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

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

机构信息

The Second Affiliated Hospital of Chongqing Medical University Cardiology Department, Chongqing, China.

出版信息

Biochim Biophys Acta Mol Cell Res. 2024 Dec;1871(8):119844. doi: 10.1016/j.bbamcr.2024.119844. Epub 2024 Sep 10.

DOI:10.1016/j.bbamcr.2024.119844

PMID:39260747

Abstract

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.

摘要

背景

Ras 鸟嘌呤核苷酸释放蛋白 2（RASGRP2）是一种重要的调节因子，介导内皮细胞功能。然而，RASGRP2 是否通过调节内皮细胞功能来介导糖尿病（DM）相关动脉粥样硬化（AS）的进展尚不清楚。

方法

用人心脏微血管内皮细胞（HCMECs）用高糖（HG）和氧化低密度脂蛋白（oxLDL）处理。用实时定量 PCR 和蛋白质印迹（WB）检测 RASGRP2 和神经前体细胞表达发育下调 4 样（NEDD4L）的表达。通过 CCK8 测定、流式细胞术、transwell 测定和管形成测定检测细胞活力、凋亡、迁移和血管生成。通过 ROS 产生和细胞通透性测试来评估细胞功能。用 WB 检测 Rap1 和 R-Ras 蛋白水平。用 Co-IP 测定和泛素化测定证实 RASGRP2 和 NEDD4L 之间的相互作用。从脂肪间充质干细胞（ADMSC）转染 RASGRP2 过表达载体中分离外泌体，然后与 HG+oxLDL 诱导的 HCMECs 共培养。

结果

RASGRP2 在 HG+oxLDL 诱导的 HCMECs 中低表达。RASGRP2 过表达抑制 HG+oxLDL 诱导的 HCMECs 通透性、凋亡和 ROS 产生，同时加速细胞活力、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 蛋白稳定性并降解其表达。功能实验表明，NEDD4L 敲低抑制 HG+oxLDL 诱导的 HCMECs 功能障碍，而 RASGRP2 下调可逆转这些作用。ADMSC-Exo 过表达 RASGRP2 可促进 HG+oxLDL 诱导的 HCMECs 中的细胞活力、迁移和血管生成，同时抑制通透性、凋亡和 ROS 产生。