Clinica Chimica Acta 507 (2020) 236–241

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Clinica Chimica Acta

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Review

Butyrate-producing bacteria and the gut-heart axis in atherosclerosis T

a,1 a,1 b a a,⁎ c,⁎
Wujun Chen , Shun Zhang , Jianfeng Wu , Ting Ye , Shuai Wang , Pan Wang ,
⁎
Dongming Xingd,a,
a
Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao Cancer Institute, Qingdao, Shandong 266071, China
b
Department of Cardiovascular Medicine, Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, The Second Affiliated Hospital of
University of South China, Hengyang, Hunan 421001, China
c
Department of Thoracic Surgery, National Cancer Center, National Clinical Research Center for Cancer, Cancer Hospital, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100021, China
d
School of Life Sciences, Tsinghua University, Beijing 100084, China

A R T I C LE I N FO A B S T R A C T

Keywords: The gut microbiota plays an important role in controlling atherosclerosis progression to support the link between
Butyrate the gut and coronary heart disease. Recent studies have shown that an imbalance in the gut-heart axis due to the
TMAO gut microbiota plays an important role in atherosclerosis progression. The gut microbiota promotes the devel-
LPS opment of atherosclerosis by producing intermediate metabolites, including TMAO, LPS, PAGln and reducing
PAGln
butyrate. TMAO and PAGln might be potential biomarkers of coronary heart disease. Many studies have shown
Gut-heart axis
that butyrate-producing bacteria prevent atherosclerosis progression by producing butyrate and maintaining the
Atherosclerosis
bacterial balance, the intestinal barrier function and the expression of various genes, including those encoding
lipids and those related to immunity, inflammation, differentiation, apoptosis, phagocytosis and efferocytosis.
This review focuses on recent advances in our understanding of the interplay between butyrate-producing
bacteria and the gut-heart axis in atherosclerosis.

1. Introduction
Atherosclerosis is a major factor in coronary heart disease (CHD),
which is characterized by the formation of fat-laden plaques in large
and medium-sized vessels, and is considered a chronic inflammatory
disease of the artery wall. The development of atherosclerosis is asso-
ciated with the recruitment of monocytes and their differentiation into
macrophages, and the resulting macrophages take up lipids to become
foam cells that affect the inflammatory response. The processes of foam
cell formation and chronic inflammation are regulated by a cholesterol
imbalance and the expression of inflammatory cytokines (e.g., IL-10
and IL-1β) [1,2], and the cholesterol balance is correlated with cho-
lesterol synthesis, uptake, esterification, and efflux. An uncontrolled
uptake of oxidized low-density lipoprotein (ox-LDL) by oxidized low-
density lipoprotein receptor 1 (LOX-1), SRA, and CD36, excessive
cholesterol esterification by acetyl-CoA acetyltransferase 1 (ACAT1)
and/or impaired cholesterol efflux by ATP-binding cassette transporter
A1 (ABCA1) and ABCG1 result in the accumulation of cholesterol esters
stored as cytoplasmic lipid droplets and subsequently trigger the
formation of foam cells. Ox-LDL also regulates initial monocyte re-
cruitment and the inflammatory response, including the expression of
interleukin-7 (IL-7), annexin A1 (ANXA1), and IL-15. Therefore, ox-LDL
plays a key role in foam cell formation and the inflammatory response
[3].
The gut microbiota in the gastrointestinal tract, which comprises
trillions of bacteria, is a complex community with metabolic activities
that contributes to education of the immune response by providing
essential nutrients and the maintenance of human health by protecting
against pathogens [4]. In addition, accumulating evidence suggests that
the gut microbiota plays an important role in controlling atherosclerosis
progression to support the link between the gut and CHD [5,6]. Bacteria
with the capacity to produce butyrate, trimethylamine-N-oxide
(TMAO), endotoxin (LPS) and phenylacetyl glutamine (PAGln) have
been found to play roles in the gut-heart axis in atherosclerosis (Fig. 1).
Many studies have shown that a reduction in butyrate in the gut pro-
motes local inflammation and foam cell formation, aggravates dysbiosis
and contributes to impairments in the gut barrier function, and the
latter exacerbates the release and invasion of bacterial toxins such as

⁎
Corresponding authors at: Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao Cancer Institute, Qingdao, Shandong
266071, China and School of Life Sciences, Tsinghua University, Beijing 100084, China.
E-mail addresses: sdwftcmws@163.com (S. Wang), wangpan@cicams.ac.cn (P. Wang), xdm_tsinghua@163.com (D. Xing).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.cca.2020.04.037
Received 16 March 2020; Received in revised form 29 April 2020; Accepted 29 April 2020
Available online 04 May 2020
0009-8981/ © 2020 Elsevier B.V. All rights reserved.
W. Chen, et al.
Fig 1. Role of microbial-derived substances in gut-heart axis crosstalk during atherosclerosis progression.
LPS and TMAO and thereby further fuels local and systemic in-
flammation [7,8]. This review focuses on recent advances in our un-
derstanding of the interplay between butyrate-producing bacteria and
the gut-heart axis in atherosclerosis.
2. Microbial-derived PAGln, LPS and TMAO impair the balance of
the gut-heart axis
The gut microbiota regulate human physiology through the release
of different classes of metabolites. An imbalance in the gut microbiota
produces many harmful substances, including TMAO, LPS, PAGln, in-
doxyl sulfate (IS), and p-cresylsulfate (PCS), and reduces many bene-
ficial substances, including short-chain fatty acids (SCFAs), particularly
butyrate [9,10]. As a result, the intestinal barrier is damaged, and large
amounts of toxic substances, such as PAGln, LPS and TMAO, enter the
blood and thereby influence known risk factors for atherosclerosis, such
as platelet invasiveness and thrombosis, foam cell formation, the in-
flammatory response, and oxidative stress. PAGln has been found to be
associated with cardiovascular disease and death in an independent
cohort (n = 4,000 subjects). The majority of ingested phenylalanine,
which is the precursor for PAGln, is absorbed in the small intestines, but
unabsorbed phenylalanine is metabolized to phenylpyruvic acid and
subsequently phenylacetic acid by the gut microbiota in the large in-
testines. Following absorption into the portal system, phenylacetic acid
is readily metabolized in the liver to produce PAGln, and PAGln in-
creases platelet responsiveness and thrombosis through G-protein cou-
pled receptors, including α2A, α2B, and β2-adrenergic receptors [10].
These results suggest that PAGln is a potential biomarker of cardio-
vascular disease. In addition, the LPS levels in 516 patients aged
50–79 years were measured, and the results showed that the risk of
incident atherosclerosis was increased three fold in patients with serum
LPS levels beyond the 90th percentile [11]. High LPS levels are asso-
ciated with obesity, metabolic syndrome, diabetes and CHD events, as
determined through a 10-year follow-up study of 2452 patients, and
these findings are independent of other established risk factors [12].
Thus, high serum LPS activity due to an imbalance in the gut microbiota
is associated with the development of atherosclerosis.
237
Clinica Chimica Acta 507 (2020) 236–241
Red meat has markedly higher levels of choline, phosphatidylcho-
line and L-carnitine, which are precursors of trimethylamine (TMA).
Thus, eating a high quantity of red meat promotes the expression of
TMA precursors by gut microbiota and directly contributes to athero-
sclerosis pathology. TMA is oxidized by FMO3 in the liver to TMAO
[13], and high TMAO levels were found to be associated with an in-
creased risk of incident major adverse cardiovascular events (MACEs) in
a cohort of 4007 patients who underwent coronary angiography and
were followed-up for 3 years [14]. In another study, high concentra-
tions of TMAO and its precursors were associated with increased risks of
MACEs and all-cause mortality in 19,256 patients, independent of tra-
ditional risk factors [15]. Another study with 26,167 patients found a
positive dose-dependent association among the TMAO plasma levels,
cardiovascular risk and mortality [16]. TMAO contributes to athero-
sclerosis development by interfering with cholesterol transport, en-
dothelial and smooth muscle cell activation, foam cell formation,
myocardial and inflammatory responses and platelet aggregation
[17–20]. Taken together, the results reveal that microbial-derived
TMAO, LPS and PAGln play a key role in gut-heart axis crosstalk during
atherosclerosis progression.
3. Butyrate-producing bacteria maintain balance of the gut-heart
axis
A reduction in dietary fiber results in a reduction in the bacterial
production of butyrate. Butyrate is the major source of energy to the
colonic mucosa needed for the maintenance of human gut health and is
an important regulator of the bacterial balance, the intestinal barrier
function and the expression of various genes, including those encoding
lipids and those related to immunity, inflammation, differentiation,
apoptosis, phagocytosis and efferocytosis. Butyrate enters colonic epi-
thelial cells via solute carrier (SLC) transporters and then induces the
upregulation of tight junctions and the maintenance of gut barrier
function. These functions inhibit permeability, and thus, fewer mole-
cules of LPS and other endotoxins, including TMAO, reach the general
circulation and induce systemic inflammation, which could regulate
macrophages and contribute to the formation of atherosclerotic plaques
W. Chen, et al. Clinica Chimica Acta 507 (2020) 236–241

Table 1 expression of SP1 by inhibiting HDACs, and these effects damage the
Butyrate-producing bacteria in the human gut. structure of the complex [29,30]. HDAC3 decreases ABCA1 expression
Bacterial species/group Clostridial cluster by reducing the LXRα promoter level [31]. SP1 binds to the promoter of
miR-27a and promotes the transcription and expression of miR-27a
Clostridium acetobutylicum I [32,33]. Previous studies conducted in our and other laboratories have
Clostridium butyricum I
demonstrated that ABCA1 is a target gene of miR-27a [34–38]. A
Clostridium saccharobutylicum I
Clostridium tyrobutyricum I
bioinformatics analysis using GeneCards showed that HDAC1, HDAC2
Anaerotruncus colihominis IV and HDAC3 exert potential effects in promoting the transcriptional
Butyricicoccus pullicaecorum IV regulation of miR-27a. Thus, butyrate increases ABCA1 expression by
Clostridium orbiscidens IV inhibiting the HDAC3/SP1-miR-27a pathway. Butyrate also induces
Faecalibacterium prausnitzii IV
macrophage phagocytosis and efferocytosis by increasing ANXA1 ex-
Papillibacter cinnamivorans IV
Subdoligranulum variabile IV pression [39–41], whereas miR-27a reduces ANXA1 expression [42].
Caldocellum saccharolyticum x However, the mechanism through which butyrate and miR-27a affect
Megasphaera elsdenii IX ANXA1 is unclear. Interestingly, an important recent study showed that
Eubacterium cylindroides XVI
ABCA1 is involved in ANXA1 expression and secretion [43,44]. In
Anaerostipes butyraticus XIVa
Anaerostipes caccae XIVa
macrophages and endocrine cells, ANXA1 secretion requires the action
Anaerostipes hadrus XIVa of ABCA1 [45]. ANXA1, which acts as an anti-inflammatory protein, is
Anaerostipes rhamnosivorans XIVa localized in macrophages and endothelial cells in human coronary
Butyrivibrio crossotus XIVa atherosclerotic plaques [46–48]. Previous studies conducted in our and
Butyrivibrio fibrisolvens XIVa
other laboratories demonstrated that ABCA1 combined with apoA-I
Butyrivibrio proteoclasticus XIVa
Coprococcus catus XIVa plays a critical role in cholesterol efflux and anti-inflammatory activity
Coprococcus comes XIVa [35,49,50]. Thus, further studies on ABCA1 are likely to shed important
Coprococcus eutactus XIVa light on potential therapeutic targets for cholesterol efflux and ANXA1-
Clostridium hathewayi XIVa mediated anti-inflammatory effects. The available evidence demon-
Clostridium indolis XIVa
Clostridium nexile XIVa
strates that butyrate increases ANXA1 expression by regulating the
Clostridium symbiosum XIVa HDAC3/SP1-miR-27a-ABCA1 pathway, and it should be noted that SP1
Eubacterium hallii XIVa is a target gene of miR-27a [51], which suggests that HDAC3/SP1-miR-
Eubacterium limosum XIVa 27a could form a negative feedback loop. In addition, the effects of
Eubacterium ramulus XIVa
butyrate on cells might be mediated by G protein-coupled receptors
Eubacterium rectale XIVa
Eubacterium ruminantium XIVa (GPRs), including GRP41, GPR43, and GPR109A. Interestingly,
Eubacterium ventriosum XIVa GPR109a differs from GPR41 and GPR43 in that it is activated by longer
Roseburia cecicola XIVa SCFAs, particularly butyrate, and is also activated by niacin [52,53].
Roseburia faecis XIVa GPR109a activation stimulates ABCA1 expression in macrophages and
Roseburia hominis XIVa
Roseburia intestinalis XIVa
then promotes apoA-I-mediated cholesterol efflux [54–56], which
Roseburia inulinivorans XIVa suggests that butyrate enhances ABCA1 expression by increasing
Ruminococcus gnavus XIVa GPR109A expression. Butyrate also increases the expression of peroxi-
Ruminococcus obeum XIVa some proliferator-activated receptor-gamma (PPARγ), which increases
SS3/4, GM2/1 XIVa
ABCA1 expression [57]. However, additional studies are needed to
Shuttleworthia satelles XIVa
SSC/2, SS2/1 XIVa elucidate the mechanism underlying the relationship between butyrate
and ABCA1.
Many butyrate-producing bacteria have been identified by culture-
[21,22]. Butyrate is mainly produced in the gut, and the concentration based approaches, DNA sequence analysis and sequence-based detec-
of butyrate in the blood passing from the intestinal tract to the liver, tion methods (Table 1) [52,58,59]. Butyrate-producing bacteria play a
heart, and lungs mimics the production rate in the large intestine. In- key role in human health, and these bacteria, including Roseburia in-
creased gut production of butyrate further increases the circulating testinalis, Butyrivibriocrossotus and Faecalibacterium cf. prausnitzii, are
level of butyrate [23]. Compared with fasted human subjects, nonfasted relatively depleted in atherosclerotic cardiovascular disease (ACVD)
subjects exhibit increased absolute and relative concentrations of bu- and type 2 diabetes (T2D) samples. The butyrate-producing bacterium
tyrate in portal blood [24]. Butyrate has been shown to regulate reverse Roseburia intestinalis is inversely correlated with atherosclerotic lesion
cholesterol transport by stimulating apoA-IV-containing lipoprotein development in mice, and the addition of Roseburia intestinalis in
secretion. Butyrate treatment also reduces cholesterol absorption by combination with a high-fiber diet reduces the sizes of atherosclerotic
increasing the transcriptional activity of liver X receptor (LXR), ATP- plaques in the aorta. Exogenous butyrate administered via the diet also
binding cassette transporters G5 and G8 (ABCG5 and G8) expression reduces the plaque sizes in apoE-/- and LDLR-/- mice [60]. Roseburia
and reducing Niemann-Pick C1-Like 1 (NPC1L1) in Caco-2 cells [25]. intestinalis improves the gut barrier function by increasing the butyrate
Butyrate influences lipoprotein assembly and secretion by altering the levels and then decreases the permeability, circulating LPS, systemic
membrane fluidity, the posttranscriptional modification of apolipo- inflammation (reduced proinflammatory cytokine expression in pla-
protein (apo) B 48 and 100, modulation of microsomal triglyceride (TG) ques), and macrophage infiltration into plaques. Akkermansia mucini-
transfer protein (MTP), the expression of sterol enzyme-encoding genes, phila also produces butyrate and decreases the size of plaques through
and lipoprotein secretion [26]. Butyrate increases ABCA1 expression in reductions in the intestinal permeability, foam cell formation, circu-
macrophages by regulating the Sp1 pathway but not LXRα [27]. lating LPS levels and inflammation in apoE-/- mice [61–64]. Faecali-
However, the mechanism through which butyrate affects SP1 and SP1 bacterium prausnitzii is an anti-inflammation-associated bacterium that
on ABCA1 is unclear. Interestingly, SP1 is often compounded with produces butyrate. Treatment with atorvastatin increases the abun-
HDACs and HATs to achieve the acetylation-mediated regulation of dance of Faecalibacterium prausnitzii in 27 hypercholesterolemic pa-
genes. The HDACs that form protein complexes with SP1 are mainly tients compared with that found in 15 untreated hypercholesterolemic
HDAC1, HDAC2 and HDAC3, and the HATs are mainly CBP/p300 and patients [65]. Clostridium butyricum promotes a hepatic antioxidant
PCAF [28]. Notably, butyrate is an inhibitor of HDACs and inhibits the status and decreases the levels of serum lipids and proinflammatory
cytokines by producing butyrate to enhance PPARγ. Clostridium

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Table 2
Main metabolites produced by the gut microbiota.
Metabolites Substrate Synthesis Reference

PAGln Phenylalanine Phenylalanine by gut microbiota → Phenylacetic acid (PAA) 10

Liver: PAA conjugation to the amino acid glutamine (Gln) of Gln PA-transferase and PA-CoA-ligase → PAGln
(major product in humans) or PAGly (major product in mice)
LPS A structural component of the microbiota itself 11, 12
TMAO Choline, phosphatidyl choline, L- Choline, phosphatidyl choline, and L-carnitine by gut microbiota → TMA 13–16
carnitine in red meat Liver: oxidation of TMA by FMO3 → TMAO
Butyrate Dietary fiber Dietary fiber fermentation by the gut microbiota → Butyrate 52, 58, 59

butyricum increases the abundance of the gut microbiota and particu- LXR Liver X receptor Cholesterol efflux
ABCG5 ATP-binding cassette transporters Cholesterol efflux
larly enriches butyrate-producing bacteria [66,67]. However, the role
G5
of Faecalibacterium prausnitzii and Clostridium butyricum in athero- ABCG8 ATP-binding cassette transporters Cholesterol efflux
sclerosis is unclear. The above-mentioned results indicate that butyrate G8
plays a key role in reducing atherosclerosis development and main- NPC1L1 Niemann-Pick C1-Like 1 Cholesterol uptake
taining the gut-heart axis balance via butyrate-producing bacteria. apoB Apolipoprotein B Chylomicron assembly and se-
cretion
MTP Microsomal triglyceride transfer Chylomicron assembly and se-
4. Summary protein cretion
HDAC Histone deacetylase Histone acetylation
Atherosclerosis is known as the main cause of mortality and mor- CBP/p300 CREB-binding protein Histone acetylation
PCAF p300/CBP-associated factor Histone acetylation
bidity in patients with coronary heart disease. Recent evidence shows
ApoA-I Apolipoprotein A-I Cholesterol efflux
that an imbalance in the gut-heart axis due to the gut microbiota plays ApoA-IV Apolipoprotein A-IV Cholesterol efflux
an important role in atherosclerosis progression. The gut microbiota GPRs G protein-coupled receptors Butyrate receptors
promotes the development of atherosclerosis by producing intermediate PPARγ Peroxisome proliferator-activated Cholesterol efflux
metabolites, including TMAO, LPS and PAGln, and reducing butyrate receptor-gamma

(Table 2). Butyrate-producing bacteria prevent atherosclerosis by pro-

ducing butyrate and maintaining the bacterial balance, the intestinal 6. Statement of ethics
barrier function and the expression of various genes, including those
that encode lipids and those related to immunity, inflammation, dif- The authors have no ethical conflicts to disclose.
ferentiation, apoptosis, phagocytosis and efferocytosis. Previous studies
have shown that butyrate-producing bacteria belonging to Clostridium Declaration of Competing Interest
cluster XIVa harbor the gene needed to metabolize precursors, in-
cluding choline, phosphatidylcholine and L-carnitine, into TMA and
The authors declare that they have no known competing financial
TMAO. These bacteria might be harmful if the diet (such as a red meat-
interests or personal relationships that could have appeared to influ-
based diet) is rich in choline, phosphatidylcholine, or carnitine. How-
ence the work reported in this paper.
ever, further studies in this research area must be performed. Butyrate-
producing bacteria might offer opportunities for manipulating nutrition
Acknowledgments
and opens up interesting new prospects related to the conservative
treatment of CHD, but extensive validation is needed. Future studies
We thank colleagues in Dr. Dongming Xing and Jianfeng Wu’s la-
should assess the role of more butyrate-producing bacteria in the
boratory for the technical help provided and the stimulating discussions
treatment of CHD.
during this investigation.
5. List of abbreviations
Funding

Abbreviation Full name Main function The authors are grateful for the financial support provided by the
Qingdao Major Scientific and Technological Project for Distinguished
CHD Coronary heart disease Scholars (20170103), the Laoshan Major Scientific and Technological
IL-1β Interleukin 1β Inflammatory response
IL-10 Interleukin 1β Inflammatory response
Project for Distinguished Scholars (20181030), the China Postdoctoral
ox-LDL Oxidized low-density lipoprotein Foam cell formation and in- Science Foundation (2019M652331, 2018M642619), the Qingdao
flammatory response Postdoctoral Application Project (RZ1900013298, 2018121236,
LOX-1 Low-density lipoprotein receptor Cholesterol uptake 2018121238), the Department of Health of Shandong Province
1
(2018WS068), and the Department of Health of Hunan Province
SRA Cholesterol uptake
CD36 Cholesterol uptake (20201533).
ACAT1 Acetyl-CoA acetyltransferase 1 Cholesterol esterification
ABCA1 ATP-binding cassette transporter Cholesterol efflux References
A1
ABCG1 ATP-binding cassette transporter Cholesterol efflux
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