Gut Microbiom and Heart Disease

The intestinal microbiota and cardiovascular disease
Themistoklis Katsimichas, MD, PhD,1,2 Alexios S. Antonopoulos, MD, PhD,1 Alexandros
Downloaded from https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvz135/5510547 by Bethel University user on 05 June 2019

Katsimichas, MD, PhD,1 Tomohito Ohtani, MD, PhD,2 Yasushi Sakata, MD, PhD,2 Dimitris
Tousoulis, MD, PhD1
1 st
1 Cardiology Department, Athens Medical School, Hippokration General Hospital, Athens,
Greece.
2
Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine,
Suita, Osaka, Japan.
Category: Review.
Word count: 6,253 (main text), Figures: 5, Tables: 3, References: 107
Corresponding author:
Professor Dimitris Tousoulis, MD, PhD.
1st Cardiology Department, Athens Medical School, Hippokration General Hospital,
Vasilissis Sofias Avenue 114, PO 115 27, Athens, Greece.
Tel: (+30) 2132088099 Fax: (+30) 2132088676
Email: drtousoulis@hotmail.com
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions please email:
journals.permissions@oup.com.
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Abstract
The intestinal microbiota of human hosts is the community of microorganisms living in the

small and, mainly, the large intestine of humans. This microbial ecosystem has co-evolved with
humans across the millennia, has come to play an important interactive role in human
physiology, and has been aptly called our forgotten organ. Significant properties of the
microbiota benefiting its host include energy harvest from food sources indigestible by humans,
protection from pathogen colonization, and vitamin synthesis. Mounting evidence has linked
changes in the composition or metabolic profiles of the microbiota with human disease,
including disorders of the cardiovascular spectrum. Although cause and effect mechanisms are
as yet essentially unproven in the relevant literature, the established associations point to the
importance of the microbiota in the pathophysiology of cardiovascular disease. In this review,
we first summarize key information on the gut microbial communities and the elaborate tools
developed to analyze their structure and metabolic functions. Ecological terms are explained
and analytical techniques are simplified, to enhance the understanding of published studies.
Statistical methods used in microbial analysis are also described in simple terms. We then
present published literature on the association of the compositional and functional changes of
the microbiota in cardiovascular disease, including heart failure, hypertension, and
atherosclerosis. Each section of the review deals with the underlying pathophysiology of the
relevant associations, connecting the observational and mechanistic aspects. Finally, we
discuss the challenges that remain to be met before this field of research can generate
knowledge which can impact everyday clinical practice.
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1. Introduction
The human intestinal microbiota is the community of microorganisms living in the human

intestinal tract, primarily the large intestine. This complex and diverse ecosystem comprises
symbiotic and mutualistic bacteria, fungi, viruses, and archaea, which colonize human hosts
from birth, have co-evolved with them across the millennia, and play important roles in several
host-related physiological functions.1.2 Such functions include the synthesis of important
vitamins, like vitamins K, B12, folate, and thiamine, contributed among others by species of
the genus Bifidobacterium, energy extraction from undigested fiber through bacterial
fermentation and production of the host-absorbable short-chain fatty acids propionate, acetate,
and butyrate, and metabolic processing of various non-dietary xenobiotic compounds, i.e.,
mainly hydrolysis and reduction by multiple bacterial species of non-human origin chemical
substances entering the gut through ingestion, either as pharmaceutical compounds or industrial
chemicals.1,2
Over 99% of the microbiota in the large intestine is composed of bacteria, mostly
anaerobic, with a total number of prokaryotic cells about equal to the total number of cells in
the human body.3-5 In recent years, this bacterial society of interdependent species has become
a center point of research, with multiple studies linking changes in gut microbiota synthesis
and diversity to a variety of human disorders.6
In this review, we focus on the associations of the intestinal microbiota with
cardiovascular disease (CVD). We first briefly overview important properties of the intestinal
microbiota and describe methods of microbial analysis. We go over the absolute basics of data
analysis to facilitate the understanding of study results. We then move to present key published
research in humans, connecting the microbiota with heart failure, hypertension, and
atherosclerosis (Table 1), and discuss remaining challenges to be met.
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2. The microbial ecosystem of the human gut
The large intestine is a habitat for trillions of commensal or mutualistic microbes that form an

interdependent and interactive community of species termed the intestinal microbiota, or gut
flora. The microbiota plays a crucial role in many host-related functions, including but not
limited to the training and maturation of the host immune system, energy harvest through
fermentation of foods undigested by humans, vitamin synthesis, deconjugation of bile acids,
and, last but not least, resistance to colonization by or overgrowth of pathogenic
microorganisms (Figure 1).1,2,7
The composition of the intestinal microbiota is overwhelmingly dominated by
anaerobic bacteria and influenced by host age and genetics, geographic origin, and diet, among
others.3,4,8,9 Especially diet exerts considerable influence in the shaping of a bacterial
community. Human infants, relying on breast-fed milk for their dietary needs, have low
diversity microbiota dominated by various species of Bifidobacterium, which thrive in the
presence of human milk oligosaccharides.9 Older children and adults experience a growing
diversity of bacterial species, as breast feeding ceases and solid food is introduced into the diet.
Distinct dietary habits across different geographic regions may also explain variations in gut
bacterial community composition of diverse adult populations, e.g. between USA citizens and
rural Africans.9
In general, humans are immediately colonized at birth, the microbiota gradually
increases its diversity and, for the higher taxonomic levels, resembles an adult composition by
age 3, and it remains relatively stable until old age if no serious perturbation occurs, comprising
four major bacterial phyla (Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria) and
an average of 500 species per host.1,7,10-12 Although community synthesis is stable at the
phylum level and a core of bacterial genera is present in the majority of adult hosts, there is
huge variability at a subspecies level, to the point where a unique microbial synthesis can be
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postulated for each individual.4,6 However, key microbial metabolic functions are present
throughout all species.13 That is, the most abundant metabolic pathways in terms of the number

of bacterial genes involved (e.g. vitamin biosynthesis, carbohydrate metabolism, or ATP
synthesis) are present in all bacteria and more or less equally prevalent among all gut microbial
communities, making the functional capacity of the intestinal microbiota much less variable
than its composition. Nevertheless, in terms of abundance, Bacteroides has been shown to be
the most abundant bacterial genus in the gut of western populations,13 while other important
genera include Prevotella and Ruminococcus.4,13
Bacteroides, an obligate anaerobic bacterial genus, makes up the greater portion of the
total microbial community and includes important and potentially infective species, such as
Bacteroides fragilis.3,13 Bacteria of this genus play crucial roles in fermenting plant fiber and
extracting energy usable by their host. Species of Prevotella are also well equipped to ferment
plant carbohydrates, and have been associated with fruit and vegetable-rich dietary patterns,14
colonizing the gut in an antagonistic struggle against species of Bacteroides. Members of
Ruminococcus, another anaerobic genus, also can ferment cellulose, pectin, and, starch,
contributing to the production of short-chain fatty acids.
Crucial to the field of intestinal microbial research are the potential ramifications that a
change in the composition or functions of the intestinal microbiota may bring about for its host.
Such departure from homeostasis has been termed “dysbiosis” and numerous lines of evidence
exist for compositional changes in a variety of human disorders, including CVD.6 A proposed
underlying mechanism posits that environmental triggers cause gut mucosal inflammation,
which in subjects predisposed to chronic disorders may lead to an increase in opportunistic
microbes and the transition to bacteria that, under certain physiological conditions, may
promote local or systemic pathogenetic functional changes in susceptible individuals.6
However, with few exceptions, current research generally remains far from establishing a cause
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and effect relationship between changes in the composition and diversity of the microbiota and
human disease.

3. Methods of microbial analysis
An in-depth coverage of all aspects of microbiome analysis is beyond the scope of this review.
A few important points should nevertheless be mentioned. After the collection of a sample and
appropriate extraction of bacterial DNA, the principal methods of analyzing the intestinal
microbial composition are either amplicon sequencing of the 16S ribosomal RNA (rRNA) gene,
or metagenomic processing of all the DNA strands recovered from the sample.15
Amplicon sequencing most commonly involves the identification and sequencing of
the 16S rRNA gene, the bacterial gene coding for the 16S rRNA component of the 30S small
subunit of the prokaryotic ribosome.16 This gene features highly preserved sequences present
in all bacteria, but also possesses multiple hypervariable regions showing differences between
different microorganisms. This unique feature makes it a prime target for both discovering all
bacteria in a sample and taxonomically differentiating between them.17 16S rRNA analysis is
a cheap, simple, and arguably accurate method of analysis, but yields a relatively low amount
of information, basically resulting in the identification and proportional calculation of bacterial
genera present in a sample.18
Metagenomic analysis, on the other hand, involves the direct, next-generation shotgun
sequencing of all the fragments of DNA recovered from all the organisms present in a sample.19
This is a much more expensive and complex method, but with a considerably higher yield of
functional information.4,19 Through appropriate techniques, bacterial species and strains
present in a sample can be identified and data on genes present in each bacterial organism can
be generated.19 This enables researchers to acquire information on important metabolic
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pathways present in the bacterial community, thus highly facilitating a functional profiling of
the intestinal microbiota.

4. Data analysis and visualization
The final output of the microbial composition analysis, generated through an appropriate
bioinformatics pipeline such as QIIME (Quantitative Insights Into Microbial Ecology),20 may
have the form of a typical high-dimensional spreadsheet (e.g. analyzed samples in rows and
the measured bacteria in hundreds of columns). Each cell will show a proportion, the percent
contribution of the specific bacterial taxon to the total of the bacterial community of a given
sample, called its relative abundance. For example, the relative abundance of Streptococcus in
sample 1 may be “α%”, meaning that α% of all prokaryotic cells present in sample 1 belong to
the genus of Streptococcus. This spreadsheet, and the preceding Operational Taxonomic Unit
(OTU) table [usually a non-human readable biological observation matrix (BIOM) file
constituting a part of the analysis pipeline], may form the basis for all subsequent analysis,20
which, at the very least, follows three basic pathways: the calculation of alpha diversity, the
calculation of beta diversity (both explained below), and a comparison of groups of subjects
by each bacterial taxon. It must be stressed that microbial relative abundance data are
multivariate (there is a large number of measured variables in each sample), that they almost
never follow a normal distribution, and that they are compositional in nature (they are
percentages that must total 100%). Extreme caution must thus be exercised on the choice of
statistical tests, as an inappropriate test will invariably lead to false conclusions.
Alpha diversity
Alpha diversity refers to specific characteristics of an ecological community, such as the
number of different species (richness) or how evenly the individual organisms are distributed
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among the different species (evenness). Usually, diversity indices that incorporate and combine
this information, such as the Shannon index,21 can be calculated, averaged across subjects, and

compared between groups of subjects through a simple ANOVA or Student’s t-test. Generally
speaking, a lower alpha diversity can be thought of as a negative phenomenon in a given
ecosystem of interacting species. Although alpha diversity is a common tool of describing
ecological communities, collapsing the incredible bacterial variation to a single index is a
limited approach to comparing groups of samples.
Beta diversity, multivariate analysis, and predictive functional profiling
Beta diversity is a concept of direct comparison between different ecological communities. The
calculation of a measure of similarity, most commonly the Bray-Curtis similarity coefficient
or the UniFrac distance metric,22,23 makes possible the direct comparison of different bacterial
communities in different samples – e.g. “samples A and B are 65% similar based on their Bray-
Curtis similarity”. Multivariate permutational methods that can analyze the data en masse, such
as the non-parametric “analysis of similarities” (ANOSIM) and the semi-parametric
“permutational multivariate analysis of variance” (PERMANOVA),24,25 can then be applied to
decide whether the grouping of samples in, for example, “healthy controls” and “patients”
yields a statistically significant difference in the overall structure of the bacterial communities;
in other words, whether the “patient” samples are significantly more similar to each other than
they are to the “healthy control” samples. Importantly, these methods do not only constitute
tests for statistical significance, but also offer a measure of how big a difference between groups
really is, regardless of its statistical significance.
Of note, multivariate analysis is not restricted to comparing samples in terms of
bacterial relative abundance values, but can also be applied to gene content data, either
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measured through next-generation sequencing or inferred by appropriate algorithms from 16S
rRNA data. A key such algorithm is PICRUSt, which stands for “Phylogenetic Investigation

of Communities by Reconstruction of Unobserved States”.26 The relative software is freely
available online and serves as a zero-cost, adequately accurate alternative of estimating
bacterial gene numbers present in a sample, to actually following the more accurate but
expensive shotgun sequencing pathway. In simple terms, PICRUSt works in two steps:26 On
the first step, it relies on a reference database to calculate gene numbers in bacteria with known,
sequenced genomes, and estimate gene numbers in bacteria without a known, sequenced
genome. This estimation is based on evolutionary models and phylogenetic principles, that
basically allow for prediction of gene content in a certain organism if the gene content of a
phylogenetically related organism is already known. On the second step, PICRUSt incorporates
the sample 16S rRNA data into the process (which bacteria are present in a sample and what
their abundance is), multiplies gene numbers from the first step by bacterial abundances from
the second step and estimates how many genes of each gene family exist in the sample. The
final output is a table of gene counts per sample per functional pathway, e.g. how many
bacterial genes related to methane metabolism are predicted to exist in sample A. This table
essentially offers a functional profile of the bacterial communities present in a researcher’s
samples. Applying multivariate analysis on such data thus allows for an extension of sample
comparison to functional (i.e. metabolic) dimensions, substantially improving research efforts
based on amplicon sequencing and allowing for a more spherical view of the bacterial
communities in question.
Ordination techniques
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Similarities or differences between groups may already be apparent after appropriate
visualization of the data, usually through complex mathematical algorithms like non-metric

multidimensional scaling (MDS) and principal coordinates analysis (PCoA).27-29 These
ordination techniques are, in simple terms, tools that will take a spreadsheet of dozens of
samples and hundreds of bacteria and, through utilization of a measure of similarity and a series
of mathematical calculations, transform it into a scatterplot graph where samples that are more
similar to each other will be less distant to each other (Figure 2). Such a treatment of the data
enables the human eye to readily identify similarities and differences in the overall structure of
the bacterial communities of the analyzed samples.
Univariate analysis
A third route of analysis involves the one-by-one comparison of each microbial variable
between groups of subjects (e.g. the mean relative abundance of each bacterial genus), much
as these groups can be compared in terms of a mean body weight or the mean left ventricular
ejection fraction in common univariate analysis. As bacterial relative abundance data are
notoriously non-normally distributed, a non-parametric test must be used in order to achieve
meaningful results. Moreover, as this route will generate hundreds of individual comparisons
and, by chance alone, many false positive results, a correction for multiple comparisons must
also be made, usually by the method of false discovery rate adjustment developed by Benjamini
and Hochberg.30
5. The intestinal microbiota and cardiovascular disease
i. Gut microbiota and heart failure
Pathophysiological sequelae of heart failure, including a reduced cardiac output and peripheral
hypoxia, may alter gut function and compromise the intestinal epithelial barrier,31 allowing
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increased leakage of microbial compounds such as lipopolysaccharide (LPS) into the
bloodstream and induction of low-grade systemic inflammation in the host (Figure 3, Table 2),

which has well established links with cardiac dysfunction and heart failure development (see
the section on atherosclerosis for a more detailed discussion of the association between the
microbiota and inflammation).32,33 However, whether gut microbial composition changes in
this context was until recently not known.
The link between heart failure and changes in the intestinal microbiota was already
hinted upon by Sandek et al., 34,35 who showed that heart failure patients had altered intestinal
function, bowel wall edema, and an overgrowth of microorganisms in examined biofilms. In
these studies, microorganisms were directly sampled from intestinal parts rather than only
collected from stool, which better reflects intestinal microbial communities. Interestingly, the
total amount of bacteria in the mucosal biofilms did not correlate with stool bacteria.35
The first study that showed changes in microbial community composition in the gut of
heart failure patients was published in 2015.36 Potentially pathogenic microbes, like species of
Shigella or Salmonella, were found in higher concentrations in stool samples of chronic heart
failure patients, as compared to healthy controls. Although this effort was flawed by neglecting
to correct for multiple comparisons and limited in its scope by being based on microbial
cultures instead of DNA analysis, it was the first step taken in the relative field.
Compositional changes were further examined by Luedde et al.,37 who were the first to
move beyond stool cultures and employ 16S rRNA amplicon sequencing. A general shift in
community composition and a depletion of several microbial genera was shown to characterize
heart failure, although alpha diversity was only nominally different between groups.
Subsequent studies have corroborated both the change in beta diversity and the fact that alpha
diversity is similar between heart failure patients and controls.38-40
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A fundamental aspect of microbiota research is the examination of microbial metabolic
capacity. To this point, we have performed a predictive analysis of the metabolic gene content

of the microbiota using the PICRUSt algorithm, which unveiled a strong differentiation
between heart failure patients and controls, and numerous metabolic pathways, including
pathways in vitamin synthesis, depicting a change in the relative abundance of involved genes
between the groups.40 Although these data were suggestive only and did not constitute gene
expression results, metabolomics analyses have been performed and have established such a
differentiation.39
In fact, a specific bacterial metabolite, trimethylamine N-oxide (TMAO), which has
been implicated in atherosclerotic disease, has also been associated with heart failure.41 Stable
heart failure patients were shown to have higher fasting plasma levels of TMAO when
compared to healthy controls, and higher levels of TMAO within the heart failure group were
independently associated with a higher 5-year mortality risk.41 In this study, however, no
significant change of the intestinal microbiota was detected in the patients with a higher
mortality risk and no information was available on the use of antibiotics prior to sampling.
In summary, the composition of the intestinal microbiota in the context of human heart
failure has been mostly examined by a few small observational studies. An emerging pattern is
that alpha diversity is not significantly different in heart failure patients compared to controls,
meaning that the richness and distribution of the intestinal bacteria do not change in this context.
Although studies agree that the overall composition of the microbiota does change, the findings
on the changes in individual bacterial genera among the studies are not consistent. Some
evidence of functional alterations has also been provided,39,40 but this is only preliminary.
Contrary to evidence causally implicating gut bacteria with atherosclerotic disease, there is no
such evidence in heart failure.
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ii. Gut microbiota and arterial hypertension
A link between the gut and hypertension has long been suggested, based on epidemiological

data associating salt and fiber intake with changes in blood pressure.42-48 Dietary sodium, a
potent factor in the pathogenesis of hypertension, makes first contact with the human organism
at the gut epithelium, where it is absorbed by enterocytes. The gut harbors its own renin-
angiotensin-aldosterone system, important for the uptake of sodium in the colon, which
interacts with gut bacteria and may be influenced by metabolites produced by the latter. Various
studies have shown that an increased sodium intake is associated with increases in blood
pressure.42-45 Moreover, meta-analyses have shown that an increased consumption of fiber,
which is primarily indigestible but can be fermented by gut bacteria as a source of short-chain
fatty acids, is associated with a lowering of both systolic and diastolic blood pressure in
hypertensive patients, as well as normotensives, to a lesser extent.47,48 Although the
mechanisms underlying this effect have not been solidly established, evidence implicating the
microbiota is accumulating (Figure 4). Unfortunately, research indicating a specific association
between compositional or functional alterations of the human intestinal microbiota and
hypertension has been limited, while some evidence has come from studies on rodents.49-51 A
mechanistic link between the intestinal microbiota and hypertension might involve quantitative
or qualitative changes in microbial metabolites, such as the short-chain fatty acid butyrate, that
could influence host inflammatory processes52 or sodium intake, and favor hypertension
development – the possible pathophysiological basis has been recently reviewed by Richards
et al.53 (Figure 4, Table 2, see also section 6 for sodium effects on the microbiota).
Yang et al.50 showed that hypertension in humans may be associated with changes in
the intestinal microbiota. Hypertensive patients reportedly had lower alpha diversity and a clear
visual separation of the hypertensive and control groups was shown in PCoA (based on the
weighted UniFrac distance metric). Although this study50 did not use healthy subjects as
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controls, it set the foundation for further research in the field, which yielded similar findings.54
Interestingly, human hypertensive hosts might transfer a hypertensive profile to germ-free mice

receiving the hosts’ intestinal microbiota, pointing to as yet unexplained causative links
between the microbiota and hypertension (Figure 4).54 Yet another study comparing
hypertensive patients to healthy controls showed differentially enriched bacterial genera
between the groups and a visual separation in distance-based redundancy analysis, although
particulars about the statistical significance of multivariate analysis were not reported.55
In summary, the concept of microbial alterations in human hypertension has not yet
been fully addressed, as there are very few studies in the literature with small sample sizes.
Contrary to the heart failure field, evidence suggests that the alpha diversity of the intestinal
microbiota in hypertensive disease may be lower. Again, causative links between gut bacteria
and the development of hypertension have not been established yet.
iii. Gut microbiota and atherosclerosis
The association of intestinal bacteria with atherosclerosis has received considerable attention,
particularly since the reported link between bacterial metabolite TMAO and CVD. This
particular link (explained and referenced below) has drawn much scientific attention and stirred
cardiovascular research on gut microbiota. It should be mentioned that much evidence exists
linking the microbiota with obesity,56,57 an important risk factor for diabetes mellitus and
atherosclerosis.58,59 However, here we focus on studies directly addressing microbial changes
in human atherosclerotic subjects.
The pathophysiology underlying the links between the intestinal microbiota and
atherosclerosis has been recently reviewed by Jonsson and Bäckhed.60 The mechanisms may
include infection, inflammation, lipid metabolism, and, possibly, production of harmful
metabolites (Figure 5, Table 2).
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Infection, regardless of site, leads to a pro-inflammatory profile that may affect plaque
stability.61 Microbial DNA has been recovered from atherosclerotic plaques, indicating a

potential link between the presence of symbiotic or pathogenic bacteria that may translocate
from the gut, the oral cavity, or other body locations to the plaque and the atherosclerotic
process.
Epidemiological evidence linking LPS with atherosclerosis exists for more than two
decades.62 Nonetheless, in trials where antibiotic use was tested, it did not show any
cardiovascular benefit.63 However, the interaction of the microbiota with the host immune
system is well established, and disruption of immune system homeostasis may influence CVD.
Human adaptive immunity has coevolved with gut bacteria, has matured in their presence and
has “learnt” to tolerate them.64 Maternal transfer of secretory IgA (sIgA) to the infant through
breast milk promotes regulatory immune networks favoring mutualism, since antigens bound
by sIgA are handled in a tolerogenic way.64 Gut associated lymphoid tissues (GALT) rely on
microbial signals to develop, as microbe-associated molecular patterns (MAMPs) are sensed
by pattern-recognition receptors, including Toll-like receptors (TLR), on epithelial and
dendritic cells, stimulating the maturation of cryptopatches.64 Under homeostatic conditions,
MAMPs lead to production of cytokines that promote tolerogenic macrophages and dendritic
cells. Innate immunity mechanisms, including secretion of antimicrobial peptides and tight
barrier function of the epithelium, mediate the confinement of bacteria in the lumen, but the
microbiota interacts with the host in regulating innate processes, as LPS and butyrate are
drivers of mucin production and maintenance of the mucus layer. Butyrate, in particular, has
strong anti-inflammatory properties through inhibition of nuclear factor kappa-light-chain-
enhancer of activated B cells (NF-κB),65 while another bacterial product, indole, increases
expression of genes that play a role in strengthening the intestinal barrier, as well as expression
of anti-inflammatory IL-10.66 Conceivably, a dysbiotic microbiota may interrupt the
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aforementioned interactive pathways and initiate inflammatory responses that may affect
atherosclerosis and CVD risk. C-C chemokine receptor type 2 (CCR2) expressed in monocytes

is associated with arterial wall inflammation,67 and has been shown to both have a lower
expression after Roux-en-Y gastric bypass surgery in obese diabetic patients and negatively
correlate with an increase in Bifidobacterium.68 TLR4 signaling, induced by LPS, may mediate
myocardial inflammation and has been shown to be inhibited by adiponectin in rodents, 69 the
expression of which in Paneth cells is regulated by a strain of Lactobacillus in mice.70 Moreover,
advanced atherosclerotic plaques in apoE-/- mice have increased expressions of miR-10b, and
bacterial metabolite cyanidin-3-O-glucoside down-regulates miR-10b and leads to plaque
regression.71
Lastly, lipid metabolism influenced by the microbiota might lead to changes in blood
cholesterol levels, indirectly contributing to atherosclerosis, but a clear association between
changes in gut microbiota and shifts in LDL cholesterol levels has not yet been reported.72 Still,
certain microbial compounds, such as LPS, or metabolites, such as TMAO, may exert a
proatherogenic effect to the host (as explained below), while microbial substances are involved
in aspects of platelet activation, as depletion of gut flora resulted in reduction of platelet-
produced soluble P-selectin concentrations in a certain strain of mice.73 Such substances may
not include only LPS, but viral particles as well. Microbial compounds could activate platelets
by acting as ligands for TLR, as platelets, like macrophages or dendritic cells, express TLR on
their surface.
Koren et al.74 were the first to have examined the composition of the intestinal
microbiota in atherosclerotic patients. They assessed the microbiome recovered from both
atherosclerotic plaques and stool samples of patients with clinical atherosclerosis and found
that they were distinct from each other, i.e., plaque and intestinal bacterial communities are
different in composition. When they compared the gut microbiome of patients to non-
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atherosclerotic controls they found no significant difference between them, although it is
unclear whether they performed a beta diversity analysis beyond the phylum level. In contrast,

other studies have found a significant difference in terms of beta diversity, or various individual
taxa (Table 1).75-78 More importantly, metabolic features of the microbiota differ between
atherosclerotic patients and controls.75,79 For example, the potential for butyrate synthesis by
the microbiota was shown to be lower in patients with atherosclerosis compared to healthy
subjects.79
TMAO and the TMAO paradox
Data on the bacterial metabolite TMAO and evidence that it may promote atherosclerosis were
first published in 2011 by Wang et al.80 The authors first isolated circulating plasma metabolites
that predict cardiovascular events from a cohort of human patients and controls and identified
TMAO and its precursor, choline, as linked to CVD risk. They next showed in mice that gut
flora presence is required for the formation of TMA from choline, before TMA is converted to
TMAO by enzymes in the liver. Choline itself is derived from dietary phosphatidylcholine
found in various foods, including red meat. The authors verified that TMAO levels predict
CVD risk in a large independent cohort of 1,876 subjects, even showing that TMAO levels are
proportionate to the number of stenotic coronary arteries. Finally, they proposed a mechanistic
link between TMAO and the atherosclerotic process through promotion of macrophage foam
cell formation, based on experiments on atherosclerotic prone mice fed with a choline enriched
diet.
Much work has followed on TMAO and its relation to CVD, with not always consistent
results. Tang et al.81 verified the predictive role of TMAO for CVD risk in a large cohort of
over 4,000 patients, on top of conventional risk factors. A recent meta-analysis supports these
results.82 In contrast, TMAO was comparatively decreased in the blood of over 300 Chinese
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patients that had suffered an ischemic stroke or a transient ischemic attack, contrary to
expectations.78

Beyond conflicting results, the TMAO paradox arises from the fact that the main dietary
source of free TMAO and the main food source capable of generating a high urinary excretion
of TMAO in humans is fish, which can yield significantly bigger amounts of absorbable TMAO
than choline contained in meat or eggs.83,84 Consumption of fish is widely accepted to be
beneficial for cardiovascular health.85-87 Not all fish have the same amounts of TMAO and
generalized epidemiological evidence might miss this distinction. However, this counter
argument should probably be taken into serious consideration. It has been suggested that
TMAO may simply be a confounding factor of a different causal relation, namely changes in
the composition of TMA-producing gut bacteria.88 TMA producers are generally of low
abundance and have been identified as Clostridium XIVa strains, Eubacterium species strain
AB3007, and Escherichia coli, although many more are expected to exist.89
To summarize, much evidence exists on the association of the intestinal microbiota with
atherosclerosis and ischemic heart disease. Studies in human subjects have yielded some
conflicting results, but a general pattern of differences in beta diversity between patients and
controls has nevertheless emerged. Importantly, evidence exists about specific microbial
metabolic products that are associated with increased CVD risk. However, data are again
conflicting and more research is therefore necessary.
6. Microbial associations with dietary sodium and carbohydrate intake, and probiotic use
in cardiovascular disease
Dietary habits play important roles in gut microbial composition, as has already been
mentioned. Two key dietary elements affecting the microbiota that merit further attention are
dietary sodium intake and carbohydrate consumption. A high salt intake has been shown to
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negatively affect species of Lactobacillus in the human gut, and supplementation of
Lactobacillus murinus in mice (a species not found in humans) prevented the development of

hypertension by inhibiting salt-sensitive generation of TH17 cells.90 This finding is in line with
the small reduction of systolic blood pressure seen with use of Lactobacillus plantarum in
humans.91 Evidence also suggests that a high salt intake alters gut microbial composition in
mice, disrupts the epithelial barrier by affecting tight junction protein levels, and promotes
bacterial translocation from the gut to the kidney.92 Changes in microbial composition have
been linked to kidney injury, which may become the basis of a later rise in blood pressure.92 It
has been suggested that the mechanism underlying high salt intake-induced changes in
microbial composition is an increase in intestinal epithelial Na+/H+ exchange, since the latter
has been found to affect gut bacterial composition.93 Although these data are intriguing, they
are based on animal models and caution must be exercised when extrapolating to human
subjects.
Carbohydrate consumption is a major factor affecting the microbiota, as dietary fiber is
a key energy source for gut bacteria. In the context of CVD, evidence from mice harboring
synthetic human gut microbiota suggests that a diet low on fiber, which may be typical for
western populations, can result in gut mucus layer degradation and pathogen susceptibility, as
some of the microbiota turns to glycoproteins secreted by its host to use as energy source. 94
Conceivably, such a pro-inflammatory state can have consequences promoting the
atherosclerotic process. Human studies have also supported the role of a high fiber intake in
reducing cardiovascular risk,95 and it can be suggested that the underlying mechanism resides
partly in gut microbial metabolism and the production of anti-inflammatory short-chain fatty
acids. Conversely, a diet rich in refined carbohydrates, also typical in the western world, has
long been suggested to increase the risk of CVD.96
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The use of probiotics has been explored as a method to modulate intestinal microbiota
properties in the context of CVD and other diseases. Probiotics are ingestible microorganisms

that reach the intestinal lumen, where they exert mainly functional effects – i.e., they generally
do not affect the composition and diversity of the resident microbiota, especially in healthy
subjects,97,98 although some evidence suggests that gut colonization by probiotics is possible in
a person- and strain-specific manner,99 and that a probiotic effect on the host gut microbiome
may be seen in susceptible individuals.99 To date, beneficial effects have been acknowledged
for some bacterial species, when these are ingested in adequate amounts.100 There is also
evidence of strain-specific benefits for certain disorders, mostly of the gastrointestinal
tract.97,100 However, most health claims for probiotics rely on poor evidence and the notion that
probiotic use can correct dysbiosis is not well supported for the majority of the examined
products.101 There is some evidence that probiotic use can confer short-lived benefits in healthy
humans, but certainly more research is required to explore any long-term effects.102 Such
research is made imperative given evidence that probiotics may adversely affect gut microbiota
recovery after experimental antibiotic use in healthy humans.103 Some probiotic strains may
take advantage of gut microbial depletion following broad spectrum antibiotic use, to
effectively colonize the intestinal mucosa. Once such a colonization takes place and while
probiotic use is continued, the new resident bacteria may antagonize and resist a
reestablishment of the homeostatic bacterial composition and diversity in multiple ways and
with conceivable negative implications for the host, in the same way that some of them are
expected to antagonize pathogen colonization in the context of preventing antibiotic-associated
diarrhea.
More specifically, there is limited data available for the use of probiotics in CVD. One
study reported that consumption of preparations containing Saccharomyces boulardii may
increase the left ventricular ejection fraction in heart failure patients.104 A larger trial is
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underway to verify this result.105 Further, a recent meta-analysis of 9 randomized, placebo-
controlled human trials showed that probiotic consumption has a weak lowering effect on both

systolic and diastolic blood pressure, reducing systolic pressure by a mean 3.56mmHg and
diastolic pressure by a mean 2.38mmHg.91 However, the included studies and the effect on
blood pressure were relatively small, and more evidence is probably required.
Lastly, probiotic administration has also been utilized to counter the possible
proatherogenic effects of the intestinal microbiota, with some promising results.106 Potential
mechanisms include the induction of bile acid deconjugation, which will increase their
excretion and force the host to use up more cholesterol to balance the effect.106 While additional
evidence is required, consumption of Lactobacillus acidophilus may have a greater effect in
lowering cholesterol than other probiotic preparations.107
Although the research mentioned above points to potentially beneficial effects of
probiotics in CVD, caution must be exercised when contemplating probiotic use in critically
ill hospitalized patients. In particular, patients with a compromised gut epithelial barrier may
theoretically suffer a translocation of ingestible microbes from the gut to other body areas,
whereas patients with central venous catheters might infrequently suffer fungemia from
Saccharomyces boulardii.108
7. Remaining challenges in microbial research
Despite some promising evidence on the role of intestinal microbiota in cardiovascular
diagnostics and therapeutics, major challenges in the field must be overcome, before an impact
at the clinical level is felt.
Such challenges may relate to technical issues that hinder the generalizability of study
results. Although a common set of methodological tools is broadly used, a consensus on how
to conduct a microbiome study, from study design and sample collection to data analysis and
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interpretation, is still missing. As a result, differences in methodology may render different
studies incomparable. For example, some studies differ from others on which hypervariable

regions of the 16S rRNA gene they have targeted for sequencing. Such a difference will
generate variation in the identification and proportional calculation of the assessed
microorganisms because of technical issues alone.
Moreover, studies involving heterogeneous patient populations may be biased by
confounders such as the microbial variation in the context of different disease states (e.g.
stable/decompensated heart failure), disease aetiology (e.g. ischemic vs. non-ischemic heart
failure), as well as differences due to the geographic origin of subjects, rendering comparisons
difficult at best. Confounding factors such as diet and antibiotic use must also be thoroughly
addressed before including subjects in a study. Often, variation in medication, age, and dietary
habits will affect results and complicate a conclusion. Especially regarding medication,
research suggests that widely prescribed drugs other than antibiotics, such as metformin and
proton pump inhibitors,109,110 are associated with alterations in gut microbial composition.
However, research regarding the direct impact of medication to gut microbial communities is
generally limited and faces various experimental challenges.111 There are virtually no studies
indexed in PubMed showing an association, causal or otherwise, of cardiovascular medication,
such as β-blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers,
diuretics, and mineralocorticoid receptor antagonists, with changes in gut microbial
composition in humans.
Further, findings on the relative abundance of specific microorganisms are usually
inconsistent between studies, as can be seen in Table 1. This could be attributed either to the
study design and sample size, or to background characteristics of subjects. Alarmingly, it could
also be attributed to chance. However, this fact may nevertheless support the notion that the
real finding of major importance is the overall shift in bacterial community composition seen
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in the context of CVD and not the changes in individual genera or species per se. Importantly,
changes in the relative abundance do not necessarily suggest a change in the actual abundance

of a given microorganism, since the rise in the relative abundance of a certain bacterium may
be the result of the actual decrease of another. This factor complicates both the outcome and
interpretation of study findings, as well as the comparison between studies. Employment of
direct quantitative PCR measurement of the bacterial genera of interest could provide more
reliable data on this issue.
Another important point relates to the statistical analysis of the data. The nature of
microbial data requires special attention and rigorous statistical methodology, which is
generally different from the common analytical tools used in the bulk of clinical cardiovascular
research.
Furthermore, prospective studies in human patients with serial sampling and reports on
the change of microbial communities as a function of time (e.g. before and after an
intervention) are absent from the relative literature. Such studies are urgently needed, to
elucidate critical questions on cause and causation.
Finally, it should also be stressed that the compositional differences of gut microbial
communities between humans and rodents make animal study findings not directly translatable
to humans.112
8. Conclusions
Ample evidence has highlighted the important functions of the gut microbiota that benefit
human hosts, as well as its compositional and functional alterations associated with heart
failure, atherosclerotic disease, and hypertension. Although associations between gut
microbiota and CVD have been reported, the existing evidence includes mostly cross-sectional
studies, with small sample sizes and inconsistent results. The overarching pathophysiological
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link between gut bacteria and CVD seems to be the induction of inflammation in the host via
structural or functional alterations of the microbiota. However, whether such changes in the

gut microbiota are causally associated with CVD, are influenced by CVD, or are simple
bystanders, remains to be proved. Future research is therefore warranted to incorporate the
wealth of information on gut microbiota into clinical decision pathways and personalized
medicine.
24
25
Conflict of interest: None declared.
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Table 1. Summary of studies on the association of the intestinal microbiota with cardiovascular disease.
Authors Subjects Study design Analysis Main findings Suggested mechanisms

Heart failure
Sandek et Caucasians; Germans by Cross- FISH of biopsy Higher mean density in An abnormal mucus barrier allows
al.34 inference. 22 patients, 22 sectional films mucus and higher adherence bacteria to penetrate the mucus and reach
controls to the mucosa for bacteria in the mucosa in patients
patient samples
Sandek et Race and ethnicity not Cross- FISH of biopsy Concentrations and Alterations of the local mucosal
al.35 stated; Germans by sectional films; FISH and proportions of bacteria similar environment caused by reduced
inference. 65 patients, 25 epifluorescence in stool samples of patients mesenteric blood flow and endothelial
controls microscopy of and controls. Higher dysfunction favor the growth of bacteria
stool samples concentration of mostly
anaerobic bacteria in biofilms
of patients
Pasini et Race and ethnicity not Cross- Stool cultures Higher concentrations of 4 Changes in fluid balance affecting
al.36 stated; Italians by sectional genera of pathogenic bacteria gastrointestinal motility and transit time
inference. 60 patients, 20 (Shigella, Salmonella, favor stasis and bacterial overgrowth in
healthy controls Campylobacter, Yersinia) and patients. Acid-suppressing therapy,
1 genus of fungi (Candida) in nutrient deprivation, and hypoxemia also
patient samples suggested as factors
Luedde et Race and ethnicity not Cross- Amplicon Significant difference in beta Diet, fluid imbalance, bowel dysmotility,
al.37 stated; Germans by sectional sequencing (16S diversity between patients and and hypoxia may favor changes of
inference. 20 patients, 20 rRNA analysis) controls. A few bacterial bacterial composition in patients
controls genera significantly decreased
in patients (Blautia,
Collinsella, unclassified
Erysipelotrichaceae,
unclassified
Ruminococcaceae)
Kamo et Race and ethnicity not Cross- Amplicon Alpha diversity similar in HF-related epithelial dysfunction or
al.38 stated; Japanese by sectional sequencing (16S patients and controls. confounders, such as acid-suppressing
inference. 22 patients, 12 rRNA analysis) Differences in beta diversity medication, induce changes in bacterial
healthy controls between groups. Two composition. Age-related confounders
bacterial genera (Clostridium may explain age-related differences;
and Dorea) less abundant in currently uncertain how much HF
patients. Differences in beta influences such differences
diversity, phyla, and few
bacterial genera between
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younger (<60) and older

(>60) patients
Cui et al.39 Chinese; 53 patients, 31 Cross- Metagenomics 86 bacterial genera Bacterial dysbiosis contributes to host
controls sectional and metabolomics differentially enriched inflammation, thus aggravating HF status.
analysis between groups. Differences Increased TMAO leading to renal
extending to metabolic dysfunction may also aggravate HF
profiles. Genes for TMAO
synthesis enriched in patients
Katsimichas Japanese; 28 patients, 19 Cross- Amplicon Groups differing in beta but Gut endothelial dysfunction in patients
et al.40 healthy controls sectional sequencing (16S not alpha diversity. Few may be responsible for bacterial dysbiosis
rRNA analysis) bacterial genera significantly
different between groups
(Streptococcus, Veillonella,
SMB53). Metabolic profiles
significantly different, various
gene families differentially
enriched or depleted in
patients
Tang et al.41 Race and ethnicity not Prospective Liquid chromato- Fasting plasma TMAO levels Overt or subclinical atherosclerosis
stated; Americans by patient graphy/mass higher in patients. Adjusted influenced by proatherogenic TMAO may
inference. 720 patients, cohort/Cross- spectrometry for higher TMAO levels aggravate HF status
300 healthy controls sectional TMAO associated with increased
comparison measurement mortality risk
to healthy
controls
Hypertension
Yang et Race and ethnicity not Cross- Amplicon Alpha diversity lower in Gut dysbiosis-related host inflammation
al.50 stated; Americans by sectional sequencing (16S patients. Groups also different contributes to the establishment and
inference. 7 patients, 10 rRNA analysis) in beta diversity maintenance of high blood pressure
controls.
Li et al.54 Race and ethnicity not Cross- Metagenomics Alpha and beta diversity Host inflammation induced by bacterial
stated; Chinese by sectional and metabolomics different between healthy dysbiosis contributes to hypertension
inference. 99 hypertensive analysis controls and prehypertensive/ pathophysiology
patients, 56 prehypertensive subjects. Fecal
hypertensive controls, 41 transplantation of
healthy controls. hypertensive subject
microbiota resulted in higher
blood pressure in mice
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Yan et al.55 Race and ethnicity not Cross- Metagenomics Beta diversity visualization None provided
stated; Chinese by sectional analysis different between healthy
inference. 60 hypertensive controls and hypertensive
patients, 60 healthy subjects. Klebsiella,
controls Clostridium, Streptococcus,
Parabacteroides, Eggerthella,
and Salmonella enriched in
patients, Faecalibacterium,
Roseburia, and Synergistetes
enriched in controls.
Lipopolysaccharide
biosynthesis and TMA
production genes enriched in
patients, SCFA genes
depleted
Atherosclerosis
Koren et Caucasians; Swedes by Cross- Amplicon Plaque and gut microbiomes Bacteria phagocytosed by macrophages in
al.74 inference. 15 patients, 15 sectional sequencing (16S different in beta diversity; the gut migrate to atherosclerotic plaques
healthy controls rRNA analysis) several bacterial phylotypes
common between
atherosclerotic plaques and
gut microbiota within the
same individual. Patient and
control groups similar in
terms of gut microbiota
Karlsson et Caucasians; Swedes by Cross- Metagenomics Patients and controls different Anti-inflammatory properties depleted in
al.75 inference. 12 patients, 13 sectional analysis in beta diversity. A few the gut microbiome of patients may
controls genera significantly different influence atherosclerotic procedure
in relative abundance between
groups (Collinsella,
Roseburia, Eubacterium).
Metabolic profiles also
different between groups,
based on KEGG orthologs
Emoto et Race and ethnicity not Cross- Terminal- The order Lactobacillales was Gut microbiota affects CAD through
al.76 stated; Japanese by sectional restriction increased in patients, the immune system-related mechanisms
inference. 39 patients, 30 fragment length phylum Bacteroidetes was
non-CAD controls, 50 polymorphism decreased
healthy controls analysis of
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bacterial DNA
from stool
samples
Yin et al.78 Race and ethnicity not Cross- Amplicon Alpha diversity greater in Stroke itself or stroke treatment reduces
stated; Chinese by sectional sequencing (16S patients, beta diversity TMAO levels by unknown pathways
inference. 322 patients, rRNA analysis) adequately separated two
231 controls for gut microbiota subgroups of 47 age- and sex-
and liquid matched pairs of patients and
chromato- controls. TMAO levels lower
graphy/mass in patients than in controls
spectrometry for
TMAO
measurement
Jie et al.79 Chinese; 218 patients, 187 Cross- Metagenomics Groups distinct in beta Patient gut microbiota linked to
healthy controls sectional analysis diversity, similar in alpha inflammatory profiles that may influence
diversity. Several species atherosclerosis
differentially abundant
between groups. Metabolic
profiles also different
between groups, based on
KEGG orthologs
Wang et Race and ethnicity not Observational Liquid chromato- TMAO predicts CVD risk in TMAO influences macrophage foam cell
al.80 stated; Multiracial by for human graphy/mass humans; TMAO promotes formation
inference. For TMAO- subjects/Int- spectrometry and atherosclerosis in
based CVD prediction: erventional other metabolo- atherosclerosis-prone rodents
cohort of 1,876 patients for rodents mics techniques
for TMAO
measurement
Tang et al.81 Race and ethnicity not Prospective Liquid chromato- TMAO levels predict TMAO enhances the accumulation of
stated; Americans by patient cohort graphy/mass increased CVD risk cholesterol in macrophages and that of
inference. 4,007 patients for main spectrometry for independently of other risk foam cells in arterial walls.
for main prospective study study leg TMAO factors
measurement
FISH: fluorescence in situ hybridization, rRNA: ribosomal RNA, HF: heart failure, TMAO: trimethylamine N-oxide, SCFA: short-chain fatty
acid, KEGG: Kyoto Encyclopedia of Genes and Genomes, CAD: coronary artery disease, CVD: cardiovascular disease.
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Table 2. Pathophysiological mechanisms linking the intestinal microbiota with
cardiovascular disease.

Disorder Pathophysiological mechanisms Key molecules
Heart failure • Reduced cardiac output may cause gut epithelial dysfunction • Cytokines (e.g. IL-1, TNF)
• Gut epithelial dysfunction may alter gut epithelial-microbial • Short-chain fatty acids
interactions, leading to dysbiosis (acetate, butyrate,
• A compromised intestinal epithelial barrier allows leakage of propionate)
bacterial LPS into the bloodstream, causing systemic inflammation • LPS
and aggravating heart failure status
Hypertension •Bacterial short-chain fatty acids affect immune and epithelial • Short-chain fatty acids
functions important in modulation of blood pressure (acetate, butyrate,
•Toxic bacterial byproducts may affect blood pressure physiology propionate)
•Microbiota-related host systemic inflammation may affect blood • TMAO
pressure levels •Tryptophan
•Enteric nervous system influenced by dysbiosis may affect blood •GABA
pressure by contributing to autonomic dysregulation •Aldosterone
•Dysbiosis modulating the gut renin-angiotensin-aldosterone •ACE2
system may affect blood pressure levels
Atherosclerosis • Bacterial infections activate inflammatory responses • Cytokines

• Bacterial compounds (LPS) leaking into the host's bloodstream • Toll-like receptors
cause systemic inflammation • Short-chain fatty acids
• Bacterial metabolites may be involved in proatherogenic (acetate, butyrate,
pathways. TMAO may inhibit reverse cholesterol transport and propionate)
increase foam cell formation • Bile acids
• Reduced bacterial short-chain fatty acid production may favor • LPS
host systemic inflammation • TMAO
LPS: lipopolysaccharide, IL-1: interleukin-1, TNF: tumor necrosis factor, TMAO: trimethylamine N-oxide,
GABA: gamma-aminobutyric acid, ACE2: angiotensin-converting enzyme 2.
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Table 3. Glossary of terms.
Term Description

Amplicon sequencing The analysis involving the identification and PCR amplification of a certain genetic
sequence, e.g. sequences of the 16S ribosomal RNA gene of the prokaryotic organisms
recovered from a stool sample.
Commensalism Non-harmful co-existence of two organisms in which only one of the two gains benefit.
Dysbiosis A change in microbial composition relative to the healthy state or homeostasis.
KEGG Kyoto Encyclopedia of Genes and Genomes. A database for genes and related gene
products involved in various cellular functions, among others. It is accessible at
www.kegg.jp.
Metabolomics The identification and measurement of the metabolites (products or intermediates of
cellular metabolic processes) found in a given sample.
Metagenomics The analysis involving the direct identification of all microorganisms and their genomes
in a given sample, without the need for cultures.
Microbiome A collective term for all the genomes of all the microorganisms in a microbial community
or sample.
Microbiota The community of microbes living in a given ecological site.
Mutualism Mutually beneficial relationship between host and hosted organisms.
Next generation Modern DNA sequencing techniques, as opposed to earlier efforts (Sanger sequencing),
sequencing that greatly reduce the time and cost required to sequence entire genomes.
Operational Roughly speaking, an alternative term for a taxon in microbiome studies, usually referring
taxonomic unit to species.
(OTU)
Probiotic The official WHO definition is “live microorganisms which when administered in
adequate amounts confer a health benefit on the host”.
Relative abundance The proportional abundance of an organism relative to the total abundance of all (similar)
organisms in a given ecosystem.
Taxon A taxonomic level for an organism, e.g. genus or family.
39
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Figure legends

Figure 1. Key host-related functions of the intestinal microbiota. The figure shows six
important functions of the intestinal microbiota that benefit its human host, as well as examples of
bacteria associated with these functions and factors influencing gut microbial composition. Many
more such functions have been described in the literature.
Figure 2. Visualization and analysis of microbial data. The visualization of microbial data may
include stacked bar charts, scatterplot graphs, shade plots, and network graphs. On the upper left
(A), a stacked bar chart depicts a hypothetical classification of the microbiota among two groups
of samples on a certain taxonomic level. Each vertical composite bar represents a sample from a
subject, each particular colored bar within the composite bar represents a taxon (e.g. a bacterial
genus), and the height of each colored bar reflects that taxon’s relative abundance. On the upper
right (B), a 3D scatterplot graph depicts a hypothetical output of non-metric multidimensional
scaling (MDS) analysis for two different groups of samples. The positioning of the samples reflects
the ranking of their similarity based on the metric used for analysis – e.g. the Bray-Curtis similarity
coefficient. The important feature is not sample positioning per se, but rather the distances between
the samples and their position relative to each other. In this hypothetical case, the samples tend to
cluster close to other samples of the same color and a clear partitioning in three-dimensional space
is apparent. Hence, the visualized data clearly suggest that the “average” microbial composition
of the red samples is considerably different from the “average” microbial composition of the green
samples. This visual difference would next be tested in formal statistical analysis, e.g. by
PERMANOVA.
40
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On the lower left (C), a shade plot (heatmap) depicts the relative abundance of 8 bacterial genera
for two groups of samples (10 samples for each group). In this hypothetical example, relative

abundances are reflected by the colored shading, which ranges from white (0%) to black (60%, the
highest relative abundance seen in these data). The differences are readily apparent between the
groups. Bacterial genera 1 to 4 are scarcely present in group 2, while the opposite is true for
bacterial genera 5 to 8. Again, such a group separation, although clear-cut, would next be tested
by an appropriate statistical method. On the lower right (D), an interaction network of bacterial
genera (nodes) and their correlations (lines) is presented. Such a visualization is generally an
advanced way of depicting the sample data, based on complex statistical software that should take
into account the compositionality of relative abundance values. Comparing networks of different
groups of samples may reveal different patterns of associations, which may be caused by differing
properties of the microbiota in each group. A discussion of networks and their applications can be
found in the supplementary text of our original study (reference 40).
41
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Figure 3. The heart-gut axis in heart failure. The pathophysiology of heart failure may lead to
increased leakage of gut microbial compounds into the bloodstream and cause low grade systemic

inflammation, which may aggravate heart failure status and complete a vicious cycle.
Accumulating evidence also suggests that the composition of the intestinal microbiota is altered in
the context of heart failure. LPS: lipopolysaccharide.
Figure 4. Evidence linking the intestinal microbiota with arterial hypertension. In an
interesting experiment by Li et al.54 (A), the intestinal microbiota of human donors with or without
hypertension was transplanted to germ-free mice, i.e. mice manipulated so that no microbes would
exist in their intestinal tracts. Ten weeks after transplantation, blood pressure was measured by the
tail cuff method and it was shown that the mice that had received the microbiota of hypertensive
subjects had significantly higher blood pressure than the mice that had received the microbiota of
the control subject. As this experiment involved a very small number of donors (2 hypertensives
and 1 normotensive), more evidence is required to solidify these findings. Part B of the figure
shows a schematic representation of how a healthy microbiota may modulate blood pressure and
reduce the risk of hypertension under a fiber rich diet. HTN: hypertension, SCFA: short-chain fatty
acids, BP: blood pressure.
Figure 5. Microbial links to atherosclerosis. The association of the intestinal microbiota with
atherosclerosis is based on infection, changes in lipid metabolism, induction of inflammatory
pathways, and production of bacterial metabolites that may exert proatherogenic effects. Overall,
inflammation caused by bacterial products or compounds may characterize the pathway linking
the microbiota with the atherosclerotic process. LPS: lipopolysaccharide, LBP: lipopolysaccharide
42
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binding protein, CD14: cluster of differentiation 14, TLR: Toll-like receptor, NF-κB: nuclear
factor kappa-light-chain-enhancer of activated B cells, TMAO: trimethylamine N-oxide.
43
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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The intestinal microbiota and cardiovascular disease

Themistoklis Katsimichas, MD, PhD,1,2 Alexios S. Antonopoulos, MD, PhD,1 Alexandros

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Tousoulis, MD, PhD1

Suita, Osaka, Japan.

Word count: 6,253 (main text), Figures: 5, Tables: 3, References: 107

Professor Dimitris Tousoulis, MD, PhD.

1st Cardiology Department, Athens Medical School, Hippokration General Hospital,

Vasilissis Sofias Avenue 114, PO 115 27, Athens, Greece.

Tel: (+30) 2132088099 Fax: (+30) 2132088676

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importance of the microbiota in the pathophysiology of cardiovascular disease. In this review,

the microbiota in cardiovascular disease, including heart failure, hypertension, and

relevant associations, connecting the observational and mechanistic aspects. Finally, we

knowledge which can impact everyday clinical practice.

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host-related physiological functions.1.2 Such functions include the synthesis of important

and diversity to a variety of human disorders.6

In this review, we focus on the associations of the intestinal microbiota with

atherosclerosis (Table 1), and discuss remaining challenges to be met.

2. The microbial ecosystem of the human gut

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fermentation of foods undigested by humans, vitamin synthesis, deconjugation of bile acids,

and, last but not least, resistance to colonization by or overgrowth of pathogenic

microorganisms (Figure 1).1,2,7

The composition of the intestinal microbiota is overwhelmingly dominated by

others.3,4,8,9 Especially diet exerts considerable influence in the shaping of a bacterial

diversity microbiota dominated by various species of Bifidobacterium, which thrive in the

In general, humans are immediately colonized at birth, the microbiota gradually

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genera include Prevotella and Ruminococcus.4,13

colonizing the gut in an antagonistic struggle against species of Bacteroides. Members of

contributing to the production of short-chain fatty acids.

which in subjects predisposed to chronic disorders may lead to an increase in opportunistic

promote local or systemic pathogenetic functional changes in susceptible individuals.6

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Amplicon sequencing most commonly involves the identification and sequencing of

of information, basically resulting in the identification and proportional calculation of bacterial

genera present in a sample.18

functional information.4,19 Through appropriate techniques, bacterial species and strains

be generated.19 This enables researchers to acquire information on important metabolic

the intestinal microbiota.

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statistical tests, as an inappropriate test will invariably lead to false conclusions.

Alpha diversity refers to specific characteristics of an ecological community, such as the

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speaking, a lower alpha diversity can be thought of as a negative phenomenon in a given

ecosystem of interacting species. Although alpha diversity is a common tool of describing

ecological communities, collapsing the incredible bacterial variation to a single index is a

limited approach to comparing groups of samples.

Beta diversity, multivariate analysis, and predictive functional profiling

calculation of a measure of similarity, most commonly the Bray-Curtis similarity coefficient

as the non-parametric “analysis of similarities” (ANOSIM) and the semi-parametric

“permutational multivariate analysis of variance” (PERMANOVA),24,25 can then be applied to

really is, regardless of its statistical significance.

Of note, multivariate analysis is not restricted to comparing samples in terms of

measured through next-generation sequencing or inferred by appropriate algorithms from 16S

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