The epigenetic and epitranscriptomic regulation of Brd4-Mettl3 axis on STING mediated vascular calcification

Aims: Vascular calcification (VC) predicts cardiovascular risk in diabetes, chronic kidney disease (CKD) and atherosclerosis patients and is closely linked to the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Our previous work revealed that cGAS-STING signalling pathway in VSMCs increases CKD-associated atherosclerotic plaque vulnerability and recent studies elucidated the involvement of STING in vascular calcification, but its upstream modulation mechanism remains to be elucidated.

Methods and results: DNA damage and robust upregulation of STING occurred in high phosphate (Pi)-stimulated VSMCs, calcified aortic tissues from 1,25(OH)₂VitD₃ (VitD₃)-overloaded mice and radial arteries from CKD patients, and these changes were accompanied by the activation of the cGAS‒STING signalling pathway. STING deficiency alleviated vascular calcification in CKD model mice. STING knockdown suppressed calcium deposition and the expression of osteogenic transdifferentiation markers in VSMCs, whereas the overexpression of STING or its agonist 2',3'-cGAMP exacerbated VC. Mechanistically, Brd4 was identified as a critical epigenetic regulator of Pi-induced DNA damage, STING activation and VC. Furthermore, the N6-methyladenosine (m6A) modification of STING mRNA was significantly increased in Pi-stimulated VSMCs, increasing its stability. Brd4 depletion decreased the m6A modification of STING and accelerated its degradation. The m6A modification of STING was mediated by the core methyltransferase METTL3, whose transcription was regulated by Brd4. Finally, C-176 showed translational potential by effectively suppressing vascular calcification and osteogenesis markers expression in both VitD₃-overloaded mice and Pi-stimulated VSMCs.

Conclusions: Our study elucidated the interplay between Brd4-mediated epigenetic and Mettl3-dependent epitranscriptomic mechanisms governs STING m6A modification and STING may be a potential therapeutic target for vascular calcification.