Marco Witkowski1,2, Mario Witkowski3, Julian Friebel2,4, Jennifer A Buffa1, Xinmin S Li1, Zeneng Wang1, Naseer Sangwan1, Lin Li1, Joseph A DiDonato1, Caroline Tizian3, Arash Haghikia2, Daniel Kirchhofer5, François Mach6, Lorenz Räber7, Christian M Matter8,9, W H Wilson Tang1,10, Ulf Landmesser2,4, Thomas F Lüscher8,11, Ursula Rauch2, Stanley L Hazen1,10. 1. Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, 9500 Euclid Ave, Cleveland, OH 44195, USA. 2. Department of Cardiology, Charité Centrum 11, Charité-Universitätsmedizin, Hindenburgdamm 30, 12203, Berlin, Germany. 3. Department of Microbiology, Infectious Diseases and Immunology, Laboratory of Innate Immunity, Charité-Universitätsmedizin Berlin, Berlin, Germany. 4. Berlin Institute of Health, Anna-Louisa-Karsch-Straße 2, 10178, Berlin, Germany. 5. Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. 6. Department of Cardiology, University Hospital Geneva, Rue Gabrielle-Perret-Gentil 4 1205, Geneva, Switzerland. 7. Department of Cardiology, Inselspital Bern, Freiburgstrasse 18 CH-3010, Bern, Switzerland. 8. Center for Molecular Cardiology, University of Zurich, Wagistrasse 12, CH-8952 Schlieren, Switzerland. 9. Department of Cardiology, University Heart Center, University Hospital Zurich, Raemistrasse 100 8091, Zurich, Switzerland. 10. Department of Cardiovascular Medicine, Heart, Vascular & Thoracic Institute, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH, USA. 11. Department of Cardiology, Royal Brompton and Harefield Hospitals, Imperial College, Sydney St, London SW3 6NP, UK.
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 = 2989) on anti-platelet therapy undergoing elective diagnostic cardiovascular evaluation at a single-site referral centre, TMAO levels were associated with an increased incident (3 years) risk for major adverse cardiovascular events (MACE) (myocardial infarction, stroke, or death) [4th quartile (Q4) vs. Q1 adjusted hazard ratio (HR) 95% confidence interval (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 = 2793]. 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 year) MACE risk in the multi-site Swiss Acute Coronary Syndromes Cohort, focusing on the subset (n = 1469) 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). CONCLUSION: 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. Published on behalf of the European Society of Cardiology. All rights reserved.
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 = 2989) on anti-platelet therapy undergoing elective diagnostic cardiovascular evaluation at a single-site referral centre, TMAO levels were associated with an increased incident (3 years) risk for major adverse cardiovascular events (MACE) (myocardial infarction, stroke, or death) [4th quartile (Q4) vs. Q1 adjusted hazard ratio (HR) 95% confidence interval (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 = 2793]. 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 year) MACE risk in the multi-site Swiss Acute Coronary Syndromes Cohort, focusing on the subset (n = 1469) 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). CONCLUSION: 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. Published on behalf of the European Society of Cardiology. All rights reserved.
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