Abstract Body (Do not enter title and authors here): 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 < 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 < 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.