, Liping Guo 3,*1 Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 301617 Tianjin, China
2 Graduate School, Tianjin University of Traditional Chinese Medicine, 301617 Tianjin, China
3 Department of Cardiology, Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital, 300120 Tianjin, China
† These authors contributed equally.
Academic Editor: Gianluca Rigatelli
Abstract
As a potential causative factor in various cardiovascular diseases, the gut microbe-generated metabolite trimethylamine N-oxide (TMAO) has courted considerable research interest as a potential biomarker. TMAO is a small molecule considered to be beneficial for the health of deep-water animals due to its ability to protect proteins against hydrostatic pressure stress. However, it may cause deleterious effects in humans as mounting evidence suggests that TMAO may enhance atherosclerosis, independent of traditional risk factors. This may be mediated by its capacity to enhance inflammation, platelet activation and thrombosis, and inhibit reverse cholesterol transport. In humans, circulating levels of TMAO have been found to be associated with increased risk of developing atherosclerotic diseases such as carotid atherosclerosis, coronary atherosclerotic heart disease, stroke, and peripheral arteriosclerosis. This review aims to discuss the current role of TMAO in the atherosclerosis process, using animal models and clinical studies, with special attention to determining whether TMAO could be used as a marker for monitoring severity and prognosis in atherosclerosis and to evaluate evidence for its role as a mediator in the pathogenesis of atherosclerotic vascular disease.
Keywords
- Atherosclerosis
- Trimethylamine N-oxide
- Coronary heart disease
- Cardiovascular risk
Atherosclerosis is a chronic inflammatory state and the main pathological basis of cardiovascular disease (CVD) [1]. Within the past ten years, the intestinal flora has been recognized as an independent and important metabolic organ, which closely links the metabolism of nutrients with molecules that cause chronic diseases [2]. Intestinal micro-ecological imbalance can promote the development of atherosclerosis by increasing systemic inflammation [3, 4, 5] and recently, a metabolite of intestinal flora TMAO, has received widespread attention as a marker for and potential mediator of many diseases.
Professor Tang WH of the Cleveland Medical Center, was the first researcher to
report a role for elevated TMAO levels as a predictor of major adverse cardiac
events such as myocardial infarction, stroke, and mortality [6]. Since then many
comparable studies have shown that increased concentrations of TMAO and its
precursors (L-carnitine, choline, and betaine) are associated with increased
risks of cardiovascular adverse events and mortality [7, 8, 9, 10, 11, 12]. Furthermore, TMAO is
thought to be more accurate at predicting these events than traditional CVD risk
factors such as blood lipid, C-reactive protein (CRP) levels, or renal function
[13]. In a meta-analysis of 17 clinical studies, an every 10
Here we review the dietary sources, synthesis, and metabolic pathways of TMAO, and provided the latest evidence on the ability of TMAO to be used as a potential marker for atherosclerotic vascular disease, and finally tested TMAO as a target for the treatment of atherosclerosis.
The main sources of TMAO are dietary choline, carnitine, and betaine. Choline is found in beef, chicken liver, bacon and soybeans, and betaine is found in wheat bran, wheat germ and spinach [17]. Meat products such as lean pork, lamb, and beef are the main sources of carnitine [18]. TMAO can also be obtained directly from fish [19] and high protein diets [20]; for example, two large eggs (65 grams) contain 320 mg of phosphatidylcholine, which represents the same amount of TMAO precursor as a 12-ounce beef burgers [21]. Trimethylamine (TMA) is an intermediate of TMAO which is mainly metabolized by the gut microbiota from dietary nutrients having a TMA like structure. It quickly reaches the liver through the portal circulation and flavin-containing monooxygenase 3 (FMO3) can oxidize TMA to form TMAO where it is released into the circulatory system and finally excreted by the kidney [6, 22] (Fig. 1). Consuming 50 mg of deuterium-labeled methyl d9-TMAO can be detected as early as 15 min in plasma, and 96% of the dose is excreted through urine instead of feces by 24 h [23]. The remaining TMAO is reduced to TMA by the action of TMAO reductase [24].
Fig. 1.Role of TMAO in atherosclerosis progression. Annotation: Trimethylamine (TMA) is generated by the action of TMA lyases in the gut microbiota from Choline, Carnitine and betaine. TMA quickly reaches the liver through the portal circulation, flavin-containing monooxygenase 3 (FMO3) significantly oxidize TMA to form TMAO into the circulatory system. TMAO promotes inflammation of blood vessels, enhances the formation of foam cells, exacerbates platelet hyperreactivity and thrombosis potential, alteres bile acids and cholesterol transport. These factors are associated with increased risk of developing Atherosclerotic disease such as Carotid atherosclerosis, Coronary atherosclerotic heart disease, Stroke, and Peripheral arteriosclerosis.
Microbial enzymes involved in the formation of TMA involve two main pathways,
one is the TMA lyase system (CUTC/D) which degrades choline [25]. The pathway
encoded by the CUTC gene cluster is the main pathway for TMA production from
human choline [26, 27] and the identified CUTC amplicons are associated with
various taxa especially Clostridium XIVa strains and Eubacterium sp [28].
Furthermore, Kymberleigh A. Romano identified eight species representing two
different phyla (Firmicutes and Proteobacteria) that showed significant choline
consumption and TMA accumulation [29]. The other pathway is the decomposition of
carnitine (CntA/B and YeaW/X) [30]. Here, CntA amplicons displayed high identity
(~99%) to Gammaproteobacteria-derived references, primarily from
Escherichia coli [28]. Recently, Robert A Koeth proposed that the conversion of
dietary carnitine to TMAO also involves the
L-carnitine
Inflammatory injury of the vascular endothelium is widely regarded as the
initial stage of atherosclerosis [32]. In vitro studies have shown an
up-regulated IL-6, CRP, TNF-
Fig. 2.Putative molecular mechanisms underlying TMAO-induced atherogenic effects. Annotation: High concentration of TMAO plays a vital role in endothelial hyper permeability and inflammation, oxidative stress, cholesterol metabolism, plaque formation, activation and thrombosis.
In young mice, six months of dietary supplementation with TMAO can induced aging
of the arterial endothelium. This effect was accompanied by increased vascular
nitrotyrosine, a marker of oxidative stress [36]. In a different study, aged rats
exhibited higher plasma TMAO, TNF-
It has been reported that there are several mechanisms underlying the activation
of inflammation, including lysosomal rupture, altered K
It is well known that loss of integrity of tight junctions found in the endothelium helps to enhance the permeability of the quasi-cellular endothelium. Stimulating ECs with TMAO can reduce the expression of the tight junction protein ZO-1 in the ECs monolayer and change ECs permeability, which was prevented by silencing NLRP3 in the ECs [39]. The high-mobility basal box protein 1 (HMGB1) is also an inflammatory mediator, which can destroy cell-to-cell connections, resulting in vascular endothelial hyper permeability [42]. In a study conducted by Singh, it was discovered that TMAO significantly reduced the expression of junctional proteins such as ZO-2, VE-kadrin, and Okrudin in the EC monolayer, while this effect was markedly attenuated by glycyrrhizin, an HMGB1 binding compound [42]. Furthermore, TMAO can upregulate HMGB1, and HMGB1 causing further activation of toll-like receptor 4(TLR4, an important receptor for HMGB1 binding where HMGB1 can activate inflammatory pathways). TLR4 siRNA can protect ECs from the interference by TMAO-related tight junction proteins [42] (Fig. 2).
High TMAO levels were also found to be accompanied by low HDL levels in patients
with cardiovascular disease [43]. The accumulation of oxidized low-density
lipoprotein (ox-LDL) in LDL cholesterol macrophages can be transported to the
liver for recirculation or excreted in the form of bile acid [44]. Previous
studies have confirmed that the RCT of choline-added APOE
Several experimental models have demonstrated a role for TMAO in the process of
atherosclerosis. A TMAO-diet significantly promoted plaque progression in
APOE
Platelets contribute to the formation of thrombus and foam cells and play an
important role in the occurrence and development of atherosclerosis [49]. After 2
months of choline supplementation, healthy subjects showed a significant
TMAO can induce the release of calcium from platelets by changing the inositol
1,4,5-triphosphate (IP
The role of TMAO in atherosclerotic vasculopathy has been demonstrated in various models of atherosclerotic risk factors, namely: diabetes, hypertension, and chronic kidney disease.
High levels of circulating TMAO are associated with an increased risk of both
type 1 and type 2 diabetes (T2DM) [56, 57, 58], as well as pre-diabetes prevalence
[59], and diabetic complications such as retinal neuropathy [60].
Miao found that LIRKO mice suffered from severe hyperglycemia
after 4 months Pigen diet, which was completely prevented by the knockdown of
FMO3 (TMAO synthase) [61]. Similar results were obtained on obese/insulin
resistant subjects, where FMO3 was significantly increased in the morbidly obese
group (P
The exact biological mechanism of how plasma TMAO concentration is involved in glucose metabolism remains to be clarified. PI3K is a key protein that transduces insulin signals into the regulation of glucose metabolism, while Akt is an important molecule downstream of the PI3K pathway, and its stimulation will in turn activate glycogen synthetase (a critical regulatory enzyme for the regulation of hepatic glycogen storage) [62]. In a study conducted by Gao it was shown that dietary TMAO reduces the mRNA levels of PI3K and Akt, indicating that dietary TMAO may exacerbate the blocked insulin signaling pathway [63].
Another mechanism involving TMAO promotes the regulation of transcription factor FoxO1 caused by PERK, and inhibition of FMO3 or manipulation of the gut microbiota can hinder metabolic dysfunction mediated by the PERK-FoxO1 axis [64]. Specifically, FoxO1 drives glucose gene expression and inhibited by insulin, the deletion of Foxo1 in the liver can reduce excessive glucose production caused by general ablation of insulin receptors [65]. PERK is a key sensor of intracellular stress and has previously been shown to induce FoxO1 by phosphorylation on serine 298 [66]. These data indicate that TMAO may be central to the pathogenesis of metabolic syndrome.
Research conducted by Yang observed a significant decrease in microbial
richness, diversity, and reproducibility in the spontaneously
hypertensive rat [67], suggesting that the intestinal flora may be involved in
the occurrence of hypertension, although the mechanism has not yet been fully
elucidated. Meta-studies have shown that for every 5
A different study demonstrated that patients with hypertension have an increased abundance of the cutC gene in their intestines, which in turn produces more TMAO [69]. The integrity of the intestinal barrier is essential for maintaining host health and preventing inflammation and atherosclerotic processes. Kinga Jaworska confirmed that the permeability of the colon to TMA in hypertensive patients is increased and promotes its penetration into the circulatory system [70], representing a novel point of view that the high concentration of TMAO in cardiovascular diseases may depend upon the increased permeability of the colon to TMA, and changes in colon permeability rather than TMAO levels are the indicators of cardiovascular risk [70].
The clearance of TMAO depends largely upon its excretion by the kidneys [71]. Serum TMAO concentrations substantially increase with deterioration of kidney function, and this effect is reversed by renal transplantation [72]. Meta-analyses of a total of 32 eligible clinical studies involving 42,062 participants showed that TMAO was strongly inversely correlated with glomerular filtration rate(GFR) and positively associated with the urine albumin-to-creatinine ratio, serum creatinine, and urine albumin excretion rates [73]. Jason R reported that the increase in TMAO concentration in CKD patients receiving coronary angiography was related to the load of coronary atherosclerosis and may be related to long-term mortality in CKD patients receiving coronary angiography [72]. Moreover, it was observed that TMAO predicts a poor overall survival rate in subjects with CKD [74, 75, 76].
TMAO is an independent predictor of CKD phase 3–5 mortality rate [77] and in
animal models, TMAO levels significantly increase renal fibrosis compared with
control (P
As discussed so far TMAO has a role in the pathological process of atherosclerosis, but can it also be used to clinically monitor the development and prognosis of patients with atherosclerosis.
TMAO is a significant predictor of carotid plaque formation which is more
significant than gender, diastolic blood pressure, total cholesterol, or diabetes
mellitus [80]. A study from southern Germany showed that higher TMAO levels can
predict thickness of the carotid intima-media (cIMT) [81]. TMAO is also an
independent predictor of new lesions as seen by MRI after carotid artery stenting
(CAS) [82]. In patients with severe carotid stenosis (
In patients with non-ST-segment elevation myocardial infarction (NSTEMI) and
ST-segment elevation myocardial infarction (STEMI), TMAO was found to be
independently related to the severity and prognosis of coronary atherosclerosis
load [83, 84, 85]. TMAO is also significantly related to the severity of the culprit
segment of calcification, including maximum calcification arc, maximum
calcification thickness, and calcification length, and can be used as an
indicator to the calcium load of the culprit plaque [86]. Studies have shown that
repeated rupture or erosion of a plaque followed by healing, is related to the
vulnerability of the plaque and a high level of inflammation, which helps to
increase the high risk of coronary thrombosis [87, 88]. The study reported that
in patients with STEMI, elevated TMAO levels indicated a higher SYNTAX score
(response to coronary atherosclerosis load) and the presence of multivascular
disease [89], and TMAO levels in patients with plaque rupture was significantly
higher than those with no rupture [90]. While TMAO levels in patients with
repaired plaques were significantly higher than those with unrepaired plaques,
and can be used as a potential biomarker to predict healed plaque presence with a
cutoff value of 2.9
TMAO not only plays a role in the process of primary atherosclerosis, but also contributes to late stent thrombosis after revascularization. A recent study found that an area under the curve (AUC) value for plasma TMAO of 0.85 can distinguish patients with new atherosclerosis and old atherosclerosis in STEMI patients who underwent PCI surgery, by using OCT to evaluate the culprit plaque [92]. The above studies indicate that high TMAO levels can be used as a useful biomarker to distinguish between plaque instability and rupture and represents a promising diagnostic marker for the prediction of new atherosclerosis in a stent.
TMAO can cross the blood-brain barrier and cause oxidative stress, thus
potentially leading to brain damage [93, 94]. Research has found that higher TMAO
levels are associated with increased risk of a first stroke [95, 96, 97], and are
closely related to a poor prognosis, such as post-stroke cognitive impairment
(PSCI). When predicting moderate to severe stroke, a critical value for plasma
TMAO levels has been found to be 4.95
Dr. Vichai Senthong first reported that elevated plasma TMAO level are an
important prognostic marker for patients with PAD [107]. In these patients, the
increase of TMAO levels was related to an increased risk of death by 2.7 times,
after adjusting for traditional risk factors, such as inflammatory biomarkers and
a history of coronary artery disease, where the highest quartile for TMAO can
still predict 5-year mortality [107]. PAD is characterized by atherosclerotic
stenosis of the lower extremity vessels, leading to ischemic muscle pain in the
elderly. Compared to patients with intermittent claudication, patients with
advanced chronic severe limb ischemia (CLI) showed higher serum carnitine and
TMAO levels and poorer long-term survival [108], whereas PAD patients with TMAO
Taken together the experimental data on TMAO it might be suggested that TMAO is a potent atherogenic cytokine and increased risk of developing atherosclerotic diseases such as CAS, coronary atherosclerosis, stroke, and PAD (Table 1, Ref. [80, 81, 82, 83, 84, 85, 86, 89, 90, 91, 92, 95, 96, 97, 98, 99, 100, 102, 103, 104, 105, 107, 108, 109]).
| TMAO’s significance | References | |
| CAS | TMAO is a significant independent predictor of carotid plaque burden. | [80] |
| Higher TMAO levels predicted increased cIMT. | [81] | |
| Plasma level of TMAO can be a predictor of ischemic brain injury secondary to CAS. | [82] | |
| The optimal cutoff value of TMAO in patients with |
[82] | |
| Coronary atherosclerosis | High Plasma TMAO is associated with coronary atherosclerotic burden in patients with STIME or NSTEMI. | [83],[84],[85],[89] |
| TMAO is positively correlated with the incidence of calcification in the culprit lesion segment. | [86] | |
| Plasma TMAO level is associated with plaque rupture. | [85],[90],[92] | |
| Plasma TMAO level is associated with healed culprit plaques. | [91] | |
| A cutoff value of TMAO was 2.9 µmol/L in patients with STEMI can be used as a potential biomarker to predict healed plaque presence. | [91] | |
| AUC value for plasma TMAO of 0.85 can distinguish patients with new atherosclerosis and old atherosclerosis in STEMI patients. | [92] | |
| Stroke | Serum TMAO concentration exhibited higher risk of first Stroke in Chinese patients. | [95],[96],[97] |
| TMAO level |
[96],[98] | |
| Higher TMAO levels correlate with worse neurological deficit . | [96],[100] | |
| TMAO is an independent predictor for cognitive impairment in post-stroke patients. | [99] | |
| TMAO is an independent predictor of functional outcome and mortality of patients with ischemic stroke. | [102],[103],[104] | |
| TMAO is also associated with poor prognosis in patients with cerebral hemorrhage at 3 months. | [105] | |
| PAD | TMAO is associated with PAD severity and prognosis. | [107],[108] |
| PAD patients with TMAO |
[109] |
Choline and carnitine are the main sources of TMAO production related to intestinal microbiota, therefore, diet regulation is a reasonable and cost-effective intervention strategy. Studies have shown that a healthy diet (vegetarian or low calorie diet) significantly improves TMAO levels [111, 112, 113]. Interestingly, microwave cooking can reduce the content of L-carnitine in some seafood [114]. In addition, exercise can change the diversity and distribution of the human microbial community [115], and voluntary exercise for 8 weeks can inhibit the increase in plasma TMAO in obese mice induced by a high-fat diet and prevent heart dysfunction also [116]. Some plant extracts and fruits, such as oolong tea [117], hawthorn [118], resveratrol [119], and ginkgolide B [120], have been shown to reduce the levels of circulating TMAO, which is beneficial for the improvement of atherosclerosis.
The use of prebiotics and probiotics may also help to produce a positive impact on the composition of the intestinal flora. Thus an in-depth study of the CutC/D gene responsible for the synthesis of TMA lyase and the development of a strain that antagonizes the synthesis pathway of TMAO represents a promising avenue of research [121]. For example, the TMA blockers DMB and IMC can inhibit the activity of microbial choline TMA enzymes in the body, increase the excretion of neutral sterols in mouse feces in the form of fecal sterols (bacterial metabolite of cholesterol), and prevent the accumulation of liver cholesterol driven by the diet [122]. Akkermansia muciniphila plays a protective role by improving intestinal barrier function to resist atherosclerosis [123]. Fecal microbiota transplantation (FMT) can reduce fecal TMA content and the content of serum TMAO [124]. These strategies that have focused on gut microbiota and microbial metabolites provide promising new insights into the treatment of atherosclerosis and CVDs.
Emerging data has established a direct link between diet, the intestinal flora metabolite TMAO, and atherosclerosis. TMAO is involved in the complex process of atherosclerosis and plays an important role in the pathogenesis of atherosclerosis caused by various risk factors such as diabetes, hypertension, and chronic kidney disease. In humans, it has been proven that TMAO is a reliable marker for predicting the development and prognosis of arterial vascular disease. Taken together this convincing evidence has emerged from preclinical studies suggest a strong relationship between TMAO and atherosclerosis. Further studies have focused on molecular mechanisms and the development of safe, effective, and feasible dietary strategies to use as an anti-atherosclerotic, involving TMAO reduction and this has led to potentially significant public health benefits. Innovative therapeutic approaches targeting gut microbiota and TMAO, including lifestyle modifications, TMA inhibitors, prebiotics, probiotics, and Chinese herbs, have shed new light on the great potential of targeting TMAO to elucidate the fundamental mechanisms underlying the disease. We propose to test TMAO levels clinically to fully assess its role as a potential marker for atherosclerosis.
GX and AY conceptualized the study and prepared the original draft wrote; GX and PL revised the manuscript; PL, LG and YW provided language help and writing assistance.
Not applicable.
Thanks to all those who helped us during the writing of this manuscript, and the peer reviewers for their opinions and suggestions.
Tianjin Postgraduate Research and Innovation Project (2020YJSB196): Study on the Mechanism of Jiangzhi Ruanmai Recipe in Improving Atherosclerosis by Regulating Cholesterol Reverse Transport.
The authors declare no conflict of interest.