CLOCK Promotes Endothelial Damage by Inducing Autophagy through Reactive Oxygen Species

Abstract

A number of recent studies have implicated that autophagy was activated by reactive oxygen species (ROS). Our previous report indicated that CLOCK increased the accumulation of ROS under hypoxic conditions. In this study, we investigated the mechanisms by which CLOCK mediated endothelial damage, focusing on the involvement of oxidative damage and autophagy. Overexpression of CLOCK in human umbilical vein endothelial cells (HUVECs) showed inhibition of cell proliferation and higher autophagosome with an increased expression of Beclin1 and LC3-I/II under hypoxic conditions. In contrast, CLOCK silencing reversed these effects. Interestingly, pretreatment with 3-methyladenine (3-MA) resulted in the attenuation of CLOCK-induced cell autophagy and but did not influence the production of intracellular reactive oxygen species (ROS). Furthermore, Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt), a ROS scavenger, significantly attenuated CLOCK-induced cell autophagy. In addition, we found that overexpression of CLOCK had no significant effects on the production of ROS and expression of Beclin1 and LC3-I/II under normoxic conditions in HUVEC. In this present investigation, our results suggested a novel mechanism of action of CLOCK in HUVECs, opening up the possibility of targeting CLOCK for the treatment of vascular diseases.

The Reactive Oxygen Species–Mitophagy Signaling Pathway Regulates Liver Endothelial Cell Survival During Ischemia/Reperfusion Injury

“…Although ROS‐dependent ATG7 activation of autophagy has been reported in other endothelial cell types, this is the first study to demonstrate this function of autophagy in primary human LEC. Importantly in vascular endothelial cells, the ROS‐autophagy signaling pathway induces endothelial damage, which highlights that autophagy may function differently in cells of different endothelial lineage. Interestingly, even in normoxic conditions, autophagic puncta are present within primary human LEC that may reflect the inherent scavenging function that LEC perform within the liver.…”

TCS2 Increases Olaquindox-Induced Apoptosis by Upregulation of ROS Production and Downregulation of Autophagy in HEK293 Cells

“…Nevertheless, the current results revealed that pretreatment with NAC efficiently reduced ROS generation and blocked olaquindox-induced autophagy as well ( Figure 4 ). However, there are a number of reports related to inhibition of ROS-mediated autophagy [ 31 , 32 ]. In short, we speculate that ROS might play an upstream role in olaquindox-induced autophagy.…”

A Positive Feedback Loop of Profilin‐1 and RhoA/ROCK1 Promotes Endothelial Dysfunction and Oxidative Stress

“…Intracellular ROS was measured using 2,7-dichlorodihydrofluorescein diacetate dye (H2DCFDA, Life Technologies) as described previously [ 19 ].…”

“…Western blotting was performed using standard methods as previously reported [ 19 ]. Antiprofilin-1 antibody (#3237), anti-ROCK1 (#4035), and anti-p-eNOS antibodies (#9570) were purchased from Cell Signaling Technology (Beverly, MA, USA).…”