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Curr Hypertens Rep (2017) 19:33

DOI 10.1007/s11906-017-0725-2

GUT MICROBIOME, SYMPATHETIC NERVOUS SYSTEM, AND HYPERTENSION (M RAIZADA AND E M. SUMNERS, SECTION EDITORS)

Oral Microbiome and Nitric Oxide: the Missing Link


in the Management of Blood Pressure
Nathan S. Bryan 1 & Gena Tribble 2 & Nikola Angelov 2

# Springer Science+Business Media New York 2017

Abstract Having high blood pressure puts you at risk for Hypertension
heart disease and stroke, which are leading causes of death
in the USA and worldwide. One out of every three Americans Human blood pressure regulation is complex, and despite de-
has hypertension, and it is estimated that despite aggressive cades of research into the variables affecting resting blood
treatment with medications, only about half of those medicat- pressure, there is still a significant knowledge gap.
ed have managed blood pressure. Recent discoveries of the Hypertension is highly prevalent in the adult population in
oral microbiome that reduces inorganic nitrate to nitrite and the USA, especially among persons older than 60 years of
nitric oxide provide a new therapeutic target for the manage- age and affects approximately one billion adults worldwide
ment of hypertension. The presence or absence of select and [1, 2]. In the USA, about 77.9 million (one out of every three)
specific bacteria may determine steady-state blood pressure adults have high blood pressure. Among persons 50 years of
levels. Eradication of oral bacteria through antiseptic mouth- age or older, isolated systolic hypertension is the most com-
wash or overuse of antibiotics causes blood pressure to in- mon form of hypertension [4, 5], and systolic blood pressure
crease. Allowing recolonization of nitrate- and nitrite- becomes more important than diastolic blood pressure as an
reducing bacteria can normalize blood pressure. This review independent risk predictor for most all cardiovascular-related
will provide evidence of the link between oral microbiota and disease including end-stage renal disease (ESRD) [69]. In
the production of nitric oxide and regulation of systemic blood some age groups, the risk of cardiovascular disease doubles
pressure. Management of systemic hypertension through for each increment of 20/10 mmHg of blood pressure, starting
maintenance of the oral microbiome is a completely new par- as low as 115/75 mmHg. In addition to coronary heart diseases
adigm in cardiovascular medicine. and stroke, complications of raised blood pressure include
heart failure, peripheral vascular disease, renal impairment,
Keywords Gut microbiome . Blood pressure . Hypertension . retinal hemorrhage, and visual impairment.
Nitric oxide . Diet . Nitrate . Nitrite Worldwide, raised blood pressure is estimated to cause 7.5
million deaths, about 12.8% of the total of all deaths. This
accounts for 57 million disability adjusted life years
This article is part of the Topical Collection on Gut Microbiome,
(DALYS) or 3.7% of total DALYS. Globally, the overall prev-
Sympathetic Nervous System, and Hypertension alence of raised blood pressure in adults aged 25 and over was
around 40% in 2008. The proportion of the worlds population
* Nathan S. Bryan with high blood pressure, or uncontrolled hypertension, fell
Nathan.bryan@bcm.edu modestly between 1980 and 2008. However, because of pop-
ulation growth and aging, the number of people with uncon-
1
Department of Molecular and Human Genetics, Baylor College of
trolled hypertension rose from 600 million in 1980 to nearly
Medicine, The University of Texas Health Science Center at one billion in 2008 [10].
Houston, Houston, TX, USA Despite major advances in understanding the pathophysi-
2
Department of Periodontics, The University of Texas Health Science ology of hypertension and availability of antihypertensive
Center at Houston, 7000 Fannin, Houston, TX 77030, USA drugs, suboptimal blood pressure control is still the most
33 Page 2 of 8 Curr Hypertens Rep (2017) 19:33

important risk factor for cardiovascular mortality. According Human Oral MicrobiomeBacterial Nitrate
to the AHA 2013 Statistics Fact Sheet, although 75% of peo- Reduction
ple that know they have hypertension and are under current
treatment, only about 52% of those have it controlled. The The human microbiome is composed of many different bac-
Systolic Blood Pressure Intervention Trial (SPRINT) showed terial species, which outnumber our human cells ten to one
that among adults with hypertension but without diabetes, and provide functions that are essential for our survival. The
lowering systolic blood pressure to a target goal of less than microbiota of the lower intestinal tract is widely recognized as
120 mmHg, as compared with the standard goal of less than playing a symbiotic role in maintaining a healthy host physi-
140 mmHg, resulted in significantly lower rates of fatal and ology [24] by participating in nutrient acquisition and bile acid
nonfatal cardiovascular events and death from any cause [11]. recycling, among other activities. In contrast, although the role
Because blood pressure remains elevated in 50% of all treat- of oral microbiota in disease is well studied, specific contribu-
ed hypertensive patients [12, 13], and more effective blood tions to host health are not well defined. A human nitrogen
pressure management can save lives, novel, efficacious, and cycle has been identified. This pathway, termed the
cost-effective therapeutic strategies are urgently required for enterosalivary nitrate-nitrite-nitric oxide pathway, can posi-
the treatment of hypertension. tively affect nitric oxide homeostasis and represents a poten-
tial symbiotic relationship between oral bacteria and their hu-
man hosts [25, 26]. It is now recognized that the oral com-
mensal bacteria provide an important metabolic function in
Nitric Oxide human physiology by contributing a nitric oxide synthase
(NOS)-independent source of NO. This process is analogous
The discovery of endothelium derived relaxing factor (EDRF) to the environmental nitrogen cycle, whereby soil bacteria
in 1980 [14] that was later identified as nitric oxide (NO) convert atmospheric nitrogen oxides to usable forms for plant
[15, 16] revolutionized vascular biology and identified new growth. Human nitrate reduction requires the presence of
strategies for controlling blood pressure. NO is produced en- nitrate-reducing bacteria as mammalian cells cannot effective-
dogenously from the five-electron oxidation of the guanidino ly reduce this anion.
nitrogen of L-arginine by the enzyme isoform endothelial ni- Inorganic nitrite (NO2) and nitrate (NO3) are known as
tric oxide synthase (eNOS) [17]. NO produced or generated in food additives to cured meats and naturally occurring in green
the vasculature then diffuses into the underlying smooth mus- leafy vegetables [27, 28] but also as oxidative end products of
cle causing these muscles to relax. This results in vasodilation, endogenous NO production [29]. However, from research per-
causing a reduction in systemic blood pressure and an increase formed over the past decade, it is now apparent that nitrate and
in blood flow and oxygen delivery to specific vascular beds. nitrite are physiologically recycled in blood and tissue to form
In healthy subjects, activation of eNOS causes vasodilation in NO and other bioactive nitrogen oxides [30, 3133] and now
both muscular conduit vessels and resistance arterioles. In considered essential nutrients [34, 35]. As a result, they should
contrast, in subjects with atherosclerosis or endothelial dys- now be viewed as storage pools for NO-like bioactivity to be
function, similar stimulation yields attenuated vasodilation in acted upon when enzymatic NO production from NOS is insuf-
peripheral vessels and causes paradoxical vasoconstriction in ficient, such as during anaerobic conditions or uncoupling of
coronary arteries, thus indicating a decrease in the production NOS. The bioactivation of nitrate from dietary (mainly green
and/or bioavailability of NO [18, 19]. Emerging evidence leafy vegetables) or endogenous sources (oxidation of NO) re-
shows that endothelial dysfunction and subsequent NO defi- quires its initial reduction to nitrite, and because mammals lack
ciency are critically associated with the development of hy- specific and effective nitrate reductase enzymes, this conversion
pertension and other forms of cardiovascular disease [20]. is mainly carried out by commensal bacteria [36]. Dietary
Hypertension accelerates atherosclerosis. Interestingly, endo- nitrate is rapidly absorbed in the upper gastrointestinal tract. In
thelial dysfunction can be demonstrated in patients with risk the blood, it mixes with the nitrate formed from the oxidation of
factors for atherosclerosis in the absence of atherosclerosis endogenous NO produced from the NOS enzymes. After a meal
itself [21, 22]. Experimental and clinical studies provide evi- rich in nitrate, the nitrate levels in plasma increase greatly and
dence that detective endothelial NO function is not only asso- remain high for a prolonged period of time (the plasma half-life
ciated with all major cardiovascular risk factors, such as hy- of nitrate is 56 h). The nitrite levels in plasma also increase after
perlipidemia, diabetes, hypertension, smoking, and the sever- nitrate ingestion after approximately 90 min [37] provided it can
ity of established atherosclerosis but also has a profound pre- be reduce by oral nitrate-reducing bacteria. Although much of
dictive value for future atherosclerotic disease progression the nitrate is eventually excreted in the urine, up to 25% is
[23]. These results indicate that essential hypertension is char- actively taken up by the salivary glands and is concentrated up
acterized by an age-related reduction of nitric oxide produc- to 20-fold in saliva [37, 38]. In the mouth, commensal faculta-
tion and bioavailability. tive anaerobic bacteria reduce salivary nitrate to nitrite during
Curr Hypertens Rep (2017) 19:33 Page 3 of 8 33

anaerobic respiration by the action of nitrate reductases [26, 36].


After a dietary nitrate load, the salivary nitrate and nitrite levels
can approach 10 mM and 12 mM, respectively [37]. When
saliva enters the acidic stomach (11.5 L per day), much of
the nitrite is rapidly protonated to form nitrous acid (HNO2;
pKa 3.3), which decomposes further to form NO and other
nitrogen oxides [32, 33]. A simplified human nitrogen cycle is
illustrated in Fig. 1. More recent studies indicate that nitrite does
not have to be protonated to be absorbed and is about 98%
bioavailable when swallowed in an aqueous solution [39].
Salivary nitrate is metabolized to nitrite via a two-electron
reduction, a reaction that mammalian cells are unable to per-
form, during anaerobic respiration by nitrate reductases pro-
duced by facultative and obligate anaerobic commensal oral
bacteria [40, 41]. As illustrated in Fig. 2, identification of
specific partial denitrifying bacteria in the oral cavity can pro-
vide optimal conditions for nitrate reduction with nitrite accu-
mulation in the saliva. Although a few nitrate-reducing bacte-
ria have been identified in the oral cavity [42], we have ana-
lyzed nitrate reduction by bacterial communities present in
tongue scrapings from healthy human volunteers during 4 days
of in vitro growth and performed a parallel metagenomic anal-
ysis of these samples to identify specific bacteria associated Fig. 2 The biological nitrogen cycle. Nitrate is reduced all the way down
to elemental nitrogen by a series of enzymatic steps from nitrate reductase
with nitrate reduction. Through 16S rRNA gene pyrosequenc- (NR), nitrite reductase [3], NO reductase (NOR), and nitrous oxide
ing and whole genome shotgun (WGS) sequencing and anal- reductase (N2OR). Identifying bacteria or communities that only reduce
ysis, we identified specific taxa that likely contribute to nitrate nitrate and/or nitrite will allow for nitrite accumulation and more efficient
reduction. Biochemical characterization of nitrate and nitrite NO
reduction by four candidate species indicates that complex
community interactions contribute to nitrate reduction [43]. worst reducing sample that belonged to the genera of interest
The bacterial communities had varying potential for nitrate and were identified through 16S rRNA gene pyrosequencing
reduction, and our study identified 14 candidate species that and analysis were Granulicatella adiacens, Haemophilus
were present in communities with the best nitrate reduction parainfluenzae, Actinomyces odontolyticus, Actinomyces
activity. The 14 species present at an abundance of at least viscosus, Actinomyces oris, Neisseria flavescens, Neisseria
0.1% in the best nitrate-reducing sample and at the highest mucosa, Neisseria sicca, Neisseria subflava, Prevotella
abundance in this sample compared to the intermediate and melaninogenica, Prevotella salivae, Veillonella dispar,
Veillonella parvula, and Veillonella atypica. Additionally,
Fusobacterium nucleatum and Brevibacillus brevis were des-
DIET ignated as species of interest even though they were not at a
NO3- relative abundance of at least 0.1% in the WGS best nitrate-
reducing sample [43]. Previously, the Doel et al., 2005, study
isolated and identified five genera of oral nitrate-reducing
BACTERIA bacterial taxa on the tongues of healthy individuals:
2 e- reduction
Veillonella, Actinomyces, Rothia, Staphylococcus, and
Oxyheme -
proteins NO2 Propionibacterium [42]. In our investigation, Veillonella spe-
cies were the most abundant group of nitrate reducers isolated
Oxygen, BACTERIA from the tongue, followed by Actinomyces spp. Veillonella
ceruloplasmin 1 e- reduction
Acid was the most abundant nitrate-reducing genus detected in
Deoxy-hemoproteins the original tongue scrapings, although Prevotella, Neisseria,
NO and Haemophilus were all found at a higher abundance than
NOS Actinomyces, highlighting the higher resolution of our study.
oxidation L-arginine
reduction This difference in resolution is likely due to our use of a
Fig. 1 The human nitrogen cycle whereby nitrate is serially reduced to sequencing-based approach, which allowed us to survey the
nitrite and NO providing the host with a source of bioactive NO native bacterial environment on the dorsal surface of the
33 Page 4 of 8 Curr Hypertens Rep (2017) 19:33

tongue without depending on the growth requirement neces- nitrosothiols (RSNOs) under both normoxic and hypoxic con-
sary for classic culture-based techniques. Metagenomic data ditions [61], and a recent study by Bryan et al. demonstrates
are important and informative in that one can determine what a that steady-state concentrations of tissue nitrite and S-nitroso
bacterial community is capable of doing, yet it is limited in the species are affected by changes in dietary NOx (nitrite and
sense that it cannot inform what the community is actually nitrate) intake [30]. Furthermore, enriching dietary intake of
doing, which can vary under different circumstances. Thus, nitrite and nitrate translates into significantly less injury from
while each community had the same capacity for nitrate re- myocardial infarction [44]. Previous studies demonstrated
duction, the true activity clearly differed between these com- that nitrite therapy given intravenously prior to reperfusion
munities. Furthermore, as not all healthy donors had nitrate- protects against hepatic and myocardial ischemia/reperfusion
reducing bacteria in their oral cavity, there may be a significant (I/R) injury [62]. Additionally, experiments in primates re-
number of individuals who may not have a functioning vealed a beneficial effect of long-term application of nitrite
nitrate-nitrite-NO enterosalivary pathway to support systemic on cerebral vasospasm [63]. Moreover, inhalation of nitrite
health. The presence or absence of these select bacteria may selectively dilates the pulmonary circulation under hypoxic
be a new determinant of nitrite and NO bioavailability in conditions in vivo in sheep [64]. Topical application of nitrite
humans and, thus, a new consideration for cardiovascular dis- improves skin infections and ulcerations [65]. Furthermore, in
ease risk. Further studies on multi-species biofilms integrating the stomach, nitrite-derived NO seems to play an important
biochemical, metagenomic, and metatranscriptomic data will role in host defense [36] and in regulation of gastric mucosal
answer these important questions and provide more informa- integrity [66]. All of these studies, along with the observation
tion regarding the community dynamics that contribute to oral that nitrite can act as a marker of NOS activity [49], have
nitrate reduction that results in nitrite accumulation. opened new avenues for the diagnostic and therapeutic appli-
cations of nitrite, especially in cardiovascular diseases, using
nitrite as a marker as well as an active agent. Oral nitrite has
Nitrite-Based Signaling also been shown to reverse L-NAME (NOS inhibitor)-in-
duced hypertension and serve as an alternate source of NO
The production of nitrite from nitrate-reducing bacteria may in vivo [67]. In fact, a report by Kleinbongard et al. [68]
have profound implications on the health of the human host. demonstrates that plasma nitrite levels progressively decrease
Numerous studies have shown that nitrite produced from bac- with increasing cardiovascular risk. Since a substantial portion
terial nitrate reduction is an important storage pool for NO in of steady-state nitrite concentrations in blood and tissue are
blood and tissues when NOS-mediated NO production is in- derived from dietary sources [30], modulation of nitrate in-
sufficient [44, 4548]. Nitrite is also an oxidative breakdown take along with optimal nitrate-reducing microbial communi-
product of NO that has been shown to serve as an acute marker ties may provide a first line of defense for conditions associ-
of NO flux/formation [49]. Nitrite is now recognized as a cell ated with NO insufficiency [50].
signaling molecule that can act as a storage from of NO as well
as a NO independent signal [30, 50]. Nitrite is in steady-state
equilibrium with S-nitrosothiols [30, 51] and has been shown Eradication of Nitrate-Reducing Bacteria Eliminates
to activate soluble guanylyl cyclase (sGC) and increase cGMP Health Benefits of Nitrate-Based Diets
levels in tissues [30] and lead to vasodilation [52, 53]. and Interventions
Therefore, it is an ideal candidate for restoring both cGMP-
dependent and cGMP-independent NO signaling. In addition In various animal models and in humans, dietary nitrate sup-
to the oxidation of NO, nitrite is also derived from reduction of plementation has shown numerous beneficial effects, includ-
salivary nitrate by commensal bacteria in the mouth and gas- ing a reduction in blood pressure, protection against ischemia-
trointestinal tract [54, 55], as well as from dietary sources, reperfusion damage, restoration of NO homeostasis with as-
such as meat, vegetables, and drinking water. sociated cardioprotection, increased vascular regeneration af-
Since nitrate reduction by microbial communities generates ter chronic ischemia, and a reversal of vascular dysfunction in
nitrite, it is of great importance to be able to recognize specific the elderly [34, 35, 69, 70]. Some of these benefits were re-
bacteria in and on the body that are capable of generating duced or completely prevented when the oral microbiota were
nitrite from nitrate. Once nitrite is formed, it can be utilized abolished with an antiseptic mouthwash [69, 71]. Plasma and
as a substrate for NO production. Although largely inefficient, salivary nitrite levels are abolished after a dietary nitrate load
there exists a number of nitrite-reducing system in mammals. in healthy subjects taking an antiseptic mouthwash [72]. It has
Nitrite reductase activity in mammalian tissues has been been reported that dietary nitrate reduces blood pressure in
linked to the mitochondrial electron transport system [56, healthy volunteers [46, 73], and that the effects are abolished
57], protonation [31], deoxyhemoglobin [58], and xanthine after rinsing with oral antiseptic mouthwash [74]. Both strong
oxidase [59, 60]. Nitrite can also transiently form S- and weak antibacterial agents suppress the rise in plasma
Curr Hypertens Rep (2017) 19:33 Page 5 of 8 33

nitrite observed following the consumption of a high nitrate suggest that the link between oral health issues such as chronic
diet and stronger antiseptics can influence the blood pressure periodontitis and cardiovascular disease may be due in part to
response during low-intensity exercise [75]. Additionally, it decreased abundance of nitrate reducers and concurrent increase
was recently shown that in the absence of any dietary modifi- of pathogenic bacterial species in the oral cavity. Disruption of
cations, a 7-day period of antiseptic mouthwash treatment to nitrite and NO production in the oral cavity may contribute to
disrupt the oral microbiota reduced both oral and plasma ni- the oral-systemic link between oral hygiene and cardiovascular
trite levels in healthy human volunteers and was associated risk and disease. The identification of new biomarkers for NO
with a sustained increase in both systolic and diastolic blood insufficiency and the exploitation of the oral microbiota to in-
pressure [74]. Oral nitrite exerts antihypertensive effects in crease cardiovascular health will be enabled by further charac-
the presence of antiseptic mouthwash that disrupts the terization of the enzymatic activities of native oral bacterial
enterosalivary circulation and reduction of nitrate [76]. communities from larger healthy cohorts and specific patient
Altogether, these studies firmly establish the role for oral populations. These cohorts should consist not only of specific
nitrate-reducing bacteria in making a physiologically relevant US population but also of other around-the-world (European,
contribution to host nitrite and thus NO levels, with Asian) populations. It is likely that the oral microbiomes of
measureable physiological effects. It appears that providing different ethnic groups, even those within different regions of
nitrite can overcome the absence of microbial nitrate the US, vary widely. It will be important to determine whether
reduction. different nitrate-reducing communities are more prevalent in
geographically dispersed healthy populations; likewise, it will
also be important to determine whether different nitrate-
Conclusion reducing communities are lacking in specific patient populations
from around the world. If certain patient populations lack spe-
Clearly, the potential for the enterosalivary nitrate-nitrite-NO cific nitrate-reducing bacteria, personalized treatments to enrich
pathway to serve as a NO bioavailability maintenance system for nitrate reducers may be warranted. It may be time to discour-
by harnessing the nitrate reductase activity of specific commensal age the use of antiseptic mouthwash. Additionally, while antibi-
bacteria calls for studies that may be profound and truly transfor- otics are sometimes used to target specific bacterial species, it is
mative. These studies will have the potential to (1) redefine the possible that potential deleterious effects of antibiotic usage on
meaning of healthy oral microbiome to include microbes asso- nitrate-reducing communities may preclude the use of antibi-
ciated with NO production, (2) provide a new target for NO-based otics in specific patient populations.
therapies and open a new direction in cardiovascular research, and For the past 30 years, scientists have focused on NO
(3) allow development of new diagnostics targeted at specific oral production/regulation at the level of nitric oxide synthase
microbial communities or select bacteria, the absence of which (NOS), through the five-electron oxidation of L-arginine.
may reflect a state of NO insufficiency and change the treatment However, this pathway becomes dysfunctional with age and
strategies for NO restoration in a number of different diseases. disease [78]. The notion that this deficiency can be overcome
With the loss of NO signaling and homeostasis being one of the by targeting oral bacteria is profound and revolutionary.
earliest events in the onset and progression of cardiovascular dis- Therapeutically, then, perhaps an effective strategy to promote
ease, targeting microbial communities early in the process may NO production and overcome conditions of NO insufficiency
lead to better preventative interventions in cardiovascular medi- may not be targeted at eNOS, but rather to target specific oral
cine. This may also affect the way oral health professionals rec- nitrate-reducing bacterial communities and increasing the con-
ommend oral hygienic practices. Manipulation of the human sumption of nitrite and nitrate enriched foods and vegetables.
microbiome as a therapeutic target for disease management is From a public health perspective, we may be able to make
on the near horizon. The oral cavity is an attractive target for better recommendations on diet and relevant host-microbe
probiotic and/or prebiotic therapy because of the ease of access. communities to affect dramatically the incidence and severity
A full understanding of the enterosalivary nitrate-nitrite-NO path- of a number of symptoms and diseases characterized by NO
way will require the generation and integration of a complete set insufficiency. Development of novel therapeutics or strategies
of data from metagenomic, metatranscriptomic, metaproteomic, to restore NO homeostasis can have a profound impact on
and metametabolomic studies coupled to biochemical functional disease prevention [79]. These new discoveries in the oral
assays. The potential to restore the oral flora as a means to provide bacterial microbiome suggest that an effective strategy to pro-
NO production is a completely new paradigm for NO biochem- mote therapeutically NO production and overcome conditions
istry and physiology as well as for cardiovascular medicine and of NO insufficiency may not solely be to target NOS, but,
dentistry. rather, to focus on understanding specific oral bacterial com-
There is a known correlation between oral health and sys- munities and the optimal conditions for efficient oral nitrate
temic disease [77]. Importantly, because oral NO production is reduction. It appears from early studies that many individuals
dependent on oral nitrate-reducing bacteria, these observations may not have the optimal microbial communities for
33 Page 6 of 8 Curr Hypertens Rep (2017) 19:33

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