Shan Shengshuai, Alanazi Abdulaziz H, Han Yohan, Zhang Duo, Liu Yutao, Narayanan S Priya, Somanath Payaningal R
Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA 30912, USA.
James and Jean Culver Vision Discovery Institute, Augusta University, Augusta, GA 30912, USA.
Biomedicines. 2024 Sep 10;12(9):2053. doi: 10.3390/biomedicines12092053.
Diabetic retinopathy (DR) is a leading cause of blindness, yet its molecular mechanisms are unclear. Extracellular vesicles (EVs) contribute to dysfunction in DR, but the characteristics and functions of vitreous EVs are unclear. This study investigated the inflammatory properties of type 2 diabetic (db) vitreous EVs. EVs isolated from the vitreous of db and non-db donors were used for nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunogold staining, Western blotting, and proteomic analysis by mass spectrometry. Intracellular uptake of vitreous EVs by differentiated macrophages was evaluated using ExoGlow membrane labeling, and the impact of EVs on macrophage (THP-1) activation was assessed by cytokine levels using RT-qPCR. NTA and TEM analysis of db and non-db vitreous EVs showed non-aggregated EVs with a heterogeneous size range below 200 nm. Western blot detected EV markers (Alix, Annexin V, HSP70, and Flotillin 1) and an upregulation of Cldn5 in db EVs. While the db EVs were incorporated into macrophages, treatment of THP-1 cells with db EVs significantly increased mRNA levels of TNFα and IL-1β compared to non-db EVs. Proteomic and gene enrichment analysis indicated pro-inflammatory characteristics of db EVs. Our results suggest a potential involvement of EC-derived Cldn5+ EVs in triggering inflammation, offering a novel mechanism involved and presenting a possible therapeutic avenue for DR.
糖尿病视网膜病变（DR）是导致失明的主要原因，但其分子机制尚不清楚。细胞外囊泡（EVs）在DR功能障碍中起作用，但玻璃体液EVs的特征和功能尚不清楚。本研究调查了2型糖尿病（db）玻璃体液EVs的炎症特性。从db和非db供体的玻璃体液中分离出的EVs用于纳米颗粒跟踪分析（NTA）、透射电子显微镜（TEM）、免疫金染色、蛋白质印迹以及质谱蛋白质组学分析。使用ExoGlow膜标记评估分化巨噬细胞对玻璃体液EVs的细胞内摄取，并通过RT-qPCR检测细胞因子水平来评估EVs对巨噬细胞（THP-1）激活的影响。对db和非db玻璃体液EVs的NTA和TEM分析显示，EVs呈非聚集状态，大小范围在200 nm以下且具有异质性。蛋白质印迹检测到EV标记物（Alix、膜联蛋白V、HSP70和小窝蛋白1），且db EVs中紧密连接蛋白5（Cldn5）上调。虽然db EVs被巨噬细胞摄取，但与非db EVs相比，用db EVs处理THP-1细胞可显著提高肿瘤坏死因子α（TNFα）和白细胞介素-1β（IL-1β）的mRNA水平。蛋白质组学和基因富集分析表明db EVs具有促炎特性。我们的结果表明，内皮细胞来源的Cldn5+ EVs可能参与引发炎症，这提供了一种新的相关机制，并为DR提出了一条可能的治疗途径。
