The role of intestinal microbiota in cardiovascular disease

Abstract Accumulating evidence has indicated that intestinal microbiota is involved in the development of various human diseases, including cardiovascular diseases (CVDs). In the recent years, both human and animal experiments have revealed that alterations in the composition and function of intestinal flora, recognized as gut microflora dysbiosis, can accelerate the progression of CVDs. Moreover, intestinal flora metabolizes the diet ingested by the host into a series of metabolites, including trimethylamine N‐oxide, short chain fatty acids, secondary bile acid and indoxyl sulfate, which affects the host physiological processes by activation of numerous signalling pathways. The aim of this review was to summarize the role of gut microbiota in the pathogenesis of CVDs, including coronary artery disease, hypertension and heart failure, which may provide valuable insights into potential therapeutic strategies for CVD that involve interfering with the composition, function and metabolites of the intestinal flora.

anatomical locations of the gut does not change significantly. Eckburg et al performed metagenomic analysis to determine that the gut microbial community consists of six families, namely the Firmicutes, Bacteroidetes, Proteobacteria, Actino-bacteria, Fusobacteria and Verrucomicrobia phyla, the majority of which are anaerobic organisms. 15 In the healthy bacterial community, the phyla Firmicutes and Bacteroidetes are the main dominant flora, accounting for more than 90% of the total population. 16 Unlike the composition of the intestinal microflora, the numbers of microbes residing in different regions of the gut vary greatly. The ascending colon has the highest number of microorganisms, followed by the distal ileum with 10 11 cells/g and 10 7-8 cells/g microorganisms respectively. The content in the proximal ileum and jejunum is less, only 10 2-3 cells/g. 17 The host provides a proper environment and essential nutrients for the intestinal microflora. In turn, the intestinal microflora is involved in the regulation of various human functions, such as providing metabolic nutrition to the host, participating in growth promotion and immune regulation, eliminating pathogenic microorganisms and maintaining the integrity of intestinal barriers and normal homeostasis. 18 Intestinal microbial ecology can be affected by improper dietary patterns, high stress, life events and use of antibiotics, leading to gut dysbiosis. 12

| Coronary artery disease
The composition and functions of the gut microbiome are affected by external factors that are associated with increased CVD risks, including aging, obesity, a sedentary lifestyle and dietary patterns. In turn, the composition of the gut microbiome may affect the development of CVD. 19 The observation that DNA from various species of bacteria is found in atherosclerotic lesions and in the gut of the same individuals suggests the gut microbiota may be a potential source of atherosclerotic bacteria and is therefore likely to participate in the pathogenesis of coronary artery disease. 20,21 Jie et al demonstrated the relationship between the gut microbiota and atherosclerotic CVD. 9 They identified that the composition of gut microbiome, including members of the Enterobacteriaceae and Streptococcus spp, was higher in atherosclerotic CVD than in the healthy controls. 9 Karlsson et al used shotgun sequencing of the gut metagenome to reveal that intestinal microbial communities in patients with symptomatic atherosclerosis were different from those in healthy controls. 22 Patients had increased numbers of the genus Collinsella, while the gender-and age-matched controls had an increased abundance of Eubacterium and Roseburia. 22 Other evidence in humans also implicated the role of the gut microbiota in the development of atherosclerosis. 23,24 In addition to these studies in humans, there are a series of animal studies. Chan  They fed germ-free ApoE−/− mice a low-or high-cholesterol diet for 3-4 months. Atherosclerotic plaques were detected in the aorta of germ-free mice fed the low-cholesterol diet. Their study supports the protective effect of bacteria on atherosclerosis. Conversely, Kasahara et al showed that the absence of microbiota could cause an increase in atherosclerotic lesion formation compared with conventionally raised controls. 27 Other bacteria, including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, have been validated to be associated with the acceleration of atherosclerosis in animal models after dietary intervention or intravenous infusion. [28][29][30] In the light of these findings, some types of gut bacteria have been identified as novel contributors to the progression of atherosclerosis, while others can protect against atherosclerotic plaque lesions. It is still unknown how the microbes that reside within our bodies can drive or initiate the development of atherosclerosis. It is unclear which species play a leading role in contributing to CVD and the detailed mechanisms involved require further investigation.
One microbial metabolite, trimethylamine N-oxide (TMAO), has gained considerable attention as a major influencing factor in CVD.
The composition of the gut microbiota is altered when dietary patterns change. Trimethylamine (TMA) is generated by the altered microbiota through metabolizing choline, 31 phosphatidylcholine, 32 L-carnitine 33 and betaine 34 via a range of microbial enzymes, primarily TMA lyases.
Then TMA enters the liver through the portal circulation and is oxi- Recently, a series of clinical trials showed the relationship between gut microbiota and CVD events. A study by Li et al revealed that the TMAO level in acute coronary syndromes was an independent predictor of both short-term (30 days and 6 months) and longterm (7-year) major adverse cardiac events. 41 Other studies have also highlighted the participation of TMAO in the development of CVD in a variety of patient cohorts. [42][43][44] Collectively, mounting evidence suggests that TMAO is part of an important mechanism by which the intestinal microflora influence CVD.

| Hypertension
The gut microbiota consists of four major phyla: Firmicutes,

Bacteroidetes, Actinobacteria and Proteobacteria. Firmicutes and
Bacteroidetes account for a large part of the intestinal microflora.

The ratio of Firmicutes (F) and Bacteroidetes (B) (F/B) is considered a
biomarker for gut dysbiosis. 45 Yang et al demonstrated that microbial richness, diversity and evenness were decreased not only in spontaneously hypertensive rat models but also in a cohort of patients with high blood pressure. 46 Additionally, an increased F/B ratio and decreased numbers of acetate-and butyrate-producing bacteria were observed.
In Ang II-infused rats, minocycline intervention was able to lower the blood pressure and induce changes such as increased gut microbial diversity, decreased F/B ratio and expanded populations of acetate-and butyrate-producing bacteria. This indicates that hypertension is linked to gut dysbiosis and that improving gut microbiota may be a target for future therapies for hypertension. Adnan et al 10 found that blood pressure can be altered through exchanging the gut microbiota between spontaneously hypertension/stroke-prone rats (SHRSP) and Wistar-Kyoto rats (WKY). Systolic blood pressure and F/B ratio were both increased in WKY rats after gavage with SHRSP microbiota. Conversely, systolic blood pressure was decreased in SHRSP rats after gavage with WKY microbiota, although this was not statistically significant. Similarly, the importance of gut microbiota in hypertension formation is evident from a study showing elevated blood pressure in germ-free mice after transferring faecal material from hypertensive patients to the mice. 47 A recent study demonstrated that there were more opportunistic pathogenic taxa (Klebsiella spp, Streptococcus spp and Parabacteroides merdae) involved in the pathogenesis of hypertension and that these were related to the severity of disease. 48  Olfr78 is expressed in olfactory neurons, renal afferent arterioles as well as in vascular smooth muscle cells, where it plays a role in blood pressure regulation. [54][55][56] Olfr78 was found to elevate renin levels, resulting in increased blood pressure, while GPR41 had an antagonistic effect. 55 Pluznick et al treated Olfr78 ˗/˗ and wild-type mice with antibiotics for a set time period to determine whether metabolites from the microbiota mediate blood pressure via Olfr78. 55 The results showed that blood pressure was increased in Olfr78 -/mice after antibiotic treatment, but no effect was seen in the control mice. This indicated that Olfr78 contributes to the hypertensive effects by means of SCFAs. Pluznick et al indicated that GPR41 and Olfr78 had opposing functions in the modulation of blood pressure after responding to propionate (a type of SCFA). 56 In addition, SCFAs have also been shown to induce vasorelaxation, 57 while other studies have revealed that GRP41 appears to decrease cAMP levels through Gαi. 58,59 Thus, blood pressure is closely linked to the diversity, richness and evenness of the microbiome living in the gut and it is affected by the F/B ratio. The hypertensive and hypotensive effects of SCFAs are mediated by binding to Olfr78 and GPR41 respectively. The discovery that drug intervention has an influence on blood pressure modulation by changing the species as well as the metabolites of the gut microbiota may provide new ideas for the treatment of hypertension. Moreover, how the intestinal microflora influences blood pressure deserves further exploration. Collectively, changes in the intestinal microflora exist in patients with HF. The aforementioned metabolite TMAO generated by the gut microbiota has a certain significance in HF patients. Two cohort studies, which enrolled hundreds of participants, demonstrated that elevated TMAO levels were predictive of the long-term mortality risk in patients suffering from not only CHF, 69 but also acute HF. 70 TMAO is likely to provide a basis for risk stratification of HF.

| Heart failure
Organ et al used transverse aortic constriction surgery to induce HF in C57BL6/J mice and found that in mice fed with either TMAO or choline supplemented diets, worse symptoms and signs of HF were observed compared with mice fed a control diet. 66 71 The mechanism of increased circulating TMAO levels in patients with HF remains to be determined. Some other gut-derived metabolites have also been shown to have an impact on HF. Secondary bile acid, transformed by the gut microbiota, was reported to increase in CHF patients, 64 and indoxyl sulfate has been linked with myocardial fibrosis and ventricular remodelling. 72 In addition to gut microbiota metabolites mentioned above, p-cresyl sulfate (PCS) and phenylacetylglutamine (PAG) are involved in CVDs as well. 73,74 PCS is a component of phenolic end products generated by gut microorganism via metabolizing aromatic amino acids, like tyrosine and phenylalanine, in the intestine. 75 PCS levels have been shown to predict cardiovascular events and all-cause mortality in elderly haemodialysis patients. 73 Likewise, PAG is one of the colonic microbial metabolites produced by glutamine conjugation of phenylacetic acid, high levels of which were known as a strong and independent risk factor for CVD and mortality in patients with chronic kidney disease. 74

| THER APEUTI C S BA S ED ON THE MICROB IOTA
Recent studies have shown that intestinal microbiota is critically involved in cardiovascular health and diseases. 76,77 For the treatment of CVD, researchers have gradually turned their attention to the intestinal microflora and related metabolites. Consequently, the gut microbiome, as a novel regulator of CVD, has become a potential target for therapeutics.

| Antibiotics
Broad-spectrum antibiotics are commonly used in cardiovascular experiments targeting the gut microbiota. Galla et al administered three types of oral antibiotics (neomycin, minocycline and vancomycin) to Dahl salt-sensitive (S) rats and spontaneously hypertensive rats (SHR) to investigate any changes in blood pressure. They found alterations in the intestinal microflora accompanied by increased systolic blood pressure in the S rats and decreased systolic blood pressure in the SHR after minocycline and vancomycin intervention, but not neomycin intervention. 78 Rune et al found that ampicillin could reduce LDL and VLDL cholesterol levels, which were risk factors for atherosclerosis in the mice model, leading to decreased aortic atherosclerotic lesion areas. 79 In trials on patients, whether the use of antibiotics has protective effects against CVD remains unanswered. Some studies showed beneficial effects, 71 while others did not. 80 Therefore, strategies for treating CVD with antibiotics remain controversial, because broad-spectrum antibiotics mediate a wide range of effects. Thus, the potential benefits of antibiotics need to be weighed against the potential side effects.

| Faecal microbiota transplantation
Faecal microbiota transplantation (FMT) is capable of contributing nutrients, inhibiting the growth of pathogenic bacteria and regulating the immune system of the host through transplanting functional bacteria from healthy individuals into the gastrointestinal tract of patients, thereby helping patients reconstruct the normal functions of the gut microbiota. 81 FMT is effective in treating recurrent Clostridium difficile infection, 82 inflammatory bowel disease 83 and irritable bowel syndrome. 84 However, the potential for treating CVD needs further investigation.
In a double-blind randomized controlled pilot study, the composition of the gut microbiota in metabolic syndrome patients changed significantly after transplantation of vegan-donor faecal microbiota. 85 However, there was no change in the gut-derived metabolite TMAO. As mentioned above, germ-free mice showed increased blood pressure following FMT from patients with hypertension. 47 For now, FMT remains a promising therapy for CVD, although more studies are needed.

| Dietary intervention
Several studies have examined the effects of diet on the gut flora and disease by administering mice a high-salt diet (HSD). [91][92][93] Bier et al investigated the relationship between a HSD and microbial variation, as well as metabolite levels. 91 The composition of the gut microbiota accompanied by SCFAs was shown to be altered in the HSD-fed mice, inducing hypertension. Three genera including the members of the families Erwinia, Christensenellaceae and Corynebacteriaceae were significantly increased in the HSD-fed mice compared with mice fed the control diet, while a reduction in the genera Anaerostipes was also observed in the HSD-fed mice. Moreover, seven taxa were found to be associated with blood pressure. Marques et al treated mice with a high-fibre diet or a diet supplemented with acetate, leading to a prominent alteration in gut microbes and elevation of SCFAs levels, which had a protective effect on hypertension and HF. 94 Furthermore, resveratrol, found in grapes and berries, had favourable effects on atherosclerosis via attenuating TMAO by inhibiting TMA generation. 95 Thus, it is important for patients to make adjustments for their diet to delay the progression of CVD.

| OTHER THER APIE S
Some research has been conducted on protecting cardiovascular system with chemicals that inhibit microbial metabolic processes.
For example, 3, 3-dimethyl-1-butanol, a non-lethal inhibitor of TMA formation, reduced atherosclerotic lesions by decreasing the levels of TMAO that were converted from TMA in ApoE−/− mice. 96 To the best of our knowledge, TMA is oxidized into TMAO by hepatic FMO3. Therefore, some studies have focused on chemical substances that could act as potential FMO3 inhibitors to prevent or treat atherosclerosis. [97][98][99] Gao et al found that the binding of methimazole and indole could provide evidence for the development of human FMO3 inhibitors. 97 Indole-3-carbinol (I3C) and its acid condensation products, I33' and LT, were reportedly responsible for the inhibitory activity of human FMO3. 98 Therapeutically, the aim was to develop targeted drugs for the intervention of certain CVDs.

| SUMMARY
Trillions of bacteria reside in the human gut, mainly divided into probiotics, neutral bacteria and pathogenic bacteria. Among

ACK N OWLED G EM ENTS
This study was financially supported by the National Natural Science Foundation of China (no. 81770370) and Scientific Research Program for Young Talents of China National Nuclear Corporation (no. 51001).

CO N FLI C T S O F I NTE R E S T
The authors declare that there are no conflicts of interest.