SENP3 regulates high glucose-induced endothelial dysfunction via ROS dependent signaling

Abstract

Background: The current study aimed to explore the role of SENP3 in endothelial cell dysfunction in a high-glucose setting.

Methods: The gene and protein expressions of SENP3 in high-glucose cultured HAECs were examined using quantitative PCR and western blotting. The effects of SENP3 on HAEC viability, apoptosis, migration, and endothelial-monocyte adhesion were evaluated in vitro by knockdown. Moreover, a mouse streptozotocin-induced type I diabetes model was established for SENP3 expression assessment. In addition, the effects of SENP3 on ROS-related signaling pathways were investigated in high-glucose cultured HAECs.

Results: Significantly increased levels of SENP3 mRNA and protein were found in high-glucose cultured HAECs in a time-dependent manner. SENP3 knockdown reversed high glucose-induced HAEC viability, apoptosis, and migration reduction. SENP3 knockdown attenuated the high glucose-induced intercellular adhesion of THP-1 monocytic cells and HAECs via downregulation of ICAM-1 and VCAM-1 expression. Increased levels of SENP3, ICAM-1, and VCAM-1 expression were observed in the aorta tissue of mice with type I diabetes. Downregulation of SENP3 expression was observed in HAECs cultured with high glucose levels using the free radical scavenger N-acetyl-L-cysteine or NOX4 siRNA.

Conclusions: SENP3 was involved in high glucose-induced endothelial dysfunction, and ROS-dependent signaling served as the mechanism.

Keywords: ROS; SENP3; diabetes mellitus; endothelial dysfunction; high glucose.

Figure 1.

Enhanced SENP3 mRNA and protein expression in high glucose-treated HAECs: (a) at 70% confluency, HAEC was treated with EBM-2 and 25 mM d-glucose at the indicated time. SENP3 mRNA level determination by qRT-PCR and (b) SENP3 protein expression determination by western blotting. *p < 0.05; **p < 0.01 versus control (normal glucose).

Figure 2.

SENP3 was involved in high glucose-induced endothelial cell dysfunction: (a) the efficacy of SENP3 knockdown by siRNA was verified by western blotting, (b) high glucose-induced HAEC death was reversed by SENP3 knockdown, (c) high glucose-induced HAEC apoptosis was reversed by SENP3 knockdown, and (d) high glucose-induced HAEC migration suppression was reversed by SENP3 knockdown. All the experiments were repeated at least thrice. ^*p < 0.05 versus normal glucose, ^#p < 0.05 versus high glucose + control siRNA.

Figure 3.

SENP3 was involved in high glucose-induced endothelial–monocyte adhesion and related adhesion molecules expression: (a) high glucose-induced endothelial–monocyte adhesion was reversed by SENP3 knockdown, (b) SENP3 was involved in high glucose-induced ICAM-1 expression, and (c) SENP3 was involved in high glucose-induced VCAM-1 expression. ^*p < 0.05 versus normal glucose, ^#p < 0.05 versus high glucose + control siRNA.

Figure 4.

Enhanced SENP3 expression in aortas from type I diabetes mice induced by streptozotocin (STZ): (a) increased blood glucose levels after STZ administration, which confirmed the type I diabetes mice model establishment, (b) increased level of SENP3 protein expression in aortas from STZ-treated type I diabetes mice, and (c) increased level of ICAM and VCAM-1 mRNA expression in aortas from STZ-treated type I diabetes mice. ^*p < 0.05 versus control.

Figure 5.

ROS related signaling is involved in the effects of SENP3 on HAECs in the setting of high glucose: (a) ROS dependent SENP3 expression in the setting of high glucose, (b) efficacy of Nox4 knockdown by siRNA, (c) decreased SENP3 level could be also achieved by Nox4 siRNA, and (d) intracellular ROS level measurement. ^*p < 0.05 versus normal glucose, ^#p < 0.05 versus high glucose.