Herrera-Zelada Nicolas, Zúñiga-Cuevas Úrsula, Ramírez-Reyes Andrés, Norambuena-Soto Ignacio, Venegas-Zamora Leslye, Troncoso Mayarling F, Hernández Alejandra, Sánchez Gina, Pedrozo Zully, Lavandero Sergio, Riquelme Jaime A
Advanced Center for Chronic Disease (ACCDiS), Facultad de Ciencias Químicas y Farmacéuticas & Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Programa de Fisiopatología, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Front Pharmacol. 2023 Mar 27;14:1143888. doi: 10.3389/fphar.2023.1143888. eCollection 2023.
Small extracellular vesicles are nanosized vesicles (30-200 nm) that can ferry proteins, nucleic acids, and lipids between cells and therefore, have significant potential as biomarkers, drug delivery tools or therapeutic agents. SEVs of endothelial origin have been shown to -among other functions-reduce ischemia/reperfusion (I/R) injury in cardiomyocytes, but whether a pro-inflammatory state of the endothelium impairs the functionality of these SEVs remains to be elucidated. To test this, human umbilical vein endothelial cells cells were treated with TNF-α 10 ng/mL and the expression of the pro-inflammatory parameters VCAM-1, ICAM-1 and eNOS were determined by Western blot. SEVs were isolated from endothelial cells treated with or without TNF-α 10 ng/mL using size exclusion chromatography. The size and concentration of SEVs was measured by Nanoparticle Tracking Analysis. The expression of the surface marker CD81 was determined by immunoassay, whereas their morphology was assessed by electron microscopy. The function of endothelial SEVs was assessed by evaluating their cardioprotective effect in an model of global I/R using isolated hearts from adult C57BL/6 mice. Treatment of HUVECs with TNF-α induced the expression of VCAM-1 and ICAM-1, whereas eNOS levels were decreased. TNF-α did not affect the production, size, morphology, or expression of CD81. SEVs significantly reduced the infarct size as compared with untreated mice hearts, but SEVs isolated from TNF-α treated cells were unable to achieve this effect. Therefore, a pro-inflammatory state induced by TNF-α does not alter the production of endothelial SEVs but impairs their function in the setting of I/R injury.
小细胞外囊泡是纳米级囊泡（30 - 200纳米），能够在细胞间运输蛋白质、核酸和脂质，因此，作为生物标志物、药物递送工具或治疗剂具有巨大潜力。内皮来源的小细胞外囊泡已被证明——除其他功能外——可减轻心肌细胞的缺血/再灌注（I/R）损伤，但内皮的促炎状态是否会损害这些小细胞外囊泡的功能仍有待阐明。为了验证这一点，用人脐静脉内皮细胞用10纳克/毫升的肿瘤坏死因子-α（TNF-α）处理，并通过蛋白质印迹法测定促炎参数血管细胞黏附分子-1（VCAM-1）、细胞间黏附分子-1（ICAM-1）和内皮型一氧化氮合酶（eNOS）的表达。使用尺寸排阻色谱法从用或不用10纳克/毫升TNF-α处理的内皮细胞中分离小细胞外囊泡。通过纳米颗粒跟踪分析测量小细胞外囊泡的大小和浓度。通过免疫测定法测定表面标志物CD81的表达，而通过电子显微镜评估其形态。通过评估内皮小细胞外囊泡在使用成年C57BL/6小鼠离体心脏的整体I/R模型中的心脏保护作用来评估其功能。用TNF-α处理人脐静脉内皮细胞（HUVECs）诱导了VCAM-1和ICAM-1的表达，而eNOS水平降低。TNF-α不影响小细胞外囊泡的产生、大小、形态或CD81的表达。与未处理的小鼠心脏相比，小细胞外囊泡显著减小了梗死面积，但从TNF-α处理的细胞中分离的小细胞外囊泡无法达到这种效果。因此，TNF-α诱导的促炎状态不会改变内皮小细胞外囊泡的产生，但会损害其在I/R损伤环境中的功能。
