Epitranscriptomic profiling of VSMC phenotypes reveals uridine modifications linked to post-transcriptional regulation

Background: Vascular smooth muscle cells (VSMCs) phenotypic plasticity can modulate atherosclerosis progression. Although several gene regulatory steps towards pro-inflammatory phenotypes have been well-studied, epitranscriptomic changes during this transition and their regulatory roles remain unexplored. Methods and Results: Primary human VSMCs were stimulated with TGF-{beta}1 to induce an atheroprotective, contractile, and matrix-producing state and with IL1-{beta} plus PDGF-BB to induce a highly energetic, pro-inflammatory state, confirmed by Illumina bulk RNA sequencing and proteomics. Untargeted screening of mRNA base modifications using Oxford Nanopore Technologies direct RNA sequencing and xPore analysis revealed enhanced uridine modification within a GUUUU motif in pro-inflammatory VSMCs. Modified uridines were enriched in 3'-UTR and accessible RNA structures, with implications on Poly(A) tail dynamics and miRNA binding. Conclusions: Atheroprotective and pro-atherogenic treatments induce distinct epitranscriptomic landscapes composed of different modification types, often co-localized in the same transcript. Modified uridines in mRNAs are abundant in a high-energy, pro-inflammatory VSMC state and associated with post-transcriptional regulation. In summary, epitranscriptomics adds a novel regulatory layer to VSMC phenotypic transitions critical for atherosclerosis development and progression.