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. 2022 Jul 27;118(10):2367-2384.
doi: 10.1093/cvr/cvab263.

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

Marco Witkowski  1   2 , Mario Witkowski  3 , Julian Friebel  2   4 , Jennifer A Buffa  1 , Xinmin S Li  1 , Zeneng Wang  1 , Naseer Sangwan  1 , Lin Li  1 , Joseph A DiDonato  1 , Caroline Tizian  3 , Arash Haghikia  2 , Daniel Kirchhofer  5 , François Mach  6 , Lorenz Räber  7 , Christian M Matter  8   9 , W H Wilson Tang  1   10 , Ulf Landmesser  2   4 , Thomas F Lüscher  8   11 , Ursula Rauch  2 , Stanley L Hazen  1   10
Affiliations

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

Marco Witkowski et al. Cardiovasc Res. .

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.

Keywords: Cardiovascular disease; Microbiome; Thrombosis; Tissue factor; Trimethylamine N-oxide.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1
Figure 1
The association of TMAO levels with thromboembolic clinical outcomes in patients on anti-platelets in the Cleveland and Swiss ACS Cohorts. Kaplan–Meier estimates and the risk of incident MACE (MI, stroke, or death) over follow-up periods ranked by quartiles of TMAO levels in (A) GeneBank patients with anti-platelet therapy (aspirin or ADP-receptor antagonists) as well as (C) Swiss ACS patients with dual anti-platelet therapy. P-values by log rank test are indicated. Forest plots indicating the risks of (B) incident MACE at 3 years for GeneBank and (D) at 1 year for Swiss ACS subjects stratified by quartiles of TMAO levels (unadjusted in grey), multivariable Cox model for HR included adjustments for traditional risk factors including age, gender, hypertension, smoking, diabetes, HDL, LDL, TG (adjustment 1, in black); and traditional risk factors plus renal function (adjustment 2, in red), as described in Section 2. The 5–95% CI is indicated by line length.
Figure 2
Figure 2
Effects of TMAO exposure on TF and VCAM1 expression in human endothelial cells. HMEC-1 was left untreated or exposed to 200 µM TMAO for 2, 4, and 6 h and mRNA expression for (A) flTF, (B) asTF, and (C) VCAM1 analysed. In addition, HMEC-1 was treated with vehicle or TMAO at different concentrations as indicated for 2 h and mRNA expression of (D) flTF, (E) asTF, and (F) VCAM1 assessed. (G) Protein amounts of flTF, asTF, and VCAM1 in HMEC treated with 200 µM TMAO for 6 h quantified via western blot. Human monocytic THP-1 cells were treated with TMAO or vehicle for 2 h and mRNA expression of (H) flTF and (I) asTF analysed. Results are presented as mean±SEM. Global P-values shown were obtained by non-parametric Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test to compare different treatments. Differences between two groups were assessed using a Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 3
Figure 3
TMAO acutely raises TF and VCAM1 expression in aortic tissue in vivo. C57/BL6 mice were injected with TMAO or vehicle intraperitoneally for 1.5 h. Next, (A) plasma TMAO levels were quantified by LC/MS/MS and arotic mRNA expression of (B) flTF, (C) asTF, and (D) VCAM1 quantified via TaqMan rtPCR. To assess protein expression, the animals were injected with vehicle or TMAO for 6 h. Subsequently, (E) TMAO plasma levels and (F) protein amounts of TF and VCAM1 were quantified via western blot and (G) density of the protein bands quantified. (H) To analyse localization within the vessel wall, aortic tissue of TMAO-injected mice was probed for TF and VCAM1 expression 6 h post injection using immunofluorescence staining (TF red, upper panel, VCAM1 red, lower panel). The tissue was counterstained with DAPI (blue) and an f-actin probe (green). Results are presented as mean±SEM. Pairwise comparison was performed using a Mann–Whitney test.
Figure 4
Figure 4
TMAO chronically induces endothelial TF and VCAM1 expression in mouse aortas in vivo. C57/BL6 mice were either fed a control chow diet or a choline diet. After 10 days of diet, (A) plasma levels of TMAO were quantified and aortic mRNA expression for (B) flTF, (C)asTF, and (D) VCAM1 analysed. (E) Protein amounts of TF and VCAM1 in aortic tissue were measured via western blot and related to the corresponding plasma levels of TMAO. Aortas of LPS-injected mice (15 mg/kg for 6 h) as well as recombinant mouse TF were used as positive controls. (F) Density of the detected bands was quantified using an imaging software. (G) Plasma of the same animals was analysed with respect to TAT complexes via ELISA. LPS-injected animals served as a known positive control for TAT induction (H) Immunohistochemistry experiments using specific antibodies were used to assess protein expression of TF, VCAM1, and the endothelial marker CD31 in aortic tissue. (I) Mean OD of endothelial protein expression from three different anatomical sides was quantified using an imaging software and three data points for each animal were plotted. Results are presented as mean±SEM. Pairwise comparison was performed using a Mann–Whitney test.
Figure 5
Figure 5
A small molecule TMA-lyase inhibitor reverses TMAO-stimulated increase in aortic TF and VCAM1. C57/BL6 mice were put on a choline diet with and without FMC in the drinking water. (A) Aortic tissue was subjected to immunohistochemistry using antibodies against TF, VCAM1, and the endothelial marker CD31. (B) Mean OD was quantified from three different anatomical sides and three data points are plotted for each animal. (B, lower panel) Levels of plasma TMAO, TMA, and choline were quantified via LC/MS/MS and (C) correlated with endothelial TF and VCAM1 expression. Results are presented as mean±SEM. Pairwise comparison was performed using a Mann–Whitney test. Correlation of TMA and TMAO levels with TF or VCAM1 mean endothelial OD was performed using non-parametric Spearman correlation.
Figure 6
Figure 6
A TF-inhibitory antibody prevents the TMAO-associated enhancement in thrombus formation following arterial injury. C57/BL6 mice were put on a chow or choline diet for 10 days. The animals then received either an isotype control antibody (IgG2a) or the TF-inhibitory antibody (1H1) prior to a ferric chloride injury in vivo thrombosis model. (A) Intravital microscopy of rhodamine-labelled platelets during the in vivo thrombosis model was used to monitor thrombus formation in the animals on a chow diet with a control antibody, choline diet with a control antibody, and choline diet with a TF-neutralizing antibody and (B) time to cessation of flow assessed. Carotid thrombi of animals on chow or choline diet that received the control antibody were stained for TF and the platelet marker CD41 via immunohistochemistry. (C) Mean OD for vessel wall-associated thrombus TF and thrombus CD41 of three different sides (three data points for each animal are plotted) quantified by an imaging software. (D) Representative Immunohistochemistry images. Results are presented as mean±SEM. Global P-values shown were obtained by non-parametric Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test to compare occlusion time. Differences between two groups were assessed using a Mann–Whitney test.
Figure 7
Figure 7
The gut microbiota choline TMA-lyase inhibitor FMC shifts the choline diet-induced changes in caecal microbial community associated with vascular TF and VCAM1. (A) Shannon diversity indices distinguishing chow, choline, and choline+FMC samples. Statistical analysis was performed using ANOVA. (B) NMDS based on Bray–Curtis index between the caecal microbiota recovered from mice that were on indicated diets. Statistical analysis was performed using permutational multivariate ANOVA with R2 values for % variance explained by diet being the variable of interest. (C, upper panel) Statistically significant (Benjamini–Hochberg false discovery rate; P < 0.05) genera differentiating three groups (chow, choline, and choline+FMC). Plotted are interquartile ranges (IQRs) (boxes). The dark line in the box is the median, lower whiskers represent smallest observation (≥25% quantile—1.5×IQR), upper whiskers largest observation (≤75% quantile—1.5×IQR) with outliers as dots outside of the box. (C, second panel) Scatter plots based on linear regression showing correlation between abundance of indicated genera with plasma TMAO (μM) levels, (C, third panel) endothelial TF protein and (C, fourth panel) endothelial VCAM1 protein in mouse aortas on the indicated diets, expressed as OD within the annotated endothelial layer quantified by immunofluorescence (as described in Section 2). R2 and P-values are indicated in each panel. For all panels, the same colour scheme was used for data to indicate animal diet: chow (green), choline (purple), and choline+FMC (red). The grey area shows the 95% CI.
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References

    1. Aron-Wisnewsky J, Clément K. The gut microbiome, diet, and links to cardiometabolic and chronic disorders. Nat Rev Nephrol 2016;12:169–181. - PubMed
    1. Kasahara K, Rey FE. The emerging role of gut microbial metabolism on cardiovascular disease. Curr Opin Microbiol 2019;50:64–70. - PubMed
    1. Tang WHW, Bäckhed F, Landmesser U, Hazen SL. Intestinal microbiota in cardiovascular health and disease: JACC state-of-the-art review. J Am Coll Cardiol 2019;73:2089–2105. - PMC - PubMed
    1. Warmbrunn MV, Herrema H, Aron-Wisnewsky J, Soeters MR, Van Raalte DH, Nieuwdorp M. Gut microbiota: a promising target against cardiometabolic diseases. Expert Rev Endocrinol Metab 2020;15:13–27. - PubMed
    1. Witkowski M, Weeks TL, Hazen SL. Gut microbiota and cardiovascular disease. Circ Res 2020;127:553–570. - PMC - PubMed

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