Abstract Body: Introduction: Vascular calcification is an actively regulated process, featuring osteogenic differentiation and calcification of vascular smooth muscle cells (VSMC). The N6-methyladenosine (m⁶A) mRNA modification, regulating target mRNA stability and translation, has been implicated in cardiovascular remodeling while its function in vascular calcification remains largely unknown.
Hypothesis: We found upregulation of m⁶A modification during VSMC calcification, supporting the hypothesized role of m⁶A in VSMC osteogenic reprogramming and calcification.
Methods: Transcriptomic and epitranscriptomic regulations during VSMC calcification were assessed using RNA sequencing and single nucleotide-resolution m⁶A microarray analyses. Combined fluorescence in situ RNA hybridization and immunofluorescence staining were performed to visualize m⁶A modification and target gene expression. The essential role of m⁶A modification on VSMC calcification was determined using gain- and loss-of-function approaches, using a pharmacologic inhibitor, shRNA, and enforced expression of the methyltransferase (Mettl3). All experiments include at least 3 biological replicates.
Results: Exposure of VSMC to osteogenic media induced a time-dependent increase in total RNA m⁶A levels accompanied by elevated Mettl3, a key regulator for m⁶A modification. Pharmacological inhibition (STM2457) or shRNA-mediated knockdown of Mettl3 markedly attenuated VSMC calcification, whereas Mettl3 overexpression promoted calcification in vitro. Integrated RNA-seq and m⁶A microarray analyses of differentially regulated genes in osteogenic conditions identified Inhibitor of DNA binding 1 (Id1) as a top m⁶A-modified mediator of VSMC calcification. Id1 knockdown reduced VSMC calcification and decreased expression of the master osteogenic marker Runt-related transcription factor 2 (Runx2). In contrast, Id1 overexpression suppressed SMC marker genes and induced Runx2 upregulation and VSMC calcification, which was attenuated by Runx2 deficiency.
Conclusions: In conclusion, our studies have uncovered that RNA m⁶A modification promotes VSMC osteogenic differentiation and calcification through Id1-dependent regulation of Runx2. The elucidated mechanisms provide novel insights into epitranscriptomic regulation of vascular calcification and support the use of m⁶A-modifying signals as potential therapeutic targets.