Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

Abstract

Aims Gut microbiota and their generated metabolites impact the host vascular phenotype. The metaorganismal metabolite trimethylamine N-oxide (TMAO) is both associated with adverse clinical thromboembolic events, and enhances platelet responsiveness in subjects. The impact of TMAO on vascular tissue factor (TF) in vivo is unknown. Here, we explore whether TMAO-enhanced thrombosis potential extends beyond TMAO effects on platelets, and is linked to TF. We also further explore the links between gut microbiota and vascular endothelial TF expression in vivo. Methods and Results In initial exploratory clinical studies, we observed that among sequential stable subjects (n = 2,989) on anti-platelet therapy undergoing elective diagnostic cardiovascular evaluation at a single-site referral center, TMAO levels were associated with an increased incident (3 yr) risk for major adverse cardiovascular events (MACE, myocardial infarction, stroke or death) [4th quartile(Q4) versus Q1 adjusted hazard ratio(95% confidence interval) HR(95%CI), 1.73(1.25-2.38)]. Similar results were observed within subjects on aspirin mono-therapy during follow-up [adjusted HR(95%CI) 1.75(1.25-2.44), n = 2,793). Leveraging access to a second higher risk cohort with previously reported TMAO data and monitoring of anti-platelet medication use, we also observed a strong association between TMAO and incident (1 yr) MACE risk in the multi-site Swiss Acute Coronary Syndromes (ACS) Cohort, focusing on the subset (n = 1,469) on chronic dual anti-platelet therapy during follow-up [adjusted HR(95% CI) 1.70(1.08-2.69)]. These collective clinical data suggest that the thrombosis-associated effects of TMAO may be mediated by cells/factors that are not inhibited by anti-platelet therapy. To test this, we first observed in human microvascular endothelial cells that TMAO dose-dependently induced expression of TF and vascular cell adhesion molecule (VCAM)1. In mouse studies, we observed that TMAO enhanced aortic TF and VCAM1 mRNA and protein expression, which upon immunolocalization studies, was shown to co-localize with vascular endothelial cells. Finally, in arterial injury mouse models, TMAO-dependent enhancement of in vivo TF expression and thrombogenicity were abrogated by either a TF-inhibitory antibody or a mechanism-based microbial choline TMA lyase inhibitor (fluoromethylcholine, FMC). Conclusions Endothelial TF contributes to TMAO-related arterial thrombosis potential, and can be specifically blocked by targeted non-lethal inhibition of gut microbial choline TMA lyase. Translational Perspective The pro-thrombotic effects of the gut microbial TMAO pathway are shown to extend beyond enhancement of platelet responsiveness and include heightened vascular Tissue Factor(TF). In clinical studies, TMAO is shown to predict event risk in patients in the presence of anti-platelet drugs. In animal studies, TMAO elevation is shown to promote vascular endothelial TF expression and a TF-dependent pro-thrombotic effect. Pharmacological targeting of gut microbial choline TMA lyase reduced host TMAO, vascular TF and abrogated the pro-thrombotic TMAO-associated phenotype. These studies suggest inhibiting the TMAO pathway may be a rational target for reducing residual risk in patients on antiplatelet therapy.

Pleiotropic Effects of the Protease-Activated Receptor 1 (PAR1) Inhibitor, Vorapaxar, on Atherosclerosis and Vascular Inflammation

“…Direct PAR1 inhibition via provision of vorapaxar reduced the expression of VCAM-1 and E-selectin ( Figure 5 A,B) in aortic specimens from ApoEko mice (relative mean fluorescence intensity of VCAM-1 in aortic plaques ± SD control vs. vorapaxar; n = 6: 1.0 ± 0.2 vs. 0.14 ± 0.08, p < 0.0001). Tissue factor contributes critically to in vivo atherothrombosis initiation and a procoagulant state [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. In our model, treatment with vorapaxar also affected the aortic expression of TF ( Figure 5 A).…”

Immune Mechanisms of Plaque Instability

“…Notably, in two clinical studies using optical coherence tomography to assess coronary plaque characteristics, increased plasma trimethylamine-N-oxide levels were linked to increased incidence of plaque rupture supporting a critical role of TMAO in vascular inflammation ( 227 , 242 ). Beyond the pathomechanistic link between TMAO and atherosclerotic plaque development, TMAO has also been reported to promote atherothrombosis by increased platelet activation ( 243 ) and tissue factor expression in endothelial cells ( 244 ). In recent studies, microbial enzymes that process the generation of TMA have been targeted using small molecule inhibitors that can modulate TMA/TMAO levels and detrimental cardiovascular effects in experimental settings ( 245 ).…”

Targeting Trimethylamine N-Oxide: A New Therapeutic Strategy for Alleviating Atherosclerosis

“…Recent investigations have demonstrated that TMAO enhances platelet reactivity by promoting ERK1/2 and Jun N-terminal kinase (JNK) phosphorylation ( 49 ). In vivo and in vitro experiments have revealed that TMAO increases thrombotic potential by strengthening aortic vascular tissue factor (TF) and vascular cell adhesion molecule (VCAM)1 expression ( 50 ).…”

“…Existing reports show that IMC can suppress choline diet-enhanced platelet aggregation and the in vivo rate of thrombus formation without increased bleeding time ( 16 ), and it can also alter host cholesterol and bile acid metabolism in preclinical animal models ( 82 ). In addition, a TF-inhibitory antibody or FMC can abrogate TMAO-dependent enhancements of in vivo TF expression and thrombogenicity ( 50 ). Reportedly, Meldonium lowers the production of TMA by inhibiting the L-carnitine metabolism intermediate γ-butyl betaine ( 83 ).…”