Ambient air pollution is one of the top ten leading risk factors contributing to the global burden of disease. It increases the risk of cardiovascular diseases such as ischemic heart disease and stroke - the top two leading causes of death worldwide. Atherosclerosis can cause ischemic heart disease and stroke, and is associated with air pollution exposure. However, biological mechanisms underlying air pollution-related atherosclerosis are not fully understood. This proposal addresses whether DNA methylation mediates the association between air pollution and atherosclerosis. DNA methylation is a potential mediator because 1) exposure to particulate matter is associated with alterations in DNA methylation, and 2) alterations in DNA methylation are found in atherosclerotic lesions and are associated with ischemic heart disease and blood pressure. Past studies show that exposure to air pollutants led to genome-wide reduction in DNA methylation or was associated with candidate gene methylation. However, these studies have significant methodological limitations, such as air pollution measurement error and potential exposure misclassification that make their findings uncertain. Another limitation is the inability to study cell-specific DNA methylation due to the use of mixed cell types that combine distinct methylation profiles. It is more relevant to study a single cell type, especially monocytes because they promote chronic inflammation that drives atherosclerosis and may differentiate into macrophages that accumulate in plaques. We propose a novel investigation to test the association between long-term air pollution exposure and DNA methylation (epigenome-wide and of candidate sites) in purified monocytes, and the association between air pollution-associated methylation patterns and subclinical atherosclerosis and gene expression. We will use predictions of ambient fine particulate matter (PM2.5) and oxides of nitrogen (NOX) estimated at participants' homes using sophisticated spatio-temporal models that incorporate both monitoring data and geographical variables. In preliminary studies, we identified 267 differentially methylated sites (DMSs) associated with long-term PM2.5 exposure. Aim 1 will expand our repertoire by identifying DMSs associated with NOX, a measure of traffic-related air pollution, and will additionally identify differentially methylated regions (DMRs) associated with PM2.5 and NOX. Aim 2 will test whether DMSs and DMRs are associated with subclinical atherosclerosis, as measured by carotid intima-media thickness and coronary artery calcium. If so, we will test whether DNA methylation is a potential mediator. Aim 3 will test whether DMSs and DMRs are associated with cis-gene expression profiles. To address these aims, we propose a cross-sectional analysis of 1,207 participants from the Multi-ethnic Study of Atherosclerosis (MESA). This work will be the first to study monocyte-specific methylation patterns, air pollution, subclinical atherosclerosis, and gene expression within the same framework. The results may elucidate biological mechanisms behind air pollution-related atherosclerosis and identify important genes to be targeted for pharmacological and preventative interventions.