Identification of biomarkers of plaque vulnerability in carotid artery disease for the prevention of brain ischemic injury.

Carotid atherosclerosis is a leading cause of cerebrovascular events, yet risk stratification, in severe asymptomatic stenosis, remains limited. Despite advances in imaging and clinical scoring, prediction of prone to rupture plaques remains challenging. This study aimed to identify potential biomarkers of plaque vulnerability by exploring both systemic and local molecular pathways involved in inflammation, oxidative stress, structural remodelling, and neuro-immune interactions. The integration of multiple markers provided a more comprehensive understanding of plaque biology and identified targets for future diagnostic and therapeutic strategies. Tissue and peripheral blood mononuclear cells (PBMCs) were obtained from 68 male patients with symptomatic carotid stenosis (n=22) or severe asymptomatic stenosis (n=46) enrolled in the Genoa Vascular Biobank. Biomarkers were analyzed, carefully selected based on previous evidence of involvement in atherosclerosis, grouped as: immuno-inflammatory (CD163), oxidative and stress-response (HO-1, AhR, BMAL1), structural and remodelling (CD147, Fibronectin-EDB), and neuro-immune (Neurofilament 200, Tyrosine Hydroxylase). Gene expression in PBMCs and plaques were analized, and immunohistochemistry evaluated focusing on plaque shoulders prone to instability. Statistical comparisons used the Mann-Whitney test, correlations were assessed by Spearman’s rank. Symptomatic patients exhibited a higher CD163 and AhR expression in PBMCs, correlates with HO-1 and BMAL1, indicating systemic activation of inflammatory and antioxidant pathways. These findings suggest that circulating immune cells reflect the ongoing vascular stress. Gene expression in plaques showed AhR strongly correlated with CD147 and Fibronectin-EDB, particularly in asymptomatic patients, suggesting local remodelling and early destabilization. AhR immunopositivity was higher in symptomatic lesions (whole lesion p=0.064), mainly localized in neovasa and inflammatory infiltrates of the media and plaque shoulder, the most active and vulnerable sites of carotid stenosis. CD147 immunopositivity inversely associated with circulating stress markers in symptomatic patients. Preliminary results of immunohistochemistry with neuro, immune and cardiovascular biomarkers revealed localization of CD3⁺ T cells, CD163⁺ macrophages, and nerve fibers expressing NF200 and Tyrosine Hydroxylase in AhR-rich regions of the plaque shoulder, indicating a complex neuro-immune interface contributing to local inflammation and tissue remodelling. Taken together these results show that systemic CD163 and AhR, linked to BMAL-1 and HO-1, associate with plaque vulnerability, while AhR, CD147, Fn-EDB correlations, especially in asymptomatic patients, suggest early vascular remodeling. Circulating AhR, CD163, and HO-1 inversely correlate with local CD147, highlighting biomarker potential. AhR expression by neuronal and immune cells supports a neuro-immune interface in plaque inflammation. Overall, these findings identify a stress-related molecular axis as a biomarker panel for vulnerable plaques. Larger multicentric studies including women and healthy controls are needed to validate these results.