Gut microbiota and atherosclerotic cardiovascular diseases. Prevention of atherosclerosis via altering gut microbiota or modulating its products.

Abstract

Cardiovascular diseases (CVDs) have become a major health problem, because of the associated high morbidity and mortality among patients. Gut microbiota have been recently implicated as a novel endocrine organ that play critical roles in regulating the host cardiometabolic function through producing bioactive metabolites. This review investigated the evidence from several clinical and experimental studies indicating an association between gut microbiota and atherosclerotic CVDs. We mainly focused on the anti-atherogenic gram-negative bacteria, Bacteroides vulgatus and dorei, and their producing lipopolysaccharide. Further, we would pay attention to gut microbiota-derived deleterious metabolites, trimethylamine N-oxide, and described the present status of its related research. We believe some of the methods to change the gut microbiota or reduce the deleterious metabolites could be clinically applied to prevent CVDs in the near future.

 1. Introduction

Cardiovascular diseases (CVDs) remain the leading cause of death in developed countries. Although the gut is not the first organ that we would think about the pathophysiology of CVDs, it is not only the organ handles digestion and absorption but also the largest immunologically active organ in the body. The gut regulates the differentiation of immune cells and protects the host from invading microorganism, which are collectively known as the gut microbiota. The host-gut microbiota interaction has been the focus of increasing interest in recent years. Although these microorganisms sometimes cause infectious diseases, they also have beneficial functions for host including nutrient absorption and vitamin production. Recent studies demonstrated that gut microbiota produces and releases many metabolites and toxins, some of which are absorbed into the host systemic circulation and serve as a mediator of microbial influence on the host. Thus, the gut microbiota works as virtual endocrine organs, communicates with host distal organs including heart and vasculature through metabolism- and immune-dependent pathways. Dysbiosis, imbalanced condition of gut microbiota, is shown to be related with host many diseases including CVDs. This review aims to highlight the relationship between gut microbiota and CVDs with emphasis on the possible molecular mechanism.

 2-1. Coronary artery disease and gut microbiota

Several studies have been conducted to elucidate which gut microbiota is involved in the incidence and progression of atherosclerotic CVD (Table 1)^1–5). At first, we tried to clarify the specific profile of gut microbiota in coronary artery disease (CAD) patients to investigate a diagnostic and therapeutic potential of gut microbiota³⁾. We used terminal restriction fragment length polymorphism (T-RFLP) analysis to detect the profile of gut microbiota in CAD and control patients who have diabetes, hypertension, and/or dyslipidemia.

Table 1 

Characteristics of gut microbiota in atherosclerotic cardiovascular diseases.

Year (Reference No.)	Study population	Country	Analytic method	Results
2012 Nat commun. Karlsson FH et al. (1)	12 patients with cerebrovascular symptomatic atherosclerosis and 13 healthy conrols	Sweden	Metagenome shotgun sequencing	Collinsella ↑, Eubacterium ↓, Roseburia ↓ in patients with symptomatic atherosclerosis
2015 J Am Heart Assoc. Yin J et al. (2)	141 patinets with stroke/transient ischemic attack (TIA) and 94 controls	China	16SrRNA random sequencing	Enterobacter ↑, Magasphaera ↑, Oscillibacter ↑, Desulfovbrio ↑, Bacteroides ↓, Prevotella ↓, Faecalibacterium ↓ in stroke/TIA patients.
2016 J Atheroscler Thromb. Emoto T et al. (3) (2018 Circulation Yoshida N et al.) (5)	“39 coronary artery disease (CAD) patients, 30 no-CAD controls with coronary risk factors, and 50 healthy volunteers.”	Japan	Terminal restriction fragment length polymorphism (16SrRNA random sequencing)	Lactobacillales ↑, Bacteroidetes ↓ (Bacteroides vulgatus and Bacteroides dorei ↓) in CAD patients
2017 Nat commun. Jie Z et al. (4)	218 patinets with atherosclerotic cardiovascular disease (ACVD) and 187 healthy controls.	China	Metagenome shotgun sequencing	Enterobacteriaceae ↑, Streptococcus ↑, Lactobacillus salivarius ↑, Solobacterium moorei ↑, Atopobium parvulum ↑, Ruminococcus gnavus ↑, Roseburia intestinalis ↓, Faecalibacterium prausnitzii ↓, Bacteroides ↓, Prevotella copri ↓ in ACVD patients.

We found that the order Lactobacillales (Lactobacillus, Streptococcus, and Enterococcus) was significantly increased and the phylum Bacteroidetes (Bacteroides + Prevotella) was significantly decreased in the CAD group compared with the Control group (Figure 1A).

Figure 1 

Characteristics of gut microbiota in coronary artery diseases (CAD).

A. Phylum Bacteroidetes is decreased in CAD patients.

B. Several papers indicated Bacteroides species are decreased in atherosclerotic CVD and obesity.

C. We found Bacteroides vulgatus and dorei were decreased in CAD patients. *p<0.05

Another paper based on a shotgun sequencing metagenome-wide association study of fecal samples from 218 CAD patients and 187 healthy subjects in China was reported⁴⁾. The abundance of the genus Streptococcus was significantly higher and the abundance of the genus Bacteroides was found to be decreased in the patients with CAD than in the healthy subjects. These results were almost the same as ours. Given that Bacteroides species are known to have an important role in maintaining a healthy gut ecosystem⁶⁾, and that the abundance of Bacteroides species was found to decrease in patients with atherosclerotic ischemic stroke and transient ischemic attack (TIA)²⁾ and obesity⁷⁾ (Figure 1B), we hypothesized that some of the Bacteroides species may have the potential to regulate or may help to prevent coronary atherosclerosis progression. We further analyzed the gut microbiota using 16S ribosomal RNA random sequencing and found that Bacteroides (B.) vulgatus and dorei were significantly decreased in CAD patients compared with controls⁵⁾ (Figure 1C).

 2-2. Bacteroides treatment could inhibit atherosclerosis in mice.

To investigate the effect of oral administration of B. vulgatus and B. dorei on atherosclerotic lesion formation in atherosclerosis-prone apolipoprotein E-deficient mice, we gavage the cultured Bacteroides 2 species and assessed the lesion. Oral administration of Bacteroides 2 species significantly reduced atherosclerotic lesion⁵⁾ (Figure 2A). Taken these, we could say that Bacteroides 2 species are anti-atherogenic bacteria at least in mice. To clarify the anti-atherogenic mechanisms, we assessed the inflammatory status of Bacteroides 2 species-treated mice and found decreasing plasma TNF-α levels and lipopolysaccharide(LPS) activities determined by the limulus amoebocyte tests⁵⁾ (Figure 2B).

Figure 2 

Effects of treating Bacteroides (B) species on mouse atherosclerotic lesion formation and structure of LPS and lipid A.

A and B. Oral administration of B vulgatus and B dorei prevented atherosclerosis in apolipoprotein E-deficient mice, and reduced the plasma and fecal LPS activities. *p<0.05, **p<0.01

C. Oral administration of B vulgatus and B dorei decreased the bacterial LPS biosynthesis gene, especially lipid A, in mice.

*p<0.05

D. LPS consists of O-antigens, inner and outer core polysaccharides, and lipid A moiety. Escherichia coli has hexa-acylated lipid A that induces strong immune responses via activating TLR4. Bacteroides species have tetra- or penta-acylated lipid A which elicits less inflammation.

Bacteroides is dominant gram-negative rod bacteria in human gut and has LPS in its body. Therefore, we measured fecal LPS levels as an indicator of LPS produced by gut microbiota and found that the fecal LPS activities were significantly lower in mice gavaged with Bacteroides 2 species than in control mice (Figure 2B). We next predicted gut bacterial functional genes based on 16S rRNA gene sequence using PICRUSt and found that the expression of genes involved in LPS biosynthesis, especially lipid A biosynthesis gene, was significantly decreased in mice gavaged with Bacteroides 2 species (Figure 2C).

The basic chemical structure of LPS is described as hydrophilic sugar moieties bound to a hydrophobic region known as lipid A⁸⁾ (Figure 2D). The hydrophilic region of LPS is composed of the O-antigens (species-specific repeating oligosaccharide subunits) and the core polysaccharide, and this region has only a minimal effect on the pro-inflammatory activity of LPS. On the other hand, the hydrophobic lipid A region is structurally conserved among various gram-negative bacteria, and consists of a phosphorylated diglucosamine backbone with four to seven attached acyl chains⁸⁾. The lipid A, the ligand of toll-like receptor 4 (TLR4) and the most critical toxic portion of LPS, activates the innate immune system including monocyte-macrophage and provokes the inflammatory action in the host^9,10). More interestingly, structure of lipid A moieties of LPS are different among bacterial species and the number of acyl chains are critical for activating TLR4. This structural difference might mainly determine LPS activity^8,9). Generally, the tetra- and penta-acylated lipid A moieties, for example the LPS derived from B. vulgatus and B. dorei, elicits reduced TLR4 responses compared to the E. coli-derived hexa-acylated lipid A moiety^9,10) (Figure 2D).

Our research findings suggest that Bacteroides treatment may serve as a novel and attractive therapeutic strategy for suppressing inflammatory response in CVD, and pave the way for further studies investigating fecal LPS levels and prevention of CVD.

 2-3. Future perspective on clinical application of LPS research findings

Accumulating evidence suggest that systemic endotoxemia and gut microbiota-derived LPS are involved in the onset and progression of not only CVD but also many inflammatory diseases, such as inflammatory bowel disease, obesity and related metabolic diseases^10,11). High fat diet definitely increases plasma LPS levels at least in mice and plasma LPS is basically considered as “toxin” in inflammatory diseases, and is involved in the pathophysiology of progression of CVD. Until now, therapy which directly reduces blood or fecal LPS do not exist. Development of clinical application of managing endotoxemia or gut microbial modulation using microbial drugs could be a novel therapeutic option.

 3-1. Trimethylamine N-oxide (TMAO) as a risk for CVDs

Dr. Hazen and his colleagues reported that the gut derived metabolite, trimethylamine N-oxide (TMAO), is an independent predictor for cardiovascular events in a large clinical cohort of patients with CVDs by using a metabolomics approach^12,13). Phosphatidylcholine, a dietary component found in food sources such as cheese, egg, and meat, is converted to choline in the gut, and subsequently metabolized to trimethylamine (TMA) by gut microbial enzyme, TMA lyase. TMA is absorbed from the gut into portal vein and then converted to TMAO via flavin-containing monooxygenases, a host liver enzyme¹²⁾. L-carnitine, an abundant nutrient in red meat containing a TMA structure similar to that of choline, is also the source of TMA and contributes the elevation of plasma TMAO and accelerates atherosclerosis¹⁴⁾. TMAO is shown to be elevated in cardiovascular patients with CAD, thrombosis¹⁵⁾, chronic kidney disease¹⁶⁾ and heart failure (HF)¹⁷⁾ and linked to adverse cardiovascular events and all-cause mortality¹³⁾ (Figure 3).

Figure 3 

Metabolism of gut microbiota-derived TMAO and its biological functions.

This panel indicates the metabolic pathways of producing TMA by gut microbial enzymes and TMAO from TMA by our host liver enzyme. TMAO was shown to induce deleterious effects on CVD including heart failure.

Oral administration of phosphatidylcholine promoted up-regulation of multiple macrophage scavenger receptors and enhanced atherosclerosis in apolipoprotein E-deficient mice, and additional treatment with antibiotics could cancel the deleterious effects. TMAO was shown to increase the form cell formation, decrease the cholesterol reverse transport, and activate the platelet aggregation^12,15). Taken these, TMAO is thought to aggravate atherosclerosis and also increase the cardiovascular events, and it is reasonable to conclude that TMAO acts as a gut microbiota-derived uremic or cardiovascular toxin contributing to systemic inflammation. However, the cause and result relationship of increasing the plasma TMAO levels and worsening CVDs have not been clarified yet. Further, the intervention to TMAO could improve the prognosis of CVDs also still remains unknown.

 3-2. The composition of gut microbiota in HF and plasma TMAO

Alterations in gut microbial composition have been described in patients with HF^18–22), especially with reduced diversity and depletion of core gut microbiota. However, the causal involvement of gut microbiota composition and HF pathophysiology remains unclear. We can suspect that heterogeneity of HF makes us difficult to focus on specific gut microbiota from the data of comparison between controls and patients with HF and that intestinal edema caused by HF affects the composition of gut microbiota²¹⁾. At the genus level, Bifidobacterium, Gordonibacter, and Bilophila was abundant in HF patients compared with controls²²⁾. We also assessed the plasma-related metabolites both in patients with decompensated HF when admitted to the hospital and in the same patients after treating HF, compensated phase. Plasma TMAO concentrations were elevated in HF patients compared with controls, but there were no significant difference between decompensated and compensated phase of HF, which was completely different pattern of plasma brain natriuretic peptide (BNP), sophisticated biomarker of HF²¹⁾. These data suggested that TMAO elevation was neither temporal nor was a therapeutic target after hospitalization to prevent a next event of HF. Although it was difficult to assess how this change of gut microbiota affected plasma TMAO, we assessed three key microbial functional genes involved in the production of TMA using shotgun sequencing metagenome analysis. CutC/D, bacterial genes of choline TMA lyase, were shown to be increase in HF patients compared with controls²³⁾, but cntA/B, genes of carnitine TMA lyase, and genes of betaine reductase were not changed (Figure 3). Bliophila, increased in HF patients in our study²²⁾, is shown to have cutC/D and probably produces TMA by choline TMA lyase. Therefore, the increasing Bilophila might be related with the high plasma TMAO levels in HF patients. Although our data was confused, additional analysis of shotgun sequencing metagenome analysis revealed that the amount of cntA/B genes (but not cutC/D genes) was correlated with plasma TMAO in HF patients and it probably provoked the most critical contribution to the increase in TMAO levels in HF²³⁾. Genus Escherichia and Klebsiella have cntA/B and depletion of these bacteria might be effective to reduce the plasma TMAO levels in HF patients. We should clarify what kinds of bacteria are involved in increasing TMAO in HF and how to intervene the bacteria for decreasing the plasma TMAO.

 3-3. Therapeutic candidates to reduce plasma TMAO production

From clinical perspective, a new intervention to suppress plasma TMAO in patients with CVDs is desired. A natural structural analog of choline, 3,3-dimethyl-1-butanol (DMB), has been shown to non-lethally inhibit TMA production from cultured bacteria²⁴⁾. However, despite the high dose of DMB provided, choline induced TMAO elevations, increased platelet aggregation, and shortened thrombus formation time were not fully rescued in mice. They, therefore, tried to develop second-generation TMA lyase inhibitors with improved therapeutic potential²⁵⁾. Especially, a new choline TMA lyase inhibitor, iodomethylcholine, improved remodeling and cardiac function in heart failure murine model with transverse aortic constriction²⁶⁾.

Probiotics treatment or alteration of gut microbiota component are also another option to reduce plasma TMAO by suppressing TMA production in the gut via modulation of gut microbiota or gut microbiota-derived metabolites.

 4. Summary and Conclusion

Many human and animal studies support that gut microbiota and their products can influence host health and diseases. The identification of bacterial function that can modulate host physiological and pathophysiological processes has opened the possibility for numerous microbial pathways as therapeutic targets for preventing CVDs. Especially, to remove or reduce the detrimental bacterial products, LPS and TMAO, could be hopeful and effective therapeutic strategies to improve the prognosis of CVD patients. However, we have not yet clarified how the toxin levels are regulated, decided, and can be changed. Taken these, further studies are needed to clear the causal relationship between the gut microbiota and CVDs, to know whether the intervention can be effective or not, and what kind of intervention can be therapeutic method. We hope future active studies in association with gut microbiota and their metabolites could improve the prognosis of CVD patients.

 

Sources of Funding: This work is supported by the PRIME from the Japan Agency for Medical Research and Development (18069370; T.Y.), the Japan Society for the Promotion of Science KAKENHI (grant No. 17K09497, 20H03676; K.H., 19H03653, 20K21603; T.Y.), and Senshin Medical Research Foundation (T.Y.).

 

Disclosures: Authors have no potential conflicts of interest to declare.

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