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

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.