Circadian misalignment promotes vascular smooth muscle cell apoptosis via defective autophagy

Defective Autophagy in vascular smooth muscle cells (VSMCs) in response to oxidative stress can lead to cellular Apoptosis and plaque instability. Previous studies have revealed that the circadian clock system is involved in autophagic regulation and plaque progression. However, the mechanism by which circadian rhythmicity influences VSMC Autophagy and plaque stability remains unclear. Our study described the circadian profiles in atheromatous plaques and verified the role of circadian misalignment in VSMC Autophagy and Apoptosis. We found that the mRNA expression levels of circadian locomotor output cycles protein kaput (CLOCK) and Beclin 1 were significantly decreased in unstable plaques compared with stable plaques. No significant differences were observed in other circadian rhythm genes. VSMCs treated with oxidized low-density lipoprotein (ox-LDL, 80 μg/ml) exhibited abnormal circadian rhythmicity and impaired Autophagy, as evidenced by consistent decreases in CLOCK and Beclin 1 expression, suggesting a correlation between CLOCK and Autophagy. CLOCK protein expression was inhibited by ox-LDL, accompanied by defective Autophagy and an increased Apoptosis rates (P < 0.05). Administration of rapamycin (10 nM) reversed the effect of ox-LDL on VSMC Autophagy and Apoptosis. Finally, CLOCK silencing led to a considerable decrease in Autophagy. VSMCs with stable CLOCK silencing also showed an increased Apoptosis rate. In addition, gene silencing of CLOCK in VSMCs counteracted the effects of moderate rapamycin concentrations on Autophagy and Apoptosis. In conclusion, these findings suggested that the CLOCK-dependent rapamycin signaling pathway is a critical mediator in ox-LDL-induced VSMCs with defective Autophagy that exacerbates plaque destabilization.