Background/Objectives: Vascular smooth muscle cells (VSMCs) reside in the medial layer of the arterial wall and are involved in the regulation of vascular tone and remodeling. However, VSMCs possess phenotypic plasticity, which plays a role in both subclinical and advanced atherosclerosis. We investigated the transition from atheroprotective to atherogenic SMC phenotypes and the impact of disease-relevant stimuli on gene regulation.
Methods: Primary human SMCs were cultured on a collagen I hydrogel and stimulated with TGF-β1 and a mixture of IL1-β and PDGF-BB. These pro-inflammatory SMCs were further stimulated with PDGF-BB to induce inflammation and oxidative stress, which was confirmed using CellRox™ assays. Transcriptome profiling of phenotypic transitions was performed using Illumina RNA sequencing, and epitranscriptomic profiling was conducted using Oxford Nanopore Technologies direct RNA sequencing. Potential functional post-transcriptional regulatory effects of RNA modifications were assessed by small RNA sequencing and proteomics.
Results: Transcriptome profiling confirmed distinct atheroprotective and atherogenic SMC states, characterized by elevated expression of genes involved in extracellular matrix organization and pro-inflammatory pathways, respectively. One distinct atherogenic SMC phenotype was susceptible to oxidative stress upon treatment with a high dose of PDGF-BB after pre-treatment with TGF-β1 and the IL1-β/PDGF-BB mixture. Although we initially hypothesized that enhanced production of reactive oxygen species might also oxidize mRNAs, we could not detect 8-oxoguanine on RNA molecules using direct RNA sequencing. Instead, we found significant differential RNA modifications, predominantly between TGF-β1 and PDGF-BB/IL1-β treatments. These modifications mainly affected uracil, typically within a GUUUU motif, located mostly in the 3’-UTR and significantly enriched in accessible RNA structure elements. In addition, we propose that RNA base modifications can affect poly(A) tail lengths and miRNA binding. Transcripts with differentially modified RNA bases were enriched for TCF21 regulation, a key transcription factor that promotes SMC transitions in atherosclerosis.
Conclusion: We have identified pro-inflammatory SMC phenotypes that are susceptible to oxidative stress, which could be detrimental in the late stages of human atherosclerotic plaque development. Furthermore, we identified RNA base modifications that may play an important role in post-transcriptional gene regulation.
