Abstract

Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel–Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca²⁺. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H₂O₂ formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H₂O₂ on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H₂O₂ for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H₂O₂ acts as a mediator that activates exocytosis of Weibel–Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.

中文翻译：

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荧光标记的ARC1779适体在评估H 2 O 2对von Willebrand因子胞吐作用的影响中的应用

摘要

在这里，我们提出了一种新方法，用于使用与vWF A1结构域相互作用的ARC1779适体对暴露在内皮细胞（EC）表面的血管性假血友病因子（vWF）进行定量估计。为了可视化vWF和适体之间的复合物形成，将适体与Cy5荧光标记偶联。用ARC1779-Cy5缀合物对培养的人脐静脉内皮细胞（HUVEC）进行染色，并用荧光显微镜成像。用CellProfiler软件分析图像。从Weibel-Palade体中释放的vWF被观察为圆形和不规则形状的亮点状结构，在HUVEC暴露于组胺或凝血酶后其数量增加了数倍。用ARC1779-Cy5染色还显示了活化的HUVEC表面上的长丝状vWF结构。²⁺。有证据表明，过氧化氢在EC中也充当第二信使。另外，在白细胞中形成的外源H ₂ O ₂可以进入EC。我们研究的目的是使用提出的方法确定H ₂ O ₂对HUVEC表面vWF暴露的影响。结果表明，浓度为100 µM的过氧化氢低于培养的HUVEC的H ₂ O ₂的细胞毒性阈值，使HUVEC质膜上暴露的点状结构数和vWF总量增加了数倍，建议H ₂ O ₂ 充当介质，在炎症过程中以及在ECs中内源性活性氧生成增加时，激活Weibel-Palade体的胞吐作用和血管内皮中vWF的分泌。