Proteomic profiling reveals a higher presence of glycolytic enzymes in human atherosclerotic lesions with unfavourable histological characteristics

Aims Molecular characterization of vulnerable atherosclerotic plaques often relies on transcriptomic data. However, RNA expression may not consistently align with protein expression. The proteomic landscape linked to plaque vulnerability is underexplored in human lesions. In this study, we analyzed a large mass spectrometry-based proteomics dataset from the plaque tissue of 320 patients to identify the molecular mechanisms associated with vulnerable plaques. Previous studies have shown significant differences in cell metabolism in murine atherosclerosis models, prompting an in-depth description of expression of key enzymes in glycolysis in human atherosclerotic plaques. Methods and results Atherosclerotic lesions from 320 patients undergoing carotid endarterectomy surgery were collected (200 discovery set and 120 for the validation set) and underwent proteomic analyses. Plaque samples were digested, enriched for extracellular matrix proteins, and processed for untargeted proteomics analysis. The resulting protein levels were linked to pathological plaque characteristics, bulk and single cell transcriptomics, and clinical data. Proteomic analysis of 200 human atherosclerotic carotid lesions detected 1499 proteins with most showing poor correlation with RNA levels. We identified 240 proteins associated with plaque vulnerability index (FDR < 0.05), including key glycolysis enzymes: Hexokinase 3 (HK3) (P = 0.003, FDR = 0.03), PKM (P = 0.008, FDR = 0.05), and LDHA (P = 0.006, FDR = 0.04). The observed associations were mainly driven by macrophage content and fat content, reflected the severity of pre-operative symptoms, exhibited significant sex differences, and correlated with plaque haemorrhage biomarker BLVRB. Validation in 120 patients confirmed HK3 and PKM's association with plaque progression and clinical symptoms (all P < 0.001). Conclusion Enzymes involved in the glycolysis process are more abundant in plaques with vulnerable histological characteristics and are significantly associated with plaque haemorrhage biomarker BLVRB. This implies that plaque destabilisation may be driven by higher glycolysis metabolism, which may contribute to plaque haemorrhage. This association was stronger in women, underlining the important role of energy metabolism in sex-specific mechanisms of atherosclerotic disease.