Circulating cell-free RNA signatures associate with plaque calcification in advanced carotid atherosclerosis

Abstract Vascular calcification (VC) is a hallmark of advanced atherosclerotic plaque biology. Circulating cell-free RNA (cfRNA) provides a non-invasive window into tissue transcriptional activity and may reflect plaque composition. We examined whether plasma cfRNA profiles are associated with carotid plaque calcification. Plasma cfRNA was profiled by RNA sequencing in 333 patients undergoing carotid endarterectomy, split into discovery ( n = 216) and internal validation ( n = 117) cohorts. Plaque calcification was quantified histologically. Differential expression analysis was performed using generalized linear models, followed by pathway enrichment. A cfRNA-derived gene score was constructed, and its incremental value beyond clinical risk factors was evaluated. Cellular deconvolution was applied to explore potential cfRNA origins. In this exploratory analysis, 13 genes showed nominal association with calcification in the discovery cohort ( p < 0.001). Of these, 11 (84.6%) demonstrated concordant directionality in validation (Spearman ρ = 0.71, p = 0.008). Pathway analysis suggested potential involvement of oxidative phosphorylation and calcium signaling. The 11-gene score remained associated with calcification after adjustment for clinical risk factors and improved model performance. Plasma cfRNA was associated with calcification in carotid atherosclerotic plaques and may reflect aspects of underlying plaque biology. These findings are exploratory and require validation in larger, independent populations.