Chen Guozhu, Pei Yisong, Jiang Peng, Ye Qiaoling, Xie Zulong, Gyawali Laxman
Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, No. 74, Linjiang Road, Yuzhong District, Chongqing, 400030, China.
Department of Cardiology, The Beibei Affiliated Hospital of Chongqing Medical University, No. 69, Jialing Village, Beibei District, Chongqing, 400030, China.
Cell Biol Toxicol. 2025 Jan 7;41(1):23. doi: 10.1007/s10565-024-09973-3.
Vascular endothelial cell-derived exosomes are thought to mediate disease progression by regulating macrophage polarization. However, its mechanism in diabetes mellitus (DM)-related atherosclerosis (AS) progress is unclear.
High-glucose (HG) and oxLDL were used to induce human cardiac microvascular endothelial cells (HCMECs) to mimic DM-related AS model. The conditioned medium (CM) from HG+oxLDL-induced HCMECs was incubated with THP1-M0 monocytes treated with LPS or oxLDL. The mRNA levels of macrophage M1/M2 polarization markers, NEDD4L, IκBα and PPARγ were determined by qRT-PCR. Flow cytometry was used to analyze macrophage marker. Dil-labeled oxLDL and oil red O staining were performed to assess oxLDL uptake by THP1-M0 cells. The levels of inflammatory factors were examined using ELISA. Transmission electron microscope was used for observing foam cell formation and exosome morphology. The protein levels of p-Smad1/Smad1, p-Smad2/Smad2, p-IκBα/IκBα, p-P65/P65, anti-lipid metabolism-related markers, and NEDD4L were tested by western blot. The interaction between NEDD4L and IκBα or PPARγ was assessed by Co-IP assay.
The CM of HG+oxLDL-induced HCMECs could promote macrophage M1 polarization, oxLDL uptake and foam cell formation, and exosome inhibiter GW4869 eliminated these effects. NEDD4L was overexpressed in exosomes from HG+oxLDL-induced HCMECs, which could be taken up by THP1-M0 cells. Exosomal NEDD4L knockdown inhibited macrophage M1 polarization, oxLDL uptake and foam cell formation by reducing the protein levels of p-Smad1/Smad1, p-Smad2/Smad2, p-IκBα/IκBα and p-P65/P65. NEDD4L could reduce IκBα and PPARγ expression through ubiquitination.
HG+oxLDL-induced HCMECs-derived exosomal NEDD4L could enhance the ubiquitination of IκBα and PPARγ to facilitate macrophage M1 polarization and oxLDL uptake, thus accelerating DM-related AS.
血管内皮细胞衍生的外泌体被认为通过调节巨噬细胞极化来介导疾病进展。然而，其在糖尿病（DM）相关动脉粥样硬化（AS）进展中的机制尚不清楚。
使用高糖（HG）和氧化低密度脂蛋白（oxLDL）诱导人心脏微血管内皮细胞（HCMECs）以模拟DM相关的AS模型。将HG + oxLDL诱导的HCMECs的条件培养基（CM）与用脂多糖（LPS）或oxLDL处理的THP1-M0单核细胞一起孵育。通过qRT-PCR测定巨噬细胞M1/M2极化标志物、NEDD4L、IκBα和PPARγ的mRNA水平。流式细胞术用于分析巨噬细胞标志物。进行Dil标记的oxLDL和油红O染色以评估THP1-M0细胞对oxLDL的摄取。使用酶联免疫吸附测定（ELISA）检测炎症因子水平。透射电子显微镜用于观察泡沫细胞形成和外泌体形态。通过蛋白质免疫印迹法检测p-Smad1/Smad1、p-Smad2/Smad2、p-IκBα/IκBα、p-P65/P65、抗脂质代谢相关标志物和NEDD4L的蛋白质水平。通过免疫共沉淀（Co-IP）测定评估NEDD4L与IκBα或PPARγ之间的相互作用。
HG + oxLDL诱导的HCMECs的CM可促进巨噬细胞M1极化、oxLDL摄取和泡沫细胞形成，外泌体抑制剂GW4869消除了这些作用。NEDD4L在HG + oxLDL诱导的HCMECs的外泌体中过表达，其可被THP1-M0细胞摄取。外泌体NEDD4L敲低通过降低p-Smad1/Smad1、p-Smad2/Smad2、p-IκBα/IκBα和p-P65/P65的蛋白质水平来抑制巨噬细胞M1极化、oxLDL摄取和泡沫细胞形成。NEDD4L可通过泛素化降低IκBα和PPARγ表达。
HG + oxLDL诱导的HCMECs衍生的外泌体NEDD4L可增强IκBα和PPARγ的泛素化，以促进巨噬细胞M1极化和oxLDL摄取，从而加速DM相关的AS。
