Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA.
J Extracell Vesicles. 2022 Jan;11(1):e12178. doi: 10.1002/jev2.12178.
Extracellular vesicles (EVs) are potent signalling mediators. Although interest in EV translation is ever-increasing, development efforts are hampered by the inability to reliably assess the uptake of EVs and their RNA cargo. Here, we establish a novel qPCR-based method for the detection of unmodified EVS using an RNA Tracer (DUST). In this proof-of-concept study we use a human-specific Y RNA-derived small RNA (YsRNA) we dub "NT4" that is enriched in cardiosphere-derived cell small EVs (CDC-sEVs). The assay is robust, sensitive, and reproducible. Intravenously administered CDC-sEVs accumulated primarily in the heart on a per mg basis. Cardiac injury enhanced EV uptake in the heart, liver, and brain. Inhibition of EV docking by heparin suppressed uptake variably, while inhibition of endocytosis attenuated uptake in all organs. In vitro, EVs were uptaken more efficiently by macrophages, endothelial cells, and cardiac fibroblasts compared to cardiomyocytes. These findings demonstrate the utility of DUST to assess uptake of EVs in vivo and in vitro.
细胞外囊泡 (EVs) 是有效的信号转导介质。尽管人们对 EV 翻译的兴趣与日俱增，但由于无法可靠地评估 EV 及其 RNA 货物的摄取，因此开发工作受到了阻碍。在这里，我们使用一种新型的基于 qPCR 的方法，使用 RNA Tracer (DUST) 来检测未修饰的 EVs。在这项概念验证研究中，我们使用了一种在人心肌细胞衍生的细胞外囊泡 (CDC-sEVs) 中富集的人类特异性 Y RNA 衍生的小 RNA (YsRNA)，我们将其称为“NT4”。该测定方法具有稳健性、灵敏度和可重复性。静脉内给予的 CDC-sEVs 主要以每毫克为基础积累在心脏中。心脏损伤增强了心脏、肝脏和大脑中 EV 的摄取。肝素抑制 EV 对接可抑制摄取的变异性，而内吞作用的抑制则可减弱所有器官的摄取。在体外，与心肌细胞相比，巨噬细胞、内皮细胞和心肌成纤维细胞更有效地摄取 EV。这些发现证明了 DUST 用于评估体内和体外 EV 摄取的效用。
