Vulnerable Carotid Artery Plaques Exhibit Proteolysis, Metabolic Reprogramming and Inflammation

Carotid plaque vulnerability is a current feature that aids in the decision making for ischemic stroke risk prevention. Proteomic analysis of plaque tissue can reveal molecular indicators of instability that complement imaging findings. We sought to identify a proteomic signature distinguishing vulnerable from stable carotid plaques in patients undergoing endarterectomy, with the aim of uncovering candidate biomarkers for potential diagnostic and therapeutic targets. 28 carotid plaque specimens were collected from 27 patients (including one patient with bilateral endarterectomy). Samples were classified as vulnerable (n=14) or non-vulnerable (n=14) based on pre-operative magnetic resonance angiography (MRA) with vessel wall imaging (VWI). Tandem mass tag-based multiplexing strategy followed by mass spectrometry analysis was used to profile the proteomes of all samples. Normalized and log2-transformed protein intensities were compared using two-sample t-tests with unequal variances, and P-values were adjusted for multiple testing with the Benjamini–Hochberg method to obtain false discovery rate q-values. Proteins with q≤0.25 were designated high-confidence candidates, and those with p<0.05 but q>0.25 were considered exploratory. From 3267 proteins identified, 398 reached nominal significance (p<0.05). From those, 29 reached at least log₂ fold change of 1, and fifteen were significant for q≤0.25. All were up-regulated in vulnerable lesions and these included: matrix-degrading enzymes (MMP7, MMP9, MMP1, ADAMDEC1); neutrophil-derived effectors (AZU1, CAMP, LTF, MPO); inflammatory regulators (IL1RN, IL4I1); glycolytic enzymes (HK3, HK2); and lipid-handling proteins (PLA2G7, APOB, PON1). An additional 17 exploratory proteins showed nominal significance (p<0.05, q>0.25) with at least log₂ fold change of 1, and 366 proteins with nominal significance but with a log₂ fold change less than 1 are reported. Our proteomic profiling delineates a robust vulnerability signature marked by enhanced proteolysis, neutrophil activation, inflammatory signaling, metabolic reprogramming, and lipid dysregulation. The high-confidence proteins offer promising tissue biomarkers for identifying unstable carotid plaques, while exploratory candidates warrant further validation.