PROJECT SUMMARY Cardiovascular disease (CVD) remains the leading cause of death in the US but traditional risk factors, such as elevated lipid levels and hypertension, account for less than 50% of the risk for CVD. We have recently identified a novel mechanism for atherosclerosis where trimethylamine N-oxide (TMAO), a metabolite derived from gut microbiome and hepatic-mediated metabolism of dietary choline and L-carnitine, increases aortic lesion formation in mice and is associated with elevated risk of CVD in humans. Our data further indicate that TMAO levels are regulated through complex interactions between dietary substrates and host genetic factors in the liver and other organ systems. However, the biological pathways that regulate TMAO at the level of hepatic production and whether these factors interact with dietary choline or L-carnitine to affect atherosclerosis are not known. Furthermore, many questions remain unanswered with respect to the biological mechanisms by which TMAO promotes atherogenesis and whether the association between TMAO and CVD in humans represents a causal relationship. The integrative strategies proposed herein directly address these critical gaps in knowledge. In Specific Aim 1, we will determine the biological mechanisms underlying the pro- atherogenic properties of TMAO using a genetically modified mouse model that we recently created for deficiency of flavin-containing monooxygenase 3 (Fmo3), the major enzyme responsible for hepatic TMAO production. Fmo3 null mice will be comprehensively characterized for aortic lesion development in the context of a atherogenic high choline diet. We will also use pharmacological and genetic perturbations strategies in mouse models to determine in vivo whether the inflammatory processes TMAO promotes at the level of the vessel wall are mediated through the NF-B pathway. In Specific Aim 2, we will use comparative systems genetics strategies with >41,000 subjects and a panel of ~100 inbred mouse strains to identify genetic factors influencing plasma TMAO levels through main effects and/or gene-dietary interactions. The results of these synteny mapping studies will be used for in silico and Mendelian randomization analyses in >500,000 subjects to establish a causal relationship between TMAO and risk of CVD. In combination, the proposed studies have the potential to 1) elucidate the inflammatory mechanisms through which TMAO promotes atherosclerosis; 2) identify the genetic determinants of a novel and clinically important risk factor for CVD as well as provide a better understanding of how interactions between genes and dietary factors mediate changes in plasma TMAO levels and CVD risk; and 3) provide genetic evidence that the relationship between TMAO and CVD is causal. Taken together, results from our studies would support the notion that targeting TMAO is a novel therapeutic strategy that may decrease CVD risk independent of known biological pathways and risk factors.