Pollutants in the Atherosclerotic Plaque and Cardiovascular Events

Among patients with carotid stenosis (according to North American Symptomatic Carotid Endarterectomy Trial classification) enlisted to undergo carotid endarterectomy for extracranial high-grade (>70%) internal carotid artery stenosis, the investigators will enroll 300 consecutive patients with asymptomatic disease. All patients will undergo a baseline clinical examination, with computed tomography or MRI to assess cerebral lesions, and health records will be collected. Clinical variables will be measured with standard procedures after overnight fasting. Patients with evidence of heart failure, valvular defects, malignant neoplasms, or secondary causes of hypertension will be excluded.

Specimens of the portion of carotid bifurcation that showed maximum disease will be obtained from atherectomy and will be cut in two halves and frozen in liquid nitrogen or fixed in in 10% buffered formalin for subsequent analyses.

Plaque specimens will be then used to assess the presence and the amount of volatile organic compounds and microplastics through pyrolysis-gas chromatography-mass spectrometry.

To corroborate these findings, plaque specimens will be also observed with transmission electron microscopy to visually identify microplastics within the atheroma and obtain further evidence and structural insights.

Plaques will be also analysed through immunohistochemistry, ELISAs, and Western Blot to quantify inflammatory, i.e. NLRP3, caspase-1, IL-1β, IL-6, TNFα, NF-kB, and CD68, and plaque stability markers, i.e. MMP-9 and collagen, in order to explore whether the presence of microplastics is associated with a poorer plaque phenotype. The groups (composed post-hoc) of patients with polluted plaques vs non-polluted plaques will be compared for the expression of all these markers.

After carotid endarterectomy, patients will be followed-up and monitored for the incidence of a composite outcome constituted by non-fatal myocardial infarction, non-fatal stroke, and all-cause mortality, which represent the main and the only hard endpoint of the study.

Patients will be categorized (post-hoc) as having polluted plaques (having detectable levels of at least one pollutant) or non-polluted plaques. Cox regression analysis will be used to examine the association between the presence of microplastics and the incidence of the composite outcome, adjusted for age, sex, BMI, systolic and diastolic blood pressure, the prevalence of diabetes, dyslipidaemia, and smoking. Among those with polluted plaques, the abundance of pollutants, i.e. the sum, will be used to categorize patients into terciles. Cox models, adjusted for the same variables, will be run to explore the relationship between the burden of microplastics and the outcome. P values < 0.05 will be considered statistically significant.

Inclusion criteria

• patients with carotid stenosis (according to North American Symptomatic Carotid Endarterectomy Trial classification) enlisted to undergo carotid endarterectomy for extracranial high-grade (>70%) internal carotid artery stenosis.

Exclusion criteria

• All patients with clinical or laboratory evidence of heart failure, valvular defects, malignant neoplasms, or secondary causes of hypertension will be excluded from the study.