1521-0103/358/1/103–108$25.00
233296
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
Minireviews
New Insight into the Dietary Cause of Atherosclerosis:
Implications for Pharmacology
Reynold Spector
Department of Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey
Received March 2, 2016; accepted April 21, 2016
ABSTRACT
At present, the guideline approach to the medical treatment and
prevention of atherosclerotic cardiovascular disease (ASCVD) is to
classify patients by risk and treat the known risk factors (contrib-
utory causes), e.g., hypertension, diabetes, obesity, smoking, and
poor diet, as appropriate. All high-risk patients should receive
statins. This approach has had substantial success but ASCVD
still remains the number one cause of death in the United States.
Until recently, the underlying cause of ASCVD remained unknown,
although a potential dietary cause was suggested by the fact that
vegetarians, especially vegans, have a much lower incidence of
ASCVD than animal flesh eaters. Recently, consistent with the
vegetarian data, substantial evidence for a cause of ASCVD in
animals and humans has been discovered. Trimethylamine (TMA)-
Introduction
In the United States, atherosclerosis in the coronary and
cerebral arteries is generally a causal factor in over 600,000
cardiac and 130,000 stroke deaths per year (Mozaffarian et al.,
2016). In most cases, the critical event (the “coup de grace”) is
a clot that forms over a rough or cracked, nonobstructive
atheromatous lesion, acutely obstructing blood flow and
causing distal tissue necrosis, although, less commonly,
emboli can also obstruct blood flow (Blumenthal and Kapur,
2006). Depending on where this occurs, the result may be a
fatal or nonfatal myocardial infarction (MI), ischemic stroke,
bowel infarction, or other less common syndromes. On the
basis of careful clinical research, seven prominent contrib-
utory causes (risk factors) for atherosclerosis have been
identified: increased serum cholesterol and blood pressure,
diabetes, obesity, a positive family history, smoking, and
an atherogenic diet (Mozaffarian et al., 2016). However, the
pathophysiology in each case is complex; for example, in-
creased blood pressure not only accelerates atherosclerosis
in the large arteries but also causes arteriosclerosis with
dx.doi.org/10.1124/jpet.116.233296.
ABBREVIATIONS: ASCVD, atherosclerotic cardiovascular disease; BMI, body mass index; CAD, coronary artery disease; DMB, 3, 3-dimethyl-1-
butanol; FMO3, flavin mono-oxygenase 3; HDL, high-density lipoprotein; HRT, hormone replacement therapy; LDL, low-density lipoprotein; MI,
myocardial infarction; RCT, reverse cholesterol transport; TMA, trimethylamine; TMAO, trimethylamine N-oxide.
http://dx.doi.org/10.1124/jpet.116.
J Pharmacol Exp Ther 358:103–108, July 2016
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containing dietary compounds in meat, milk, and other animal
foods (e.g., lecithin, choline, and carnitine) are converted by closely
related gut bacterial TMA lyases to TMA, which is absorbed and
converted predominantly by flavin mono-oxygenase 3 to the toxic
trimethylamine N-oxide (TMAO). TMAO causes atherosclerosis in
animals and is elevated in patients with coronary heart disease.
Inhibition of bacterial lyases in mice prevents TMA and secondarily
TMAO formation and atherosclerosis, strong evidence for the
TMAO hypothesis. At present, the challenge for the pharmaceutical
industry is to discover and develop a potent “broad spectrum”
bacterial lyase inhibitor that, along with diet and exercise, could, if
the TMAO hypothesis is correct, revolutionize the preventive
treatment of ASCVD.
arteriolar hyaline change or even medial necrosis in malig-
nant hypertension (Mozaffarian et al., 2016).
However, none of these “contributory causes” fully explains
the underlying cause and pathophysiology of the atheroscle-
rotic process in most cases. Yet, a somewhat successful ap-
proach has been to control these risk factors when present:
lowering blood pressure with safe and effective drugs and
blood cholesterol with statins; controlling type II diabetes
and obesity with drugs, diet, and weight loss; stopping smok-
ing and improving diets, e.g., with a vegetarian-type diet
(Mozaffarian et al., 2016). All these measures are useful and
have lowered age-adjusted mortality resulting from atheroscle-
rotic cardiovascular disease (ASCVD) by ∼30% from 2003 to
2013 (Mozaffarian et al., 2016). As a consequence, in 2016 for
the first time cancer (with more than 600,000 projected deaths)
may overtake heart disease as the leading cause of death in the
United States. Stroke, which used to be the second leading
cause of death, has now descended to fifth place after cancer,
heart disease, respiratory ailments, and accidents, thus attest-
ing to the success of pharmacologic control of risk factors,
primarily hypertension in this case. (Mozaffarian et al., 2016).
Notwithstanding these successes, myocardial and cerebral
infarctions (ASCVD) together remain the leading cause of
103
104 Spector
death in the United States and developed world (Mozaffarian
et al., 2016). On the basis of convincing epidemiology work,
there has been a strong suspicion that a dietary factor(s) might
be an underlying cause, i.e., a necessary and sufficient cause
exacerbated by the risk factors (contributory causes) enumer-
ated above. This hypothesis, described in detail below, has
recently been supported by substantial evidence in animals
and humans (Wang, et al., 2011; Koeth et al., 2013; Tang
et. al., 2013; Jonsson and Bäckhed; Shih, et al., 2015; Troseid,
et al., 2015; Wang et al., 2015; Warrier, et al., 2015). Briefly,
trimethylamine (TMA)–containing dietary compounds, found
mainly in meat, milk, some fish, and other animal foods (e.g.,
lecithin, choline, betaine, carnitine) are converted by TMA
lyases (lyases) in gut bacteria to TMA, which is absorbed
and then oxidized by hepatic flavin mono-oxygenase 3 (FMO3)
to the toxic trimethylamine N-oxide (TMAO). TMAO causes
atherosclerosis in animals and probably humans, and is
elevated in patients with atherosclerotic coronary artery
disease (CAD). Before reviewing the TMAO evidence, I will
review the cholesterol hypothesis, which for decades has been
widely accepted as the principal cause of atherosclerosis. I will
also briefly comment on the management of diabetes and
hypertension, since this is relevant to prevention of athero-
sclerosis, and finally the epidemiology work on dietary causes
of atherosclerosis.
The Cholesterol Hypothesis
Initially, the cholesterol hypothesis as a cause of athero-
sclerosis had three components: elevated high-density lipo-
protein (HDL) was protective and elevated low-density
lipoprotein (LDL) and triglycerides in blood were harmful.
The HDL and triglyceride components of the hypothesis are
not well supported and will not be discussed further (Spector,
2013; Medical Letter, 2013). In favor of the LDL component of
the cholesterol hypothesis are: 1) the higher the LDL choles-
terol, the greater the risk of CAD; 2) certain patients with
rare genetic disorders and very high serum cholesterol have
accelerated ASCVD; 3) feeding large quantities of cholesterol
to rabbits and other animals causes atherosclerosis; and 4)
statins, which lower cholesterol, are unequivocally beneficial
(Scandinavian Simvastatin Survival Study Group, 1994; Spector
2013; Mozaffarian et al., 2016).
There is, however, persuasive evidence that the LDL cho-
lesterol is not the underlying causal factor in most patients
with ASCVD:
1) More than one-third of MI patients have normal or even
low serum cholesterol, and many patients with elevated LDL
never manifest ASCVD, thus showing that increased serum
LDL is not a necessary or sufficient cause of ASCVD (Spector
2013).
2) In fact, although statins lower serum cholesterol and LDL
cholesterol, they do not work primarily by slowing or regress-
ing the atherosclerotic process (Blumenthal and Kapur, 2006).
In a large 2-year placebo-controlled randomized study in pa-
tients with CAD treated with high-dose lovastatin (80 mg)
or placebo, there was no difference in changes in coronary
anatomy as measured by quantitative coronary angiography
between drug and placebo groups, both at baseline and after 2
years of treatment (Blankenhorn et al., 1993). With maximal
dose rosuvastatin (40 mg) and atorvastatin (80 mg), in two
studies employing ultrasound, there was approximately a 1%
decrease in coronary atheroma volume compared with base-
line over 2 years (Nissen et al., 2006; Nicholls et al., 2011);
however, there were no controls in the these atheroma studies
and no luminal measurements, so these data are difficult to
interpret, unlike the Blankenhorn et al. (1993) study referred
to above with lovastatin (Blumenthal and Kapur, 2006). But
what can be said from these studies is that statins do not alter
the atheroma process much if at all. Yet, in a carefully done
double-blind, randomized clinical trial of secondary preven-
tion, there was approximately a 30% decrease in all-cause
mortality, as well as similar decreases in MI and stroke, in
patients randomized to moderate-dose simvastatin compared
with placebo patients (Scandinavian Simvastatin Survival
Study Group, 1994; Spector 2013, Mozaffarian et al., 2016).
This trial included 100% follow up for mortality, so there is
absolutely no ambiguity about the results. The mechanism
of this effect we now know is not attributable to decreasing
atherosclerosis per se but to stabilizing nonobstructing ath-
eromatous plaques and preventing clot formation on them
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when these plaques are rough or rupture (Blumenthal and
Kapur, 2006). Antiplatelet agents (e.g., aspirin) also help pre-
vent clots on such lesions (Mora, 2012).
3) Statin efficacy is independent of baseline serum choles-
terol. This has been shown in studies of both simvastatin (Heart
Protection Study Collaborative Group, 2002) and pravastatin
(Sacks et al., 2000).
4) Feeding huge amounts of cholesterol to rabbits is not a
good model of human ASCVD, unlike the TMAO data discussed
below.
5) Many drugs that lower serum LDL cholesterol and raise
HDL [e.g., hormone replacement therapy (HRT) and niacin]
either have no effect on ASCVD or are actually harmful
(Hulley et al., 1998; Boden et al., 2011; Spector, 2013). For
example, in the first large placebo-controlled trial of HRT in
women who had CAD, there was no cardiovascular benefit but
significantly more gall bladder and thromboembolic disease
in the HRT arm (Hulley et al., 1998). Subsequent controlled
trials of HRT confirmed no cardiovascular benefit or actual
harm (Spector and Vesell, 2002).
6) On February 15, 2016, the FDA made its decision about
the utility of Vytorin, a drug that combines 40 mg of simvastatin
with 10 mg of ezetimbe, a drug which blocks cholesterol
absorption. As expected, Vytorin lowers serum total and LDL
cholesterol more than simvastatin (40 mg) alone. However,
after a careful review of a 7-year, approximately 18,000-
patient randomized controlled trial of Vytorin versus sim-
vastatin (40 mg) alone (Cannon, et al., 2015) and other data,
the FDA and its advisory committee concluded that Vytorin
(40/10) was no better than simvastatin (40 mg) in preventing
cardiovascular death, stroke, and MI. In short, Vytorin,
which lowers total and LDL serum cholesterol more than
simvastatin alone, is not more effective clinically. This result
is consistent with a recent analysis of the statin data from
extant randomized clinical trials (Takagi and Umemoto,
2013). This analysis does not make unjustified linear as-
sumptions, suggests that the clinical benefit of statins is
achieved with doses of 40 mg of atorvastatin or simvastatin,
and that further lowering of blood cholesterol achieves very
little (Spector and Snapinn, 2011; Takagi and Umemoto,
2013). For all the above reasons, serum cholesterol must
be considered a contributory cause of ASCVD in some cases
only.

It is worth noting that the new national guidelines (by the
Veterans Administration, US Department of Defense, Amer-
ican Heart Association, American College of Cardiology) for
the treatment and prevention of ASCVD have finally recog-
nized these facts about statins (Downs and O’Malley, 2015).
On the basis of the current treatments available, the guide-
lines now correctly focus on risk: BP and weight control;
cessation of smoking; exercise; and statins and aspirin (Mora,
2012) for high-risk patients, with the use of moderate-dose
inexpensive generic statins (e.g., 20 mg or preferably 40 mg
of atorvastatin or simvastatin) for almost all high-risk pa-
tients irrespective of serum cholesterol. The guidelines have
eschewed hypothetical unproven schemes like treatment to
arbitrary LDL goals (Downs and O’Malley, 2015). In other
words, the guidelines implicitly recognize that statins do not
appreciably alter atherosclerosis but do prevent MI, stroke,
and death by the mechanism noted above, and thereby reduce
risk (Krumbolz and Hayward, 2010; Spector 2013; Downs and
O’Malley, 2015).
Hypertension and Diabetes
Hypertension and diabetes have in common the ability to
accelerate the development of atherosclerosis in large arteries
and, in their more severe forms, to cause arteriolosclerosis, a
separate problem. Arteriolosclerosis can manifest itself as la-
cunar infarcts in brain, kidney damage, retinal destruction,
and, in diabetes, neuropathy owing to decreased blood flow to
peripheral nerves. Current treatment of hypertension with
inexpensive safe drugs (ACE inhibitors, angiotensin-receptor
blockers, b-blockers, calcium channel blockers, and diuretics)
is effective in lowering blood pressure; in many patients these
drugs drastically reduce the risks of arteriolosclerosis and
stroke (Mozaffarian, et al., 2016). Treatment of hypertension
also slows the atheromatous process in large arteries. How-
ever, good control of diabetes slows the arteriolar damage but,
for unclear reasons, does not have much effect on large artery
atherosclerosis. In diabetes, statins and other drugs are needed
to combat the risks of atherosclerosis (Downs and O’Malley,
2015; Mozaffarian, et al., 2016).
The Role of Diet
A tremendous number of epidemiology/observation stud-
ies and a few controlled trials have been published on the
relationship between diet and ASCVD. The vast majority of
these epidemiologic/observation studies suffer from multiple
methodological deficiencies and the results are often ambig-
uous, misleading, or actually quantitatively incorrect, as pointed
out by ourselves, and many others (Spector and Vesell, 2000,
2002, 2006a; Campbell and Campbell 2006). These methodolog-
ical deficiencies include poor validation of food questionnaires,
infrequent or no assessment of change in diet, inattention to the
Hill criteria for proving causality, improper use of statistics in
nonrandomized studies, futile attempts to focus on one variable,
“data dredging” (also termed data mining, i.e., looking for
correlations in large data sets without a predetermined hypoth-
esis, a procedure that leads to many false positive correlations),
and many other errors (Spector and Vesell, 2000; 2002, 2006a;
Campbell and Campbell 2006). Two important examples of
false positives studies include those that concluded that HRT
and mega-vitamin E prevented ASCVD (Spector and Vesell,
Cause of Atherosclerosis: Implications for Pharmacology 105
2002). However, subsequent controlled trials show they were
ineffective or harmful (Spector and Vesell, 2002). Another
example is the advice some decades ago to switch from butter
(saturated fat) to margarine on the basis of incorrect epidemiology/
observation studies suggesting that saturated fat was harmful
(Siri-Tarino et al., 2010); this was terrible advice since most
margarine contains trans-fats, now known to be atherogenic. In
recent years it has been recognized that eating saturated fat
is in fact not harmful per se and the original studies were
incorrect. Stampfer, the head of epidemiology at the Harvard
School of Public Health, which carried out many of these ear-
lier epidemiology/observation studies admitted, “This has been
our greatest failure and disappointment—that we have not
learned what people can do to lower their risk” [quoted in
Campbell and Campbell (2006), who provided a detailed critique
of such studies].
Because of the tremendous variability in such studies, sev-
eral authors have published meta-analyses of nonrandom-
ized epidemiology/observation studies in an attempt to answer
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certain questions. For example: Does red meat increase the
risk of ASCVD? A recent meta-analysis of 20 epidemiology/
observation studies with over one million individuals sug-
gests it does not (Micha et al., 2010). Is this correct? See
below.
However, there have been several, I believe, more revealing
epidemiology studies. During and after the Second World
War in England, there was more than a 50% decline in the
incidence of diabetes and deaths from heart disease (Trowell,
1974). This was ascribed to the meager diets during and after
the war. Consistent with the dietary hypothesis were studies
of CAD and diabetes in Japanese men in Japan, Hawaii, and
California (Kagan et al., 1974; Marmot et al., 1975; Worth
et al., 1975; Tsunehara et al., 1990). At the time of these
studies in Japanese men, there was a gradient by location,
with CAD and diabetes being lowest in Japan, next Hawaii,
and highest in California. The investigators in these studies
suggested that the diet in Japan at the time was mainly a
vegetarian-type diet and not the “richer” animal protein/fat
diet of California. However, from these studies of Japanese
men in Japan, Hawaii, and California, only qualitative con-
clusions can be drawn. Third the “China Study” showed that a
mainly plant-based diet in China was associated with much
less ASCVD than a nonvegetarian animal-based Western
diet (Campbell and Campbell, 2006). Moreover, there have
been several small controlled trials that have also suggested a
vegetarian-type diet is better with respect to morbidity and
mortality than a standard Western diet in patients with CAD
(Campbell and Campbell, 2006).
A recent analysis of other studies in homogenous popula-
tions provides further powerful evidence for the efficacy of
vegetarian diets in minimizing ASCVD mortality and mor-
bidity. Unlike many of the negative or ambiguous studies that
compared subjects who ate lesser or greater quantities of animal
products noted above, e.g., the Nurses Study, these vegetarian
studies compared vegetarians versus animal-product eaters
(Fraser 2009). The results are clear and consistent: Vegetarians
enjoy lower overall mortality and 32% lower incidence of coronary
heart disease mortality than nonvegetarians. Moreover, there is a
clear gradient in morbidity from vegans (no animal products
permitted), to lacto-ovo vegetarians (eggs and milk permit-
ted), to pesco-vegetarians (eggs, milk, and fish permitted), to
semivegetarians, to nonvegetarians (Fraser, 2009).
106 Spector
However, causal interpretations are complex; between vegans
and nonvegetarians there is also a gradient favoring vegans in
body mass index (BMI), diabetes, and hypertension. Vegans in
one study had a BMI 18% lower than nonvegetarians and a
prevalence of diabetes and hypertension 22% and 25%, re-
spectively, of nonvegetarians. Therefore, these results only allow
one to conclude that a vegetarian (especially a vegan diet) is
helpful in preventing ASCVD, but not to conclude exactly why. Is
it the diet, the decreased BMI, blood pressure, diabetes, or a
combination?
New Information
Thus, in 2010, by lowering risk factors we could effectively
treat some patients with established ASCVD or prevent clinical
ASCVD in those with risk factors. However, the underlying
cause of atherosclerosis in the larger arteries remained un-
known, although as noted above, there was convincing evidence
that a vegetarian diet (especially a vegan diet) was healthier
than the Western (animal-based) diet. By 2010, the best
advice was Aristotle’s: “a sound mind in a sound body” and
“moderation in all things.” This was translated into: Keep
one’s weight at a level that maintained BMI between 20 and
25, be sure the diet contains the known essential nutrients
(ions, vitamins, and essential fatty and amino acids), enjoy
a balanced diet even including an occasional egg or two (Virtanen,
et al., 2016), and tend toward a vegetarian diet; finally, do not
forget to exercise.
However, beginning in 2011, investigators in the Cleveland
Clinic and other venues published a series of groundbreaking
papers suggesting TMAO formed from dietary TMA-containing
nutrients, especially animal products, might, in fact, be the
“toxic” metabolite, a prime cause of atherosclerosis (Wang
et al., 2011; Koeth et al., 2013; Tang et al., 2013; Jonsson
and Bäckhed, 2015; Shih et al., 2015; Troseid et al., 2015;
Wang et al., 2015; Warrier et al., 2015). Basically, what
these investigators have shown is that the TMA covalently
incorporated in lecithin, choline, betaine, or carnitine can be
metabolized in gut by bacterial lyases to release TMA, a gas.
TMA is readily absorbed by mice and humans and converted to
TMAO by liver FMO 3; TMAO can cause atherosclerosis in
mice (Wang et al., 2011). This process however is variable and
complex. First, different gut bacteria have different abilities
to form TMA from TMA-containing nutrients. This is in part
because there are two distinct bacterial lyases; one consists
of the cut C and cut D gene products and is termed cut C/D
choline TMA lyase. The other consists of the Yea W/X gene
products (Yea W/X TMA lyase). This promiscuous lyase can
convert lecithin, choline, betaine, carnitine, and butyrobetaine
to TMA (Falony et al., 2015; Wang et al., 2015). Both of these
lyases are inhibitable to a greater or lesser extent by 3, 3
dimethyl-1-butanol (DMB) (Wang et al., 2015). There is also
a third enzyme in human gut bacteria consisting of the gene
products Cnt A and Cnt B termed carnitine Cnt A/B oxygen-
ase/ reductase that converts carnitine to TMA and malic
semialdehyde (Falony et al., 2015; Wang et al., 2015). The
quantitative importance of this enzyme remains uncertain. It
is not inhibited by DNB (Wang et al., 2015). Some gut bacteria
contain no TMA-forming enzymes; others contain one or two
or all three of these enzymes (Falony et al., 2015). Further
complicating matters, different individuals contain different
levels of gut bacteria that can form TMA in vivo.
When TMA formation in mice is blocked with either DMB or
antibiotics, TMAO is not formed, and in mice atherosclerosis
is greatly diminished especially on a high-choline diet (Wang
et al., 2015). Moreover, especially interesting is the finding
that DNB blockade of lyase activity leads to gut bacteria with
less lyase activity (Wang et al., 2015). It is worth noting the
nontoxic DMB is found in balsamic vinegars, red wines and
extra-virgin olive, and grapeseed oils (Wang et al., 2015).
After absorption, TMA is N-oxidized mainly by FMO3
(Wang et al., 2015; Warrier et al., 2015). Congenital absence
of FMO3 leads to “fish odor syndrome,” a result of the inability
to metabolize TMA; TMA smells like rotting fish (Wang, et al.,
2015). To avoid the odor, such patients avoid TMA-containing
foods. In normal humans, TMA is almost completely N-oxidized
to TMAO after a 600-mg TMA base “load” (Zhang et al., 1995).
However, obligate heterozygotes (parents of fish odor–affected
children) on a normal diet oxidize more than 90% of the TMA
to TMAO, but after a 600-mg TMA base load they N-oxidize
only 76 6 3% to TMAD, clearly different than normal (96 6 2%.)
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(Zhang et al., 1995). Furthermore in a study of one-hundred
volunteers challenged with a 600-mg base TMA, only one was
within the range found among obligate heterozygotes (Zhang
et al., 1995). Interestingly, in three normal Japanese women
during menstruation, the ratio of TMA to TMAO in the urine
increased to levels seen in obligate heterozygotes, suggesting a
hormonal effect (Shimizu et al., 2007). If this observation can be
confirmed and extended, it might explain why menstruating
women are protected from ASCVD, i.e., lower production of the
toxic TMAO.
In mice, there are large effects on FMO3 activity, with cholic
acid causing upregulation and testosterone downregulation;
inhibition of FMO3 has effects on reverse cholesterol transport
(RCT), hepatic inflammation, and bile formation (Bennett
et al., 2013; Warrier et al., 2015). Thus, inhibition of FMO3 to
block formation of TMAO would not only cause fish odor
syndrome but also might have effects on RCT, exacerbate
hepatic inflammation, and have other complex effects. How-
ever, humans lacking FMO3, beside fish odor, are said to be
healthy. Moreover, as noted above, normal humans (with the
possible exception of menstruating women) efficiently convert
TMA to TMAO, and the relevance of the mouse studies dealing
with regulation of FMO3 (Bennett et al., 2013; Warrier et al.,
2015) remains to be determined. In any case, it may be more
acceptable to reduce dietary precursors of TMA than to reduce
FMO3 activity.
In human studies, consistent with animal studies, TMAO
was elevated in patients with ASCVD and could be suppressed
with antibiotics, presumably by killing gut bacteria (Wang
et al., 2015). Especially revealing was a 3-year prospective
cohort study of 4007 patients with CAD in which an elevated
TMAO concentration was associated with an increased risk
of major ASCVD events (the risk in the highest quintile of
TMAO was 2.5 times the lowest quintile; 95% confidence in-
terval, 2–3.3; p..001) (Tang et al., 2013). Likewise, TMAO
was increased in heart-failure patients with ischemic heart
disease (Troseid et al., 2015).
This work has important implications for understanding
much previous data. Assuming TMAO is the principal “culprit
agent” (cause) of atherosclerosis, then it is clear why patients
with normal cholesterol, weight, blood pressure, and blood
sugar (i.e., without diabetes) and who don’t smoke could have
severe ASCVD. They may produce excess TMAO because of

diet and lyase-containing intestinal bacteria. In a steady-
weight individual, it is also clear that saturated fats and
sugar, which are metabolized to CO2 and H2O for energy (ATP
formation), should not be harmful per se (Siri-Tarino et al.,
2010). Moreover, there is no evidence that dietary cholesterol
is harmful (Virtanen et al., 2016). Thus, if the TMAO hypothesis
is correct, the recent focus of dietary therapy (beyond a balanced
diet containing essential nutrients) should not be on dietary
sugar, cholesterol, and saturated fat, if preventing atherosclero-
sis is the objective, but on TMA-containing nutrients like lecithin,
carnitine, choline and betaine; in other words, a diet more like a
vegetarian-type diet should be followed. The potential benefit of
a vegetarian-type diet which minimizes TMAO formation thus
becomes clear. However, a word of caution is necessary; there
might be other unknown dietary factors and metabolites not
present or formed in vegetarian-type diets that, along with
TMAO, are toxic.
The TMAO hypothesis also is consistent with the fact that
high doses of statins, which lower LDL cholesterol by 40–60%,
have no or minimal effect on coronary atherosclerosis (by arteriog-
raphy or ultrasound) (Blankenhorn et al., 1993; Blumenthal
and Kapur, 2006). As noted above, statins stabilize plaques
thereby preventing clotting, obstruction, and distal ischemia/
necrosis. Aspirin and other anti-platelets agents also prevent
platelet clotting (Mora, 2012).
Implications for Pharmacology
One of the most important decisions in drug discovery and
development is the selection of the correct target (Spector
and Vesell, 2006b). When this is done, the pharmaceutical
industry has made remarkably safe and effective drugs and
vaccines. In the current case, we have very strong preclinical
and clinical data that TMA lyases in gut bacteria are a worthy
target, although there are different forms of this lyase in
different bacterial species as noted above (Wang et al., 2015).
DMB, which has an IC50 of ∼10 mM in lysates of bacterial cells,
is a weak competitive inhibitor but has been able to provide
proof of principle (Wang et al., 2015; Jonsson and Bäckhed,
2015). The IC50 is probably much higher in intact bacterial
cells. Extrapolating from the mouse work, probably 10–20 g a
day of DMB in divided doses would be required in humans to
inhibit intestinal bacterial lyase activity. What is needed is
a full-throttle approach by the pharmaceutical companies
to make a potent “broad-spectrum” bacterial lyase inhibitor,
an inhibitor with an IC50 of more like 10 nM, preferably one
not absorbed, so it “worked” only on bacteria in the gut or, if
ultimately absorbed, rapidly metabolized. If such a compound
was discovered, phase I and II trails in humans could be
performed to test the compound’s ability to block TMAO pro-
duction in vivo. Further development in large long-term
phase III clinical trials versus placebo could be done to test (on
top of statins and aspirin) whether such a compound could
decrease MI, strokes, and cardiovascular deaths. If the TMAO
hypothesis is correct, there should be a major effect. What is
not known is exactly how TMAO might “cause” ASCVD in
humans. There is speculation that TMAO may interfere with
RCT (Wang et al., 2015; Warrier et al., 2015). There are also
convincing data that TMAO enhances platelet hyper-reactivity
in animals and humans, and thrombosis risk in mice (Zhu et al.,
2016). Such an effect if it occurs in vivo in humans could at least
potentially explain the ability of TMAO to “cause” ASCVD in
Cause of Atherosclerosis: Implications for Pharmacology 107
humans. With a potent lyase inhibitor as a tool, these hypotheses
(RCT inhibition, platelet hyper-reactivity, and others) could be
tested to help uncover the mechanism of TMAO toxicity if, in
fact, TMAO is a causative factor in ASCVD.
In summary, in the past five years, several investigative
groups have brought forward an exciting hypothesis for the
actual cause of atherosclerosis. These investigators have also
potentially explained the benefits of vegetarian-type diets.
Moreover, they have provided strong evidence for a pharma-
cological target for the treatment and prevention of ASCVD
(Wang et al., 2015; Zhu et al., 2016). If an effective broad-
spectrum bacterial lyase inhibitor (or blocker of TMAO toxicity)
can be discovered and developed that can prevent ASCVD,
especially when coupled with a prudent vegetarian-type diet
and exercise, we may at long last have effective tools to decrease
substantially the morbidity and mortality currently associated
with ASCVD.
Acknowledgments
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2021
The author thanks Michiko Spector for preparation of the
manuscript.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Spector.
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