CLOCK Stabilizes Vulnerable Plaques by Regulating Vascular Smooth Muscle Cell Phenotype Switching via RhoA/ROCK Signaling in Atherosclerosis

Abstract

Background: It has been known that circadian locomotor output cycles kaput protein (CLOCK) can not only regulate circadian rhythm but also participates in various biological processes. However, the association between CLOCK expression and plaque stability has not been well examined. This study aims to explore the role of CLOCK in the stability of atherosclerotic plaque and its potential molecular mechanism. Methods: The expression of CLOCK in stable plaque and ruptured plaque in the GSE41571 dataset was analyzed. The qRT-PCR (quantitative real-time polymerase chain reaction) and western blot were used to analyze the mRNA (messenger ribonucleic acid) and protein expression levels of CLOCK and phenotypic transformation markers of vascular smooth muscle cells (VSMCs) in human carotid atherosclerotic plaque samples or HAVSMCs (Human aortic vascular smooth muscle cells) cells, respectively. Cell Counting Kit-8 (CCK-8) and Transwell® assays were used to evaluate the cell proliferation and migration ability, respectively. Results: This study revealed that the CLOCK mRNA levels were reduced in unstable plaques (USP), following a similar pattern as the expression of vascular smooth muscle cells contractile markers such as calponin, α-smooth muscle actin (α-SMA), and smooth muscle protein 22α (SM22-α). Meanwhile, vascular smooth muscle cell synthetic phenotypic marker osteopontin (OPN) was increased. In addition, we found that oxidized low-density lipoprotein (ox-LDL) exposure accelerated phenotypic switching in VSMCs, a process promoted by knocking down the expression of CLOCK and inhibited by CLOCK overexpression. Further mechanisms studies showed that the Y-27632-mediated suppression of the RhoA/ROCK (Rho-Rho kinase) pathway rescued the phenotype induced by CLOCK deletion in VSMCs. Conclusions: Our results showed that decreased expression of CLOCK could contribute to phenotype switching of VSMCs and could be associated with atherosclerotic plaque instability.