Ang II-induced oscillation of clock genes can attenuate phenotypic transformation in vascular smooth muscle cells by activating the AT1R/PLC/Ca2+/PKC/p-CREB pathway.

Abstract

This study aimed to investigate the role of clock genes Per1/Per2 in angiotensin II (Ang II)-induced vascular smooth muscle cells (VSMCs) phenotypic transformation and the underlying mechanisms. Primary rat VSMCs were treated with Ang, valsartan or other inhibitors. Assays included PCR, Western blot (Per1, Per2, p-MLC₂₀, p-CREB, AT1R), cell viability (MTT, Ki67, binuclear count), cell cycle, calcium and IP3. 50% fetal bovine serum shock significantly reduced the proliferation-promoting effect of Ang. Ang significantly increased the expression of Per1/Per2 mRNA at ZT3 but decreased it at ZT19 and ZT23 correlating with p-MLC₂₀ changes. Valsartan (AT1R inhibitor), Calphostin C (PKC inhibitor), U73122 (PLC inhibitor), 2-APB (IP3R blocker) and dantrolene sodium salt (Calcium channel protein inhibitor) significantly blocked the effects of Ang on Per1/Per2 genes. Ang significantly increased the p-CREB expression, IP3 and [Ca²⁺]i concentration transiently but decreased it in the long term. However, Ang significantly decrease Per1, Per2, and AT1R proteins expression transiently but increased it in the long term. Finally, silencing Per1 and Per2 enhances Ang-induced proliferation of VSMCs. Ang II triggers Per1/Per2 oscillation via the AT1R/PLC/Ca²⁺/PKC/p-CREB axis. Remarkably, the resultant AT1R-Per1/Per2 feedback loop counteracts Ang II-driven VSMC phenotypic transformation.