Abstract Body: Introduction: Carotid Artery Stenosis (CAS) is a cause of ischemic stroke. CAS management is based on the presence of symptoms (stroke) and the degree of stenosis. Asymptomatic CAS patients are at risk for an unpredictable ischemic stroke event, and we need to better delineate risk within this heterogeneous group.

Hypothesis: We hypothesize that epigenetic and proteomic profiling can inform aging clocks and may uncover high-risk asymptomatic CAS patients who warrant more aggressive management.

Methods: Symptomatic CAS (27), asymptomatic CAS (146), subclinical (37; no CAS, minor carotid plaque), and healthy control patients (28; varicose vein-related clinic visits) were retrospectively enrolled with institutional research ethics board approval. DNA methylation was profiled in peripheral blood mononuclear cells (PBMCs) (Infinium, Illumina) and used to calculate epigenetic age (DunedinPACE: pace of aging; IntrinClock: accounts for cell-type heterogeneity of PBMCs). Differentially methylated regions (DMRs) were mapped to their corresponding genes. Deep plasma proteome profiling was performed (Proteograph XT Assay, SEER), with analysis of differentially enriched proteins (DEPs). Pathway enrichment analyses were then performed for DMRs and DEPs.

Results: DunedinPACE was higher in CAS patients compared to healthy controls (P < 0.05). Epigenetic age was greater than chronological age in symptomatic CAS patients (P < 0.05). Pathway analysis of symptomatic vs asymptomatic CAS DMRs revealed enrichment of cellular senescence, MAPK, mTOR, notch and Ras signalling pathways (q < 0.05). Pathway enrichment of symptomatic vs asymptomatic CAS DEPs revealed immune response pathways (q < 0.05). To interrogate CAS at an earlier disease stage, DEP analysis was performed in non-severe CAS (< 70% stenosis) patients. K-means clustering of non-severe CAS patients using DEPs from symptomatic non-severe CAS patients revealed a “high-risk” asymptomatic population that clustered closely with symptomatic CAS patients (cluster 1), separate from entirely asymptomatic patients (cluster 2) that may be “low-risk”. Pathway analysis of cluster 1 vs 2 DEPs revealed enrichment of iron-ion homeostasis, complement activation, cell-substrate adhesion, endothelial cell migration and angiogenesis pathways (q < 0.05).

Conclusion: Epigenetics and proteomics can identify high-risk CAS patients. Future work aims to use these insights to develop a multimodal tool to risk-stratify asymptomatic CAS patients.