Abstract 4141953: Genetic deficiency of flavin containing monooxygenase 3 ( <i>Fmo3</i> ) lowers circulating trimethylamine N-oxide level and protects against atherosclerosis

Introduction: Our groups have previously identified trimethylamine N-oxide (TMAO) as a risk factor for cardiovascular diseases (CVD). In humans, elevated plasma TMAO levels are associated with a ~2.5-fold increased risk for major adverse cardiac events, such as death, myocardial infarction, and stroke. Bacterial metabolism of dietary choline (and L-carnitine) in the intestine leads to an intermediate, trimethylamine (TMA), which is absorbed from the gut and oxidized primarily by the hepatic flavin-containing monooxygenase 3 (FMO3) to generate TMAO. Hypothesis: We hypothesize that decreased TMAO generation caused by FMO3 deficiency in mice leads to decreased circulating TMAO levels and atherosclerosis. Approach and Results: We generated FMO3 knockout (KO) mouse on a C57BL/6J background via CRISPR/Cas9 technology. We then crossed the FMO3KO mice with LDL receptor knockout (LDLRKO) mice to generate FMO3 heterozygous (FMO3Het)/LDLRKO mice and FMO3KO/LDLRKO mice for the study. Female FMO3Het/LDLRKO and FMO3KO/LDLRKO mice were fed a 1% choline, a TMAO precursor, and 0.5% cholesterol diet for 3 months to increase circulating TMAO and total cholesterol levels. After diet feeding, FMO3KO/LDLRKO mice had 51% lower TMAO levels compared to FMO3Het/LDLRKO mice (18.3 μM vs. 38.3 μM, p &lt; 0.0001), but no effects on plasma lipids, glucose, or insulin levels. Most notably, atherosclerotic lesion at the aortic root was decreased by 24% (p &lt; 0.01) in the FMO3KO/LDLRKO mice as compared to the FMO3Het/LDLRKO mice. The mRNA levels of inflammatory genes, such as Cox2 and E-selectin, in the aorta were significantly decreased by more than 50% in FMO3KO/LDLRKO mice when compared to the control mice. Western blot analysis revealed decreased NF-κB activation in the aorta samples of the FMO3KO/LDLRKO mice as compared to those of the controls, suggesting reduced inflammation in the former. We also measured circulating IL-6 and TNF-α levels but did not observe any differences in plasma levels of these inflammatory biomarkers. Conclusions: Taken together, our data demonstrate that Fmo3 deficiency reduces TMAO levels and aortic lesion development in the context of an appropriate pro-atherogenic diet that contains also high levels of a TMAO precursor. These results also suggest that one mechanism for the pro-atherogenic effects of TMAO is through localized upregulation of inflammatory pathways at the level of the vessel wall, which do not necessarily manifest systemically.