Abstract Body: Introduction: Neointimal hyperplasia is a major cause for failure of interventions such as balloon angioplasty, stenting, and surgical bypass. Gut microbe-derived metabolites from dietary components may contribute to the host arterial remodeling response after injury. Flavin containing monooxygenase 3 (FMO3) catalyzes the oxidation of trimethylamine (TMA), which is produced from the catabolism of dietary choline by gut microbiota, to trimethylamine-N-oxide (TMAO), which is associated with cardiovascular disease.
Hypothesis: We hypothesize that the TMA/FMO3/TMAO axis regulates endothelial denudation and other cell mechanisms leading to neointimal hyperplasia.
Methods: Fourteen-week-old female transgenic mice overexpressing human FMO3 (hFMO3-Tg+) and sex- and age-matched wild-type (WT) mice were fed a diet supplemented with 1% choline for 3 weeks prior to undergoing left femoral artery wire injury. After 5 days, Evans blue dye was injected intravenously. Bilateral femoral arteries were collected for quantification of endothelial denudation at the site of arterial injury. Plasma was analysed by metabolomics. Cultured human aortic smooth muscle cells (HAoSMC) were treated with 100 µM TMA or 200 µM TMAO. MTT and transwell migration assays were used to assess cell viability and migration. Immunofluorescence for alpha-smooth muscle actin (αSMA) was performed to assess for changes in cell morphology at 3, 5, and 7 days.
Results: Metabolomic analysis demonstrated that plasma TMA was significantly lower (4.3 μM ± 0.5 vs. 360.9 μM ± 49.7; p<0.0001) and TMAO was significantly higher in choline-treated hFMO3-Tg+ (501.4 μM ± 68.92 vs. 368.7 μM ± 14.81; p=0.02) compared to WT mice. Femoral arteries of choline-treated hFMO3-Tg+ mice also had more Evans blue staining (8.6% ± 0.8 vs. 368.7% ± 0.7; p=0.01), indicating more vascular leakage and endothelial denudation. In vitro, HAoSMC treated with TMAO had more viability compared to controls at 24, 48 and 72 hours (p=0.0008) and greater migration (p=0.03) at 6 hours. HAoSMCs treated with TMA and TMAO also exhibited changes in morphology from spindle to hypertrophic appearance with differential expression of αSMA.
Conclusions: The TMA-FMO3-TMAO axis plays an important role in neointimal hyperplasia development by modulating the endothelial and vascular smooth muscle cell responses to injury.