International Journal of Molecular Sciences Review Gut Microbiota-Derived TMAO: A Causal Factor Promoting Atherosclerotic Cardiovascular Disease? Marina Canyelles 1,2 , Carla Borràs 1,2,3 , Noemí Rotllan 1,2 , Mireia Tondo 2,4, * , Joan Carles Escolà-Gil 1,2, *,† and Francisco Blanco-Vaca 2,3,4,† 1 Institut de Recerca de l’Hospital Santa Creu i Sant Pau, Institut d’Investigacions Biomèdiques IIB Sant Pau, 08041 Barcelona, Spain 2 CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 28029 Madrid, Spain 3 Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08041 Barcelona, Spain 4 Department of Clinical Biochemistry, Hospital de la Santa Creu i Sant Pau, IIB Sant Pau, 08041 Barcelona, Spain * Correspondence: mtondo@santpau.cat (M.T.); jescola@santpau.cat (J.C.E.-G.) † These authors contributed equally to this work. Abstract: Trimethylamine-N-oxide (TMAO) is the main diet-induced metabolite produced by the gut microbiota, and it is mainly eliminated through renal excretion. TMAO has been correlated with an increased risk of atherosclerotic cardiovascular disease (ASCVD) and related complications, such as cardiovascular mortality or major adverse cardiovascular events (MACE). Meta-analyses have postulated that high circulating TMAO levels are associated with an increased risk of cardiovascular events and all-cause mortality, but the link between TMAO and CVD remains not fully consistent. The results of prospective studies vary depending on the target population and the outcome studied, and the adjustment for renal function tends to decrease or reverse the significant association between TMAO and the outcome studied, strongly suggesting that the association is substantially mediated by renal function. Importantly, one Mendelian randomization study did not find a significant association between genetically predicted higher TMAO levels and cardiometabolic disease, but another found Citation: Canyelles, M.; Borràs, C.; a positive causal relationship between TMAO levels and systolic blood pressure, which—at least Rotllan, N.; Tondo, M.; Escolà-Gil, in part—could explain the link with renal function. The mechanisms by which TMAO can increase J.C.; Blanco-Vaca, F. Gut this risk are not clearly elucidated, but current evidence indicates that TMAO induces cholesterol Microbiota-Derived TMAO: A Causal metabolism alterations, inflammation, endothelial dysfunction, and platelet activation. Overall, there Factor Promoting Atherosclerotic is no fully conclusive evidence that TMAO is a causal factor of ASCVD, and, especially, whether Cardiovascular Disease? Int. J. Mol. TMAO induces or just is a marker of hypertension and renal dysfunction requires further study. Sci. 2023, 24, 1940. https://doi.org/ 10.3390/ijms24031940 Keywords: atherosclerosis; TMAO; mortality; CVD; renal function; prospective Academic Editor: Ida Daniela Perrotta Received: 22 December 2022 Revised: 13 January 2023 1. Introduction Accepted: 16 January 2023 Atherosclerosis is a condition characterized by plaque build-up within the arteries Published: 18 January 2023 and the thickening of the arterial walls. It is widely regarded as the leading cause of cardiovascular disease (CVD), which is currently the main cause of death in the world [1]. In recent years, the concept of pathological variation in the gut microbiota as a cause of several cardiometabolic diseases has emerged due to studies performed in both rodents and Copyright: © 2023 by the authors. humans. Trimethylamine-N-oxide (TMAO), the main diet-induced metabolite produced by Licensee MDPI, Basel, Switzerland. the gut microbiota, has been correlated with an increased risk of atherosclerosis, thrombosis, This article is an open access article stroke, heart disease, type 2 diabetes, and obesity [2]. distributed under the terms and TMAO is the product of trimethylamine (TMA) oxidation in the liver. TMA can conditions of the Creative Commons Attribution (CC BY) license (https:// be generated by the action of the gut microbiota using dietary precursors with betaine, creativecommons.org/licenses/by/ L-carnitine, or choline as the main sources [3]. The first publication linking TMAO and 4.0/). CVD was an untargeted metabolomic study published in 2011. In this study, following Int. J. Mol. Sci. 2023, 24, 1940. https://doi.org/10.3390/ijms24031940 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 1940 2 of 17 the adjustment for traditional cardiac risk factors and medication usage, three gut-related metabolites (TMAO, choline, and betaine) were significantly correlated with CVD [4]. Since then, the number of studies aiming to link TMAO with CVD has increased exponentially. A recent systematic review and meta-analysis of prospective studies concluded that elevated TMAO concentrations were associated with a 62% increased risk of all-cause mortality [5] as well as a 23% higher risk of cardiovascular events and a 55% higher risk of all-cause mortality [6]. However, despite these assumptions, the link between TMAO and CVD remained limited and not all the studies found a significant association [6]. Indeed, the mechanisms by which TMAO can cause an increased risk of CVD are not clearly elucidated and encompass a diverse range of pathological processes. The studied mechanisms range from cholesterol metabolism disruption, affecting atherosclerotic plaque formation, to platelet activation, leading to enhanced thrombosis activity, inflammation, and endothelial dysfunction [4,7,8]. In the present narrative review, we aimed to describe and update the causal relation- ship between TMAO and atherosclerotic cardiovascular disease (ASCVD), analyzing the results of three different approaches: prospective, genetic, and mechanistic studies. A literature review was performed looking for relevant published studies available on the 15 November 2022 using three electronic databases: PubMed, Google Scholar, and Web of Science. For selecting the prospective studies, we used the following combination of terms: “Cardiovascular Disease”, “Mortality”, “Cardiovascular Mortality”, or “MACE” with “TMAO” or “Trimethylamine-N-oxide” and “Prospective” or “Genetic.” The literature review was performed based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement and the process was agreed upon by all authors. 2. Trimethylamine-N-Oxide (TMAO) 2.1. Microbial Pathways Leading to TMA Production in the Gut Various gut microorganisms can form TMA from dietary precursors. In the gut micro- biota, several families of bacteria, such as Firmicutes, Proteobacteria, and Actinobacteria phyla, isolated from commensal bacteria in the human intestine, have been identified as choline and carnitine consumers and, therefore, potential TMA producers. Bacteroidetes, however, appear to be unable to produce TMA [9]. The choline utilization gene cluster, which is responsible for anaerobic choline degradation, has been shown to contain a gene coding for the specific glycyl radical enzyme choline TMA-lyase and its corresponding radical S-adenosyl-L-methionine activating protein, which uses choline as a substrate [10]. Another microbial metabolic pathway via which TMA is generated is a two-component oxygenase/reductase system, which uses carnitine as a substrate [11] Lastly, another bac- terial lyase is responsible for TMA production, using choline, betaine, L-carnitine, and γ-butyrobetaine (γBB) as a substrate [12]. Inter-individual variability in the composition of the gut microbiota can be influenced by dietary patterns. A greater increase in post-prandial plasma TMAO was observed following consuming both eggs and beef compared to fruit, which was used as a control. High TMAO producers had a higher number of Firmicutes than Bacteroidetes and a less diverse gut microbiome. More specifically, Clostridiaceae, Lachnospiraceae and Veillonellaceae, Clostridiales of the Firmicutes phylum were the most abundant genre. Conversely, low TMAO producers had a greater number of Bacteroidales of the Bacteroidetes phylum, of which Bacteroidaceae and Prevotellaceae were predominant [13]. 2.2. Dietary Precursors of TMA and TMAO TMAO can be ingested directly from food. It is found in fish and marine invertebrates such as mussels or other seafood. Concentrations in fresh seafood can vary considerably between species, habitat, depth, and season. Compared to other dietary sources, fish has the biggest impact on TMAO concentrations. The consumption of fish yielded 50 times higher postprandial circulating TMAO concentrations than the consumption of other foods rich in choline or carnitine [14]. Furthermore, TMAO can also be produced by the 2.2. Dietary Precursors of TMA and TMAO TMAO can be ingested directly from food. It is found in fish and marine invertebrates such as mussels or other seafood. Concentrations in fresh seafood can vary considerably between species, habitat, depth, and season. Compared to other dietary Int. J. Mol. Sci. 2023, 24, 1940 sources, fish has the biggest impact on TMAO concentrations. The consumption of fish 3 of 17 yielded 50 times higher postprandial circulating TMAO concentrations than the consumption of other foods rich in choline or carnitine [14]. Furthermore, TMAO can also beoxidation produced of by TMAthegenerated oxidation by of the TMAgutgenerated by thedietary using different gut using differentL-carnitine, precursors: dietary precursors: L‐carnitine, choline, N6,N6,N6‐trimethyl‐L‐lysine choline, N6,N6,N6-trimethyl-L-lysine (trimethyllysine, (trimethyllysine, TML), betaine, TML), A and ergothioneine. betaine, and ergothioneine. A schematic presentation of the major microbial pathways schematic presentation of the major microbial pathways leading to TMA/TMAO synthesis leading to TMA/TMAO from dietary precursorssynthesis is shownfrom dietary in Figure 1. precursors is shown in Figure 1. Figure 1. Schematic representation of TMA/TMAO formation and TMAO excretion. TMA is obtained from dietary Figure precursors, 1. Schematic such as ergothioneine, representation of TMA/TMAO choline, betaine,and formation L-carnitine, γ-butyrobetaine, TMAO excretion. TMA is and TML metabolized obtained from dietaryby microbial enzymes. precursors, such TMA crosses the intestinal as ergothioneine, choline,barrier and, in betaine, the liver, is L‐carnitine, γ‐butyrobetaine, converted into TMAOand TML metabolized by FMO3. TMAObycan microbial enzymes. be directly obtainedTMAfromcrosses fish andthemarine intestinal barrier invertebrates. and, in the the It crosses liver, is converted intestinal into barrier andTMAO by FMO3. is mainly removed TMAO can excretion. by renal be directlyTMAO obtained from could fish and interact with marine invertebrates. endothelial It crossesduring cells in circulation the intestinal barrier and its metabolism, afterisoxidation mainly removed byand in the liver, renal excretion. before entering TMAO into thecould interact kidneys. with The endothelial potential cells effects of in circulation circulating duringonitsatherogenic TMAO metabolism,processes after oxidation in are shown the liver, and before entering into the kidneys. The potential effects of circulating TMAO on in Figure 2. TMA: Trimethylamine-mine; TMAO: Trimethylamine-N-oxide; TML: Trimethyllysine; atherogenic processes are shown in Figure 2. TMA: Trimethylamine‐mine; TMAO: FMO3: flavin monooxygenase Trimethylamine‐N‐oxide; TML: 3; OCT2: organic cation Trimethyllysine; FMO3:transporter 2. flavin monooxygenase 3; OCT2: organic cation transporter 2. L-carnitine is synthesized endogenously from methionine and lysine in the liver, kidneys, heart, testes, L‐carnitine and brain.endogenously is synthesized In certain conditions, an exogenous from methionine and source lysine may be liver, in the needed to supplement endogenous synthesis. Foods of animal origin (especially meat and dairy kidneys, heart, testes, and brain. In certain conditions, an exogenous source may be products) are rich in L-carnitine. It can also be found in grains and vegetables but in lower needed to supplement endogenous synthesis. Foods of animal origin (especially meat concentrations [15]. Choline can also be considered a conditionally essential nutrient. As and dairy products) are rich in L‐carnitine. It can also be found in grains and vegetables the endogenous production by the liver may not be enough for human requirements, it but in lower concentrations [15]. Choline can also be considered a conditionally essential needs to be obtained from the diet. Choline is present in foods in different forms, mainly in nutrient. As the endogenous production by the liver may not be enough for human water-soluble forms that include free choline, phosphocholine, and glycerophosphocholine, requirements, it needs to be obtained from the diet. Choline is present in foods in and lipid-soluble forms that include phosphatidylcholine and sphingomyelin. The main different forms, mainly in water‐soluble forms that include free choline, phosphocholine, sources of choline are eggs and liver, followed by fish, whole-grain cereals, vegetables, and glycerophosphocholine, and lipid‐soluble forms that include phosphatidylcholine fruit, milk, fat, and oils [16]. TML is a non-protein amino acid with an important role as a and sphingomyelin. The main sources of choline are eggs and liver, followed by fish, precursor of L-carnitine. TML is present as a free amino acid in legumes, grains, leafy and Solanaceae vegetables, and fruit [17]. 2.3. TMAO Metabolism Once the gut microbiota has produced TMA from dietary precursors, it crosses the intestinal lumen. TMA can only be produced and absorbed by the small intestine, and it is then transported via portal circulation to the liver [18]. TMA is afterward converted into TMAO by flavin monooxygenase (FMO) in the liver of the host. Among the five members Int. J. Mol. Sci. 2023, 24, 1940 4 of 17 of the FMO family, only FMO1 and FMO3 have the ability to oxidize TMA to TMAO. FMO3 is the main isoform expressed in the human liver (Figure 1) [19]. Interestingly, a sexually dimorphic expression pattern of FMO3 has been observed in both mice and humans, with females showing higher expression than males [20]. This gender difference may be explained by hormonal regulation, as testosterone is responsible for the lower hepatic FMO3 expression in males and estrogen induces FMO3 gene expres- sion in females [21]. Studies on humans have produced divergent results on gender-related differences in plasma TMAO concentrations. Several studies investigating TMAO expres- sion in humans did not find significant differences between the sexes in terms of plasma TMAO levels [22–24]; others, however, reported higher levels either in females [19,25] or in males [26,27]. Urinary excretion is the major elimination pathway of TMAO from systemic circulation. TMAO is almost exclusively eliminated by the kidneys in an unmodified form via filtration and active secretion mechanisms (Figure 1). A kinetic study in 40 healthy men with d9- TMAO revealed that TMAO can be detected in plasma only 15 min after oral ingestion. The elimination of TMAO was greatest during the first six hours (showing an enrichment of 67%) after consumption of the tracer. An estimated 96% of the administrated d9-TMAO was excreted unmodified in the urine after 24 h, leaving only 4% retained in the body, with none detected in the feces [28]. Active secretion mechanisms, which are implicated in TMAO excretion, occur through different transporters. The most important transporters are the organic cation transporters (OCT). Specifically, OCT1 to 3 are TMAO transporters, and OCT1 and OCT2 are decisive in TMAO kinetics in mice. TMAO can be absorbed by OCT2 in humans, but its contribution to tubular secretion is not clearly observed under normal conditions [29]. OCT1/2 knockout mice present higher levels of TMAO than wild-type mice. OCT1 is mainly located in the liver and OCT2 is in the kidney. The increase in TMAO levels is a consequence of low renal excretion; the liver/plasma ratio of TMAO is not altered, but the kidney/plasma ratio of TMAO is decreased. Moreover, OCT2 is physiologically relevant to TMAO renal tubular uptake across all the concentrations observed in humans [30]. 3. TMAO in Prospective Studies After Wang et al. linked TMAO with CVD in 2011, several studies have been carried out, with somewhat controversial results. While some reported that TMAO independently predicted mortality or major adverse cardiovascular events (MACE), others did not find independent associations between TMAO levels and cardiovascular outcomes. In this review, we focused on prospective studies, since they are more powerful in assessing the risk association between an exposition or molecule and an outcome. The first meta- analysis of published prospective studies concluded that higher circulating levels of gut- related metabolites, including TMAO, were associated with an increased risk of MACE, regardless of conventional risk factors [5]. However, only one year later, another meta- analysis including 11 prospective studies found that, despite high circulating TMAO being associated with an increased risk of cardiovascular events and all-cause mortality, the link between TMAO and CVD remained limited and inconsistent [6]. Moreover, the most recent systematic review, which examined the relationship of TMAO with MACE, cardiovascular mortality, and all-cause mortality, also found that TMAO was positively associated with these outcomes in some but not all of the included studies [31]. There are several limitations to be considered in systematic reviews and meta-analyses. One of them is the high heterogeneity of the studies, which, in some cases, may not equally take into account the type of cohort included (general population, pre-existing, cardiovascular risks, etc.). Another is the different definition of elevated TMAO levels or confounding factors, such as environment (diet or lifestyle), not being taken into account. Table 1 includes larger published prospective studies evaluating the association of TMAO and adverse outcomes (cardiovascular-related and mortality-related) in CVD-free individuals, patients with CVD or coronary artery disease (CAD), patients with acute Int. J. Mol. Sci. 2023, 24, 1940 5 of 17 coronary syndrome (ACS), and patients with chronic kidney disease (CKD). The table includes analyses of individuals free of CVD, as it is a population unbiased by any specific disease or condition; the CAD and ACS patients are a representation of systemic CVD; and the CKD patients were included because TMAO is almost exclusively cleared via kidney and thus, renal function is the most well-known confounder. Only studies with clear results in terms of hazard ratio, confidence interval, and p values were included in the table. Depending on the studied population, the outcome evaluated, or the covariates included in the adjustment, different results were found. In community-based studies, adjustment for estimated glomerular filtration rate (eGFR) [32] and age and sex [33] showed a non-significant association between TMAO and all-cause mortality. Moreover, no significant association was found between TMAO and ASCVD [34]. In patients with prevalent CVD or CAD the most significant association was found with all-cause mortality [35,36] rather than MACE [26] or ASCVD [34,35]. In patients with ACS, a significant association with major outcomes was also found, but renal function was not included in the adjustment analysis [37]. Only short-term outcomes, with a follow-up of one or two months, maintained a significant association after adjustment for eGFR [38]. Last, the CKD patient results also showed a significant association when eGFR was not included in the adjustment [39], while adjustment for renal function led to divergent results [40,41]. Renal function, mainly calculated as the eGFR, is the main covariate used in adjust- ment. Correction for renal function almost always translated into a decrease or loss in the significant association between TMAO and the outcome—independent of the target popu- lation. Considering this observation, it could be argued that TMAO’s association with the outcomes may be mediated—at least partially—by renal function [42]. Moreover, TMAO does not seem to add an additional risk to that already provided by impaired renal function or CKD alone. TMAO could be just a marker of renal impairment or directly contribute to decreased renal function, as it was observed in mice [43]. This rather suggests that elevated circulating concentrations of TMAO may have a direct effect on renal impairment, which, in turn, is a determinant of CVD risk [44]. An important limitation of these studies is that the use of antibiotics and modifications in dietary habits were not always recorded as confounding factors. Table 1. Prospective studies about TMAO and adverse outcomes. Hazard Ratio (95% CI) and Year N Follow-Up Outcomes Covariates in the Adjusted Models Ref. p Value Individuals free of known CVD: community-based population 1: age, sex, smoking status, SBP, LDLc, HDLc, diabetes, CRP UA: 2.54 (1.96–3.28) p < 0.001 2: model 1 + myeloperoxidase level, 1: 1.88 (1.44–2.44) p < 0.001 2013 4007 3 years MACE [45] eGFR, total white-cell count, BMI, 2: 1.49 (1.10–2.03) p = 0.01 medication 3: 1.43 (1.05–1.94) p = 0.02 3: model 2 + extent of the disease Age, sex, race, study site, education, Adj: 1.07 (0.90–1.27) p = 0.516 income, health status, smoking Adj: 1.37 (0.98–1.90) p = 0.084 status, alcohol intake, physical (individuals with eGFR activity, BMI, WC, lipid-lowering 2021 4131 15 years ASCVD < 60 mL/min 1.73 m2 ) [34] medication, antihypertensive Adj: 0.98 (0.80–1.20) p = 0.903 medication, antibiotics, T2D, (individuals with eGFR HDL-C, LDL-C, TG, CRP, SBP, DBP, ≥ 60 mL/min 1.73 m2 ) diet, eGFR Int. J. Mol. Sci. 2023, 24, 1940 6 of 17 Table 1. Cont. Hazard Ratio (95% CI) and Year N Follow-Up Outcomes Covariates in the Adjusted Models Ref. p Value 1: age, sex, race, and enrollment site 2: model 1 + education, household income, smoking, BMI, physical activity, treated hypertension, 1: 1.36 (1.24–1.51) p < 0.001 All-cause instrumental activities of daily 2: 1.30 (1.17–1.44) p < 0.001 mortality living, self-reported health status, 3: 1.07 (0.96–1.19) p = 0.51 Cardiovascular systolic blood pressure, HDL mortality cholesterol, prevalent atrial 1: 1.50 (1.27–1.77) p < 0.0001 2022 5333 13.2 years fibrillation, prevalent coronary heart 2: 1.35 (1.14–1.60) p < 0.0001 [32] disease, myocardial infarction, 3: 1.09 (0.91–1.30) p = 0.65 Non- prevalent diabetes, prevalent cardiovascular chronic obstructive pulmonary 1: 1.29 (1.14–1.46) p < 0.0001 mortality disease, and reported daily intake of 2: 1.27 (1.12–1.44) p = 0.0003 eggs, fish, liver, non-processed red 3: 1.06 (0.93–1.21) p = 0.61 meat, processed meat, and total calories 3: model 2 + eGFR UA: 3.07 (2.48–3.80) p < 0.001 All-cause 1: 1.09 (0.88–1.37) p = 0.43 mortality 2: 1.11 (0.88–1.40) p = 0.38 1: age and sex Cardiovascular UA: 3.75 (2.56–5.49) p < 0.001 2: model 1 + HT, diabetes mellitus, 2022 6393 10.5 years mortality 1: 1.13 (0.77–1.70) p = 0.54 [33] total cholesterol, smoking, BMI, and 2: 0.97 (0.65–1.46) p = 0.88 eGFR Non- cardiovascular UA: 2.77 (2.14–3.60) p < 0.001 mortality 1: 1.07 (0.82–1.40) p = 0.64 2: 1.17 (0.88–1.54) p = 0.28 Patients with CVD or CAD UA: 1.01 (0.98–1.04) p = 0.493 2015 339 8 years MACE eGFR [26] Adj: 0.991 (0.98–1.00) p = 0.062 1: age, sex, SBP, LDL-C, HDL-C, smoking, and diabetes mellitus 2: model 1 + hsCRP and eGFR UA: 3.90 (2.78–5.48) p < 0.0001 3: model 2 + medications All-cause 1: 2.61 (1.82–3.76) p < 0.0001 2016 2235 5 years (angiotensin-converting enzyme [36] mortality 2: 1.95 (1.33–2.86) p = 0.003 inhibitor, angiotensin-receptor 3: 1.71 (1.11–2.61) p = 0.0138 blocker, β-blocker, aspirin, or statin), number of stenotic vessels, myeloperoxidase, and BNP Age, sex, race, study site, education, income, health status, smoking status, alcohol intake, physical activity, BMI, WC, lipid-lowering 2021 1287 15 years ASCVD 1.10 (0.87–1.39) p = 0.179 [34] medication, antihypertensive medication, antibiotics, T2D, HDL-C, LDL-C, TG, CRP, SBP, DBP, diet, eGFR Int. J. Mol. Sci. 2023, 24, 1940 7 of 17 Table 1. Cont. Hazard Ratio (95% CI) and Year N Follow-Up Outcomes Covariates in the Adjusted Models Ref. p Value 1: BMI, diabetes status, eGFR 2: model 1 + age, sex, smoking UA: 1.61 (1.36–1.92) p < 0.001 All-cause status, SBP, DBP, hsCRP, HDL-C, 2021 449 9 years 1: 1.30 (1.07–1.58) p = 0.009 [35] mortality LDL-C, white blood cell count, and 2: 1.24 (1.01–1.51) p = 0.040 in LIFE-CAD additionally for presence of CAD UA: 1.42 (1.29–1.58) p < 0.001 1: age, gender, and history of CAD 1: 1.28 (1.13–1.43) p < 0.001 2: pre-defined patient 1 *: 1.16 (1.03–1.30) p = 0.013 All-cause characteristics, cardiovascular risk 2: 1.23 (1.09–1.38) p < 0.001 mortality factors, and medical history, 2 *: 1.11 (0.99–1.26) p = 0.084 including age, gender, body mass index, smoking history, positive UA: 1.60 (1.39–1.84) p < 0.001 cardiovascular family history, 1: 1.44 (1.4–1.68) p < 0.001 2022 1726 5 years Cardiovascular hypertension, hypercholesterolemia, 1 *: 1.28 (1.10–1.48) p < 0.001 [46] mortality history of diabetes, history of stroke, 2: 1.36 (1.16–1.58) p < 0.001 history of CAD, previous AMI, 2 *: 1.19 (1.01–1.40) p = 0.032 history of heart failure and adjudicated functionally relevant UA: 1.38 (1.17–1.65) p < 0.001 AMI coronary artery disease * 1: 1.33 (1.11–1.60) p = 0.002 Both models also adjusted adding 1 *: 1.22 (1.01–1.48) p = 0.043 Cystatin-C 2: 1.32 (1.08–1.60) p = 0.006 2 *: 1.17 (0.95–1.45) p = 0.146 UA: 2.29 (1.88–2.79) p < 0.001 All-cause 1: 1.32 (1.08–1.62) p = 0.01 mortality 2: 1.07 (0.86–1.32) p = 0.56 1: age and sex Cardiovascular UA: 2.73 (2.00–3.72) p < 0.001 2: model 1 + HT, diabetes mellitus, 2022 4132 9.8 years mortality 1: 1.52 (1.11–2.09) p = 0.01 [33] total cholesterol, smoking, BMI, and 2: 1.16 (0.83–1.62) p = 0.38 eGFR Non- cardiovascular UA: 2.02 (1.57–2.61) p < 0.001 mortality 1: 1.20 (0.92–1.56) p = 0.18 2: 1.02 (0.77–1.34) p = 0.90 ACS patients Age, sex, SBP, heart rate, past All-cause histories of MI/angina, increased UA: 1.21 (0.98–1.48) p = 0.074 mortality 2017 1079 2 years BP and diabetes, Killip score, STEMI UA: 1.4 (1.26–1.55) p < 0.0005 [37] class, revascularization, and Adj: 1.21 (1.03–1.439) p = 0.023 Death/MI medication UA: 2.29 (1.39–3.79) p < 0.01 MACE Age, gender, HDL-C, LDL-C, Adj: 6.3 (1.89–21.00) p < 0.01 30 days (30 days and smoking, presence or absence of a 6 months) history of diabetes mellitus, HT, 2017 UA: 2.48 (1.51–4.09) p < 0.001 530 6 months hyperlipidemia, revascularization or [38] Adj: 5.65 (1.91–16.74) p < 0.01 All-cause CAD, CRP level, eGFR, initial cTnT 7 years mortality (7 level, diagnosis of either STEMI, UA: 3.72 (2.23–6.20) p < 0.001 years) nonSTEMI, or unstable angina Adj: 1.81 (1.04–3.15) p < 0.05 1: BMI, diabetes status, eGFR 2: model 1 + age, sex, smoking UA: 1.39 (0.98–1.97) p = 0.066 All-cause status, SBP, DBP, hsCRP, HDL-C, 2021 326 9 years 1: 1.10 (0.74–1.65) p = 0.638 [35] mortality LDL-C, white blood cell count, and 2: 1.07 (0.70–1.63) p = 0.771 in LIFE-CAD additionally for presence of CAD Int. J. Mol. Sci. 2023, 24, 1940 8 of 17 Table 1. Cont. Hazard Ratio (95% CI) and Year N Follow-Up Outcomes Covariates in the Adjusted Models Ref. p Value Prior MI, diabetes, age, gender, Cardiovascular UA: 21.33 (4.7–96.5) p < 0.001 2021 292 7 years diabetes, hemoglobin and creatinine [47] mortality Adj: 11.62 (2.3–59.7) p = 0.003 levels at admission 1: age, sex, BMI, smoking, HT, dyslipidemia, type 2 diabetes, unstable angina, STEMI, nonSTEMI, UA: 2.63 (1.69–4.08) p < 0.001 2022 309 6.7 years MACE statin medication, beta-blockers, 1: 1.83 (1.08–3.09) p = 0.043 [42] oral antidiabetic medication, insulin 2: 1.66 (0.98–2.82) p = 0.119 medication, diuretics, aspirin 2: model 1 + eGFR Stable CKD patients Age, sex, systolic blood pressure, All-cause UA: 2.76 (1.74–4.37) p < 0.001 2015 521 5 years LDL-C, HDL-C, smoking, diabetes [41] mortality Adj: 1.93 (1.13–3.29) p < 0.05 mellitus, hsCRP, and eGFR 1: age, sex, race, diabetes and cardiovascular comorbidities at baseline UA: 1.45 (1.28–1.64) 2: model 1 + traditional 1: 1.38 (1.21–1.57) p < 0.0001 cardiovascular risk factors 2016 2529 3 years Ischemic events 2: 1.39 (1.22–1.59) p = 0.0055 [39] 3: model 2 + chronic kidney 3: 1.24 (1.07–1.43) p = 0.065 disease-specific risk factors 4: 1.23 (1.06–1.42) p = 0.0059 4: Adjusted for all covariates with statically significant association with ischemic events 1: age, race, sex All-cause 1: 2.45 (1.09–5.50) p = 0.02 2016 339 3.3 years 2: model 1 + SBP, LDL-C, HDL-C, [40] mortality 2: 1.25 (0.48–3.28) p = 0.37 smoking, CRP, eGFR Adj: adjusted; AMI: acute myocardial infarction; ACS: acute coronary syndrome; BNP: brain natriuretic peptide; BMI: body mass index; CAD: coronary artery disease; CKD: chronic kidney disease; cTnT: cardiac troponin T; CVD: cardiovascular disease; DBP: diastolic blood pressure; eGFR: estimated glomerular filtration rate; HDL-C: high- density lipoprotein cholesterol; HR: hazard ratio; hsCRP: high sensitivity C reactive protein; HT: hypertension; LDL-C: low-density lipoprotein cholesterol; MACE: major adverse cardiovascular event; SBP: systolic blood pressure; STEMI: ST-segment elevation myocardial infarction; TG: triglycerides; UA: unadjusted; WC: waist circumference. Other target populations were also analyzed in other prospective studies, such as heart failure, type 2 diabetes (T2D), peripheral artery disease, and hemodialysis treatment, among others. The results of these studies are summarized in Supplementary Table S1. As previously noted, the association tends to remain only significant when renal function is not included in the adjustment analysis. Focusing on the relationship of TMAO with atherosclerotic burden, there are also controversial results. The TMAO levels were independently associated with high coronary atherosclerotic burden in NSTEMI [43] and STEMI [44] patients. A specific gut microbiota profile was found to be associated with coronary artery calcium (CAC) in patients without previously reported CVD [45]. However, TMAO was not associated with different measures of atherosclerosis, including CAC, in another population-based study [46]. 4. TMAO in Genetic Studies The results of observational studies are not always consistent, and, therefore, it is at times difficult to establish the causality relation between TMAO and CVD. Mendelian randomization (MR) uses measured variation in genes of known function, in this case influ- encing TMAO plasma concentration on a disease or on several diseases. An MR method is somewhat similar to a randomized controlled trial, where genetic alleles are randomly Int. J. Mol. Sci. 2023, 24, 1940 9 of 17 assorted during conception, and is thus less likely to be affected by confounding or reverse causation. For this reason, it has been widely accepted as a method to explore the potential causal effect of exposure on disease [48]. Only three MR studies were performed focus- ing on the role of TMAO. The first one assessed the association of gut-microbiota-related metabolites and cardiometabolic health [49]; the second was in relation to Alzheimer’s disease [50]; and the most recent was in relation to blood pressure [51]. In the study of Jia et al., they did not find a significant association of genetically predicted higher TMAO levels with cardiometabolic disease (including T2D, atrial fibrillation, CAD, MI, stroke, and CKD). They also found that T2D and CKD were causally associated with an increase in TMAO levels, suggesting that the association of TMAO and CVD may be due to confounding or reverse causality [49]. On the other hand, Wang et al. found a positive causal relationship between TMAO and systolic blood pressure—but not with diastolic blood pressure [51]. One recent meta-analysis also found a significant positive dose-dependent association between circulating TMAO levels and hypertension prevalence regardless of different stratifications [52]. The specific mechanism by which TMAO could affect blood pressure is currently unknown, and all proposed are also linked to the arteriosclerosis-induced capacity of TMAO. 5. Mechanisms Related to TMAO Atherogenic Effect 5.1. TMAO, Cholesterol Metabolism Disruption, and Atherosclerotic Plaque Formation Experimental evidence indicates that TMAO-mediated foam cell formation in atherosclerotic plaques may occur through multiple pathways. Early evidence demon- strated that TMAO enhanced macrophage cholesterol uptake via receptor cluster of dif- ferentiation (CD) 36 and scavenger receptor A upregulation [4]. Furthermore, in another study, TMAO-mediated enhancement of CD36 expression and foam cell formation was also induced by oxidized LDL, and this was dependent on the MAPK/JNK pathway [53]. Furthermore, TMAO induces the expression of stress-inducible heat shock proteins (HSP) such as glucose-regulated protein 94 and HSP70 in mouse macrophages, leading to the induction of endoplasmic reticulum stress [54]. On the other hand, some human studies reported low HDL cholesterol levels in high TMAO-expressing individuals with CVD [36,55], thereby indicating that HDL dysfunction could be mechanistically linked to the proatherogenic effects of TMAO. However, other reports found a positive correlation or neutral relationship between HDL cholesterol and CVD [22,56]. The ability of HDL to induce macrophage cholesterol efflux, the first step of reverse cholesterol transport (RCT), is considered the main HDL atheroprotective function. Several reports have shown divergent results regarding the TMAO-mediated effects on macrophage transporters involved in cholesterol efflux, specifically indicating enhancing [57], neutral [58], or negative effects [59,60] on ATP binding cassette (ABC) A1. We recently reported that TMAO and its major gut microbial precursors did not produce significant effects on HDL-mediated macrophage cholesterol efflux [61], indicating that the first step in the RCT process was not affected by TMAO. In contrast, the suppression of the intestinal microbiota was correlated with a two-fold stimulation of the overall macrophage-to-feces RCT in mice. This change was concomitant with the enhancement of the last RCT step, the biliary secretion of bile acids [62]. In line with these findings, dietary supplementation with TMAO impaired the overall RCT transport from macrophages in mice, and antibiotic treatment reversed this TMAO-dependent downregulation of RCT. In this report, TMAO mainly affected the major pathways for bile acid synthesis and liver transport that control their elimination from the body [57]. Mechanistically, farnesoid X receptor (FXR), a nuclear receptor that acts as a sensor of intracellular bile acid levels, plays a critical role in TMAO-mediated bile acid disruption, since liver FMO3 expression is upregulated by dietary bile acids through an FXR-mediated pathway [19]. Importantly, FMO3 was also reported to be a negative regulator of transintestinal cholesterol export [63], a process responsible for 30% of the total cholesterol loss through the feces in mice [64]. Consistent with these findings, liver FMO3 knockdown also reduced liver and plasma lipid major pathways for bile acid synthesis and liver transport that control their elimination from the body [57]. Mechanistically, farnesoid X receptor (FXR), a nuclear receptor that acts as a sensor of intracellular bile acid levels, plays a critical role in TMAO‐mediated bile acid disruption, since liver FMO3 expression is upregulated by dietary bile acids through an FXR‐mediated pathway [19]. Importantly, FMO3 was also reported to be a Int. J. Mol. Sci. 2023, 24, 1940 10 of 17 negative regulator of transintestinal cholesterol export [63], a process responsible for 30% of the total cholesterol loss through the feces in mice [64]. Consistent with these findings, liver FMO3 knockdown also reduced liver and plasma lipid levels, the bile acid pool size, andlevels, glucosetheand bileinsulin levels acid pool andand size, prevented glucose atherosclerosis in LDL and insulin levels and receptor‐deficient prevented atherosclerosis micein LDL[65], suggesting receptor-deficient multiple mice mechanistic [65], suggesting processes multiple involved mechanistic processesininvolved TMAO/FMO3‐mediated in TMAO/FMO3-mediated foam cell formation foam in vivo. inThe cell formation main vivo. Thepathophysiological main pathophysiological mechanisms mechanisms involved in atherosclerotic involved plaqueplaque in atherosclerotic formation are shown formation are shown in2.Figure 2. in Figure Figure 2. TMAO enhances proatherogenic and prothrombotic mechanisms. TMAO promotes Figure 2. TMAO enhances proatherogenic and prothrombotic mechanisms. TMAO promotes macrophage macrophage foamfoam cell formation, cell formation, impairsimpairs liver cholesterol liver cholesterol metabolism, metabolism, affectscholesterol affects reverse reverse cholesterol transport, transport, andand induces induces endothelialdysfunction endothelial dysfunctionandand platelet platelet aggregation aggregation bybya variety of mechanisms. a variety of mechanisms. TMAO: Trimethylamine‐N‐oxide. TMAO: Trimethylamine-N-oxide. 5.2. 5.2. TMAO,TMAO, Inflammation, Inflammation, and Endothelial and Endothelial Dysfunction Dysfunction Endothelial Endothelial cell dysfunction cell dysfunction in the in the vasculature vasculature is considered is considered a key a key factor infactor in the early the early stages stages of atherosclerosis. of atherosclerosis. This process This process is characterized is characterized by higherby production higher production of reactive of reactive oxygen oxygen species species (ROS)(ROS) and pro-inflammatory and pro‐inflammatory factorsfactors but alsobut also a deficiency a deficiency in nitric in nitric oxide oxide (NO) bioavailability, (NO) which bioavailability, regulates which the vascular regulates tone [66]. the vascular toneIndeed, TMAO elevated [66]. Indeed, TMAO elevated interleukin 6, C6,reactive interleukin protein, C reactive tumor protein, tumor necrosis factor necrosis alpha, factor andand alpha, ROSROSin endothelial in endothelial cells, cells, whereas whereas it reduced it reduced NO NO production[67]. production [67].TMAO TMAOalso alsoinduced inducedthetheexpression expressionofofcytokines cytokines and adhesion molecules in endothelial cells, thereby and adhesion molecules in endothelial cells, thereby promoting monocyte promoting monocyte adherence [68]; this induction required the nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway [8] (Figure 2). Other lines of evidence indicate that TMAO is inversely related to artery endothelial function in vivo, as assessed in one study via brachial artery flow-mediated dilation in elderly patients. In the same work, TMAO supplementation also induced endothelial dysfunction via reduced NO bioavailability and increased superoxide-driven oxidative stress in young mice [69]. In line with these findings, 3,3-Dimethyl-1-butanol, an inhibitor of TMAO production, normalized the expression of pro-inflammatory cytokines and super- oxide production, whereas it reduced endothelial nitric oxide synthase (eNOS) expression in the aorta of old rats [70]. Int. J. Mol. Sci. 2023, 24, 1940 11 of 17 Mechanistically, strong evidence indicates that TMAO promotes different inflam- matory signaling pathways, particularly the NOD-, LRR-, and pyrin domain-containing protein 3 (NLPR3), which is also involved in enhanced oxidative stress, endothelial cell apoptosis, and hyperpermeability (Figure 2). Accordingly, TMAO significantly enhanced oxidative stress and activated the TXNIP-NLRP3 inflammasome in human umbilical vein endothelial cells, and these effects were reversed by the ROS inhibitor N-acetylcysteine [71]. These data were confirmed by another study that demonstrated TMAO induced the forma- tion and activation of NLRP3 and enhanced IL-1β production in carotid artery endothelial cells. These effects were also blunted by ROS scavengers and the inhibition of lysosomal cysteine protease cathepsins B [72], thereby suggesting that TMAO induces the inflamma- some via both lysosomal destabilization and ROS release. It has also been reported that TMAO upregulated NLRP3, pyroptosis, and succinate dehydrogenase complex subunit B (SDHB) in vascular endothelial cells of dyslipidemic apoE-deficient mice. TMAO also impaired the mitochondria’s structure and function and increased ROS generation by upregulating SDHB expression in cultured vein endothelial cells [73], thereby identifying an alternative TMAO-mediated proatherogenic mechanism. The integrity of the endothelium is maintained by tight junction proteins. In this context, TMAO downregulated the tight junction protein-1 expression on the endothelial cells, and this was prevented by silencing NLRP3. Furthermore, the TMAO-mediated increase in endothelial cell permeability was also blunted by NLRP3 siRNA transfection [72]. On the other hand, TMAO treatment also caused endothelial cell disjunction via high- mobility group box 1, and this caused the further activation of pro-inflammatory toll-like receptor (TLR) 4. Interestingly, TLR4 inhibition attenuated TMAO-induced junction protein disruption [74]. 5.3. TMAO, Platelet Activation, and Thrombosis The first proposed mechanism by which TMAO enhances thrombosis was related to its capacity to directly increase platelet sensibility to multiple agonists, also increasing thrombosis in vivo and stimulating platelet hypersensitivity, enhancing the release of intracellular Ca2+ (Figure 2). Dietary precursors of TMAO, such as choline, also increase platelet sensitivity as well as thrombosis in vivo, with a key role played by the microbiota [7]. Moreover, oral choline supplementation causes an increase in platelet aggregation responses to the platelet agonist adenosine diphosphate (ADP) and strikes the dose-dependent association between TMAO levels and platelet function [75]. 3,3-dimethyl-1-butanol (DMB), a drug that inhibits microbial choline TMA-lyase activ- ity in vitro and in vivo [76], was shown to be a therapeutic strategy for reducing thrombosis potential. DMB significantly reduced both the TMAO levels and ADP-dependent platelet aggregation in platelet-rich plasma (PRP); this reduction was completely reversed by the direct injection of TMAO [77]. Indeed, germ-free mice colonized with a microbiome from a high TMAO producer donor presented a significantly increased platelet aggregation response and platelet responsiveness [78], supporting the link among the microbiome, TMAO, and thrombosis in vivo (Figure 2). TMAO also increases thrombosis in vitro, since mouse PRP incubated with high levels of TMAO enhanced platelet aggregation through two different agonists—ADP and collagen. In addition, PRP from FMO3-knockdown mice showed a significant decrease in platelet aggregation response induced by ADP stimulation. FMO3 suppression significantly caused decreased collagen-dependent platelet adhesion to the matrix in whole blood, decreased platelet responsiveness, slower clot formation, and significantly longer times to vessel occlusion [79,80]. Furthermore, decreased expression levels of inflammatory genes were found, but no differences were observed in terms of other inflammatory or thrombotic-related biomarkers, such as E-selection, intercellular adhesion molecule 1, and Von Willebrand factor [80]. On the other hand, FMO3 overexpression in mice induced heightened platelet responsiveness to ADP, faster clot formation, and a significantly shorter time to vessel occlusion [79]. Int. J. Mol. Sci. 2023, 24, 1940 12 of 17 The most recent proposed mechanism was the ability of TMAO to induce tissular factor (TF) expression and activity in endothelial vascular cells as a cause of atherosclerotic thrombosis (Figure 2). Incubating TMAO with human coronary endothelial cells causes an increase in TF expression (mRNA, protein, and surface expression) as well as enhances TF activity and thrombin production [81]. Moreover, TMAO causes a dose-dependent overexpression of TF and vascular cell adhesion molecule type 1 in both human and mouse cells. This TMAO-dependent enhancement in TF expression was shown to cause an increase in thrombogenicity in mouse models and could be reverted through a TF-inhibitory antibody or TMA-lyase inhibitor. Overall, endothelial TF contributes to TMAO-related arterial thrombosis potential and can be specifically blocked by the targeted non-lethal inhibition of gut microbial choline TMA-lyase [82]. 6. Conclusions During the last decade, the number of studies focusing on the association of TMAO with CVD increased year after year. However, the results of prospective studies failed to establish a clear relationship between TMAO and CVD or related events, with a key role of renal function as a potential confounding factor. However, it is also possible that increased TMAO causes renal dysfunction—at least in part through hypertension. Experimental data have proposed several TMAO-mediated proatherogenic and prothrombotic mechanisms, including the disruption of cholesterol metabolism, inflammation, endothelial dysfunction, and platelet activation. However, the impact of these detrimental effects of TMAO by itself may be not enough to be clinically meaningful. Overall, the potential of TMAO as a causal agent of ASCVD in humans remains to be fully clarified. Supplementary Materials: The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms24031940/s1. References [55,83–95] are cited in the Supplementary Materials. Author Contributions: Writing—original draft preparation, M.C., C.B. and J.C.E.-G.; writing—review and editing, M.C., N.R., M.T., J.C.E.-G. and F.B.-V. All authors have read and agreed to the published version of the manuscript. Funding: This work was partly funded by the Instituto de Salud Carlos III and FEDER “Una manera de hacer Europa” grants PI18-00164 and PI21-00140 (to F.B.-V. and M.T.), PI19-00136 (to J.C.E.-G.), and Ministerio de Ciencia, Innovación y Universidades PID2019-104367RB-100 to N.R. and M.C. was supported by a Rio Hortega contract CM20/00033; and N.R. by the Subprograma Ramón y Cajal (RYC-201722879). This work was also funded by the Agencia Estatal de Investi- gación (AEI/10.13039/501100011033) within the Subprograma Ramón y Cajal (RYC-201722879) to N.R. CIBERDEM is an initiative of the Instituto de Salud Carlos III. J.C.E.-G. is member of Red de Investigación en “Enfermedades Metabólicas y Cáncer” (RED2018-102799-T), Ministerio de Economía y Competitividad (MINECO), Madrid, Spain. Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau is accredited by the Generalitat de Catalunya as Centre de Recerca de Catalunya (CERCA). C.B. was funded by the FPU (Formación de Profesorado Universitario) grant FPU20/07440 from Ministerio de Universidades, Spain. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results. Abbreviations ATP binding cassette (ABC); acute coronary syndrome (ACS); adenosine diphosphate (ADP); atherosclerotic cardiovascular disease (ASCVD); coronary artery calcium (CAC); coronary artery disease (CAD); cluster of differentiation (CD); chronic kidney disease (CKD); cardiovascular disease (CVD); 3,3-dimethyl-1-butanol (DMB); estimated glomerular filtra- tion rate (eGFR); endothelial nitric oxide synthase (eNOS); flavin monooxygenase (FMO); farnesoid X receptor (FXR); heat shock proteins (HSP); major adverse cardiovascular events (MACE); Mendelian randomization (MR); NOD-, LRR-, and pyrin domain-containing protein 3 (NLPR3); nitric oxide (NO); organic cation transporters (OCT); platelet-rich Int. J. Mol. 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