Introduction: Atherosclerotic cardiovascular disease remains the leading cause of death worldwide, underscoring the urgent need to identify markers that prospectively associate with disease progression. Here, we explore the molecular mechanisms of the RNA-binding protein HuR in subclinical atherosclerosis and its clinical relevance as a potential biomarker for early atherosclerotic progression.
Methods: To investigate the mechanistic role of endothelial HuR, we conducted silencing and overexpression experiments, RNA stability assays, and pharmacological inhibition. The HuR–mRNA interactome was delineated using iCLIP-seq, RIP-seq, and bulk RNA sequencing. To assess the clinical relevance of HuR expression, we analyzed single-cell RNA sequencing data and HuR levels in peripheral blood mononuclear cells from 977 individuals with and without atherosclerotic cardiovascular disease. Carotid and femoral artery ultrasound was performed to evaluate intima-media thickness, maximum wall thickness, plaque burden, and disease progression, with participants prospectively followed for major adverse cardiovascular events (MACE).
Results: Mechanistic studies employing iCLIP-seq, RIP-seq, and transcriptomics in endothelial cells under basal and TNF-α–stimulated conditions identified multiple HuR binding sites within AUUUA- and UUUU(G/U)-rich 3′UTRs of pro-inflammatory and pro-thrombotic transcripts involved in adhesion, TGF-β signaling, migration, and differentiation. Pharmacological inhibition or siRNA-mediated knockdown of HuR suppressed endothelial inflammatory gene expression, whereas HuR overexpression induced a pro-inflammatory endothelial phenotype through stabilization of its mRNA targets. Expression levels of HuR-regulated transcripts strongly correlated with HuR abundance in vascular disease tissues. Single-cell analysis revealed increased HuR expression in infiltrating immune cells from Ldlr⁻/⁻ mice fed a high-fat diet compared to chow, as well as in human carotid plaques. In patients, elevated HuR expression was independently associated with coronary artery disease (OR = 2.67, highest vs. lower tertiles), elevated C-reactive protein, and greater mean carotid intima-media thickness, after adjusting for traditional risk factors (P < 0.05). Among healthy individuals, baseline HuR levels predicted accelerated progression of subclinical atherosclerosis, evidenced by increased carotid and femoral plaque counts (P < 0.001) and greater maximum wall thickness progression (P for interaction = 0.045). High HuR expression also correlated with increased incidence of major adverse cardiovascular events over a median follow-up of 48 months (log-rank P = 0.009).
Conclusion: HuR inhibition represents a promising therapeutic strategy for early intervention in subclinical atherosclerosis.
