Microbiota-metabolic effects on the vascular thrombogenicity and inflammation
Final Report Abstract
Cardiovascular diseases (CVD) affect more than 400 million people worldwide and their thromboembolic complications, including myocardial infarction or stroke, are the leading cause of death. Tissue Factor (TF) is the initiator of extrinsic clotting cascade and essential for hemostasis. Physiologically expressed outside the vasculature, TF mediates activation of Factor(F)VII and FXa leading to thrombin accumulation and clot formation in case of injury. However, in the presence of CVD, TF can be induced in the vessel wall, where it leads to a pathological pro-thrombotic state and triggers thrombosis. A central therapeutic limitation is that TF activity is not targetable. Unselective blockage of the TF-pathway impairs hemostasis outside the vasculature and causes severe bleeding complications. Therefore, there is a critical need to identify drivers for pathological vascular TF expression that may serve as therapeutic targets to selectively inhibit vascular TF activity and prevent thrombotic CVD complications without bleeding. Recently, the gut microbiome has been involved in CVD and atherothrombotic complications. In particular, the metaorganismal (involving microbiota and host) metabolite trimethylamine N-oxide (TMAO) has been both associated with major adverse cardiac events (MACE) in large clinical cohorts, and mechanistically linked to heightened in vivo thrombosis potential involving platelet hyper reactivity. The majority of TMAO (from dietary choline precursor) is generated in a 2-step pathway involving gut microbial cutC/D (TMA lyase activity) and host liver enzymes. The exact mechanism how TMAO promotes thrombosis remains unclear. In initial exploratory clinical studies, we observed that among sequential stable CVD subjects on anti-platelet therapy (n=2,989), TMAO levels were associated with an increased incident (3yr) risk for MACE [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 chronic dual anti-platelet therapy during follow-up after acute coronary syndrome (ACS) [adjusted HR(95% CI) 1.70(1.08-2.69)] (n=1,469). These 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 pathophysiological levels of TMAO dose-dependently induced expression of TF. In mouse model studies, both acute and chronic TMAO exposure enhanced aortic TF mRNA and protein expression, which in immunofluorescence studies was shown to co-localize with vascular endothelial cells. In arterial injury mouse models, TMAO-dependent enhancement of thrombogenicity was abrogated by a TF-inhibitory antibody showing that the TF pathway contributes to TMAO’s pro-thrombotic phenotype. In animal models, provision of a non-lethal mechanism-based cutC/D inhibitor that targets gut microbial TMA lyase activity substantially reduced diet-induced TMAO levels in the blood and selectively blocked TMAO-induced endothelial TF expression in the aorta while perivascular TF expression remained unchanged. The studies identified a gut microbial pathway that leads to vascular TF expression in the host and thereby fosters a pro-thrombotic phenotype. TMAO-induced endothelial TF critically mediates heightened thrombosis potential and may explain – at least in part- the strong association of plasma TMAO levels with CVD event risk observed in multiple clinical cohorts. Targeting gut microbial TMA lyase activity serves as a novel approach to control aberrant vascular TF pathway activity in the host without compromising the hemostatic balance.
Publications
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Protease-activated receptor 2 deficiency mediates cardiac fibrosis and diastolic dysfunction. Eur Heart J.;40(40):3318-32.
Friebel J, Weithauser A, Witkowski M, Rauch BH, Savvatis K, Dörner A, et al.
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Gut Microbiota and Cardiovascular Disease. Circ Res. ;127(4):553-70.
Witkowski M, Weeks TL, Hazen SL.
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Vascular miR-181b controls tissue factor-dependent thrombogenicity and inflammation in type 2 diabetes. Cardiovasc Diabetol.19(1):20.
Witkowski M, Witkowski M, Saffarzadeh M, Friebel J, Tabaraie T, Ta Bao L, et al.
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Metformin Is Associated with Reduced Tissue Factor Procoagulant Activity in Patients with Poorly Controlled Diabetes. Cardiovasc Drugs Ther. 35(4):809-13.
Witkowski M, Friebel J, Tabaraie T, Grabitz S, Dörner A, Taghipour L, et al.
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Prognostic value of subclinical myocardial necrosis using high-sensitivity cardiac troponin T in patients with prediabetes. Cardiovasc Diabetol. 20(1):171.
Witkowski M, Wu Y, Hazen SL, Tang WHW.
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Vascular endothelial Tissue Factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis. Cardiovasc Res.
Witkowski M, Witkowski M, Friebel J, Buffa JA, Li XS, Wang Z, et al.
