NEDD4L 介导的 RASGRP2 通过激活 Rap1 和 R-Ras，抑制高血糖和 oxLDL 诱导的血管内皮细胞功能障碍。

IF 4.6 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Molecular cell research Pub Date : 2024-09-10 DOI:10.1016/j.bbamcr.2024.119844

Guozhu Chen , Yisong Pei , Qiaoling Ye , Zulong Xie , Laxman Gyawali , Xing Liang

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引用次数: 0

摘要

背景RAS鸟苷释放蛋白2（RASGRP2）是介导内皮细胞功能的重要调节因子。方法用高葡萄糖（HG）和氧化低密度脂蛋白（oxLDL）处理人心脏微血管内皮细胞（HCMECs）。实时定量 PCR 和免疫印迹（WB）检测了 RASGRP2 和神经前体细胞表达发育下调 4 样（NEDD4L）的表达。细胞活力、凋亡、迁移和血管生成通过 CCK8 检测法、流式细胞术、transwell 检测法和管形成检测法进行检测。通过检测 ROS 生成和细胞通透性来评估细胞功能。使用 WB 检测 Rap1 和 R-Ras 蛋白水平。RASGRP2和NEDD4L之间的相互作用通过Co-IP检测和泛素化检测得到了证实。结果RASGRP2在HG + oxLDL诱导的HCMECs中低表达。RASGRP2的过表达抑制了HG + oxLDL诱导的HCMECs的通透性、细胞凋亡和ROS产生，同时加速了细胞活力、迁移和血管生成。NEDD4L可通过泛素化与RASGRP2相互作用，从而抑制RASGRP2蛋白的稳定性，降解其表达。功能实验表明，敲除NEDD4L可抑制HG + oxLDL诱导的HCMECs功能障碍，而下调RASGRP2可逆转这些效应。我们的数据表明，靶向 NEDD4L/RASGRP2 轴或诱导 RASGRP2 修饰的 ADMSC-Exo 可能是缓解 DM 相关 AS 的有效策略。

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NEDD4L-mediated RASGRP2 suppresses high-glucose and oxLDL-induced vascular endothelial cell dysfunctions by activating Rap1 and R-Ras

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NEDD4L-mediated RASGRP2 suppresses high-glucose and oxLDL-induced vascular endothelial cell dysfunctions by activating Rap1 and R-Ras

Background

Ras guanyl-releasing protein 2 (RASGRP2) is an important regulator mediating endothelial cell function. However, whether RASGRP2 mediates diabetes mellitus (DM)-related atherosclerosis (AS) progression by regulating endothelial cell functions is unknown.

Methods

Human cardiac microvascular endothelial cells (HCMECs) were treated with high-glucose (HG) and oxidized low-density lipoprotein (oxLDL). The expression of RASGRP2 and neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) was examined by quantitative real-time PCR and western blot (WB). Cell viability, apoptosis, migration, angiogenesis were detected by CCK8 assay, flow cytometry, transwell assay and tube formation assay. ROS production and cell permeability were tested to assess cell function. Rap1 and R-Ras protein levels were examined using WB. The interaction between RASGRP2 and NEDD4L was confirmed by Co-IP assay and ubiquitination assay. Exosomes were isolated from adipose-derived MSC (ADMSC)-transfected RASGRP2 overexpression vector, and then co-cultured with HG + oxLDL-induced HCMECs.

Results

RASGRP2 was lowly expressed in HG + oxLDL-induced HCMECs. RASGRP2 overexpression inhibited HG + oxLDL-induced HCMECs permeability, apoptosis and ROS production, while accelerated cell viability, migration and angiogenesis. NEDD4L could interact with RASGRP2 by ubiquitination, thus inhibiting RASGRP2 protein stability to degrade its expression. Functional experiments showed that NEDD4L knockdown suppressed HG + oxLDL-induced HCMECs dysfunction, while these effects were reversed by RASGRP2 downregulation. ADMSC-Exo overexpressed RASGRP2 could promote cell viability, migration and angiogenesis, while suppress permeability, apoptosis and ROS production in HG + oxLDL-induced HCMECs.

Conclusion

Our data showed that targeting NEDD4L/RASGRP2 axis or inducing RASGRP2-modified ADMSC-Exo might be the efficient strategy for alleviating DM-related AS.

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来源期刊

Biochimica et biophysica acta. Molecular cell research 生物-生化与分子生物学

CiteScore

10.00

自引率

2.00%

发文量

151

审稿时长

44 days

期刊介绍： BBA Molecular Cell Research focuses on understanding the mechanisms of cellular processes at the molecular level. These include aspects of cellular signaling, signal transduction, cell cycle, apoptosis, intracellular trafficking, secretory and endocytic pathways, biogenesis of cell organelles, cytoskeletal structures, cellular interactions, cell/tissue differentiation and cellular enzymology. Also included are studies at the interface between Cell Biology and Biophysics which apply for example novel imaging methods for characterizing cellular processes.