Circulation Research

REVIEW

Gut Microbiota and Cardiovascular Disease

Marco Witkowski, Taylor L. Weeks, Stanley L. Hazen

ABSTRACT: Fecal microbial community changes are associated with numerous disease states, including cardiovascular disease
(CVD). However, such data are merely associative. A causal contribution for gut microbiota in CVD has been further supported
by a multitude of more direct experimental evidence. Indeed, gut microbiota transplantation studies, specific gut microbiota–
dependent pathways, and downstream metabolites have all been shown to influence host metabolism and CVD, sometimes
through specific identified host receptors. Multiple metaorganismal pathways (involving both microbe and host) both impact
CVD in animal models and show striking clinical associations in human studies. For example, trimethylamine N-oxide and,
more recently, phenylacetylglutamine are gut microbiota–dependent metabolites whose blood levels are associated with
incident CVD risks in large-scale clinical studies. Importantly, a causal link to CVD for these and other specific gut microbial
metabolites/pathways has been shown through numerous mechanistic animal model studies. Phenylacetylglutamine, for
example, was recently shown to promote adverse cardiovascular phenotypes in the host via interaction with multiple ARs
(adrenergic receptors)—a class of key receptors that regulate cardiovascular homeostasis. In this review, we summarize
recent advances of microbiome research in CVD and related cardiometabolic phenotypes that have helped to move the
field forward from associative to causative results. We focus on microbiota and metaorganismal compounds/pathways, with
specific attention paid to short-chain fatty acids, secondary bile acids, trimethylamine N-oxide, and phenylacetylglutamine.
We also discuss novel therapeutic strategies for directly targeting the gut microbiome to improve cardiovascular outcomes.

Key Words: atherosclerosis ◼ cardiovascular diseases ◼ gastrointestinal microbiome ◼ thrombosis ◼ vascular diseases
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T
he collection of microbes living in the human intesti- studies by Koren et al6 suggested microbiota may be linked
nal tract (gut microbiota) and their combined genetic to atherosclerosis, because human atherosclerotic plaques
capacities (gut microbiome) have influence far beyond were noted to contain bacterial DNA, though whether or
digestion. Indeed, gut microbiota generate biologically not the DNA was derived from live bacteria within the
active metabolites that impact many aspects of host phys- artery wall was not determined. The first studies reveal-
iology and collectively are widely considered the body’s ing a potential causal link between the gut microbiome
largest endocrine organ. While gut microbiota facilitate and CVD focused on trimethylamine N-oxide (TMAO)—a
many necessary and beneficial physiological processes, metaorganismal metabolite formed following ingestion of
like the digestion of macronutrients and synthesis of some dietary nutrients abundant in a Western diet (eg, lecithin,
vitamins, numerous lines of evidence show gut microbiota choline, carnitine).7–9 The microbiome field has since rap-
can play a role in the development of adverse phenotypes. idly expanded to involve many previously disparate areas
In particular, distinct changes in the microbial community of research, demonstrating the far-reaching effects of
structure and function are associated with multiple dis- gut microbiota on human health and disease. Figure 1
ease states, including cardiovascular disease (CVD).1 illustrates several of the major pathways identified link-
Early work in the gut microbiome field demonstrated ing gut microbiota to CVD, numerous related phenotypes,
that alterations in fecal microbial community composition and known molecular participants, including some of the
are associated with the development of obesity and insulin identified host receptors. We will use Figure 1 as a tem-
resistance and that microbial transplantation could trans- plate for organizing this review, first providing an overview
mit heightened adiposity in the host.2–4 Subsequently, it of the field and then focusing on several areas of more
was discovered that disruptions to the microbiome early in recent advancement. Finally, we will discuss therapeutic
life can promote heightened adiposity.5 Early sequencing targeting of the gut microbiome as a potential treatment

Correspondence to: Stanley L. Hazen, MD, PhD, Cleveland Clinic, 9500 Euclid Ave, Mailcode NC-10, Cleveland, OH 44195. Email [email protected]
For Disclosures, see page 566.
© 2020 American Heart Association, Inc.
Circulation Research is available at www.ahajournals.org/journal/res

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

have reported an association between CVD phenotypes

Nonstandard Abbreviations and Acronyms and changes in the relative abundance of specific micro-
bial taxa or gut bacterial richness or diversity. For example,
AR adrenergic receptor in early studies, bacterial DNA was detected in atheroscle-
Review

BA bile acid rotic plaques with signatures that match taxa associated
cut choline utilization with disease states,6 and microbial compositional changes
CVD cardiovascular disease have been reported in patients with numerous CVD risk
FMO flavin monooxygenase factors, including hypertension, dyslipidemia, insulin resis-
FXR farnesoid X receptor tance, and other metabolic phenotypes.10,11 Changes in
GF germ free microbiota composition, diversity, and richness are asso-
GPCR G-protein–coupled receptor ciative, making it difficult to determine whether microbial
GRP41 G-protein receptor 41 changes are driving disease states or rather being driven
ICAM intercellular cell adhesion molecule by them. Moreover, typically-used sequencing techniques
IL interleukin often do not reach species or strain level resolution, and
LPS lipopolysaccharide analyses often exclude less abundant microbes. Studies
LXR liver X receptor focusing primarily on abundance can thus neglect gain-
of-function microbial pathways that contribute to disease,
MAPK mitogen-activated protein kinase
despite arising from microbes that can represent only a
NFκB nuclear factor-kappa B
small proportion of the microbial community. A recent
Olfr78 olfactory receptor 78
example of this is shown in a series of microbial trans-
PAG phenylacetylglutamine
plantation studies designed to demonstrate a direct role
PERK protein kinase R-like endoplasmic reticu-
of gut microbial cut (choline utilization) C—a major micro-
lum kinase
bial gene responsible for choline→trimethylamine (TMA)
PXR pregnane X receptor
transformation12,13—in host thrombosis potential14 (Fig-
SCFA short-chain fatty acid
ure 2). These studies used germ-free (GF) mice colo-
TAAR5 trace amine-associated receptor 5
nized with a synthetic polymicrobial community lacking
TF tissue factor choline→TMA functional capacity, and the addition of a
TGR5 takeda G-protein–coupled receptor 5 human commensal (Clostridium sporogenes) genetically
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TLR toll-like receptor engineered to either possess or lack a functional microbial

TMA trimethylamine cutC gene. While only present at a low abundance (only
TMAO trimethylamine N-oxide ≈0.1%) within the large intestine gut microbial community,
TXNIP thioredoxin-interacting protein the gain-of-function C sporogenes mutant elevated cir-
VWF von Willebrand factor culating TMA and TMAO levels within the host, and both
enhanced rate of thrombus formation and reduced time
to cessation of blood flow following arterial injury in vivo.14
or prevention strategy for cardiovascular and metabolic
Whole genome sequencing coupled with bioinformat-
diseases, highlighting recent advances in the development
ics analyses can also be used to infer potential functional
of nonlethal small-molecule inhibitors of specific microbial
capacities of gut microbial communities. For example,
pathways as a novel approach to improve CVD outcomes.
deep sequencing and systematic analyses of biosynthetic
gene clusters identified the presence of common antibi-
GUT BACTERIAL COMMUNITY otic genes in human commensals.15 But just as in studies
in vertebrate animals, the mere presence of a gene within
COMPOSITIONAL CHANGES AND bacteria does not provide information about its expres-
ASSOCIATION WITH CVD sion and activity. What’s more, the majority of microbial
Due to advances in culture-independent sequencing tech- genes have unknown functions. Further, microbial protein
nologies and bioinformatics, the vast majority of gut micro- expression is highly dynamic, as bacterial gene regulation
biome–related studies assess gut (often fecal) microbial is greatly influenced by environmental cues. This is dem-
community compositional changes associated with vari- onstrated in recent work showing that dietary addition of
ous disease states. This has led to a wealth of associative a single type of fiber can cause dramatic changes in the
data, which, while helpful, are limited in terms of investigat- expression of genes required for bacterial metabolism of
ing causality. While it is difficult to define a truly pathogenic fiber glycans.16 So while studies and approaches that focus
bacterial community, the term dysbiosis has been used to on microbial gene abundance and inference of potential
describe an imbalance of intestinal microbiota composition functional capacity can be valuable tools for hypothesis
within a disease state or phenotype. Many investigators generation, they need to be experimentally validated.17

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

Review
Figure 1. Molecular pathways and host receptors that link gut microbiota–derived products and metabolites with
cardiovascular and cardiometabolic disease phenotypes.
ADRA indicates adrenergic receptor alpha; ADRB, adrenergic receptor beta; FXR, farnesol X receptor; GPR, G-protein–coupled receptor; LPS,
lipopolysaccharide; LXR, liver X receptor; Olfr, olfactory receptor; PAG, phenylacetylglutamine; PERK, protein kinase R-like endoplasmic reticulum
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kinase; ROS, reactive oxygen species; SCFA, short-chain fatty acid; TAAR, trace amine-associated receptor; TGR, takeda G-protein–coupled
receptor; TLR, toll-like receptor; TMA, trimethylamine; and TMAO, trimethylamine N-oxide.

enter the host circulation, where it is mainly recognized by

GUT LEAKINESS AS A POTENTIAL TLR (toll-like receptor) on the surface of immune cells.24
PORTAL FOR GUT MICROBIOTA–DERIVED Upon binding of bacterial ligands, TLR signaling induces
PRODUCTS AND INFLAMMATION the release of proinflammatory cytokines that orchestrate
In its healthy state, intestinal barrier function is maintained a proinflammatory state in the host.
by physical factors including tight junctions between epi- Enhanced levels of LPS and other bacterial wall prod-
thelial cells, mucus production, and mucosal immunity. And ucts, presumably derived from gut microbiota, have been
in heart failure patients, we often observe bowel wall edema mechanistically linked to modulation of inflammation,
and impaired barrier function.18,19 Following the leaky gut immunity, and vascular function (Figure 1). Patients with
concept, impaired gut barrier function leads to translocation decompensated heart failure have higher endotoxin levels
of bacterial products into host circulation, which can result in the blood compared with stable patients.25 Translocation
in a proinflammatory state. Multiple studies show patients of LPS from the bowel in this setting is supported by higher
with heart failure have alterations in intestinal integrity and endotoxin concentrations in the hepatic vein as compared
that elevated levels of proinflammatory cytokines in the with blood directly sampled from the ventricles.26 The
blood correlate with symptom severity and poorer out- detection of gut microbiota and metaorganismal metabo-
comes.20,21 In the presence of heart failure, venous fluid lites at heightened levels in subjects with CVD, or risk for
overload, adaptive sympathetic activation resulting in redis- incident adverse CVD events, may, in part, reflect altera-
tribution of the systemic circulation, and low cardiac out- tions of the host barrier function (Figure 1). In a recent
put contribute to bowel wall edema and reduced mucosal observational study, circulating LPS concentrations were
perfusion.22 Intestinal hypoperfusion in heart failure alters predictive of major adverse cardiac events in a cohort of
mucosal function, as evidenced by increased paracellular patients with atrial fibrillation, suggesting that endotoxin
permeability and augmented intestinal bacterial biofilm translocation impacts CVD complications.27 It is worth not-
formation.23 When the gut barrier is impaired, lipopolysac- ing that a Mediterranean diet was negatively associated
charide (LPS) originating from Gram-negative bacteria can with endotoxemia in this study, suggesting the involvement

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 Witkowski et al Gut Microbiota and Cardiovascular Disease
Review
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Figure 2. Dietary precursors, such as choline and carnitine, are metabolized into trimethylamine (TMA) by gut microbiota via
specific genes, including members of the cut (choline utilization) gene cluster C/D.
Host hepatic FMOs (flavin monooxygenases) oxidize TMA into TMA N-oxide (TMAO), which promotes metabolic and functional changes in the
host including cardiovascular and renal end-organ damage.

of the gut microbiota in gut barrier function of the host. gram-negative Bacteroides (vulgatus and dorei) that pro-
Indeed, in preclinical models, gavage of Western diet–fed duce penta- and tetra-acylated lipid A, in contrast to the
ApoE (Apolipoprotein E)−/− mice with live Akkermansia hexa-acylated lipid A of E coli, reduced colonic inflamma-
muciniphila decreased intestinal permeability and lowered tion, endotoxemia, and atherosclerosis in ApoE−/− mice.31
fecal and circulating LPS levels, which was associated with It should be noted that factors beyond gut leakiness con-
reduced aortic atherosclerosis independent of lipid metab-
tribute to CVD. There are many disease conditions under
olism.28 While the mechanism of Akkermansia-induced
which enhanced gut leakiness is present, but not all such
changes in barrier function needs to be explored, a recent
diseases show heightened associations with CVD risks.
proof-of-concept study in humans found that administra-
For example, while the presence of heart failure–induced
tion of pasteurized (but not live) A muciniphila for 3 months
led to reduction in plasma LPS in obese individuals with bowel wall edema has been linked to endotoxemia and
metabolic syndrome.29 Although the intervention did not CVD progression, gut barrier defects caused by colitis and
change body weight, the authors observed improvement inflammatory bowel diseases are not classically associated
in insulin sensitivity and dyslipidemia in the Akkermansia- with heightened CVD risks. These observations point to a
treated group. Interestingly, structural differences of LPS more complex relationship between bowel wall integrity,
subtypes from different gut microbial species have been changes in gut microbial communities, and the relationship
associated with altered TLR recognition and their effects between host systemic inflammation and altered suscepti-
on host innate immunity.30 For instance, gavage with the bility for development of CVD. We speculate that different

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

mechanisms of gut leakiness (eg, inflammatory, bowel secondary BAs in turn impact host physiology though inter-
wall edema) may differentially impact gut microbiome and action with a variety of host nuclear receptors including
pathophysiological processes that modulate susceptibility FXR, LXR (liver X receptor), PXR (pregnane X receptor),
for the development of CVD. Improved understanding of and specific GPCRs (G-protein–coupled receptors) like

Review
the role of gut bacterial proinflammatory factors in trigger- TGR5 (takeda G-protein–coupled receptor 5; Figure 1).40–
ing systemic inflammatory cascades may help to provide 44
Perturbations to the dynamic interactions among diet, gut
novel therapeutic strategies to improve care and risk strati- microbiota, and specific BAs may thus contribute to cardio-
fication among patients with CVD. metabolic phenotypes and disease susceptibly. For example,
Numerous lines of evidence link multiple different fac- altered levels of BAs in plasma are associated with insulin
ets of inflammation to heightened risks for CVD.32,33 The resistance in type 2 diabetes mellitus,45,46 and modulation
role of inflammatory pathways in CVD has recently been of BA signaling may contribute to metabolic improvements
reaffirmed by the CANTOS clinical trial (Canakinumab Anti- during some antidiabetic treatments.47 As such, measuring
inflammatory Thrombosis Outcome Study). Administration systemic levels of BAs may aid in the assessment of poten-
of canakinumab—an antibody against IL (interleukin)-1β— tial gut microbiota contributions to cardiometabolic dis-
showed that inhibition of the IL-1β pathway reduces car- eases.46 A better understanding of how perturbation in BA
diovascular event risk independent of lipid level lowering.34 profiles are associated with future development of disease
Thus, treatment and prevention of CVD with immunomodu- states or responses to therapy may enhance our under-
latory therapies seems promising. But targeting inflamma- standing of CVD pathogenesis. Functional studies to deter-
tory pathways bears the risk for opportunistic infections, mine whether candidate BAs are mechanistically linked to
which may limit its use in patients with multiple comorbidi- these processes are promising areas of future investigation.
ties, as is often observed in patients with CVD. Identification
of gut microbiota that elicit host immune responses that
Short-Chain Fatty Acids and Blood Pressure in
participate in CVD pathogenesis may provide a therapeutic
avenue to ameliorate inflammation-driven CVD phenotypes. the Host
Short-chain fatty acids (SCFAs; fatty acids with 5 carbons
or less) can be products of host metabolism (eg, ace-
BILE ACIDS ARE PREDOMINANTLY GUT tate48). However, they are also produced in large quanti-
MICROBIOTA DERIVED AND SERVE AS ties by gut microbiota through anaerobic fermentation of
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dietary fiber.49,50 The most common SCFAs include ace-

MODULATORS OF HOST METABOLISM tate, propionate, and butyrate, which have been linked to
A major function historically attributed to bile acids (BAs) alterations in host blood pressure homeostasis, myocardial
has been to facilitate emulsification and adsorption of repair, and inflammation (Figure 1). The idea that circulat-
fat-soluble dietary nutrients. However, BAs are com- ing SCFAs are produced in large part by gut commensals
posed of a diverse array of structurally specific species is supported by studies showing that free SCFA levels
whose concentrations differ by many orders of magni- are virtually undetectable in plasma recovered from GF
tude. More recent studies have shown structurally spe- animals.51 Besides acting as an energy source for large
cific and distinct BAs also play additional roles, including intestine gut epithelial cells (eg, colonocytes), SCFAs are
but not limited to modulation of host lipid metabolism, absorbed into the portal blood and participate in various
glucose/insulin metabolism, and inflammation (Figure 1). processes of the host, including lipid metabolism, glucose
Initially synthesized from cholesterol in the host liver, pri- homeostasis, gut inflammation, and neurogenesis.52 An
mary BAs, which only represent a small fraction of the total association between SCFAs and adiposity early in life has
BA pool, are then secreted into the intestinal (duodenum) been shown by Cho et al.5 Exposure to antibiotics during
lumen, where subsequent gut microbiota–dependent modi- weaning changed gut microbial communities with increas-
fications participate in the generation of a remarkably large ing metabolic capacity to produce acetate, propionate, and
array of BA species. The body maintains a large pool of butyrate in C57BL/6J mice. Influx of these SCFAs to the
hydrophobic BAs through reuptake in the ileum and through liver resulted in substantial changes in the regulation of
negative feedback. These negative feedback mechanisms hepatic lipid metabolism and an obese phenotype.5 More-
are triggered by activation of the FXR (farnesoid X recep- over, antibiotic exposure early in life has also been associ-
tor) and cholesterol 7α hydroxylase35 or by expression of ated with changes in microbiota diversity,53 lasting effects
intestinal bile transporters.36 BAs modulate gut microbial on the host immunity,54 and cardiometabolic diseases, such
composition via potent antimicrobial properties, immune as diabetes mellitus.55,56 Interestingly, transfer of antibiotic-
responses,37 and FXR,38 and bile obstruction can lead to perturbed microbiota to the next generation of mice led to
bacterial overgrowth syndromes.39 Gut microbiota modify loss of microbial richness and changes in metagenomic
primary BAs via bile salt hydrolysis and BA 7α dehydrox- gene expression and susceptibility for colitis.57
ylation, yielding secondary BAs, many of which have hor- Initial clinical intervention studies reported that fiber
mone-like functions. Following systemic adsorption, some intake is associated with a decrease in blood pressure58

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

and support the idea that SCFAs are involved in the regu- molecular species of SCFAs and the host receptors sens-
lation of blood pressure. Early mechanistic studies by ing them is an area of future investigation. Of particular
Pluznick,59 Pluznick et al,60 and Natarajan et al61 substan- interest, recent studies using untargeted metabolomics
tiated this idea by demonstrating the G-protein–coupled suggest additional gut microbiota–derived plasma metabo-
Review

SCFA receptors Olfr78 (olfactory receptor 78) and Gpr41 lites beyond SCFA may also contribute to host blood pres-
(G-protein receptor 41) participate in host blood pressure sure regulation.68 These studies found multiple structurally
regulation. Olfr78 is expressed in vascular smooth mus- specific compounds including some uremic toxins that had
cle cells and the juxtaglomerular apparatus, and mediates previously been reported to activate the renin-angiotensin-
renin release and changes in vascular resistance, contrib- aldosterone system and have been linked to heightened
uting to hypertension.60 By contrast, Gpr41 is expressed kidney injury in model systems.69–71 A growing number of
in the vascular endothelium and promotes reduction microbiota-dependent products, including uremic toxins
in blood pressure.61 Propionate administration in the like p-cresol sulfate, indoxyl sulfate, and a variety of aro-
absence of Gpr41 tended to increase blood pressure, matic amino acid metabolites, are thought to potentially
while it caused a pronounced drop in blood pressure in alter host metabolism via specific receptors, including the
Olfr78−/− mice, suggesting a differential function of both aromatic hydrocarbon receptor (Figure 3).72,73
receptors in SCFA-dependent regulation of blood pres-
sure.60 Interestingly, when the SCFA pool was depleted
by antibiotics in Olfr78−/− mice, blood pressure went up, TMAO—A METAORGANISMAL METABOLITE
further corroborating a protective role of microbial SCFA CAUSALLY LINKED TO CVD AND
generation to balance Olfr78 signaling. However, the
overall hypotensive effects in wild-type animals might be
METABOLIC DISEASE
explained by both a decline in cardiac output and the loss Almost a decade ago, Wang et al9 first causally linked
of vascular resistance exhibited by SCFAs.62 While micro- gut microbiota with oral intake of nutrient precursors,
biota suppression with antimicrobials (ie, poorly absorbed TMAO production, and CVD risk (Figures 1 and 2). A
antibiotics; Figure 3) can serve as a valuable tool for dem- combination of untargeted metabolomics and mechanis-
onstrating involvement of the gut microbiome in a host tic animal model studies was used to uncover small mol-
phenotype, it is not a viable approach for therapeutically ecules whose levels in blood associate with CVD risk in
targeting the gut microbiome to achieve a desired long- humans, and impact CVD-relevant phenotypes in animal
term outcome in the host, due to the selection of gut models. Several analytes linked to phosphatidylcholine
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microbial communities with antibiotic resistance. metabolism, including TMAO, were identified, and TMAO
Many additional studies have supported a role for gut was shown to both predict CVD risks in multiple clinical
microbiota generation of SCFAs in modulation of blood cohorts and facilitate accelerated atherosclerosis (as did
pressure in the host. For example, fecal transfer from nutrient precursors with an intact host gut microbiome)
human hypertensive (versus normotensive) donors into GF in animal models.9 Through these and other studies, gen-
mice revealed transmission of heightened blood pressure.63 eration of TMAO in humans and mice has been shown
But in another study, transplantation of feces from nor- to occur via a multistep, metaorganismal pathway start-
motensive Dahl salt-resistant rats into hypertensive Dahl ing with the dietary precursors choline,9 phosphatidylcho-
salt-sensitive rats in fact exacerbated hypertension in the line,8,9 and carnitine.7 These are most abundant in foods
recipients, suggesting that additional host genetic variables found in a Western diet, including red meat, egg yolks,
may interact with microbial factors to modulate blood pres- and other animal products (Figure 2). Notably, plasma
sure control.64 A role for SCFAs in hypertensive end-organ or serum levels of every additional TMA nutrient precur-
damage in angiotensin II–infused mice has also been sug- sor identified (ie, shown to generate TMA and TMAO in
gested.65 Thus, numerous lines of evidence show that the hosts via a gut microbiota–dependent fashion), including
gut microbial community can impact blood pressure regu- betaine,9 γ-butyrobetaine,74 and trimethyllysine,75 have all
lation in the host and that SCFAs represent at least one of similarly shown associations with incident CVD risks in
the microbial mediators that contribute to vasomotor tone large-scale clinical studies, and these associations appear
and blood pressure. Recent studies provided further evi- to be mediated by TMAO (since their clinical prognostic
dence that SCFAs are involved in other CVD processes, value becomes attenuated with TMAO in the model).
such as ischemia reperfusion injury, cardiac repair following Gut microbial metabolism of TMA-containing nutrient
myocardial infarction, and impaired arterial compliance.66,67 precursors begins with specific microbial TMA lyases that
SCFAs represent a readout of saccharidic metabolism generate TMA—an odorous gas—as a product. The major
by the whole microbial community and often serve as ter- microbial choline TMA lyase is thought to be encoded
minal end products of (poly)microbial catabolic pathways. by the microbial cutC/D genes (cut gene cluster genes
Therefore, their levels may reflect a convergence of multi- C [catalytic] and D).12 The TMA produced is then trans-
ple microbial participants and competing pathways. Further ported to liver via the portal vein and readily metabolized
understanding of the factors that contribute to individual by host hepatic FMOs (flavin monooxygenases; mainly

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

Review
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Figure 3. Illustration of current strategies to improve cardiovascular disease by manipulating gut microbiota including dietary
interventions, targeting host enzymes involved in generation of metaorganismal metabolites, fecal microbial transplantation,
pre/probiotics, bacterial enzyme inhibitors, and antimicrobials.

FMO376) into TMAO.9 In the circulation, TMA levels are strong relationship indeed exists between elevated cir-
typically negligible. When radiolabeled TMA or TMAO culating TMAO levels and both CVD risk and mortality
was orally administered to human volunteers, 95% of the in multiple cohorts on different continents.105–107 In many
dose label was excreted via the kidneys with the majority studies, a plasma TMAO cutoff value of >6 μM predicted
being TMAO.77 TMAO has been shown to enhance ath- heightened risk of adverse cardiac events.108 And in one
erosclerosis in most, but not all, mechanistic and animal recent meta-analysis comprising >25 000 subjects, a
model studies.7,9,78–82 It has also been shown to promote 7.6% increase in all-cause mortality was noted for each
platelet reactivity and thrombosis potential,14,83–85 vascular 10-μmol/L increment of TMAO.105
inflammation and inflammasome activation,86–88 height- Several factors impact circulating TMAO levels within
ened heart failure,89–91 and chronic kidney disease92– subjects.19,108 First, the gut microbial community composi-
95
–related phenotypes in animal models (Figure 2). tion is critical since there is an initial obligatory role of gut
Circulating levels of TMAO have been shown to asso- microbes in TMA(O) generation.7,9 Second, renal functional
ciate with CVD and predict outcomes in the presence decline leads to less efficient excretion and thus height-
of multiple CVD phenotypes, including peripheral artery ened levels of TMA(O).95 But elevated levels are also
disease,96 coronary artery disease,97 acute coronary syn- frequently observed among subjects with normal kidney
drome,97–99 and heart failure.100–104 Notably, the prognos- function.105 In all subjects, choline is a major and continu-
tic value of TMAO withstands adjustment for traditional ous nutrient precursor, since beyond diet, choline content
risk factors, highlighting its potential as a biomarker for in bile is remarkably high and thus baths gut microbes in
risk stratification beyond what has been considered tra- both omnivore and vegans/vegetarians alike (the origins
ditional risk. While not all studies have observed the rela- of the word choline are in the Greek word khole, for bile,
tionship between heightened TMAO levels and incident since choline was first isolated by Adolph Strecker from
CVD risks, examination of extant clinical studies with pig and ox bile in 1862).109 However, carnitine, which is
TMAO in multiple meta-analyses have concluded that a found in high levels in red meat (and some energy drinks

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

and over-the-counter supplements), also serves as a nutri- antimicrobial/antibiotics) blocked choline diet–dependent
ent precursor and can account for significant elevation in enhancement in atherosclerosis (Figures 2 and 3).9 In
TMAO levels, particularly in some omnivores.7,110,111 Large- atherosclerosis-prone ApoE−/− mice, dietary supplemen-
scale clinical observational studies show that subjects with tation with choline led to augmented atherosclerotic lesion
Review

heightened circulating carnitine levels have higher risk for burden, higher aortic expression of scavenger receptors
incident CVD events (heart attack, stroke, and death). The (CD36 [cluster of differentiation] and scavenger recep-
prognostic value for plasma carnitine levels, like other TMA tor A), increased cholesterol-laden macrophage foam cell
precursors, seems to be mediated by TMAO, as inclusion formation,9 and impaired in vivo reverse cholesterol trans-
of TMAO in statistical models attenuates the prognostic port.7 In addition, TMAO suppressed BA pool size and,
value of carnitine (but TMAO remains a robust predictor), therefore, cholesterol clearance in the host.7 Consistent
and carnitine supplementation accelerates atherosclero- and complementary to these findings, FMO3 knockdown
sis development in animal models.7,112 has been shown to impair TMA transformation into TMAO,
In a recent human dietary intervention study examining thus reducing plasma TMAO levels and concomitantly
protein source (red meat versus white meat versus non- restoring cholesterol balance.114 Early microbial transplan-
meat), substantially higher levels of circulating TMAO were tation studies of cecal microbial communities from high
observed when subjects consumed a red meat diet (equiv- TMA-producing inbred C57BL/6J mice into atheroscle-
alent to 8 oz of steak daily for 1 month).111 Although there rosis-resistant NZW/LacJ recipients demonstrated the
is a modest increase in choline content in omnivorous ver- transmissibility of dietary choline-induced TMA and TMAO
sus vegan diets, as noted above, a substantial amount of generation and atherosclerosis.115 Not all TMAO precur-
choline is introduced into the gut in the form of bile (choline sor feeding studies, however, have shown similar results,
in the form of phosphatidylcholine is a major component of supporting the notion that differences in the microbial
bile). Thus, there is far more modest overall difference in communities present in the host can impact the final phe-
choline exposure to the gut microbial community of vegan/ notypes observed.116
vegetarian versus omnivore. By contrast, omnivorous diets The striking association between circulating TMAO lev-
show markedly higher carnitine content since vegan/veg- els in subjects and thrombotic event risks, such as heart
etarian diets are virtually devoid of carnitine. In line with this, attack and stroke, has been witnessed across numerous
a major source of the observed elevation in plasma TMAO large-scale clinical cohorts.105,117 This has prompted mech-
levels in subjects following 1 month of either a red meat– anistic studies in both humans and mice to explore the
rich diet versus white meat or nonmeat diet (predominantly role of TMAO in thrombosis. Zhu et al found that TMAO
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vegetarian protein source) was shown to arise from car- alters human platelet calcium signaling, heightening their
nitine. In addition to having enhanced nutrient density of responsiveness to submaximal stimulation by agonists (eg,
TMA(O) precursors (including carnitine), isotope tracer thrombin, ADP, collagen). Consequently, heightened throm-
studies showed the red meat–rich diet induced functional bosis potential has been observed in both whole blood
remodeling of the gut microbial community to enhance and in vivo arterial injury models.14,83–85,118 In a subsequent
carnitine→TMA transformation but not choline→TMA human feeding study, healthy volunteers (both omnivore
generation.111 Interestingly, the chronic exposure to a red and vegan/vegetarian) that were orally supplemented with
meat–rich diet also induced a functional change in the kid- choline exhibited higher levels of TMAO and concomitant
neys, with reduction in the fractional renal excretion rate enhanced platelet responsiveness and aggregation.77,118
for TMAO, despite no change in glomerular filtration rate.111 Importantly, higher TMAO levels were dose dependently
It is also notable that numerous studies support a dose- associated with increased platelet aggregation respon-
dependent association between meat consumption and siveness, even in subjects on low-dose aspirin. This sug-
CVD risks and mortality.113 gests that in subjects with elevated TMAO, the antiplatelet
effects of aspirin may be attenuated, highlighting the pos-
sible involvement of TMAO in on-treatment platelet reac-
TMAO—A NEWLY RECOGNIZED tivity and so-called aspirin resistance.
PARTICIPANT IN ATHEROSCLEROSIS, The mechanistic involvement of the metaorganismal
THROMBOSIS, AND VASCULAR TMAO pathway in platelet function and in vivo throm-
bosis potential has also been examined through both
INFLAMMATION genetic gain- and loss-of-function manipulations, includ-
A causal contribution of gut microbiota to atherosclerosis ing to the host gene FMO3 (Figure 2). Multiple studies
susceptibility was first demonstrated with the discovery of have confirmed through both genetic gain- (as global
TMAO as a gut microbiota–derived factor and the initial FMO3 transgene) and loss-of-function (via antisense oli-
functional studies demonstrating both direct provision of gonucleotide to FMO3 and via global FMO3 knockout)
TMAO accelerated atherosclerosis in murine models and studies in mice that manipulation of TMA and TMAO
that suppression of gut microbiota–dependent conversion levels in vivo alters platelet responsiveness, rate of clot
of nutrient precursors (choline) into TMA and TMAO (with generation, and thrombosis potential.83,84,119 Moreover,

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

cecal microbial transplantation experiments confirmed oxidative stress.129 Recent investigations have suggested
that the prothrombotic phenotype mediated by a cho- TMAO can impact inflammation via priming and activation
line-rich diet was a transmissible trait.83 Early studies of the NLRP3 (NOD-, LRR-, and pyrin domain-containing
by Cracium et al12 first identified the cut gene cluster protein 3) inflammasome in endothelial cells, as well as

Review
in human commensals encoding the catalytic and regu- the arterial vascular wall in mice, involving mitochondrial
latory gene products CutC and CutD. The presence of reactive oxygen species production, TXNIP (thioredoxin-
the microbial cutC/D genes in human microbiota is asso- interacting protein), and lysosomal destabilization.87,130,131
ciated with the ability to generate TMA from choline120 However, the exact mechanisms by which TMAO induces
and with subsequent TMAO accumulation.121 Importantly, inflammasome activity have yet to be explored.
studies using GF mice colonized with synthetic microbial The receptor for TMA was identified long ago as the
communities±a genetically engineered gain- or loss-of- highly sensitive olfactory receptor TAAR5 (trace amine-
function cutC mutant human commensal confirmed that associated receptor 5).132 TAAR5 shows high specific-
a functional microbial cutC gene is sufficient to transmit ity for TMA and does not recognize TMAO. While TMA
TMA and TMAO generation, as well as in vivo thrombosis alone may contribute to proinflammatory signaling in the
potential. Microbial cutC may thus represent a therapeu- vasculature,86 the role of TAAR5 in CVD requires further
tic target for preventing thromboembolic complications.14 investigation. Recently, a receptor for TMAO has been
Recent studies have shown that beyond impacting reported and shown to participate in TMAO-dependent
platelet function, TMAO induces expression of TF (tissue effects on glucose and insulin metabolism.133 Chen et
factor)—the initiator of the extrinsic clotting—in endothe- al133 showed that TMAO directly binds to PERK (pro-
lial cells in vitro.122 Vascular TF promotes thrombosis and tein kinase R-like endoplasmic reticulum kinase), a main
vascular inflammation,123 particularly in patients with type component of the unfolded protein response—a signaling
2 diabetes mellitus who have higher levels of circulating pathway that adapts the cell to ER (endoplasmic reticu-
TMAO.124–126 Animal model studies are still needed to vali- lum) stress (Figure 1). Dietary supplementation of TMAO
date a contribution of gut microbiota and TMAO generation in C57BL/6J wild-type animals induced hepatic PERK
to alterations in TF pathway in vivo and to altered thrombo- expression, accompanied by increased Fox01 (forkhead
sis potential. A recent study reported that the absence of box protein 01)—a key transcription factor in insulin sin-
microbiota was associated with reduced hepatic VWF (von gling and impaired glycemic control. Genetic manipulation
Willebrand factor) synthesis and reduced thrombus growth demonstrated that the absence of hepatic PERK blunted
after carotid artery injury in C57BL/6 GF mice as compared
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the TMAO-induced increases in Fox01 expression and

with conventionally raised littermates.127 However, the role
improved glycemic indices. Whether or not PERK plays
of metaorganismal metabolites, in particular TMAO, in the
a role in TMAO-mediated effects on atherosclerosis or
VWF-depending thrombosis has yet to be determined.
thrombosis remains unknown and is an important area
Vascular inflammation is critically involved in the patho-
for further exploration. It is also interesting to note that
genesis of atherosclerosis and thrombotic complications.
ER stress is implicated in the pathogenesis of many CVD
Seldin et al86 found that acute infusion of physiological
phenotypes. Whether TMAO and PERK participate in
levels of TMAO in mice heightens vascular inflammation
these associations also remains to be explored.
(Figure 2), as supported by aortic endothelial cell activa-
tion (recovered by laser microdissection), including activa-
tion of MAPK (mitogen-activated protein kinase) signaling
THEORETICAL BENEFITS OF TMAO
and NF-κB (nuclear factor-kappa B) nuclear transloca-
tion, leading to subsequent proinflammatory gene expres- Due to its small size and combination of hydrophilic and
sion. Complementary findings (TMAO stimulated vascular hydrophobic properties, TMAO behaves as a chaotropic
inflammation; Figure 2) have been observed in vitro using agent, with the ability to alter protein conformation and
primary human aortic endothelial cells and vascular smooth potentially serve as an allosteric modulator to proteins.134
muscle cells.86 After an acute injection, mouse aortas also These features may have important physiological func-
showed increased expression of vascular adhesion mol- tions in the host (eg, impacting the protein unfolding
ecules, such as E selectin or ICAM (intercellular cell adhe- or ER stress response within cells). In some aquatic
sion molecule)-1, even when TMAO had been cleared animals, including a subset of deep sea fish, TMAO is
from circulation, implying sustained vascular inflammation. reported to act as an osmolyte and to protect against
Further, TMAO was reported to increase oxidative stress pressure-induced protein destabilization.135,136 Indeed,
and vascular senescence, which was characterized by some bony fish can use large amounts of TMAO for
impaired cell proliferation and migration in human umbilical osmoregulation, and TMAO plasma levels within the fish
vein endothelial cells.128 In other animal model studies, gut increase with depth of habitat, reaching levels up to 400
microbiota suppression with oral poorly absorbed antibiot- mmol/kg in snailfish that were caught at 7000-m depth
ics was associated with decreased TMAO levels, improved in the South Pacific Ocean.137 TMAO has been shown to
endothelial function, reduced arterial stiffness, and lower stabilize proteins to elevated hydrostatic pressure and is

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

thought to both impact protein conformational changes, increases in hepatic FMO3 expression.76 These results
like those that occur with allosteric regulation, and mod- suggest that high levels of TMAO in the terminal stages
ulate intracellular molecular crowding effects.136,138,139 of pregnancy may in theory better equip women to avoid
Thus, TMAO accumulation both in deep sea creatures severe blood loss during delivery. In addition, beyond
Review

and in mammals may represent an adaptive mechanism FMO3 expression, microbial community alterations are
to impact protein stability and intracellular signaling known to occur during pregnancy.145
processes. While purely speculative, evolutionary drive may have
Other studies have reported that TMAO is involved selected for metabolic changes in hosts that lead to
in the adaptive freeze avoidance response to extreme harboring of gut commensal communities with the abil-
cold—in other words, it acts as antifreeze—such as within ity to produce TMA or other gut microbiota–generated
Newfoundland smelt.140 These fish have the ability to prothrombotic metabolites, thus enabling hosts to better
elevate plasma osmolarity by seasonal accumulation cope with environmental stressors that lead to hemor-
of TMAO and other organic solutes, which depresses rhage (such as parturition). However, these features
the freezing point of body fluids and allows them to sur- may have become detrimental for individuals living in
vive at subfreezing temperatures. It has recently been modern Western societies associated with a CVD-
hypothesized that TMAO-induced protein stabilization prone environment and less need to survive traumatic
may play a role to protect cardiomyocytes from hydro- injuries. Rodents show a significant sexual dimorphism
static pressure fluctuations during heart failure.141 By with respect to FMO3 expression, and TMAO levels, with
contrast, high concentrations of TMAO may also impair females showing higher levels (and greater atheroscle-
function, as was reported for the activity of the actomyo- rosis capacity9,76). Although gene expression studies in
sin motor.142 humans have suggested gender differences in hepatic
TMAO may also be involved in tissue osmolality in FMO3 expression, plasma levels of TMAO thus far
vertebrates. When measuring concentrations in various reported have failed to show sex differences, and the
mouse tissues, we observed that TMAO levels in kidney prognostic value of TMAO appears to be similar in both
tissue largely exceeded those observed in corresponding men and women.105 However, it should also be noted
paired plasma samples collected at the same time from that virtually all clinical TMAO studies reported involve
the same animals (Hazen, unpublished data, 2016). In cohorts that are of postmenopausal age. Future inves-
the kidneys, an osmotic gradient arising from the cortico- tigation should include a full exploration of sex-specific
medullary boundary to the inner medullary tip is normally
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differences in TMAO levels in between younger men

maintained as a mechanism that allows reabsorption of and women.
water and concentration of the urine—a process also
called countercurrent multiplication.143 It seems plausible
that high levels of TMAO in the kidneys may function METAORGANISMAL METABOLITE
analogously to urea, playing a role in osmoregulation and PHENYLACETYLGLUTAMINE IS BOTH
renal function. Understanding the molecular participants
involved in TMAO secretion and cellular transport in the
LINKED TO CVD AND ACTS VIA ARS
kidneys and other tissues is an important potential area The pathogenesis of type 2 diabetes mellitus goes
of future investigation, as it may reveal novel targets for beyond glycemic control, and traditional risk factors,
therapeutic intervention. For example, one could theorize including level of glucose control, poorly stratify CVD
a small-molecule drug that targets renal TMAO trans- risk among diabetics. To investigate this, Nemet et al146
port/secretion might function as a diuretic, facilitating used untargeted metabolomics to identify novel metabo-
TMAO urinary excretion and reducing both blood pres- lites that associate with incident risk for major adverse
sure and CVD risks. cardiac event, are increased in type 2 diabetes mellitus
Many studies have shown that high TMAO levels are subjects, and do not significantly correlate with glyce-
associated with risk for thrombotic complications. How- mic control. The candidate analyte showing the stron-
ever, these same properties may theoretically confer gest association with major adverse cardiac events (m/z
benefits to the host during situations with high bleeding 265.1188) was subsequently identified as phenylacetyl-
risk, such as the delivery of a baby. It is thus interest- glutamine (PAG)—a phenylalanine-derived metabolite
ing that one study reported TMAO levels may increase (Figure 4). The association of PAGln with incident risk
during pregnancy.144 Studies by Bennett et al found for major adverse cardiac events like heart attack, stroke,
that human liver samples from female subjects showed and death was further validated in an independent and
higher FMO1/3 expression and, therefore, an increased nonoverlapping cohort comprised of 4000 stable cardio-
capacity for TMAO generation, than those from males. vascular subjects and shown to be independent of tradi-
Likewise, gonadectomized male mice treated with tes- tional CVD risk factors in both diabetics and nondiabetics
tosterone show reduced hepatic FMO3 expression, while alike.146 Additional functional studies revealed that the
ovariectomized female mice treated with estrogen exhibit association of PAG with incident CVD risks likely occurs

562   July 31, 2020 Circulation Research. 2020;127:553–570. DOI: 10.1161/CIRCRESAHA.120.316242

 Witkowski et al Gut Microbiota and Cardiovascular Disease

because the metabolite impacts host physiology and fos- reason to assume that the subset of ARs identified in
ters CVD-related phenotypes. Moreover, PAG was shown this study are the only receptors modulated by PAG. It
to be generated via gut microbes during metabolism of remains to be determined whether other members of the
phenylalanine, as illustrated in Figure 4. Genetic engi- AR gene family, some of which are also known to par-

Review
neering studies in microbes coupled with transplantation ticipate in the regulation of cardiovascular homeostasis
into GF mice confirmed gut microbial (and some of the are similarly modulated by PAG. It is intriguing to specu-
gut microbial genes) contribution to host platelet reactiv- late that some phenotypes exhibited by microbiota are
ity and in vivo thrombosis potential. And through multiple directly or indirectly mediated via ARs. This is indirectly
gain-of-function and loss-of-function genetic and phar- supported by the fact that the absence of microbiota
macological studies, PAG was shown to interact with alters cardiovascular homeostasis, such as blood pres-
GPCRs, including the α- and β-ARs (adrenergic recep- sure regulation,150 myocardial repair following postinfarc-
tors).146 ARs are crucially involved in heart disease147 tion cardiac repair,66 or thrombosis growth127—functions
and platelet function.148 However, until the discovery that all involve ARs. More studies are needed to further
of PAG, AR signaling had not yet been implicated in
characterize PAG-mediated functions in the host. PAG
gut microbiota-derived factors driving CVD. The new
also appears to represent another potential gut microbi-
studies by Nemet et al146 also showed that adverse
ome pharmacological target for future efforts.151
CVD-related phenotypes observed with PAG adminis-
tration at physiological levels were attenuated with the
β-blocker carvedilol.
DRUGGING THE MICROBIOME
It is striking that gut microbiota appear to elicit adverse
cardiovascular phenotypes in the host via modulation of In response to accumulating evidence that the micro-
ARs. Such a finding may help explain some of the benefi- biota affects susceptibility for CVD and cardiometabolic
cial effects of clinical β-blocker treatment. Interestingly, diseases, researchers have begun to develop microbi-
ARs may also in turn modulate microbial abundance as ota-directed interventions to improve clinical outcomes
observed in β-AR knockout mice.149 The novel discovery (Figure 3). The microbiome-host axis comprises many dif-
of a microbiota-AR signaling axis is particularly interest- ferent layers, including dietary precursors, microbial com-
ing considering the widespread implication of ARs in munities, and metaorganismal pathways that generate
cardiovascular physiology and metabolism. There is no bioactive metabolites recognized by host receptors—all
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Figure 4. The metaorganismal metabolite phenylacetylglutamine (PAG) is derived from microbial metabolism of phenylalanine
and is involved in enhancement of platelet thrombotic potential via adrenergic receptors.
α2A indicates α2A adrenergic receptor; α2B, α2B adrenergic receptor; β2, β2 adrenergic receptor; DAG, diacylglycerol; GP VI, glycoprotein
VI; IP3, inositol 1,4,5-triphosphate; P2Y12, purinergic receptor P2Y12; PAR-1, protease-activated receptor 1; PIP3, phosphatidylinositol
trisphosphate; and PLC, phospholipase C.

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

of which represent potential therapeutic targets to mod- some clinical intervention studies using either prebiotics
ulate community output and host phenotype. Figure 3 or probiotics that have shown promising results. However,
illustrates several different therapeutic approaches for these studies tend to be relatively small in size, and their
targeting the gut microbiome to exert a beneficial effect adoption into clinical practice would require further study.
Review

in the host. We have already mentioned the use of anti- The use of probiotics and prebiotics has recently been
microbial agents such as poorly absorbed antibiotics as a extensively reviewed, including discussions of the many
valuable tool for demonstrating involvement of gut micro- promises and potential challenges of their development
biota in both animal models and humans9 but as a poor and use.162–167 While a complete review of this topic is
choice as a long-term therapeutic due to the develop- beyond the scope of the present review, below we discuss
ment of antibiotic resistance. While many studies have several preclinical studies involving probiotics, particularly
reported associations between atherosclerotic plaque where a mechanistic role for alteration in gut microbi-
and the presence of pathogens such as Cytomegalo- ome related processes to improved host phenotype were
virus, Chlamydia, and Helicobactor pylori,152–155 multiple reported. One example includes a recent rodent hyper-
prospective randomized trials with antibiotics have thus tension study in which either a high-fiber diet (a prebiotic)
far failed to demonstrate clinical benefit.156–158 Further, or acetic acid alone (a gut microbial product) resulted in
the impact of antibiotic treatment on microbial communi- reduced blood pressure and adverse cardiac remodel-
ties is hard to predict, often because the microbial com- ing.168 In a different mouse model involving ApoE−/− mice
munity that recolonizes after cessation of antibiotics can on a n-3 polyunsaturated fatty acid–depleted diet for 12
be variable, depending upon many factors, including the weeks, supplementation of dietary inulin-type fructans
microbes one is exposed to as the antibiotics are metab- was shown to reverse endothelial dysfunction in carotid
olized and excreted. Currently, there is no clear evidence arteries via activation of the NO synthase/NO pathway.169
that antibiotics have efficacy in the treatment of CVD in Beyond changes in vascular function, probiotic use in ani-
humans. Use of antibiotics thus seems better suited to mal studies of heart failure has also shown promise. For
eradicating true pathogens than as a chronic long-term example, administration of Lactobacillus rhamnosus GR-1
preventive intervention. in rodents improved systolic and diastolic left ventricular
In a recent study that screened over 1000 com- function following coronary artery ligation.170 This study
monly prescribed nonantibiotic drugs for their impact on was of interest because it was shown that while the GR-1
a broad selection of human gut commensals, nearly a strain did not colonize to distal intestines where most
quarter of the tested medications demonstrated antibi- anaerobes reside, beneficial cardiac remodeling was
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otic-like activity, significantly inhibiting the growth of at none-the-less observed. In another recent study, provision
least 1 human commensal.159 These results suggest that of A muciniphila was associated with reduced aortic ath-
commonly prescribed medications may impact human erosclerosis in the hypercholesterolemic ApoE−/− mouse
gut microbial communities, thereby impacting host phe- model.28 This probiotic is of interest because of its use
notypes through indirect effects mediated by changes in human clinical investigations. For example, provision of
in the gut microbiome. One well-studied example of this A muciniphila in a randomized placebo controlled double-
is the widely used antidiabetic drug metformin. Recent blinded interventional exploratory study was reported to
studies by Bäckhed et al160 show that some of the anti- promote a reduction in plasma LPS in obese individuals
diabetic effects of metformin therapy appear to be medi- with metabolic syndrome.29
ated by alterations in gut microbiota composition, since While prebiotic and probiotic interventions have shown
microbial transplantation studies reveal transmission ability to favorably alter metabolic profiles in some human
of antidiabetic effects in recipient GF mice following studies, results are highly variable, and animal model
fecal transfer from metformin treated donors. In addi- findings have not yet been translated to evidence of clini-
tion to the potential for common medications to impact cal efficacy. It is still unclear whether most of the used
gut microbial composition and function, thus potentially microbes survive the acidity of the stomach, if they colo-
contributing to drug effects in the host, it is becoming nize the colon where most of the gut microbiota reside,
increasingly clear that there is broad diversity in gut and whether the beneficial effects are mediated by the
microbial metabolism of medications, leading to poten- ingested microbes, caused by shifts in the community
tial altered responsiveness to drugs. The diverse drug- structure or possibly even caused by secondary effects
microbiome interactions that vary between individuals on host immune education and function. One difficulty
is another area where further studies are needed, and with probiotic studies where secondary effects on micro-
shows promise for possible use in drug development bial communities likely play a role in beneficial effects if
efforts and personalized medicine.161 observed is the vastness of the gut microbial community
Defined microbial compositions (probiotics) and non- and the interindividual differences in community struc-
microbial substances that may alter microbial community ture. These factors are thought to give rise to variable
structure (prebiotics) have also been proposed to improve responses to probiotics or prebiotics in general.171 Thus,
CVD (Figure 3). Indeed, there are many preclinical and results of probiotic or prebiotic administration have been

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 Witkowski et al Gut Microbiota and Cardiovascular Disease

difficult to predict. Moreover, it is worth noting that the dimethylbutanol inhibited choline diet–dependent TMAO
current selection of probiotics seems to be primarily generation, reduced macrophage foam cell formation,
driven by abundance-based analyses of microbiota com- and inhibited aortic atherosclerotic plaque development.78
position, wherein microbial community members whose Next-generation choline TMA lyase inhibitors have since

Review
proportions are highly associated with beneficial pheno- been developed that selectively target and accumulate
types are the focus of interest. However, the keystone in microbiota, thereby limiting systemic exposure in the
commensal organizing a community architecture, or pro- host. The choline TMA lyase suicide substrate inhibitor
viding a key gain of function as discussed above, can fluoromethylcholine was shown to be over 10 000-fold
be a low abundance component and is often not easily more potent an inhibitor of cutC than 1,3 dimethylbutanol
detected by current conventional sequencing depths of and markedly blocks microbial choline catabolism.85 Inter-
analyses. estingly, owing to their highly polar nature, fluoromethyl-
Perhaps the most obvious potential therapeutic inter- choline and related halomethylcholines were shown to
vention for targeting the gut microbiome is diet (Fig- be poorly absorbed into the host, limiting systemic expo-
ure 3). The TMAO pathway is an excellent example of sure and thus chances of side effects. In addition, cutC
this, given the nutrient precursors are more abundant inhibition by fluoromethylcholine and iodomethylcholine
in a Western diet, and diets rich in phosphatidylcholine was shown to result in microbial cytosolic choline eleva-
and carnitine are associated with heightened levels of tion, which appears to be sensed as an abundant fuel
TMAO, whereas vegetarian or vegan diets have reduced source by the microbe. This then induces expression of
nutrient precursors.111,118 Notably, however, TMAO levels the entire cut gene cluster, including both cutC and the
appear to be driven more so by the gut microbial com- microbial choline transporter. This leads to active micro-
munity composition than by the dietary intervention, and bial uptake of fluoromethylcholine and the substrate of
significant variation in TMAO production among individu- cutC, choline. A positive feedback loop is thus created,
als on a given diet is observed.14,111 A low choline- or car- whereby the more microbial catabolism of choline into
nitine-containing diet can be generated using a primarily TMA is inhibited, the greater the elevation in cytosolic
vegetarian or vegan selection of food, and such diets are choline within the gut microbe (sensed as an abundant
rational recommendations for subjects with high TMAO fuel source), and the greater the sequestering of choline
levels. But such dietary recommendations are harder to from the intestinal lumen into the inhibited gut microbe.
envision with other gut microbiota-generated metabo- As intestinal luminal choline is depleted, microbial com-
lites, where either the nutrient precursors are numerous
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munity TMA production is globally inhibited, even from

(eg, SCFA) or are essential amino acids (eg, phenylala- neighboring community members who might otherwise
nine and PAG), and so cannot be easily avoided. How-
not be potently inhibited.85
ever, even with phenylalanine, there are dietary choices
Although human clinical studies with choline TMA
that can be made to reduce intake. For example, those
lyase inhibitors have not yet been reported, numerous
who experience the inborn error of metabolism called
efforts are ongoing in this area. Microbiota-targeting non-
phenylketonuria have a host enzyme deficiency that
lethal small-molecule inhibitors will require the same sort
makes eating foods abundant in phenylalanine harmful
of safety testing as any other drug. But with compounds
(ie, proteins). Consequently, throughout life, a phenylke-
that have reduced systemic absorption, there is a theo-
tonuric diet is highly recommended, which has an overall
retical potential benefit of limiting adverse side effects
low phenylalanine content.172 The impact of adopting a
from off-target inhibitory activities in the host. Thus, phar-
phenylketonuria diet in subjects with high PAG levels (eg,
macological interventions aimed at drugging the microbi-
subjects with diabetes mellitus, renal disease) has yet to
ome with nonlethal small-molecule inhibitors represents
be examined but is of considerable interest.
Recent work has examined the selective nonlethal tar- a novel therapeutic approach for both the treatment and
geting of gut microbial enzymes for TMA generation as prevention of cardiometabolic diseases that will need to
a therapeutic approach for the treatment or prevention be validated in clinical intervention studies.
of CVD (Figure 3). Key to this approach is the develop-
ment of a small-molecule inhibitor that is nonlethal to the
microbe and thus does not trigger as great a selective
CONCLUSIONS
pressure as an antibiotic for development of resistance. Although our knowledge about how microbiota impact CVD
The first study of this type targeting the gut microbiome is still rudimentary, the rate at which new discoveries are
for the treatment of CVD used a choline structural ana- emerging is impressive. As outlined, there is overwhelming
logue, 1,3 dimethylbutanol. Through a series of studies, evidence that gut microbiota-derived processes in general
1,3 dimethylbutanol was shown to serve as a nonlethal are linked to numerous CVD-relevant phenotypes, includ-
microbial enzyme inhibitor of choline→TMA transfor- ing but not limited to atherosclerosis, platelet reactivity and
mation and to reduce TMAO production in vivo without thrombosis potential, blood pressure, lipid metabolism, adi-
affecting microbial fitness.78 When fed to animals, 1,3 posity, glucose homeostasis, and vascular inflammation.

Circulation Research. 2020;127:553–570. DOI: 10.1161/CIRCRESAHA.120.316242 July 31, 2020   565

 Witkowski et al Gut Microbiota and Cardiovascular Disease

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reactivity and thrombosis potential. Circ Res. 2018;123:1164–1176. doi:
From the Department of Cardiovascular and Metabolic Sciences, Lerner Re- 10.1161/CIRCRESAHA.118.313142
search Institute (M.W., T.L.W., S.L.H.), Center for Microbiome and Human Health 15. Donia MS, Cimermancic P, Schulze CJ, Wieland Brown LC, Martin J, Mitreva
(M.W., S.L.H.), and Department of Cardiovascular Medicine, Heart and Vascular
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Institute (S.L.H.), Cleveland Clinic, OH.
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antibiotics. Cell. 2014;158:1402–1414. doi: 10.1016/j.cell.2014.08.032

Acknowledgments
16. Patnode ML, Beller ZW, Han ND, Cheng J, Peters SL, Terrapon N, Henrissat
This work is supported by grants from the NIH and Office of Dietary Supplements
B, Le Gall S, Saulnier L, Hayashi DK, et al. Interspecies competition impacts
(P01HL147823 and R01HL103866), the German Research Foundation (WI
targeted manipulation of human gut bacteria by fiber-derived glycans. Cell.
5229/1-1), and an award from the Leducq Foundation. S.L. Hazen reports being
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partially supported by a gift from the Krieger Fund. M. Witkowski, T.L. Weeks, and
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S.L. Hazen all contributed to the drafting of the manuscript and critical revisions
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Disclosures ventricular dysfunction, splanchnic hemodynamics, and the intestinal
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S.L. Hazen reports being named as coinventor on pending and issued patents held
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by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics, be-
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ing a paid consultant for Procter & Gamble, having received research funds from
and heart failure. Nat Rev Cardiol. 2019;16:137–154. doi: 10.1038/
Procter & Gamble and Roche Diagnostics, and being eligible to receive royalty
s41569-018-0108-7
payments for inventions or discoveries related to cardiovascular diagnostics or
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therapeutics from Cleveland HeartLab, Quest Diagnostics, and Procter & Gamble.
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The other authors report no conflicts.
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