The Hatter Cardiovascular Institute, University College London, London, UK.
Advanced Center for Chronic Diseases (ACCDiS), Facultad de Ciencias Quimicas y Farmaceuticas & Facultad de Medicina, Universidad de Chile, Santiago, Chile.
J Cell Mol Med. 2020 Apr;24(8):4871-4876. doi: 10.1111/jcmm.15047. Epub 2020 Feb 26.
Small extracellular vesicles (EVs) are novel players in vascular biology. However, a thorough understanding of their production and function remains elusive. Endothelial senescence is a key feature of vascular ageing and thus, is an attractive therapeutic target for the treatment of vascular disease. In this study, we sought to characterize the EV production of senescent endothelial cells. To achieve this, Human Umbilical Vascular Endothelial Cells (HUVECs) were replicated until they reached senescence, as determined by measurement of Senescence-Associated β-Galactosidase activity via microscopy and flow cytometry. Expression of the endosomal marker Rab7 and the EV marker CD63 was determined by immunofluorescence. Small EVs were isolated by ultracentrifugation and characterized using electron microscopy, nanoparticle tracking analysis and immunoassays to assess morphology, size, concentration and expression of exosome markers CD9 and CD81. Migration of HUVECs in response to EVs was studied using a transwell assay. The results showed that senescent endothelial cells express higher levels of Rab7 and CD63. Moreover, senescent endothelial cells produced higher levels of CD9- and CD81-positive EVs. Additionally, small EVs from both young and senescent endothelial cells promoted HUVEC migration. Overall, senescent endothelial cells produce an increased number of functional small EVs, which may have a role in vascular physiology and disease.
小细胞外囊泡 (EVs) 是血管生物学中的新角色。然而,它们的产生和功能仍难以理解。内皮细胞衰老 (endothelial senescence) 是血管老化的一个关键特征,因此,是治疗血管疾病的一个有吸引力的治疗靶点。在这项研究中,我们试图描述衰老内皮细胞的 EV 产生。为了实现这一目标,通过显微镜和流式细胞术测量衰老相关β-半乳糖苷酶活性,复制人脐静脉内皮细胞 (HUVEC) 直至达到衰老状态。通过免疫荧光测定内体标记物 Rab7 和 EV 标记物 CD63 的表达。通过超速离心分离小 EV,并使用电子显微镜、纳米颗粒跟踪分析和免疫测定法对其进行表征,以评估形态、大小、浓度和外泌体标记物 CD9 和 CD81 的表达。使用 Transwell 测定法研究 HUVEC 对 EV 的迁移。结果表明,衰老的内皮细胞表达更高水平的 Rab7 和 CD63。此外,衰老的内皮细胞产生更高水平的 CD9-和 CD81-阳性 EVs。此外,来自年轻和衰老内皮细胞的小 EV 均促进 HUVEC 迁移。总体而言,衰老的内皮细胞产生了更多数量的功能性小 EVs,这可能在血管生理学和疾病中发挥作用。
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