4，Flt4)、粘蛋白类型蛋白泊多普宁(PDPN)和辅助受体神经粘蛋白2(NRP2)。为了对新生儿心脏心外膜下网络进行成像和定量分析，选择了VEGFR3，因为它在淋巴管内皮细胞中唯一表达。除LEC外，LYVE1还表达于组织内巨噬细胞，PDPN表达于心外膜，NRP2表达于心脏自主神经系统。总体而言，我们描述了新生儿心脏中常用淋巴标志物的表达模式，并提供了一种图像分析管道，可用于研究其他器官和系统(例如，血管和神经系统)。
The development and maturation of the lymphatic vasculature are essential for organ function with disruption leading to severe phenotypes. For example, malfunction of cardiac lymphatics results in myocardial oedema, persistent inflammation and reduced cardiac output. Thus, it is important to study the process of cardiac lymphatic formation and growth from the early stages of fetal development to adulthood. In the murine heart the lymphatics continue to develop and expand postnatally with extensive growth and patterning occurring up to at least 2 weeks after birth. Here, we describe a protocol for whole-mount, multi-view imaging and quantification of lymphatic vessel parameters, including vessel junction number (i.e., branching density), vessel length, and number of vessel end points in the murine postnatal heart. This protocol is based on the use of reliable antibodies against key markers of lymphatic endothelial cells (LECs), specifically the glycoprotein lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), the vascular endothelial growth factor receptor 3 (VEGFR3; also known as Fms-related receptor tyrosine kinase 4, FLT4), the mucin-type protein podoplanin (PDPN), and the co-receptor neuropilin 2 (NRP2). For imaging and quantitative analysis of the sub-epicardial network in neonatal hearts, VEGFR3 was selected given its exclusive expression in the lymphatic endothelium. In addition to LECs, LYVE1 expression was detected in tissue-resident macrophages, PDPN in the epicardium, and NRP2 in the autonomic nervous system of the heart. Overall, we characterized the expression patterns of commonly used lymphatic markers in the context of the neonatal heart and provide an image analysis pipeline that can be adapted to study other organs and systems (e.g., blood vasculature and nerve system).