Nathan S. Bryan, Gena Tribble, Nikola Angelov Baylor College of Medicine, The University of Texas Health Science Center at Houston
Having high blood pressure puts you at risk for heart disease and stroke, which are leading causes of death in the United States and worldwide. One out of every 3 Americans has hypertension and it is estimated that despite aggressive treatment with medications, only about half of those medicated have managed blood pressure. Recent discoveries of the oral microbiome that reduce inorganic nitrate to nitrite and nitric oxide provide a new therapeutic target for the management of hypertension. The presence or absence of select and specific bacteria may determine steady state blood pressure levels. Eradication of oral bacteria through anti-septic mouthwash or over use of antibiotics causes blood pressure to increase. Allowing recolonization of nitrate and nitrite reducing bacteria can normalize blood pressure. This review will provide evidence of the link between oral microbiota and the production of nitric oxide and regulation of systemic blood pressure. Management of systemic hypertension through maintenance of the oral microbiome is a completely new paradigm in cardiovascular medicine.
Human blood pressure regulation is complex, and despite decades of research into the variables affecting resting blood pressure, there is still a significant knowledge gap. Hypertension is highly prevalent in the adult population in the United States, especially among persons older than 60 years of age, and affects approximately 1 billion adults worldwide [1, 2]. In the United States, about 77.9 million (1 out of every 3) adults have high blood pressure. Among persons 50 years of age or older, isolated systolic hypertension is the most common form of hypertension [4, 5] and systolic blood pressure becomes more important than diastolic blood pressure as an independent risk predictor for most all cardiovascular related disease including end stage renal disease (ESRD) [6-9]. In some age groups, the risk of cardiovascular disease doubles for each increment of 20/10 mmHg of blood pressure, starting as low as 115/75 mmHg. In addition to coronary heart diseases and stroke, complications of raised blood pressure include heart failure, peripheral vascular disease, renal impairment, retinal hemorrhage and visual impairment.
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 (DALYS) or 3.7% of total DALYS. Globally, the overall prevalence of raised blood pressure in adults aged 25 and over was around 40% in 2008. The proportion of the world’s population with high blood pressure, or uncontrolled hypertension, fell modestly between 1980 and 2008. However, because of population growth and ageing, the number of people with uncontrolled hypertension rose from 600 million in 1980 to nearly 1 billion in 2008 .
Despite major advances in understanding the pathophysiology of hypertension and availability of antihypertensive drugs, suboptimal blood pressure control is still the most important risk factor for cardiovascular mortality. According to the AHA 2013 Statistics Fact Sheet, although 75% of people that know they have hypertension and are under current treatment, only about 52% of those have it controlled. The Systolic Blood Pressure Intervention Trial (SPRINT) showed that among adults with hypertension but without diabetes, lowering systolic blood pressure to a target goal of less than 120 mm Hg, as compared with the standard goal of less than 140 mm Hg, resulted in significantly lower rates of fatal and nonfatal cardiovascular events and death from any cause.. Because blood pressure remains elevated in ≈50% of all treated hypertensive patients [12, 13], and more effective blood pressure management can save lives, novel, efficacious and cost-effective therapeutic strategies are urgently required for the treatment of hypertension.
The discovery of endothelium derived relaxing factor (EDRF) in 1980 ** that was later identified as nitric oxide (NO) [15, 16]* revolutionized vascular biology and identified new strategies for controlling blood pressure. NO is produced endogenously from the five-electron oxidation of the guanidino nitrogen of L-arginine by the enzyme isoform eNOS (endothelial nitric oxide synthase) . NO produced or generated in the vasculature then diffuses into the underlying smooth muscle causing these muscles to relax. This results in vasodilation, causing a reduction in systemic blood pressure and an increase in blood flow and oxygen delivery to specific vascular beds. In healthy subjects, activation of eNOS causes vasodilation in both muscular conduit vessels and resistance arterioles. In contrast, in subjects with atherosclerosis or endothelial dysfunction, similar stimulation yields attenuated vasodilation in peripheral vessels and causes paradoxical vasoconstriction in coronary arteries, thus indicating a decrease in the production and/or bioavailability of NO [18, 19]. Emerging evidence shows that endothelial dysfunction and subsequent NO-deficiency are critically associated with the development of hypertension and other forms of cardiovascular disease . Hypertension accelerates atherosclerosis. Interestingly, endothelial dysfunction can be demonstrated in patients with risk factors for atherosclerosis in the absence of atherosclerosisitself [21, 22]. Experimental and clinical studies provide evidence that detective endothelial NO function is not only associated with all major cardiovascular risk factors, such as hyperlipidemia, diabetes, hypertension, smoking and the severity of established atherosclerosis, but also has a profound predictive value for future atherosclerotic disease progression . These results indicate that essential hypertension is characterized by an age-related reduction of nitric oxide production and bioavailability.
Human Oral Microbiome – Bacterial Nitrate Reduction
The human microbiome is composed of many different bacterial species, which outnumber our human cells ten to one and provide functions that are essential for our survival. The microbiota of the lower intestinal tract is widely recognized as playing a symbiotic role in maintaining a healthy host physiology  by participating in nutrient acquisition and bile acid recycling, among other activities. In contrast, although the role of oral microbiota in disease is well studied, specific contributions to host health are not well defined. A human nitrogen cycle has been identified. This pathway, termed the entero-salivary nitrate-nitrite-nitric oxide pathway, can positively affect nitric oxide homeostasis, and represents a potential symbiotic relationship between oral bacteria and their human hosts [25, 26]. It is now recognized that the oral commensal bacteria provide an important metabolic function in human physiology by contributing a nitric oxide synthase (NOS)-independent source of NO. This process is analogous to the environmental nitrogen cycle whereby soil bacteria convert atmospheric nitrogen oxides to usable forms for plant growth. Human nitrate reduction requires the presence of nitrate-reducing bacteria as mammalian cells cannot effectively reduce this anion.
Inorganic nitrite (NO2 – ) and nitrate (NO3 – ) are known as food additives cured meats and naturally occurring in green leafy vegetables [27, 28] but also as inert oxidative end-products of endogenous NO metabolism . However, from research performed over the past decade, it is now apparent that nitrate and nitrite are physiologically recycled in blood and tissue to form NO and other bioactive nitrogen oxides [30-33] and now considered essential nutrients [34*, 35*]. As a result, they should now be viewed as storage pools for NO-like bioactivity to be acted upon when enzymatic NO production from NOS is insufficient, such as during anaerobic conditions or uncoupling of NOS. The bio-activation of nitrate from dietary (mainly green leafy vegetables) or endogenous sources (oxidation of NO) requires its initial reduction to nitrite, and because mammals lack specific and effective nitrate reductase enzymes, this conversion is mainly carried out by commensal bacteria **. Dietary nitrate is rapidly absorbed in the upper gastrointestinal tract. In the blood, it mixes with the nitrate formed from the oxidation of endogenous NO produced from the NOS enzymes. After a meal rich in nitrate, the nitrate levels in plasma increase greatly and remain high for a prolonged period of time (the plasma half-life of nitrate is 5–6 hours). The nitrite levels in plasma also increase after nitrate ingestion after approximately 90 minutes  provide it can be reduce by oral nitrate reducing bacteria.
Although much of the nitrate is eventually excreted in the urine, up to 25% is actively taken up by the salivary glands and is concentrated up to 20-fold in saliva [37, 38]. In the mouth, commensal facultative anaerobic bacteria reduce salivary nitrate to nitrite during 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 1–2 mM, respectively . When saliva enters the acidic stomach (1–1.5 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 Figure 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 .
Salivary nitrate is metabolized to nitrite via a two-electron reduction, a reaction that mammalian cells are unable to perform, during anaerobic respiration by nitrate reductases produced by facultative and obligate anaerobic commensal oral bacteria [40, 41]. As illustrated in Figure 2, identification of specific partial denitrifying bacteria in the oral cavity can provide optimal conditions for nitrate reduction with nitrite accumulation in the saliva. Although a few nitrate-reducing bacteria have been identified in the oral cavity , we have analyzed nitrate reduction by bacterial communities present in tongue-scrapings from healthy human volunteers during four days of in vitro growth and performed a parallel metagenomic analysis of these samples to identify specific bacteria associated with nitrate reduction. Through 16S rRNA gene pyrosequencing and whole genome shotgun (WGS) sequencing and analysis, we identified specific taxa that likely contribute to nitrate reduction. Biochemical characterization of nitrate and nitrite reduction by four candidate species indicates that complex community interactions contribute to nitrate reduction . The bacterial communities had varying potential for nitrate reduction, and our study identified 14 candidate species that were present in communities with the best nitrate reduction activity. The fourteen species present at an abundance of at least 0.1% in the best nitrate-reducing sample and at the highest abundance in this sample compared to the intermediate and worst reducing sample that belonged to the genera of interest and were identified through 16S rRNA gene pyrosequencing and analysis were: Granulicatella adiacens, Haemophilus parainfluenzae, Actinomyces odontolyticus, Actinomyces viscosus, Actinomyces oris, Neisseria flavescens, Neisseria mucosa, Neisseria sicca, Neisseria subflava, Prevotella melaninogenica, Prevotella salivae, Veillonella dispar, Veillonella parvula, and Veillonella atypica. Additionally, Fusobacterium nucleatum and Brevibacillus brevis were designated as species of interest even though they were not at a relative abundance of at least 0.1% in the WGS best nitratereducing sample .
Previously the Doel et al., 2005, study isolated and identified five genera of oral nitrate reducing bacterial taxa on the tongues of healthy individuals: Veillonella, Actinomyces, Rothia, Staphylococcus, and Propionibacterium . In our investigation, Veillonella species were the most abundant group of nitrate reducers isolated from the tongue, followed by Actinomyces spp. Veillonella was the most abundant nitrate-reducing genus detected in the original tongue scrapings, although Prevotella, Neisseria, and Haemophilus were all found at a higher abundance than Actinomyces, highlighting the higher resolution of our study. This difference in resolution is likely due to our use of a sequencing-based approach, which allowed us to survey the native bacterial environment on the dorsal surface of the tongue without depending on the growth requirement necessary for classic culture-based techniques. Metagenomic data are important and informative in that one can determine what a bacterial community is capable of doing, yet it is limited in the sense that it cannot inform what the community is actually doing, which can vary under different circumstances. Thus, while each community had the same capacity for nitrate reduction, the true activity clearly differed between these communities. Furthermore, as not all healthy donors had nitrate-reducing bacteria in their oral cavity, there may be a significant number of individuals who may not have a functioning nitrate-nitrite-NO enterosalivary pathway to support systemic health. The presence or absence of these select bacteria may be a new determinant of nitrite and NO bioavailability in humans and, thus, a new consideration for cardiovascular disease risk. Further studies on multi-species biofilms integrating biochemical, metagenomic and metatranscriptomic data will answer these important questions and provide more information regarding the community dynamics that contribute to oral nitrate reduction that results in nitrite accumulation.
Nitrite Based Signaling
The production of nitrite from nitrate-reducing bacteria may have profound implications on the health of the human host. Numerous studies have shown that nitrite produced from bacterial nitrate reduction is an important storage pool for NO in blood and tissues when NOSmediated NO production is insufficient [44-48]. Nitrite is also an oxidative breakdown product of NO that has been shown to serve as an acute marker of NO flux/formation . Nitrite is now recognized as a cell 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 to activate soluble guanylyl cyclase (sGC) and increase cGMP levels in tissues  and lead to vasodilation [52, 53]. Therefore, it is an ideal candidate for restoring both cGMP-dependent and cGMP-independent NO signaling. In addition to the oxidation of NO, nitrite is also derived from reduction of salivary nitrate by commensal bacteria in the mouth and gastrointestinal tract [54, 55], as well as from dietary sources, such as meat, vegetables, and drinking water.
Since nitrate reduction by microbial communities generates nitrite, it is of great importance to be able to recognize specific bacteria in and on the body that are capable of generating nitrite from nitrate. Once nitrite is formed, it can be utilized as a substrate for NO production. Although largely inefficient, there exists a number of nitrite- reducing system in mammals. Nitrite reductase activity in mammalian tissues has been linked to the mitochondrial electron transport system [56, 57], protonation , deoxyhemoglobin , and xanthine oxidase [60, 61]. Nitrite can also transiently form S-nitrosothiols (RSNOs) under both normoxic and hypoxic conditions , and a recent study by Bryan et al demonstrates that steady state concentrations of tissue nitrite and S-nitroso species are affected by changes in dietary NOx (nitrite and nitrate) intake . Furthermore, enriching dietary intake of nitrite and nitrate translates into significantly less injury from myocardial infarction *. Previous studies demonstrated that nitrite therapy given intravenously prior to reperfusion protects against hepatic and myocardial ischemia/reperfusion (I/R) injury . Additionally, experiments in primates revealed a beneficial effect of long-term application of nitrite on cerebral vasospasm . Moreover, inhalation of nitrite selectively dilates the pulmonary circulation under hypoxic conditions in vivo in sheep . Topical application of nitrite improves skin infections and ulcerations . Furthermore, in the stomach, nitrite-derived NO seems to play an important role in host defense  and in regulation of gastric mucosal integrity . All of these studies, along with the observation that nitrite can act as a marker of NOS activity , have opened new avenues for the diagnostic and therapeutic applications of nitrite, especially in cardiovascular diseases, using nitrite as a marker as well as an active agent. Oral nitrite has also been shown to reverse L-NAME (NOS inhibitor)-induced hypertension and serve as an alternate source of NO in vivo . In fact, a report by Kleinbongard et al.  demonstrates that plasma nitrite levels progressively decrease with increasing cardiovascular risk. Since a substantial portion of steadystate nitrite concentrations in blood and tissue are derived from dietary sources , modulation of nitrate intake along with optimal nitrate reducing microbial communities may provide a first line of defense for conditions associated with NO insufficiency .
Eradication of Nitrate Reducing Bacteria Eliminates Health Benefits of Nitrate based Diets and Interventions
In various animal models and in humans, dietary nitrate supplementation has shown numerous beneficial effects, including a reduction in blood pressure, protection against ischemiareperfusion damage, restoration of NO homeostasis with associated cardioprotection, increased vascular regeneration after chronic ischemia, and a reversal of vascular dysfunction in the elderly [34, 35, 70, 71]. Some of these benefits were reduced or completely prevented when the oral microbiota were abolished with an antiseptic mouthwash [70, 72]. Plasma and salivary nitrite levels are abolished after a dietary nitrate load in healthy subjects taking an antiseptic mouthwash . It has been reported that dietary nitrate reduces blood pressure in healthy volunteers [46, 74], and that the effects are abolished after rinsing with oral antiseptic mouthwash **. Both strong and weak antibacterial agents suppress the rise in plasma nitrite observed following the consumption of a high nitrate diet and stronger antiseptics can influence the blood pressure response during low-intensity exercise . Additionally, it was recently shown that in the absence of any dietary modifications, a seven-day period of antiseptic mouthwash treatment to disrupt the oral microbiota reduced both oral and plasma nitrite levels in healthy human volunteers, and was associated with a sustained increase in both systolic and diastolic blood pressure . Oral nitrite exerts antihypertensive effects in the presence of antiseptic mouthwash that disrupts the enterosalivary circulation and reduction of nitrate . Altogether, these studies firmly establish the role for oral nitrate-reducing bacteria in making a physiologically relevant contribution to host nitrite and thus NO levels, with measureable physiological effects. It appears that providing nitrite can overcome the absence of microbial nitrate reduction.
Clearly, the potential for the entero-salivary nitrate-nitrite-NO pathway to serve as a NO bioavailability maintenance system by harnessing the nitrate reductase activity of specific commensal bacteria calls for studies that may be profound and truly transformative. These studies will have the potential to: 1) redefine the meaning of “healthy oral microbiome” to include microbes associated with NO production, 2) provide a new target for NO-based therapies and open a new direction in cardiovascular research and 3) allow development of new diagnostics targeted at specific oral microbial communities or select bacteria, the absence of which may reflect a state of NO insufficiency and change the treatment strategies for NO restoration in a number of different diseases. With the loss of NO signaling and homeostasis being one of the earliest events in the onset and progression of cardiovascular disease, targeting microbial communities early in the process may lead to better preventative interventions in cardiovascular medicine. This may also affect the way oral health professionals recommend oral hygienic practices. Manipulation of the human microbiome as a therapeutic target for disease management is on the near horizon. The oral cavity is an attractive target for probiotic and/or prebiotic therapy because of the ease of access. A full understanding of the entero-salivary nitrate-nitrite-NO pathway will require the generation and integration of a complete set of data from metagenomic, metatranscriptomic, metaproteomic and metametabolomic studies coupled to biochemical functional assays. The potential to restore the oral flora as a means to provide NO production is a completely new paradigm for NO biochemistry and physiology as well as for cardiovascular medicine and dentistry.
There is a known correlation between oral health and systemic disease . Importantly, because oral NO production is dependent on oral nitrate reducing bacteria, these observations suggest that the link between oral health issues such as chronic periodontitis and cardiovascular disease may be due in part to decreased abundance of nitrate reducers and concurrent increase of pathogenic bacterial species in the oral cavity. Disruption of nitrite and NO production in the oral cavity may contribute to the oral-systemic link between oral hygiene and cardiovascular risk and disease. The identification of new biomarkers for NO insufficiency and the exploitation of the oral microbiota to increase cardiovascular health will be enabled by further characterization of the enzymatic activities of native oral bacterial communities from larger healthy cohorts and specific patient populations. These cohorts should consist not only of specific U.S. population, but also of other around-the-world (European, Asian) populations. It is likely that the oral microbiomes of different ethnic groups, even those within different regions of the U.S., vary widely. It will be important to determine whether different nitrate-reducing communities are more prevalent in geographically dispersed healthy populations; likewise, it will also be important to determine whether different nitrate reducing communities are lacking in specific patient populations from around the world. If certain patient populations lack specific nitrate reducing bacteria, personalized treatments to enrich for nitrate reducers may be warranted. It may be time to discourage the use of antiseptic mouthwash. Additionally, while antibiotics are sometimes used to target specific bacterial species, it is possible that potential deleterious effects of antibiotic usage on nitrate-reducing communities may preclude the use of antibiotics in specific patient populations.
For the past 30 years, scientists have focused on NO production/regulation at the level of nitric oxide synthase (NOS), through the five-electron oxidation of L-arginine. However, this pathway becomes dysfunctional with age and disease . The notion that this deficiency can be overcome by targeting oral bacteria is profound and revolutionary. Therapeutically, then, perhaps an effective strategy to promote NO production and overcome conditions of NO insufficiency may not be targeted at eNOS, but rather to target specific oral nitrate reducing bacterial communities and increasing the consumption of nitrite and nitrate enriched foods and vegetables. From a public health perspective, we may be able to make better recommendations on diet and relevant host-microbe communities to affect dramatically the incidence and severity of a number of symptoms and diseases characterized by NO insufficiency. Development of novel therapeutics or strategies to restore NO homeostasis can have a profound impact on disease prevention . These new discoveries in the oral bacterial microbiome suggest that an effective strategy to promote therapeutically NO production and overcome conditions of NO insufficiency may not solely be to target NOS, but, rather, to focus on understanding specific oral bacterial communities and the optimal conditions for efficient oral nitrate reduction. It appears from early studies that many individuals may not have the optimal microbial communities for maximum nitrate reduction, causing a disruption in a critical NO production pathway. Understanding and harnessing this alternative pathway may prove to be a viable and costeffective strategy for maintaining NO homeostasis in humans. Because NO signaling affects all organ systems and almost all disease processes described to date, this novel approach to NO regulation has the potential to affect the study and treatment of many diseases across all organ systems.
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